2008, Volume 24, Number 3, Page(s) 194-212
Pathology of malignant gliomas: Challenges of everyday practice and the WHO 2007
Tarık TİHAN1, Ayça ERŞEN2
1 University of California, San Francisco, Department of Pathology, Neuropathology Unit, SAN FRANCISCO
2 Dokuz Eylül University, School of Medicine, Department of Pathology, İZMİR
Keywords: Malignant glioma, glioblastoma, oligodendroglioma, oligoastrocytoma, ependymoma
The recent revision of the “2007 World Health Organization
Classification of Tumours of the Central Nervous
System” introduces a series of new entities and variants
in addition to bringing more clarity to existing
ones. It is critical for the practicing surgical pathologist
to be aware of these changes, especially those relating to
common primary tumors such as malignant gliomas.
This study presents a critical review of the changes and
attempts to provide practical insights for the surgical
The morphological spectrum of malignant gliomas is
quite diverse, and with definition of newer variants and
patterns, there is an increasing need to be more specific
on the type and the grade of these aggressive neoplasms.
The added value of special stains, immunohistochemistry
and molecular/genetic analysis is expected to
gradually increase in everyday practice. Thus, the surgical
pathologist must be in tune with the progress in
these fields and must be in a position to apply and interpret
Appropriate management of the patients and the correct
interpretation of the disease always depend on effective,
direct communication of the neuropathologist,
neuroradiologist and the neurosurgeon, coupled with
the application of carefully chosen ancillary techniques.
The combination of collaborative efforts and special
techniques is certain to be more critical in the future,
and effective utilization of all these elements will better
characterize these neoplasms and improve patient management.
The new “2007 World Health Organization Classification Tumors of the Central Nervous
System” (WHO 2007) clarified and revised
some of the highly diverse and diagnostically
challenging tumor categories 1. Two clarifications
effect the organization of the classification
scheme. First, WHO 2007 provides a better definition
of types and subtypes of brain tumors.
All brain tumors have now been categorized as
either Entity, Variant, or Histological Pattern.
An Entity represents a unique form of a neoplastic
disease with a defined clinicopathological
spectrum and is given its own chapter in WHO
2007. Variant represents a significant subtype
of an entity with sufficiently distinctive biological
properties and/or clinical behavior. Histological
Pattern represents a particular differentiation
pattern or phenotype that does not have a
different biological behavior or prognosis within
a specific entity.
The second important clarification is an attempt
to better define “Grading” philosophy of
WHO 2007. The grading, occasionally detached
from histological typing, reports a “stage of malignancy”
or “biological behavior”. The revised
scheme defines grades as follows:
GRADE I=typically well-circumscribed,
slowly progressing and may be cured by resection;
GRADE II=partly or mostly infiltrative
with low proliferation rates, but have a higher likelihood
of recurrence compared to grade I tumors;
GRADE III=histologically malignant and
require aggressive adjuvant therapy; GRADE
IV=highly aggressive and usually rapidly fatal
with all the histological features of malignancy.
While the improvements in the current
classification scheme are not revolutionary, they
allow better definition and description of the
primary central nervous system (CNS) tumors,
and will significantly affect everyday surgical
THE SPECTRUM OF MALIGNANT GLIOMAS
The following review focuses on the practical
and clinical aspects of malignant gliomas,
all of which require multi-modality therapy, and
are not likely to be cured by surgery alone. While
“malignant glioma” does not constitute a specific
pathological entity, the term identifies a
group of aggressive tumors that require a multidisciplinary
Malignant gliomas include a range of neoplasms
from astrocytoma to ependymoma,
with rare anaplastic forms of circumscribed
gliomas such as pleomorphic xanthoastrocytoma
with anaplastic features 2. The overwhelming
majority of malignant gliomas are WHO
grade III (anaplastic) and IV (glioblastoma) astrocytomas.
High grade oligodendrogliomas,
oligoastrocytomas and ependymomas are less
common tumors included in this general category.
Pathological features of malignant gliomas
vary based on the tumor type 3. In addition to
the typical architectural and cytological features,
molecular and genetic distinctions are increasingly
being made among types of malignant
gliomas 4,5. It is well recognized that a
significant number of infiltrating gliomas emerge
as low grade tumors and eventually progress
to high grade 6,7. An even larger group is malignant
at diagnosis with only limited options
for therapy and a short survival. The tumors in
the latter group may have “low-grade” microscopic
regions that are critical in the recognition
of the type of malignant glioma. This large body
of information will be briefly summarized for
each tumor type with only limited references to
the molecular alterations, since a detailed discussion
is beyond the scope of this article.
(Infiltrating Astrocytoma WHO GRADE III-Fig. 1)
Intraoperatively, there may be little or no
external abnormality, since most anaplastic astrocytomas
are deep hemispheric lesions with
only limited involvement of the cortex. Large
tumors may have a superficial component that
expand the gyri or cause surface discoloration.
In some examples, a striking demarcation can be
observed on imaging studies. The infiltrative
nature of a diffuse astrocytoma is most evident
to the neurosurgeon, who usually finds its boundaries
difficult or impossible to define intraoperatively.
Some anaplastic astrocytomas in the
brain stem produce enlargement of the pons without
creating a discrete mass. Macroscopically,
the anatomic details of the region are often lost,
and the structures appear distorted or occasionally
‘swollen' (Fig. 2a). Deep white matter tissue
fragments from anaplastic astrocytomas are
typically dusky, speckled gray with variable
consistency from soft to almost gelatinous. Microcysts,
which are often not visible, may give a
spongy appearance to larger tissue fragments.
Recognizing the extent of the tumor can be even
more challenging in anaplastic astrocytomas of
the spinal cord.
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|Fig 1: Anaplastic astrocytoma, typical MRI appearance with limited mass effect and significant contrast enhancement. It is important to note that a significant percentage of anaplastic astrocytomas do not show contrast enhancement. A) axial T1-weighted image; B) axial T2-weighted image; C) axial FLAIR; D) axial contrast-enhanced T1-weighted image.
