Freiburg Neuropathology Case Conference

A 58-year-old female patient with a history of left temporal glioblastoma multiforme (GBM) World Health Organization (WHO) grade IV that had been diagnosed and treated by total tumor resection 12 months earlier (Figs. 1 and 2) presented to a peripheral hospital with a new onset of dizziness, nausea and diplopia. A magnetic resonance imaging (MRI) of the head was performed, which revealed a new space-occupying contrast-enhanced prepontine lesion (Fig. 3). At the left temporal resection site, no signs of a GBM recurrence were present. The patient was immediately transferred to our hospital. At the time of admission, the patient was awake and oriented with nausea and diplopia due to a palsy of the sixth cranial nerve. There were no clinical signs of swallowing difficulties or impairment of other cranial nerves. Due to the tumor location, the mass effect on the brainstem and the unknown etiology of the prepontine lesion, the indication for an open biopsy with reduction of the tumor was established. The day before surgery, the patient deteriorated neurologically and presented with respiratory insufficiency. A computed tomography (CT) of the head identified an obstructive hydrocephalus, which required an immediate placement of an external ventricular drainage.

Open biopsy and partial tumor resection were performed the following day with the patient under general anesthesia in a park bench position using intraoperative neurophysiological monitoring. The partial tumor resection was performed via a retrosigmoid approach. Macroscopically the tumor showed a grayish color, had a soft consistency and was adherent to the cranial nerves. Due to the adherence of the tumor in relation to the cranial nerves, a complete resection could not be performed.

The postoperative course on our intensive care unit showed missing swallowing and gag reflexes. An extubation could therefore not be performed, a tracheotomy was refused by the otherwise neurologically intact patient. In the following days an acute decrease of consciousness occurred. A CT of the head demonstrated an extensive tumor progression with compression of the brainstem. In fulfilment of the patient’s will, the treatment was discontinued and the patient passed away the following day.

Initial preoperative MRI shows a space-occupying lesion of the left-sided temporal lobe (Fig. 1, arrows). T2 weighted images (Fig. 1a, arrow) show regressive changes on T1 weighted images after administration of gadolinium (Gd). The lesion displays distinct contrast enhancement (Fig. 1b, arrow and arrowhead). Early postoperative (< 24 h) MRI obtained after tumor surgery confirms complete tumor resection (Fig. 2) but 12 months later the patient had MRI for dizziness, nausea, and diplopia (Fig. 3) showing a large space-occupying lesion (arrow) located in the prepontine cistern. The lesion compresses and dislocates the brainstem and the myelon (Fig. 3b, arrowhead). On T1-weighted MRI after administration of Gd the lesion shows some degree of homogeneous enhancement of contrast medium (Fig. 3c–e, arrow). On diffusion-weighted images (b-value: 1000), the lesion shows restricted diffusion (Fig. 3f, arrow).

Nearly one year prior to the surgical removal of the clival tumor, we had assessed a tissue biopsy from the same patient from the temporal lobe. We identified a glial tumor with a diffuse infiltration into neighboring CNS tissue (Fig. 4a). The tumor cells looked pleomorphic and the nuclei appeared chromatin dense. Mitotic features and necrotic areas were visible. The tumor cells showed positivity in the immunohistochemical reaction for glial fibrillary acidic protein (GFAP, Fig. 4b). No R132H mutation of the IDH1 gene product was detectable immunohistochemically. After further immunohistochemical characterization, the patient was diagnosed with a glioblastoma, IDH-wildtype, CNS WHO grade 4.

We now obtained a small tissue biopsy for intraoperative neuropathological assessment. A cryostat section was stained with hematoxylin and eosin (H&E, Fig. 5a). A pleomorphic tumor with a high cell density and mitotic features could be identified. Therefore, it was suspected that the tissue corresponds to the pre-existing glioblastoma. Additional tissue was fixed in formaldehyde and embedded in paraffin (FFPE).

An H&E stained FFPE tissue section showed connective tissue rich in collagen fibers as well as fresh bleedings. Several small fragments of the tumor could be observed (Fig. 5b). The nuclei were chromatin dense. Mitotic features were visible. Overall, the lesion resembled a small round blue cell tumor (SRBCT).

