Radiological characteristics study on epithelioid glioblastoma, a rare subtype of GBM

Purpose Epithelioid glioblastoma (eGBM) is rare and a newly recognized subtype of GBM. Given the short of studies focusing on radiological characteristics of these tumors, we aimed to report the radiological features of eGBM deriving from six patients. Methods Six patients with pathologically diagnosed as eGBM were enrolled in this retrospective study. CT and pre-operative MR examinations with conventional and advanced sequences, such as diffusion weighted imaging and so on were analyzed. Immunohistological staining and mutation analysis of BRAF V600E was also explored.

3 and intratumoral calcium, may support the diagnosis of eGBM. Background Glioblastoma (GBM), the most common and devastating tumor in central nervous system(CNS) of adult, has a very poor prognosis in despite of intensive treatment.
Epithelioid glioblastoma (eGBM), firstly describing as "closely packed tumor cells arranged in sold trabeculae and club-like formations" [1], is formally identified as an entire subtype of GBM in 2016 World Health Organization (WHO) classification of CNS. Due to its early recurrence, rapid progression and dissemination (leptomeninge and cerebrospinal fluid), eGBM display more aggressive clinical course and with a median survival time shorter than 14.5 months in typical GBM [2].
The etiology and cell origin of eGBM remain unknown. Although in several cases, a low grade astrocytoma or pleomorphic xanthoastrocytoma (PXA) has found to be as preexisting or co-existing lesion with an eGBM, eGBM is usually recognized as a de novo neoplasm according to the presence of wild type isocitrate dehydrogenase 1 (IDH-1) and absence of ATRX mutation. To date, only one case is diagnosed as a secondary eGBM with IDH-1 mutation that develop from a oligoastrocytoma (WHO grade II) [3]. There is a close relationship between the eGBM and PXA since those two types of tumor share high overlap of genetic alteration, such as serine/threonine kinase B-Raf (BRAF) V600E mutation and TERT promoter (TERT-p) mutation. Even some authors propose that eGBM may arise from a PXA with BRAF V600E mutation [4].
Owing to insufficient recognition resulting from its rarity and intratumoral heterogeneity of eGBM, making an exact diagnosis encounter huge challenge. In the current study, we collected 6 cases initially diagnosed as eGBM. Of particular note, one case had a colocating diffuse astrocytoma (WHO grade ii), both of that two tumors harbored IDH-1 mutation. In our knowledge, that case is the second eGBM case displaying IDH-1 mutation. 4 In order to help making a more accurate diagnosis, this study was more on discovering radiological characteristics of eGBM. Beyond that, we described and analyzed clinical and histological features of the 6 cases.

Methods
For retrospective analysis, consecutive 6 patients who were pathologically diagnosed as epithelioid glioblastoma (eGBM) were enrolled. 4 cases were male, 2 were female, aged from 18-year to 52-year, the median age was 35.5 years. Durations of the clinical symptoms varied from several days to 1 year. Prior to any treatment, all the cases performed conventional MR scanning with or without contrast agent, diffusion-weighted imaging (DWI), susceptible-weighted imaging (SWI) and 1 H-MR spectroscopy ( 1 H-MRS).
This study was approved ethically by the institutional review board at Daping Hospital, Army Medical University (Chongqing, China) (IRB #201879). All the patients with suspected glioma had previously informed the possibility of using their medical imaging for research and obtained authorization for utilization of their medical records.

Imaging post-processing and analysis
6 All the DSC-PWI and DWI original data were processed with a commercial post-processing software package (SygnoMMWP VE36A, Siemens, Erlangen, Germany) to obtain cerebral blood volume (CBV) and ADC maps. The imaging analysis was performed by two experienced, board-certified neuro-radiologist with more than 5-year work experience. The ROIs with identical size were placed on the solid component and contralateral normal appearance white matter (NAWM), CBV m ax and ADC m in was determined. Then, the rCBV m ax and rADC m in were calculated by normalizing CBV m ax and ADC m in to the value of contralateral NAWM. On 1 H-MRS, Lip/Lac value was stratified: -, totally negative or can't be measured; 1+, detectable; 2+, the second peak; 3+, the largest peak [5]. According to the method provided by Park et al. [6], intratumoral susceptibility signal intensity (ITSS) on SWI was scored as follows: 0, absent; 1, number of ITSS ≤ 5; 2, ITSS ≤ 10; 3, ITSS ≥ 11.