In recent years, successful use of the ultrasonic
aspirator (CUSA) has made neuropathologists'
task even more challenging, since the tissue
obtained during these procedures are, at
best, suboptimal for pathological typing or grading.
In tissues obtained by CUSA, the macroscopic
as well as microscopic inspection is often
of little value. The gross evaluation post radiation
treatment specimens is equally challenging.
There is much more complexity to the texture
and appearance of these specimens that prevent
recognizing tumor tissue from reactive changes
8. One major caveat of CUSA material is the
extensive “fried-egg” appearance of cells in tissue
that can mislead to the diagnosis of oligodendroglioma.
Anaplastic astrocytomas often exhibit distinct
hypercellularity with readily identifiable
nuclear irregularities 9. The cell density is often
a magnitude higher than the normal brain
parenchyma, and a few fold of that seen in low
grade infiltrating astrocytoma (Fig. 2b). Most
anaplastic astrocytomas consist of cells with
small amount of cytoplasm (except for gemistocytes),
scant and short cell processes, and
markedly hyperchromatic nuclei. Typically, numerous
delicate processes in a glial cell are more
indicative of reactive astrocyte. The morphological
characteristics of the nuclei are critical in
diagnosis, yet any individual feature should be
carefully interpreted, since there is considerable
overlap between neoplastic and non-neoplastic
processes in terms of nuclear size and shape.
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|Fig 2: A) Anaplastic astrocytoma, gross appearance of anaplastic astrocytoma that distorts and expands the midbrain; B) typical hypercellularity and scattered mitoses; C) granular cell pattern of anaplastic astrocytoma; D) glioneuronal tumor with neuropil-like islands (all images H&E original magnification x200).
A critical microscopic feature of anaplastic
astrocytoma is the presence of mitotic figures
10,11. Finding even a single mitotic figure in
a surgical specimen can be associated with a
slightly poorer prognosis when compared to
typical grade II diffuse astrocytomas 12. Nevertheless,
presence of a single mitotic figure in
a well-sampled tumor specimen (when most of
the tumor is available for pathology) may not be
sufficient to increase the tumor grade to anaplastic
13. However a cut-off value for mitoses
has been elusive and there are different approaches.
Our recommendation is to identify at least
one mitotic figure for each large tissue fragment,
and one can be confident about the diagnosis
of anaplastic astrocytoma when a search
readily identifies numerous mitotic figures.
Necrosis or vascular endothelial proliferation
is not expected in anaplastic astrocytomas.
It must be noted that focal ischemic type necrosis
and linear type vascular proliferation can occur
in an astrocytic neoplasm independent of its
aggressiveness. Especially in the setting of prior
adjuvant therapy or surgical procedures (e.g.
stereotactic biopsy) such focal changes should
be interpreted with caution.
Anaplastic astrocytomas vary in architectural
and cytological composition, as to give the
tumor a “variably variable” appearance. One
common architectural element is a microcyst
containing mucinous material, typical of lowgrade
astrocytomas 11. The presence of microcysts
is not indicative of a particular tumor
grade, even though earlier reports claim a prognostic
significance to the microcystic change
14. Other uncommon architectural patterns
include perineuronal satellitosis and cortical calcifications,
which are more typical of oligodendrogliomas.
Focal myxoid/mucinous change
and cartilaginous metaplasia have also been reported
in anaplastic astrocytomas, and production
of cartilage had been related to the ability of
tumor cells to secrete mucopolysaccharides
One common cell type is the so-called gemistocytic
cell that appears swollen with round
to oval but distinctly eosinophilic cytoplasm,
small number of truncated processes, and a
hyperchromatic, eccentric nucleus. Gemistocytic
cells can be present in any infiltrating astrocytoma,
yet the tumors composed of >20%
such cells have been classified under the ‘gemistocytic
astrocytoma' category 16. It is important
to note that any given anaplastic astrocytoma
may contain gemistocytes, and the 20%
cut-off value currently accepted by WHO 2007
for “gemistocytic astrocytoma” is almost entirely
arbitrary. Gemistocytic cells have a low
proliferation index, while the small neoplastic
astrocytes that invariably accompany them
show higher proliferative capacity. Even though
suggestion was made to implicate the gemistocytic
morphology as a marker of poor prognosis,
there is insufficient data to suggest that gemistocytic
morphology is an independent prognostic
indicator 3. Gemistocytic cells can be
confused with the “minigemistocytes” commonly
seen in a subgroup of oligodendrogliomas.
The minigemistocytes of oligodendroglioma
have significant overlap with the typical gemistocytes
in astrocytomas, but are smaller with
more concentric collection of intermediate filaments
and strong GFAP staining. There is almost
no definitive criterion to separate minigemistocytes
of oligodendroglioma from astrocytic
gemistocytes, and the diagnosis should be
made by the overall histological features of the
A less common cytological variation is the
neoplastic cells with a highly granular cytoplasm
(Fig. 2c). Granular cells can be seen in
many astrocytomas, but tumors that are predominantly
composed of such cells are rare 17.
Infiltrating astrocytomas with granular cell features
(the so-called granular cell astrocytoma)
can resemble macrophage infiltrate or a reactive
“Glioneuronal tumor with neuropil-like islands”
recently described by Teo et. al. 19, has
been recognized as an aggressive glioneuronal
tumor. Most of these tumors are supratentorial,
but a case has been described in the spinal cord
20. These uncommon infiltrating tumors contain
microscopically well defined, round to oval
islands composed of a delicate, neuropil-like
matrix with synaptophysin positivity. The neuropil-like islands are
surrounded by oligodendrocyte-like cells in a rosetted fashion (Fig. 2d).
These cells as well as more atypical cellular elements
show immunoreactivity for neuronal antibodies.
The glial component consists of irregular
atypical cells and GFAP-positive fibrillary
and gemistocytic elements 21. Currently the
WHO 2007 recognizes this neoplasm as a histologic
pattern within anaplastic astrocytoma.
