Glioblastoma Multiforme: A clinico- pathological analysis

Background: Aim of the study was to classify Glioblastoma Multiforme (GBM) and its variants on histology and analyze their clinicopathological features. Better understanding of the heterogeneous nature of GBM and its variants may provide improved treatment paradigms, prognostic classification and approaches towards molecular targeted treatments. Methods: 40 cases of GBM were analyzed retrospectively and prospectively, covering a period of 8 years (January 2008July2015). Cases were analyzed on the basis of clinical presentation, histopathological features and radiological reports. Results: Of the 40 cases of GBM, the Conventional GBM, GBM with oligodendroglial component, Giant Cell GBM accounted for 26, 9 and 3 cases respectively. While there was 1 case each of Gliosarcoma and P-NET variants. Conventional GBM most commonly presented with Headache (69.2%) and paresis (57.7%) whereas GBMO patients presented with convulsions. On radiology, the variants of GBM cannot be distinguished as the findings were similar in all of them. Non-palisading necrosis was predominantly present in Conventional GBM (61.5%) and palisading necrosis in GBMO (66.7%). Conclusion: Histopathology remains the main tool for differentiating the variants as radiological differentiation is not possible due to similar appearing features.


Introduction
GLIOBLASTOMA synonym (Glioblastoma Multiforme -GBM) is the most malignant neoplasm of the central nervous system with predominant astrocytic differentiation, affecting adults and preferentially located in the cerebral hemispheres [1] . Glioblastoma can be primary (De Novo), or secondary on the background of pre-existing astrocytic neoplasm. This is a morphologically diverse neoplasm with a dismal prognosis. Though three morphologically different variants have been recognized, additional variants which have significant morphology overlap with tumors having more favourable prognosis and treatment response rates, have been described. Even though Glioblastoma Multiforme is a quite rare tumor with a global incidence rate of only 3.17 per 100,000 [2] it significantly impacts the life of the affected patients due to its poor prognosis with a median survival time of only 12-15 months from the time of diagnosis. [3]

Materials and Methods:
After approval from the institutional ethics committee for this retrospective and prospective study, 40 cases from January 2008 to July 2015 were analyzed. Retrospective study was done using the blocks and slides available. For prospective study biopsy tissue was fixed overnight in 10% buffered formalin and submitted entirely for processing.
Intraoperative tissues sent were processed as squash preparation and frozen section on cryostat while still in an unfixed state and H&E staining was performed. After frozen section diagnosis, remaining tissue was transferred to a fixative and processed routinely. Paraffin sections were cut 4 to 6 microns in thickness and routine H&E staining was performed, special stains were performed wherever necessary.
The clinical and radiological data was obtained from patient's proforma available in the Pathology Department. Anatomical location of the tumor was based on radiological imaging and or operative findings. GBM was diagnosed and classified as per the 2007 WHO.

Results
In the 8 years study period (January 2008 to July 2015), there were 433 cases of central nervous system space occupying lesions, out of which 40 were diagnosed as GBM. The overall incidence of GBM was 9.2%.The average number of GBM cases received was 5 per year.
The maximum number of cases were diagnosed in the 4 th and 5 th decade, 9 cases each (22.5 % each). In our study, a male predominance was found (Male: female=2.07:1). The youngest patient was 5 years old while the oldest one was 73 years old. GBM was found to be more common in males in the 41-50 years age group and in females in the 51-60 years age group.
Headache and paresis were the commonest symptoms encountered, 24 cases (60%) and 21 cases (52.5%) respectively. Majority of the patients (22.5%) had duration of symptoms from 15-30 days. 29 cases (72.5 %) presented in less than 3 months. 95% of cases had supratentorial GBM and 5 % had infratentorial. The most common locations of the tumour were frontal lobe and temporoparietal region. (17.5% each). 95 % (38/40) of the tumors were present in cerebral hemisphere whereas 2.5 % tumor involved cerebello-pontine angle and brain stem. Out of 38 Glioblastomas in cerebral hemisphere 18 involved only single lobe whereas 17 involved 2 lobes and 3 involved thalamocapsular ganglion.
CT findings were available in 25 cases (16 of Conventional GBM, 6 of GBMO and 3 of Giant-cell GBM). MRI findings were available for 15 cases (10 of Conventional GBM, 3 of GBMO and 1 each of GBM-PNET and Gliosarcoma). Conventional GBM and GBMO showed iso to hypodensity on CT (87.5% and 66.5% respectively). Hemorrhagic necrosis was present in all the cases. Non-palisading necrosis was predominantly present in Conventional GBM (61.5%) and palisading necrosis in GBMO (66.7%).