Mutation analysis of BRAF V600E
DNA was extracted from the paraffin-embedded specimens by using DNA extraction Kit (Amoy Diagnostics, Xiamen, China) and following the manufactural protocol. BRAF V600E ARMS-PCR Kit (Amoy Diagnostics, Xiamen, China) was applied to determine the mutation statue of the specimens, all the procedures followed the kit specification. The total reaction system was 45μL, including 5μL DNA template and 40μL reaction 7 mixture (consist of Taq DNA polymerase, scorpions primer, reaction buffer and so on).
Each reaction contained a positive and a negative control. The PCR reaction was performed as follows: Stage i 95℃for 5min; Stage ii 95℃for 25s followed 72℃for 20s, repeated 15 cycles; Stage iii 93℃ for 25s, 60℃ for 35s, 72℃ for 20s, repeated 31 cycles.
Data was automatically collected when the reaction was completed. Mutation result was concluded based on the manufacture instruction.

Clinical characteristics
The clinical symptoms depended on the location of the tumor, predominantly presented symptoms associated with intracranial hypertension including dizzy, headache and vomiting (5/6). One case demonstrated convulsion of the limbs accompanying conscious disturbance (1/6). All lesions located in supratentorial region. 3 cases were primary tumor, 2 were recurrent tumor, 1 was co-located with a diffusion astrocytoma. All cases underwent surgical resection, the post-operative radiotherapy and chemotherapy with temozolomide was applied in 3 cases. 1case died soon after surgery, 1 missed the followup data, the other 4 cases were alive until June 2018. (Table 1) Survival time ranged from 11 to 22 months, average survival time was 14.7 months.
On conventional MRI, tumors presented iso-and hypointensity (3/6) or mixed intensity (3/6) on T1WI, mild hyperintensity (3/6) or mixed intensity (3/6) on T2WI and FLAIR (Fig   2a-c). With contrast agent, majority tumors (5/6) demonstrated markedly heterogeneous enhancement with both cystic wall and solid component exhibiting striking enhancing, only one case showed slight enhancement (Fig 2d). Notably, case 4 revealed area with nodular-enhancing pattern within the tumor. Moreover, linear and patchy enhancement was observed in tentorium cerebelli and superior vermis of cerebellum in case 4, that indicated a CSF dissemination (Fig 3). Involvement of leptomeninge was discovered in 4 cases showing leptomeningeal thickening and abnormal enhancing (Fig 2d). All 6 cases performed DWI and obtained rADC m in via image post-processing, 5 cases revealed notable restricted-diffusion within the solid component of the tumor (Fig 4a-b), only 1 case (case 3) showed mild restricted-diffusion which may be attributed to the influence from the skull base near the tumor. The rADC m in values of the tumor were decreased, the average value was 0.84×10 -3 mm 2 /s (0.78×10 -3 ~ 0.93×10 -9 mm 2 /s ). SWI was not available in case 6, dotted or patchy hypointensity was noted within the other cases (Fig 4c). According to the grading method mentioned above, the 5 cases can be divided into 0 (case 3), 1 (case 5), 2 (case 1) and 3 (case 2 and 4). On 1 H-MRS, except case 3, whose baseline was unstable resulting from the influence of adjacent skull base, the other 5 cases demonstrated decreased NAA, increased Cho value and Cho/NAA ratio, the average Cho/NAA value was 12.94 (3.6 ~ 33.2). Increased Lac/Lip value also can be found in case 1, case 2, case 4 9 and case 6 with grade 2+, 1+, 1+ and 2+, respectively (Fig 4d). For 4 cases (case 2, 4, 5 and 6) with DSC-PWI examination, all without exception presented increased rCBV, the rCBV m ax differed from 4.44 to 7.55, the mean rCBV m ax was 5.94 (Fig 4e).
On CT, all the cases (case 2, 4 and 5) showed an ill-defined mass with mixed density. Solid component exhibited higher density compared to the contralateral normal brain, while cystic area within the tumor exhibited low density. The CT images also clearly exhibited the intratumoral hemorrhage and calcification.