While this may not be the final designation for
these uncommon neoplasms, their biological behavior
is similar to infiltrating astrocytomas of
Smear preparations from anaplastic astrocytomas
are often suggestive, if not diagnostic
of an infiltrating astrocytic neoplasm. However,
grading of infiltrating astrocytomas is not
recommended during intraoperative consultations
due to significant geographic variation in tumor
composition, and the possibility of a higher
grade area elsewhere not sampled for intraoperative
analysis. Typically, frozen sections from
infiltrating astrocytomas have substantial freezing
artifact that prevent identification of mitotic
figures or the extent of nuclear pleomorphism.
Therefore, proper intraoperative smear
preparations are vitally important in determining
the cytological irregularities and mitotic figures.
Intraoperative smears are also of great
help in determining the ‘background fibrillarity'
indicative of incorporated neuropil, and the fibrillarity
emanating from neoplastic cells that often
implies an astrocytic phenotype. Florid reactive
changes incited by inflammation or infectious
agents such as the JC virus may cause significant
atypia in astrocytic nuclei, and can easily
be interpreted as astrocytoma. It is critical to
have a fair understanding of the radiological and
clinical features of the case, and discuss the findings
with the neurosurgeon to avoid any misinterpretation.
Although the astrocytic processes are often
evident on routine stains, the diagnosis is aided
by identification of these processes on immunohistochemistry
for glial fibrillary acidic
protein (GFAP). GFAP staining should be considered
a marker for glial phenotype and not as
evidence of astrocytic differentiation. It should
also be noted that some astrocytomas stain poorly
with GFAP, and not all neoplastic cells in a
given astrocytoma are GFAP positive. True gemistocytes
are only weakly positive for GFAP,
whereas the minigemistocytes of oligodendrogliomas
show stronger staining. Neuronal stains
are of little value in the characterization of anaplastic
astrocytomas. The most important contribution
is probably the immunohistochemical
stains for neurofilament protein (NF) that can
assist in the recognition of invaded neuropil and
substantiate the degree of tissue infiltration.
Both Vimentin (VIM) and S-100 protein stain
most astrocytomas along with the incorporated
cells and elements of neuropil. Astrocytic neoplasms,
especially anaplastic astrocytomas have
been reported to stain more avidly with antibodies
against VIM in comparison to oligodendrogliomas,
but a statistically significant difference
could not be found 22. There is variable
data on the intensity of VIM staining in different
types and grades of gliomas 23-25. In practice,
the greatest value of VIM is to recognize the suitability
of paraffin tissue for immunohistochemistry.
VIM should stain a significant number
of normal CNS components, vessels, reactive as
well as neoplastic glial cells. A completely negative
VIM is unlikely to be accurate, and identifies
tissue or the method as inappropriate for
immunohistochemistry. Anaplastic astrocytomas
can rarely stain for cytokeratins, which is
often not a problem, but this may be challenging
in less differentiated glial neoplasms 26. As a
surrogate marker for cell proliferation, Ki-67
(MIB-1 antibody) is widely used in clinical
practice, although its impact on the final grading
or prognostication is debatable. There are more
studies than one could care to list on MIB-1 labeling
of astrocytic neoplasms. The studies on
paraffin samples are often confounded by the
fact that the determination of a particular labeling
index is highly method-dependent. Suffice
it to say that MIB-1 staining that is readily perceivable
on low power magnification is compatible
with a high-grade astrocytoma, and is
much more typical of anaplastic than grade II
astrocytomas. The percentage of MIB-1 positive
nuclei differs widely and can range from 2% to
10% or even higher for anaplastic astrocytoma.
A significant percentage of anaplastic astrocytomas,
especially the gemistocytic variant
is immunoreactive for p53 protein. Positive staining
in large number of nuclei is typically indicative
of a stabilizing mutation, even though
most antibodies recognize both the wild type
and mutant p53 protein. Since positive nuclei
have been encountered in infectious as well as
reactive conditions, rare scattered positivity
should be interpreted cautiously. Furthermore,
since a significant number of tumors are negative
with this stain, absence of p53 staining does
not exclude anaplastic astrocytoma.
Astrocytoma, WHO Grade IV-Fig. 3)
Glioblastoma (GBM) causes significant
alterations in the brain parenchyma that are readily
observed radiologically and macroscopically.
Tumors expand the gyri, create texture
and color alterations often familiar to the neurosurgeon.
The tumor tissue is variable in color,
ranging from grayish yellow suggestive of necrosis
to dark brown hemorrhage or thrombosis.
The intraoperative finding of thrombosed vessels
is a well-recognized ominous sign cited by
neurosurgeons. Despite the false impression of
circumscription intraoperatively, the tumor extends
far beyond the visible abnormality. Normal
appearing tissue submitted to pathology
will harbor significant number of infiltrating astrocytes.
GBM can extend to the contralateral
hemisphere via the corpus callosum, a structure
involved in many cases. Although most GBMs
are not well circumscribed, demarcation is a feature
of some giant cell GBMs and gliosarcomas.
These two tumor subtypes can mimic metastatic
carcinomas, and occasionally meningiomas
radiologically and intraoperatively 27,28.
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|Fig 3: Glioblastoma, typical MRI appearance: A) axial T1-weighted image; B) axial T2-weighted image; C) axial FLAIR; D) axial contrast-enhanced T1-weighted image.
The cytological and architectural features
of GBM are remarkably diverse, and there is
virtually no limit to the variations one can describe
within the spectrum of such neoplasms
(Fig. 4). It is this diversity of phenotypic characteristics
that implies GBM is not a single neoplastic
entity, but the final, and most malignant
culmination of glial neoplasms from diverse genetic
and etiological origins. Typically, the fibrillarity
and variable degree of cell processes are
readily evident, confirming the glial nature of
GBMs. The tumor cells are markedly pleomorphic
and hyperchromatic, and mitotic figures can
be readily detected. The vascular endothelial
proliferation in GBMs is polymorphous and includes
not only endothelial cells but also smooth
muscle cells and pericytes (Fig. 5a). A somewhat
less well-defined term ‘microvascular proliferation'
has been used instead of vascular proliferation.
