PMC3872647_01
Hemotoxylin and eosin staining of the three lesions; (a) primary gliosarcoma (x200); (b) meningioma (x200); (c) recurrent gliosarcoma (x200)
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F1
The pathological examination revealed two distinct lesions being the anterior a meningioma and the posterior a gliosarcoma [Figure 1]. The MRI showed a regrowth of the initial gliosarcoma and the patient was re-operated with macroscopically complete removal of the recurrent gliosarcoma [Figure 1].
Methylation-specific PCR (MSP) analyses of the MGMT promoter of the three lesions; P (primary gliosarcoma); R (recurrent gliosarcoma); M (meningioma). MSP controls reactions consisted of blood-extracted DNA from a cancer-free individual to use as umethylated DNA control (Un.), and a CpGenome  Universal Methylated DNA (Chemicon International) as methylated DNA control (m). PCR reactions in the absence of DNA (water) were performed as negative controls for both the unmethylated and methylated reactions
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F2
As illustrated in Figure 2, MGMT methylation was found in both primary and recurrent gliosarcoma. The meningioma lesion was negative for MGMT methylation [Table 1 and Figure 2].
Imunohistochemistry and Chromogenic In Situ Hybridization (CISH) analysis of the three lesions. COX2 immunohistochemistry (x200) positive for the meningioma lesion and negative for the primary and recurrent gliosarcoma. MGMT staining (x200) was only positive for the meningioma. EGFR immunostaining was negative in primary gliosarcoma and meningioma, with recurrent gliosarcoma exhibiting strong positivity. CISH analysis of EGFR confirmed these findings after EGFR amplification
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F3
In concordance with methylation status, MGMT expression was only present in the meningioma and absent in both primary and recurrent gliosarcoma [Table 1 and Figure 3]. EGFR protein expression [Table 1 and Figure 3]. EGFR immunohistochemistry showed a focal region with strong immunoreactivity in the recurrent gliosarcoma. EGFR gene copy number alterations [Table 1 and Figure 3]. EGFR gene amplification, represented by clusters of more than 5 signals per neoplastic cell, was observed only in the recurrent gliosarcoma, in the same region with strong focal positivity for EGFR protein [Table 1 and Figure 3]. We observed positivity only in meningioma lesion [Figure 3]. Both gliosarcomas (primary and recurrent) were negative for COX-2 expression [Table 1 and Figure 3].