While the tumor cells of the pre-existing glioblastoma had stained positive for glial fibrillary acidic protein (GFAP), the immunohistochemical reaction for GFAP now remained negative (Fig. 5c). The tumor cells were also negative for oligodendrocyte transcription factor 2 (OLIG2). The nuclear expression of ATRX was preserved. An R132H mutation of the IDH1 gene product could not be detected immunohistochemically. The proliferation marker MIB‑1 labelled more than 60% of the tumor cells (Fig. 5d).

The immunohistochemical reactions for the pan-cytokeratin markers MNF116 and AE1/3 as well as epithelial membrane antigen (EMA), transcription termination factor 1 (TTF1) and melan‑A remained negative. Vascular structures were labelled in the immunohistochemical reaction for alpha-smooth muscle actin (SMA). Desmin, signal transducer and activator of transcription 6 (STAT6) and S100 were negative. Nuclear expression of integrase interactor 1 (INI-1) was preserved.

Only a few cells were labelled in the immunohistochemical reactions for leukocyte common antigen (LCA, CD45), B‑lymphocyte antigen CD20 and B‑cell antigen receptor complex-associated protein alpha chain (CD79a). The tumor cells showed partial positivity in the reaction for vimentin (Fig. 5e). Positivity was also observed in the reactions for synaptophysin (Fig. 5f) and neural cell adhesion molecule (NCAM, CD56, Fig. 5g), while chromogranin remained negative. The tumor cells also showed positive signal in the immunohistochemical reaction for CD99 (Fig. 5h). With the previous histological and immunohistochemical assessment, a clinically suspected lymphoma, chordoma and a chondrosarcoma could be excluded. The tumor’s immunohistochemical profile did not match the pre-existing glioblastoma. With synaptophysin and CD56 positivity, a metastasis of a neuroendocrine carcinoma was discussed. All additional immunohistochemical reactions (CDX2, CK19, CK20 and EP4) were negative. Since the tumor cells were expressing CD99, we have also discussed a Ewing sarcoma. No translocation involving the EWSR1 gene could be detected by chromogenic in situ hybridization (CISH). Next, an 850k DNA methylation assay of the current tumor as well as the tissue from the pre-existing glioblastoma was performed [8]. The methylation pattern was compared to a reference dataset, the brain tumor classifier, version 11b4. Both the previous tissue from the temporal lobe that has histologically and immunohistochemically been classified as a glioblastoma and the current biopsy showed the highest calibrated score for the methylation class family glioblastoma, IDH wildtype. The methylation pattern of both samples matched to the methylation class glioblastoma, IDH wildtype, subclass RTK II. We then applied the most recent reference dataset, the newly developed brain tumor classifier version 12.5. With a highest calibrated score of 0.9999 and 0.99964, both samples then matched to the methylation class glioblastoma, IDH-wildtype with primitive neuronal component (novel). The copy number variation profile showed the characteristics of glioblastomas with a primitive neuronal component (Fig. 6a,b). Both samples showed a loss of chromosome 10, which is observed in more than 90% of the glioblastomas with a primitive neuronal component [9]. Moreover, there was a gain in chromosome 1, which is observed in around half of the cases [9]. The copy number variation profile also showed alterations in RB1. No CDKN2A/B alterations were observed. The MGMT promotor was predicted to be methylation in both specimens. Taken together, the two tumor samples showed a different immunohistochemical pattern. The clival tumor showed no GFAP expression. The absence of GFAP, the high proliferation index and the CD99 positivity in particular led to the exploration of further potential differential diagnoses, e.g. a metastasis of a neuroendocrine carcinoma or a Ewing sarcoma. The result of the 850k methylation assay then revealed that both tumors share the same distinct methylation pattern. Therefore, the clival tumor has to be seen as a metastasis of the tumor in the temporal lobe. The methylation pattern of both tumors matched to the novel methylation class glioblastoma, IDH wildtype with primitive neuronal component. Given the loss of GFAP and the expression of synaptophysin and CD56 in the new clival biopsy, the tumor did certainly show the immunohistochemical features of a glioblastoma with a neuronal component. The immunohistochemical reactions for synaptophysin, CD56 and CD99 were subsequently also performed on the initial biopsy tissue from the temporal lobe. The tumor cells showed a strong positivity for synaptophysin and CD56. Furthermore, some tumor cell groups were weakly positivity for CD99. Despite moderate GFAP positivity, it is therefore reasonable to diagnose a glioblastoma with a neuronal component in the temporal lobe biopsy as well.