An overwhelming majority of GBMs
exhibit necrosis, and some may display a hypercellular
ribbon of neoplastic astrocytes around
the necrotic foci, referred as pseudopalisading
necrosis. In some areas, necrosis is associated
with a linear pattern of proliferating vascular
structures. This linear pattern of vascular proliferation
can also be observed around non-neoplastic
cysts and is not a sign of aggressive biological
behavior (Fig. 5b). The vascular proliferation
and necrosis with pseudopalisading comprise
the essential histological correlates that define
GBM in addition to nuclear pleomorphism
and mitotic figures 11.
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|Fig 4: Histological diversity of glioblastoma: patterns and variants A) giant cell ; B) small cell ; C) pleomorphic; D) sarcomatoid; E) gemistocytic F) PNET-like areas in otherwise typical glioblastomas (all images H&E original magnification x200).
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|Fig 5: Glioblastoma, A) bona fide vascular endothelial proliferation B) neovascularization around non-neoplastic cyst, not considered a valid criterion in grading (all images H&E original magnification x200).
The distinctive subtypes for GBMs include
the giant cell GBM and the gliosarcoma,
which are recognized by the WHO as GBM variants.
The former is typically composed of extremely large cells with bizarre nuclei and abundant
cytoplasm, admixed with smaller and more
proliferative neoplastic astrocytes. Initial observations
suggested that one should distinguish
these neoplasms from the typical GBM 29.
The gliosarcoma, on the other hand, is considered
similar to typical GBM in terms of prognosis,
but is distinguished by both morphological
and immunohistochemical evidence of mesenchymal
differentiation. Studies support the
conclusion that gliosarcoma shares significant
clinical and genetic similarities with GBM, and
that the same principles should be applied for
patient enrollment in research protocols and treatment
for these two entities 30. Gliosarcomas
can exhibit a plethora of mesenchymal patterns
including cartilaginous 31, angiosarcoma-like
32, osteosarcoma-like 33 and MFH-like 34
features. Two added histological patterns of
GBM are important to mention; the small cell
GBM and the glioblastoma with oligodendroglial
component. Recent work by Burger et al.
concluded small cell GBMs constitute a significant
percentage of primary GBMs, and are associated
with a high rate of epidermal growth factor
receptor (EGFR) gene amplification. Small
cell GBMs exhibit striking cellular monomorphism
not typically observed in other subtypes
and may show little or inconspicuous positivity
with the GFAP antibody. Small cell GBMs can
be confused with anaplastic oligodendrogliomas,
but are distinct from these neoplasms in
discernable ways 35. The other important histologic
pattern is the glioblastoma with oligodendroglioma
component 36. This tumor has
similar histologic appearance with anaplastic
oligoastrocytoma except for the necrosis with or
without palisading. Patients with this tumor have
a worse prognosis than the patients with
anaplastic oligoastrocytoma 37. However they
have a better overall survival probability than
the patients with conventional glioblastoma
Focal, and sometimes prominent epithelioid
features may be seen in some tumors, and these
have been previously referred as ‘lipid rich
epithelioid GBMs' 39. These tumors are circumscribed
cerebral tumors with diffuse cytoplasmic
lipidization and a cohesive architectural
disposition in epithelioid nests and sheets. These
exceptional GBMs have been confused with
metastatic carcinoma 39. In addition to the lipidrich epithelioid pattern, some tumors with
epithelioid features have been termed “adenoid
GBM”. These tumors may have branching trabecula
of polygonal cells, mimicking epithelial
tubules in a myxoid stroma 40,41. Rarely, one
can observe intensely eosinophilic intracytoplasmic
inclusions within the neoplastic astrocytes
in a GBM, akin to those described for anaplastic
Histological features previously considered
among ‘secondary structures' include satellitosis,
perivascular and subpial aggregation of
tumor cells, and may be prominent in some tumors.
Perivascular pseudorosettes in some small
cell GBMs may resemble the perivascular pseudorosettes
of ependymomas. In some tumors,
subpial aggregation of neoplastic cells extend
into the leptomeningeal space or dura mater
causing a secondary ‘extra-axial' mass. Leptomeningeal
involvement by neoplastic astrocytes
can generate significant desmoplastic reaction
and cause a more spindled appearance of the tumor.
In such foci, tumor cells may retain their
glial nature, but the phenotypic alterations suggest
a mesenchymal differentiation, similar to
gliosarcoma. Rare tumors demonstrate extensive
leptomeningeal tumor spread without a discernable
parenchymal mass 43-45.
Perivascular inflammatory infiltrates can
be found in some GBMs, especially with the gemistocytic
pattern, or in the giant cell variant
46. Most GBMs also harbor numerous macrophages
both in association with necrotic foci
and as scattered or isolated cells within the tumor.
Presence of abundance of macrophages is
not entirely incompatible with the diagnosis of
GBM but one should exercise extreme caution
not to over-interpret a macrophage-rich process
such as demyelination.
Recently published examples of glioblastoma
with PNET-like features are not included
in the current WHO classification 47. These
tumors had foci resembling conventional glioblastoma,
and separate foci with convincing evidence
of neuronal or neuroblastic differentiation
36. Nevertheless, there are very few published
cases to determine the specific features of these
tumors, and the WHO group has left the final
decision on the fate of these rare tumors to the
Intraoperative smear preparations of
GBMs highlight the nuclear as well as cytoplasmic
variations. Typically, nuclear features readily
establish the neoplastic nature of the glial
process. The tandem of pleomorphism and variable
fibrillarity is very helpful in recognizing a
malignant tumor as a glial tumor on smears.
When adequate, smear preparations contain mitotic
figures, which are also helpful in establishing
an impression of a high-grade glioma. Furthermore,
both smears and frozen sections can
include clear evidence of vascular proliferation.