Histopathological findings
The tumors were consisted by epithelioid tumor cells which were round or oval, abundant eosinophilic cytoplasm, overt nuclei and mitotic figures, sometimes with intranuclear pseudoinclusions (Fig 5a). Multinucleated giant cells were found in some cases. All the cases revealed necrosis showing geographic or pseudo-palisading pattern. Local spindle cells with sarcomatoid change was seen in case 5 (Fig 5b). The spindle cells showed obvious atypia, intensive cellular proliferation and transition zone, which made a distinction between spindle cells and epithelioid tumor cells. Stromal vascular proliferation was evident. Glomeruli-like vessels were seen in periphery area of tumor necrosis and hyperplastic stromal vessels.
All cases showed positive GFAP, p53 and Syn staining with variable immunoreactivity.
Positive INI-1 staining was seen in all cases (Fig 5c-e). In case 6, IDH-1 mutation was found both in eGBM and co-existing low grade diffuse astrocytoma region (Fig 5f-g). Ki-67 labeling index varied among 20% to 30%. BRAF V600E mutation, which is less frequently found in other types of GBM, can be found in eGBM at a relatively high frequency of 56%. In our six cases, three cases (50%) harbored BRAF V600E mutation (Fig 5h).

Discussion
Since eGBM is a newly recognized subtype of GBM according to criterion of the latest WHO classification (2016), a large cohort is hard to obtain. To date, only Korshunov A, et al.
reported a series of sixty-four cases [7]. Therefore, the incidence rate of eGBM is not available. Due to its rarity, reported cases pay more attention to the histological and clinical features of eGBM rather than radiological findings. To date, only one case of eGBM with IDH-1 mutation is reported [3], although several cases report eGBM with pre-exiting or co-exiting tumor, such as PXA or low-grade diffuse astrocytoma [3,4,[8][9][10][11]. In the present study, we reported one eGBM collocating with a low-grade diffuse astrocytoma, and in that two tumor districts, IDH-1 instead of BRAF V600E mutation was found.
Moreover, we summarized and analyzed clinical, MRI/CT and histological features of six cases. The radiological findings, especially the findings obtained from the advanced MR sequences was the key attention of this study.
Unlike typical GBM tend to occur in elderly people, eGBM apt to afflict children and young adults at less than 30 years old [12,13]. eGBM usually arise in cerebral cortex and diencephalon. In adult, eGBM often develop predominantly in cerebral cortex, temporal and frontal lobe are the most common locations. While, pediatric eGBM commonly occur in diencephalon [14]. Age at initial diagnosis of this study was disagree with previous reports, only 2 in 6 cases of this study were young people at less than 20 years old, the other 4 cases ranged from 30 to 50 years old. This discrepancy suggests that eGBM may be with a wider age spectrum. There was no gender prevalence, the ratio of male versus female was 1:1 (3/3). Consistent with the reported studies, 3 cases (50%) located in temporal lobe, 2 out 6 cases involved frontal lobe, 1 case occurred in basal ganglia region. However, 2 cases (case 4, 6) involved deep brain structure, including corpus callosum and basal ganglia. That may indicate the heterogeneity of eGBM just like typical 11 GBM. The common complications, like headache (4/6), may be intracranial hyperintension associated.