Discussion
GBM or astrocytoma grade IV ( WHO classification) is the most aggressive and the most frequent of all primary brain tumors. [4] Incidence rate of GBM at our institute was 9.2% which is almost similar to the incidence stated by Ghosh et al who reported an incidence of 7.9% for GBM. WHO (2007) states an incidence of around 12-15% for GBM of all intracranial neoplasms [5] .
In our study, 45 % of CNS tumors were encountered in the fourth to sixth decade. The mean age was 46.4 years. The youngest patient was 5 year old while the oldest one was 73year old. The finding is similar to study done by Manisha Khanna et al, who found majority of the tumors in age group of 51-60 years [6] . Childhood glioblastomas are extremely rare compared to their adult counterparts [4] .Of the 40 cases, 27 were male and 13 were female. The male: female ratio in our study was found to be 2.07:1 which was comparable with Manisha Khanna et al [6] (2.38:1). Ghosh et al. observed a male/female ratio of 3.9:1 [5] .
Although the duration of symptoms was more than 3 months in 11 cases, there were no histological proven or past clinical history of high grade gliomas. Hence no case of secondary GBM was found in the study.
According to the literature, GBM is preferentially located supratentorially [4] . . In our study too about 95 % of cases were supratentorial and 5% cases showed infratentorial GBM.
In our study, the commonest site of GBM was frontal region and temporo-parietal region (17.5% each). These findings were comparable to study by Manisha Khanna et al [6]. In our study, 10% tumors were present in frontal lobe involving corpus callosum but none extended to other lobe or across the midline. In literature [7] it is said that the corpus callosum is relatively resistant to infiltration by edema or infection. Any lesion seen extending across the midline in this way, whether symmetric or asymmetric, should always be suspected of being a diffuse astrocytoma.
MRI Brain mainly revealed extensive white matter edema (97.5%) noted with significant midline shift. Non enhancing areas corresponded to haemorrhage and necrosis on histopathology were seen in 100 % of the cases. These findings is in accordance to study by Gabriel Iacob et al [4] .
In our study, morphological analysis included the degree of cellularity, pleomorphism, mitosis, type of necrosis and vascular proliferation. Of the 40 cases, 34 cases were analyzed on the frozen section .All these were given the diagnosis of high grade glioma on the basis of micro vascular proliferation and necrosis. On further histopathological examination, these were given the final diagnosis as Conventional GBM (26 cases) or GBMO (9 cases).
All the cases of glioblastoma diagnosed on squash smear and frozen section showed significantly increased population of glial cells in a fibrillary background with mitoses with microvascular proliferation and necrosis.
The study by Chandrasoma PT et al provides evidence that, with careful target placement, stereotactic biopsy can provide biopsy material that represents the entire lesion with an accuracy that is sufficient for clinical management [8] .
In present study, we had 26 cases (65%) of Conventional GBM. It was prevalent in 3 rd to 5 th decade (69.3%). Out of 26 cases of Conventional GBM , 20 were found in males and 6 in females. The ratio in Conventional GBM was 3.3:1.
Headache and paresis was the main presenting symptom in most cases of conventional GBM. Most frequent site affected in patients with Conventional GBM was frontal.
Clinical and pathological studies of GBMO are currently scarce. Its exact incidence is thus largely unknown and has ranged from 4% to 27% of all GBMs in previous studies [9,[11][12][13] with an incidence of 22.5% (9 cases) in the current study.
In present study, it was noted that GBMO was prevalent in elderly patient's i.e.in age group of 60-70 years. The youngest patient of GBMO was a 16 year old male and the oldest patient was also a male of 72 years. Out of 9 cases of GBMO , 6 were found in males and 3 in females. So GBMO was more common in male just as Conventional GBM, but the M: F ratio of GBMO was 2:1, compared to 3.3:1 in Conventional GBM.
In a study of Yongzhi Wang et al., 40 (18.3%) of the 219 primary GBMs selected fulfilled the criteria for GBMO. Fourteen patients (35%) had seizure attacks as presenting symptoms.
The patients with GBMO were more likely to present with seizures than were patients with conventional GBM. There were no significant differences in sex, tumor location, tumorrelated seizures were more frequent in the GBMO patients (35%) than in conventional GBM patients (19.7%).