This is extremely helpful in favoring a malignant
glioma over lymphoma or metastatic carcinoma,
since it is highly unusual to find true vascular
(or microvascular) proliferation in the latter
A note of caution is highly appropriate for
the interpretation of smears and frozen sections:
none of the features in isolation can be deemed
sufficient to diagnose a GBM. Intraoperative diagnosis
of malignant gliomas is a gestalt impression
of the collective features seen in all slides,
and is invariably helped by the knowledge
of the radiological and clinical features. Even
though it cannot be a mandate (for practical purposes),
we highly recommend a visit to the operating
room and direct communication with the
neurosurgeon during intraoperative consultation.
This gives the best chance for understanding
the critical details of the case. This is also helpful
in cases where the initial material is non-diagnostic,
or devoid of viable cells, precluding a
One can readily establish the presence of
glial filaments within neoplastic cells using immunohistochemical
stains for GFAP, which is
neither required nor constitutes evidence of astrocytic
differentiation. When positive, even focally,
it is helpful establishing the glial nature of
the neoplasm, and confirms an astrocytoma
when coupled with the cellular morphology. Some
GBMs show little or no GFAP positivity, or
require additional antigen retrieval techniques
not routinely used in clinical laboratories. Staining
for neurofilament proteins often highlight
the invaded neuropil within the tumor, and are
especially helpful in establishing the tumor as
an infiltrating glioma. Beyond this feature, neuronal
stains are not of particular help in the diagnosis
Immunohistochemical stains for cytokeratins,
especially ‘cocktail' antibodies can be positive
in some GBMs, and can be misleading
when metastatic carcinoma is within the differential
diagnosis 26,48. Reactive astrocytes
are often intensely labeled with cytokeratin antibodies
with the exception of low molecular
weight cytokeratins such as CAM 5.2. As mentioned
above, some GBM can contain many
macrophages and strongly stain for antibodies
such as HAM-56 or CD68. Immunohistochemical
markers of proliferation such as Ki-67
(MIB-1) has been used extensively, and the index
has varied considerably 49-56. Due to
considerable regional variation in staining, detection
and counting methods and a lack of standardization,
Ki-67 indices have been of little use
in prognostication or grading, but a low Ki-67
staining may raise suspicion of the diagnosis of
Immunohistochemical stains for p53 and
the EGFR proteins are increasingly being used
to better define the GBMs as primary, or secondary,
since alterations in these two molecules
appear to define two distinct molecular forms of
the disease based on amplification and mutation.
7,57. EGFR gene amplification and p53
mutation rarely seem to occur together. Among
many variants of EGFR gene alterations, the one
with class III deletion (EGFRvIII) is seen in more
than half of glioblastoma patients. In addition,
the increased use of drugs targeting growth
factor receptors such as EGFR necessitates recognition
of this molecular status in individual
tumors. Two commonly used antibodies, one
which recognizes the native EGFR molecule
and another which recognizes the EGFRvIII
mutant have become routine staples in the neuropathologists
armament. It will be important
to provide these stains in routine immunohistochemistry
laboratories in the future.
(Oligodendroglioma, WHO Grade III-Fig. 6)
Oligodendrogliomas involve the superficial
cortex and result in “swollen” gyri. The tumor
has at least partially gelatinous texture and yellow-gray necrosis, hemorrhagic or thrombotic
vessels. Intraoperatively, the affected cortex appears
edematous with grayish discoloration and
the architectural details become obscured. On
cross sections, effacement of the cortex-white
matter boundary can be readily identified. In some
areas, cortical calcifications can be abundant
enough to give the tumor a gritty texture.
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|Fig 6: Anaplastic oligodendroglioma, typical radiological features A) axial T1-weighted image; B) axial T2-weighted image; C) axial FLAIR; D) axial contrast-enhanced T1-weighted image.
In addition to the monotonous infiltrative
microscopic appearance of low-grade oligodendroglioma,
anaplastic examples demonstrate
vascular proliferation, markedly increased mitotic
rate and occasional necrotic foci. Vascular
proliferation can be seen as either in the form
‘glomeruloid' vessels, or endothelial hyperplasia
(Fig. 7a). Even though palisading necrosis
can be seen, necrosis is more commonly encountered
as the coagulative type without palisades.
The typical cellular monomorphism of low-grade oligodendrogliomas may not be readily
evident in the anaplastic examples, especially if
there is prior treatment. Similar to low-grade tumors,
involvement of the cortical structures produces
perineuronal satellitosis, subpial accumulation
of tumor cells, and microcalcifications.
Perineuronal satellitosis can be observed in reactive
processes or in the normal cortex, but the
extent is much more exaggerated in oligodendrogliomas,
and the cells that satellite the neurons
are atypical and more numerous than in reactive
conditions. Other secondary structures of
anaplastic oligodendrogliomas are also observed
in the white matter. The most prominent of
these is the so-called ‘filing' of tumor cells
along white matter tracts that give the impression
of a beaded chain. Another feature that can
be seen in the anaplastic examples is the hypercellular
nodules with mitotic figures, apoptosis,
and more pleomorphic nuclei (Fig. 7b). It is not
clear whether the hypercellular nodules represent
clonal growth centers with additional genetic
aberrations or simply overcrowded foci. Some
anaplastic oligodendrogliomas show densely
cellular areas composed of minigemistocytes.
The minigemistocyte, unlike the gemistocyte of
astrocytomas, has not been linked to a more aggressive
behavior 58, yet some anaplastic oligodendrogliomas
may harbor significant number
of minigemistocytes. In some tumor cells,
striking eosinophilic granular structures, also
called crystals can be seen within the cytoplasm,
which can also be positive with periodic acid-Schiff (PAS) stain 59. These neoplastic cells
are distinct from the minigemistocytes, and their
presence has not been associated with any
particular clinical variable except for uncertainty
about the diagnosis of oligodendroglioma.
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|Fig 7: Anaplastic oligodendroglioma, A) typical architectural pattern with mitotic figures and bona fide vascular endothelia proliferation (H&E original magnification X200; B) hypercellular nodules-not considered as a valid a criterion for grading (H&E original magnification x40).