BRAF V600E mutation is found in several types of CNS tumors, including PXA, PXA with anaplasia, gangliogliomas, anaplastic gangliogliomas and extracerebellarpilocyticastrocytomas [10]. Recently, a 64-cases cohort study demonstrates up to 56% eGBM can be detected BRAF V600E mutation, while this mutation is rarely found in other types of GBM [7]. BRAF V600E mutation state constitutively activates MAPK signaling pathway, which lead to the increasing of cell proliferation, apoptosis resistance and tumor progression. That may explain the more aggressive phenotype of eGBM. In the six cases reported here, BRAF V600E mutation was observed in three cases (50%) and thus coincided with the previous reports. As expected, all six cases were immunoreactive for INI-1, the focal loss of which used to be proposed as a valuable marker to identify eGBM from rhabdoid GBM [15]. However, with increasing INI-1-immunoreactive rhabdoid GBMs (rGBM) are identified, the distinguishing value of IN1-1 between eGBM and rhabdoid GBM is doubtable. IDH-1 mutation, which is often seen in secondary GBM, is uncommon to be found in eGBM. To our knowledge, besides the case 6 in this study, there is only one previously reported eGBM case with IDH-1 mutation. That reported eGBM with IDH-1 mutation is progressed from a mixed oligoastrocytoma (WHO grade II) in the resected cavity. While, in the current presentation, the case 6 was concurrent with a WHO-II diffuse astrocytoma. Taken together, those findings suggest that BRAF V600E mutation may play a vital role in tumorigenesis of eGBM. In addition, other genetic alterations including IDH-1 mutation in the context of BRAF V600E mutation could result in transformation of a lowgrade glioma into eGBM.
Radiologically, eGBM mostly showed cystic and solid appearance with mass or irregular configuration. Like the typical GBM, moderate or severe peritumoral edema, necrosis and 12 hemorrhage was commonly seen in all cases of this study. Invasion into the wall of vessels in the subarachnoid space and discohesiveness of tumor cells may be related to the extensive intratumoral hemorrhage of eGBM [12]. Calcium, which is remotely found in typical GMB, is often detected in eGBM with pre-exiting or co-exiting lesion. Therefore, calcium is supposed to accompany with secondary eGBM [16]. To our surprise, CT detected calcium in case 4, which was a primary eGBM. That finding may suggest that calcium can present both in original and secondary eGBM, and may exert distinguishing value between typical GBM and eGBM. Involvement of leptomeninge and cerebrospinal fluid dissemination are notable characteristics of eGBM, most reported cases refer to that.
Frequently locating in cerebral cortex may contribute to the invasion of leptomeninge, which demonstrate thickening and aberrant enhancement with contrast medium. In the current study, three cases revealed an abnormal enhancement of leptomeninge, but only one case showed a "dura matter tail" sign. In addition, case 4 broke through the basal ganglia resulting in dissemination in lateral ventricle, vermis and tentorium of cerebellum at diagnosis. Advanced MR sequence can supply more detailed information associate with cellularity, vascular dynamics, tumoral metabolism and so on. Similar to high grade glioma, eGBM exhibited a markedly restricted diffusion on DWI, increased Cho/NAA ratio on 1 H-MRS and rCBV m ax value on DSC-PWI. Owing to intratumoral hemorrhage or calcium, diffusely patchy or dotted hypointensity which was ranked grade 2 or 3, was found on SWI in 3 cases.

Conclusions
In summary, eGBM is a newly recognized subtype of GBM until 2016 WHO classification of CNS tumor, the more specific features including clinical, radiological and histopathological remains further study. Although radiological features have some overlaps between typical 13 GBM and eGBM, some characteristics, such as location (often in cerebral cortex), involvement of leptomeninge, intratumoral calcium, may support the diagnosis of eGBM.
In addition, young patient has a history of PXA or low grade glioma with BRAF V600E mutation should be hypervigilant for transformation into eGBM.

Consent for publication
Written informed consent from study participant for publication of their cases and accompanying images.