Microscopic examination of GBM-O showed Oligodendroglial component intermingled with or in different foci of glioblastomatous tissues ( figure-19). Majority of the cases showed marked cellularity (66.6%), moderate pleomorphism (88.9%), and presence of small cells (88.9%), palisading necrosis (66.7%) and thrombosed blood vessels (22.2%). All the cases showed microvascular proliferation (100%) and Mitosis (100%). These findings were similar to findings mentioned in literature [14,15] Giant cell GBM is a rare variant of GBM thought to encompass 2-5% of GBM diagnoses [1] , while in our study incidence is about 7.5% which is slightly higher. In the present study, patients with Giant cell GBM presented mainly with headache (66.7%).This finding was almost similar to Conventional GBM (69.2%). The other symptoms were loss of consciousness, aphasia and paresis (33.3 %) each. In contrast to the Conventional GBM, majority of the cases (66.7%) presented with duration of symptoms of more than 3 months. This finding is in contrast with the findings of Valle et al. where they found the duration of symptoms to be short (and similar to Classical GBM) [16] . The difference noted may be due to small sample of the gcGBM in the present study.
As the name implies, the tumor cells are markedly enlarged and bizarre, often appearing multinucleated. All the cases showed marked cellularity, moderate pleomorphism , Giant cells , palisading, non-palisading and pseudo palisading necrosis were found in 33.3% respectively. All cases showed mitosis and microvascular proliferation. Gemistocytes were seen in 66.7% and 33.3% showed perivascular lymphocytes.
In this study, patients with Conventional GBM and gcGBM showed similar gender and racial distributions as well as insignificant tumour size and location differences. However, age at diagnosis was significantly younger in gcGBM vs. GBM (51 vs. 62 years) and gcGBMs were more likely to undergo complete resection. [17] In the present study gliosarcoma accounted for 2.5% of all GBM case (1/40). In various series, the incidence of GS has been reported to vary from 2% to 8 % [18]. This corroborates with our study findings. In the present study, the only case of Gliosarcoma, a 55 year old female presented with psychiatric symptoms since 8 days. Tumour was located in the left fronto-parietal region. Study by Morantz [19] and colleague described that great percentage of patients with GS were older than 60 years of age and in study by Manisha et al [6] most of the patients were in the age group of 51 to 70 years and found GS most commonly in the temporal region, followed by frontal and parietal. Common site of occurrence was cerebrum and the common clinical symptoms were muscle weakness, headache and mental changes.
Microscopic examination of GS showed sarcomatous tissue intermingled with or in different foci of pre-existing glioblastomatous tissues . The case showed marked cellularity, moderate pleomorphism, small cells, nonpalisading necrosis, mitosis, microvascular proliferation and presence of secondary structure of Scherer. These findings were similar to Manisha khanna et al [6] . Narendra Kumar et al. [20] reviewed 27 gliosarcoma patients and found that all the tumors were having the biphasic histologic pattern consisting of gliomatous and sarcomatous components which is in accordance to our study.
In our study we found 1 case of GBM-PNET in a 26 year old female who presented with convulsions since 15 days. The tumor histo-morphologically consisted of undifferentiated cellular areas alongside classic GBM areas. The undifferentiated cellular areas composed of small undifferentiated cells with scant cytoplasm and oval round hyper chromatic nuclei. Immunohistochemistry revealed astrocytic component of the tumor strongly positive for GFAP and undifferentiated area stained strongly for synaptophysin. This concluded the diagnosis of GBM-PNET. [Table6] As the clinical and histopathological properties of those tumors have been described only recently, the number of cases reported in the literature is limited. The largest series published on these tumors belong to Varlet et al [ 21] (n=40) and Perry et al. (22) (n=53). These studies highlighted the clinical, radiological, and histopathological differences of GB-PNET from classic GBM. They are encountered more commonly among adults and 52.5% of tumors are localized in the temporal lobe, whereas they are rarely seen with infratentorial localization. Having a well circumscribed character facilitates the surgical excision.

Conclusion
The most common histological findings of GBM included marked cellularity, moderate pleomorphism, microvascular proliferation, non-palisading necrosis and mitosis. Clinical and histopathology remains the main tool for differentiating the variants as radiological differentiation is not possible due to similar appearing features. Newer diagnostic technique like immunohistochemistry and molecular analysis has great prognostic significance and help to decide the line treatment.