Even though formation of perinuclear haloes
or the so-called ‘fried-egg' cells are typical
of oligodendrogliomas, this feature is neither
unique nor uniformly present. Anaplastic oligodendrogliomas
are less likely to show a predominantly
fried-egg cell artifact. Since this is an
artifact of delayed fixation, haloes are absent in
tissues fixed promptly, but are especially prominent
in CUSA material. CUSA specimens from
any infiltrating astrocytoma can easily be misrecognized
Floridly anaplastic oligodendrogliomas
can lose all evidence of their oligodendroglial
nature. Even though the chicken-wire vascular
pattern may be retained, recognition of such a
neoplasm as anaplastic oligodendroglioma can
be very difficult. In such cases, the most reliable
feature is a low-grade oligodendroglioma in
an earlier biopsy, or low-grade, typical oligodendroglioma
elsewhere in the specimen.
While the current WHO categorizes oligodendrogliomas
as either grade II or III neoplasm,
one can encounter individual cases that
complicate this grading scheme. A low-grade
histology with increased mitotic activity or a
well-differentiated tumor with necrosis are such
examples. On the other hand, a cellular and focally
pleomorphic tumor with few mitoses, and
without vascular proliferation or necrosis force
the boundaries of the anaplastic category.
Earlier studies suggested that necrosis or
the mitotic count can be used to identify the
"anaplastic" oligodendroglioma in the appropriate
setting 60. A widely quoted study from
the Armed Forces Institute of Pathology used a
four-tiered grading scheme on the basis of a binary
(yes-no) assessment of five variables 61.
These included microvascular proliferation,
necrosis, nuclear/cytoplasmic ratio, cell density,
and pleomorphism. Significant differences in
survival were noted between low and high-grade
groups, but the survival curves overlapped
for patients with the two intermediate grades.
“Pleomorphism” was the only histological variable
correlated with patient survival in this
study 61. In other studies, the most compelling
feature of anaplasia was glomeruloid or microvascular
proliferation. A large study involving 7
neuropathologists identified 6 unfavorable prognostic
factors by univariate analysis: older age,
high cellularity, presence of mitoses, endothelial
hypertrophy, microvascular proliferation, and
necrosis 37. Multivariate analysis reduced the
significant factors, leaving only age and microvascular
proliferation as independent variables.
Mitoses were not significant for the group of
pathologists as a whole, but were significant for
individual pathologists' analyses. Thus, it is
suggested that the designation of anaplastic oligodendroglioma
can still be made in the absence
of vascular proliferation if there is sufficient
degree of cellular atypia and mitoses 13. One
should exercise caution in using mitoses for evidence
of anaplasia, since a universally accepted
cut-off value for mitotic rate is not available.
In short, some of the histological features
found to be of prognostic significance in one
study are not validated in another. This discrepancy
may be partly due to the variations in the
diagnostic criteria and/or inclusion of oligodendroglioma-like neoplasms such as small cell astrocytoma
There are significant challenges in recognizing
anaplastic oligodendrogliomas in frozen
sections and in smear preparations. The increased
nuclear pleomorphism, loss of typical architecture
and absence of fixation artifacts that are
used to recognize oligodendrogliomas constitute
the apparent reasons for this challenge. Furthermore,
the anaplastic oligodendrogliomas
may have more complex vascularity, and the
typical ‘chicken-wire' vascular network may be
elusive. One of the most helpful tools during intraoperative
evaluation is a well-prepared smear
with undisturbed cellular and nuclear morphology
indicating an oligodendroglial neoplasm.
Usually, smear preparations reveal a more monomorphous
tumor cell population with rounded
nuclei harboring a speckled ‘salt-and-pepper'
chromatin pattern. Unfortunately, sufficient
number of anaplastic oligodendrogliomas divert
from this typical pattern.
The search for lineage specific markers
that effectively distinguish oligodendrogliomas
from astrocytomas has been fraught with major
challenges. Antibodies that are presumed most
useful in determining cell lineage, e.g. myelin
basic protein or myelin-associated glycoprotein
have proven to be of limited or no use as specific
‘markers' 48. Although a well-differentiated
oligodendroglioma is expected to show negative
immunostaining with GFAP antibodies,
many oligodendrogliomas contain GFAP positive
tumor cells. Since oligodendrogliomas also
integrate significant number of reactive, as well
as normal astrocytes, the tumors may appear to
be GFAP positive. This issue is further confounded
by variable GFAP staining of astrocytic neoplasms,
making the distinction based on this
stain subjective. In addition to radiological and
clinical information, neuronal markers are frequently
applied to oligodendrogliomas in an effort
to distinguish them from neuronal or glioneuronal
neoplasms 62. Although synaptophysin
stains the underlying neuropil that is invaded by
an oligodendroglioma, it often fails to stain tumor
cells. However, variable degrees of neuronal
marker expression have been reported in
typical oligodendrogliomas which can confound
the diagnosis and should be interpreted with caution
63. It is unclear whether tumors that coexpress
neuronal markers represent divergent
neuronal differentiation, a distinctive form of
glioneuronal neoplasm, or a reflection of histogenesis
in oligodendrogliomas. Markers that are
typically positive in mature neuronal cells, such
as Neu-N are often negative in anaplastic oligodendrogliomas.
Stains for neurofilament proteins
can also be helpful in anaplastic oligodendrogliomas
in highlighting the extent of neuropil
Immunohistochemical stains for proliferative
markers such as Ki-67 (MIB-1) show marked
variability within anaplastic oligodendrogliomas,
and the labeling indices are rarely helpful
to determine the tumor grade. Some studies
have found survival differences in oligodendrogliomas
based on Ki-67 labeling index 64.
Variation among studies may be due to methodological
or interpretive differences 65. For
practical purposes we do not recommend altering
the grade of an oligodendroglioma based on
the Ki-67 labeling index.
Staining for p53 is present in some anaplastic
oligodendrogliomas, and occurs much
more commonly than low-grade examples 66.
The staining is often variable in intensity and is
present in a small fraction of the tumor nuclei.
The p53 staining status does not appear to correlate
with outcome in anaplastic oligodendrogliomas
One of the most helpful molecular features
in the recognition of anaplastic oligodendroglioma
is the recently recognized deletions of chromosome
1p and 19q that also implies a chemoresponsive
tumor 70. The detection of this
combined deletion is almost sine qua non for
oligodendroglial tumors and is also shared by
most oligoastrocytomas 71. A number of methods
can be easily applied to detection of this
genetic alteration, but in our opinion, the one
that is most suitable for the pathologist's interpretation
is the fluorescent in-situ hybridization
(FISH) technique. While the chromogenic alternative,
CISH appears to be equally sensitive, the
former test is currently better standardized and
should be performed in all tumors where an oligodendroglial
component is suspected. A few
words of caution are warranted for this test: 1) it
is important to identify regions of tumor in
samples before this test is executed, 2) it is critical
not to consider hyperploidy (i.e. tumors
with multiple copies of both arms) as “relative”
deletion. Most anaplastic oligodendrogliomas
will retain this genetic alteration, but occasionally
it may be helpful to retrieve an earlier, low
grade sample from the same patients to perform
the analysis. We strongly recommend addition
of this analysis as a part of routine testing in surgical
(Oligoastrocytoma, WHO Grade III )
Typical oligodendrogliomas and astrocytomas
have distinguishing features and can be
readily recognized, but there is considerable histological
overlap in some infiltrating gliomas
with features of both or neither. To some, this is
a true ‘mixed' glioma, with discrete areas corresponding
to either phenotype, or a haphazard
intersection of a typical astrocytoma with a typical
oligodendroglioma. The former is an exceedingly
rare occurrence wherein distinct fields of
classic oligodendroglioma and diffuse astrocytoma
coexist 72, while the diagnostic criteria
for the latter is virtually non-existent. Thus, there
are no definitive histological criteria for the
diagnosis of “mixed glioma”. Most tumors designated
“mixed gliomas” are grade II or III neoplasms
that contain cells resembling the rounded
nuclei of oligodendroglioma, but has an undefined
percentage of tumor cells with the nuclear
and cytoplasmic features of astrocytomas.
Some consider this vague, and highly subjective
category as a tumor with bidirectional differentiation,
even though attempts to identify to genetically
distinct neoplastic cell groups have been
unsuccessful. Since there are significant number
of tumors in which histological features are neither
of the two well-defined infiltrating gliomas,
it is difficult to be critical of the oligoastrocytoma
concept. However, the greatly variable
incidence of oligoastrocytomas in different
institutions, and increase and decrease of the incidence
of such tumors after an arrival or departure
of a pathologist from an institution imply
that there is tremendous subjectivity in the definition
and diagnosis of oligoastrocytomas. Some
reserve the “mixed” designation for truly
biphasic tumors with fibrillary or frankly gemistocytic
cells 73. Some consider this category
when tumors have features of neither oligodendroglia
nor astrocytes. Recent studies suggest
that the occurrence of mixed gliomas is not indicative
of a tumor with bi-clonal origin but rather
reflects altered gene expression, leading to a
change in the balance of growth factors influencing
glioma differentiation 74. The nosological
and therapeutic problems created by these
neoplasms are still unresolved.
There is no established percentage of “astrocytes” in a mixed glioma. The oft-quoted figure
of 20% has no scientific or experimental
basis. Fortunately, at present, the distinction
between oligodendroglioma and “mixed glioma”
makes little initial therapeutic difference,
since “oligoastrocytomas” and “pure” oligodendrogliomas
are treated similarly. For the patient,
however, the difference can be highly significant
given the more aggressive nature of diffuse
astrocytic neoplasms. Furthermore, the designation
of an astrocytic neoplasm as a mixed glioma
may generate undeserved prognostic optimism.
One might expect that “mixed gliomas”
with a 1p/19q loss would behave in an indolent
or chemo-responsive fashion expected of an oligodendroglioma,
whereas those without this
profile act biologically like astrocytic tumors,
i.e. more aggressively and more prone to high
grade transformation 75. The current WHO
scheme classifies high grade tumors showing
mixed glial features and necrosis as “glioblastoma
with oligodendroglioma component”.
(Ependymoma, WHO Grade III-Fig. 8)
The ependymal neoplasms often appear
sharply circumscribed, and their textures vary
from soft to rubbery. Some tumors are firm and
gritty due to calcification. Most tumors have variegated
color with hemorrhagic areas. Intraoperatively,
one can observe involvement of the subarachnoid
space where the tumor extends to
encase cranial nerves and blood vessels. Anaplastic
ependymomas are less well circumscribed,
much more hemorrhagic and may have
Click Here to Zoom
|Fig 8: Anaplastic ependymoma, typical radiological features A) sagittal T1-weighted image; B) sagittal contrast enhanced T1-weighted image; C) axial T2-weighted image; D) axial FLAIR image.
Anaplastic ependymomas exhibit moderate
to high cellularity, mitotic activity, and vascular
endothelial proliferation 76. Evidence of
ependymal phenotype is often critical in recognizing
anaplastic ependymomas, and the most
convincing proof is found in the typical perivascular
pseudorosettes. Even though histological
grade has been considered to be prognostically
significant, there is controversy in the use of histological
criteria. In addition, studies report
conflicting results on the prognostic significance
of histological grading in ependymoma 77-79.
Almost all ependymomas may contain mitotic
figures that are more evident in anaplastic
examples. While there is no standard cut-off value
in the frequency of mitotic figures, some studies
report mitotic count as a significant prognostic
indicator 80,81 . Vascular proliferation
is also considered a strong evidence of high-grade
ependymoma (Fig. 9a), while the presence of
necrosis has been controversial. Necrosis was
considered inconsequential in one study, while
others have found it among the prognostically
important factors 76,78,82. Necrosis in isolation
is of little value since in our experience, most
low-grade ependymomas in the posterior fossa
contain necrotic regions, when sampled adequately.
Click Here to Zoom
|Fig 9: Anaplastic ependymoma, typical histological features A) vascular endothelial proliferation B) hypercellular nodules-not considered a valid criterion for grading. (all images original magnification x200).
Ependymomas show wide variation in cell
density and differentiation. Some tumors may
appear quite cellular with hyperchromatic nuclei
and hypercellular nodules (Fig. 9b), while others may be only slightly more cellular than subependymoma.
Hypercellular nodules often demonstrate
pleomorphic nuclei and mitotic figures.
Such nodules may not justify the diagnosis
of anaplastic ependymoma, but their presence
raises doubts about a favorable prognosis 83.
The progression of anaplastic ependymoma
to GBM is problematic and largely semantic.
Unlike GBM, high-grade ependymomas usually
exhibit a solid, non-infiltrating growth pattern.
On the other hand, GBMs rarely show unequivocal
ependymal features, and then only in the
form of focal and vague perivascular pseudorosettes.
In some cases, an infiltrating malignant
glioma with focal, vague ependymal features is
much more likely to behave as a typical GBM
than an ependymoma.
Recognizing ependymomas in frozen sections
can be challenging since perivascular pseudorosettes
and the ependymal differentiation
may be difficult to recognize. The freezing artifacts
may accentuate the fibrillarity and cytoplasm
of cells resulting in an impression of an astrocytoma.
On the other hand small-blue-roundcells
with mitotic figures and a lack of distinctive
architecture can raise the possibility of medulloblastoma,
especially in the setting of a posterior
fossa tumor in a child. The intraoperative
smear of ependymomas highlight two distinctive
features: the glial quality of the cells and the
monotonous nature of the nuclei. Angiocentric
arrangement of cells may be conspicuous in
smears, and can also be observed in frozen sections
but this is less common in anaplastic
ependymomas. Vascular proliferation can suggest
a high-grade neoplasm, but grading of
ependymomas during intraoperative consultations
is discouraged unless there is unequivocal
evidence of high-grade features.
Ependymomas are variably GFAP positive.
Typically, there is an accentuation of staining
in the perivascular spaces corresponding to
cellular processes. Soma anaplastic examples
may show limited GFAP positivity. Others are
diffusely positive for GFAP as well as S-100
protein. Keratin cocktails (AE1/AE3) can be reactive
in a large percentage of cases, the pattern
being similar to that of GFAP 84. The frequency
of staining for other keratins is quite variable
and limited to rare individual cells and processes.
Epithelial membrane antigen (EMA) stains
up to half of the anaplastic ependymomas
and particularly highlights true rosettes, canals
and individual cells. Diffuse strong staining for
other keratins or for CEA is inconsistent with a
diagnosis of ependymoma. Vimentin staining is
often diffuse and strong, but this only confirms
the suitability of tissue for immunohistochemical
Recent studies suggest that high Ki-67
(MIB-1) and p53 immunostaining might be objective
indicators of high grade in ependymomas
that do not otherwise meet routine histological criteria 85,86. A similar study proposed
a cut-off value of 20% for ependymomas with
more aggressive behavior 87. Others found no
correlation between Ki-67 labeling index and
outcome 82. The practical value of these stains
in grading is currently limited.
OTHER MALIGNANT GIOMAS
PXA with Anaplastic Features
While PXAs have a more favorable outcome,
the neoplasm has a significant rate of recurrence,
some becoming histologically malignant
in time 88. Some PXAs show anaplastic features
at initial surgery. The relationship between
histological features and outcome is still debated,
as reviewed recently in a number of retrospective
studies 88-91. These studies reveal
that high mitotic rate and the extent of surgical
resection appear to be the most valuable prognostic
information, while necrosis is not of critical
importance. Typically, one should not observe
brisk necrosis, easily identifiable mitotic
cells or vascular proliferation in PXA. The presence
of these features should be viewed with
concern and should prompt a closer scrutiny of
Currently, despite the presence of such aggressive
PXAs, the WHO classification does not
recognize a separate anaplastic variant, and recommends
the use of the term “PXA with anaplastic
features”. Whether this distinction is relevant
to the management of the patient, and necessitates
further treatment is not clear. Obviously,
the concept of a grade IV PXA is even more
controversial. This concept is also often contrasted
with giant cell GBM that can be confused
with a PXA with anaplastic features. Ultrastructurally,
PXAs often reveal various epithelial
properties, such as cell junctions and interdigitations,
and prominent basal laminae surrounding
tumor nests, often distinctive from GMB. As a
rule, PXAs with anaplastic features lack the
microvascular proliferation or a predominant infiltrative
component common to GBM. The predominance
of epithelioid cells differs from the
composition of giant cell GBMs with bizarre
mitoses. The differences between PXA with
anaplastic features and giant cell GBM are not
entirely clarified, but in our experience, the former
can still be considered less aggressive.
Pilocytic Astrocytoma with Malignant Transformation
The validity of grading pilocytic astrocytomas
is controversial, but there are well-documented
cases wherein an apparently pilocytic
astrocytoma demonstrates a very aggressive clinical
course along with high grade histological
features 92-94. The classical grading schemes
used for infiltrating gliomas are of little value in
pilocytic astrocytomas. There are, nevertheless,
tumors that exhibit high cellularity, brisk mitotic
activity, microvascular proliferation and/or
necrosis with pseudopalisading, for which the
term “malignant” or “anaplastic” is unavoidable.
One should always suspect the diagnosis in
an older patient with atypical radiological features,
especially in the absence of a well documented
prior pilocytic astrocytoma. Small
samples of malignant deep seated gliomas are
potentially likely to be misrecognized as pilocytic
astrocytoma, only to be correctly diagnosed
in subsequent surgery. Histological malignancy
in pilocytic astrocytoma is quite rare and less reliably
correlates with prognosis than in patients
with fibrillary astrocytomas 95.
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