Glioblastoma (GB) recurrences are rarely removed, therefore, tissue modifications induced by radi... more Glioblastoma (GB) recurrences are rarely removed, therefore, tissue modifications induced by radiotherapy, and temozolomide chemotherapy are scarcely known. Nuclear cyclin D1 is associated with GB progression and resistance to therapy. We previously found that the expression of autophagic protein beclin-1 is a major determinant of prognosis in GB. In 31 patients with primary GB and their recurrences, we investigated the protein expression of cyclin D1 and beclin-1, before and after radiotherapy-temozolomide therapy by immunohistochemistry. Most (20/31) primary GBs were negative for nuclear cyclin D1, and highly expressed beclin-1. In their recurrences, cytoplasmic cyclin D1 positivity was observable, which co-localized with beclin-1. Eleven primary GBs instead exhibited low beclin-1 expression and were positive for nuclear cyclin D1; three of their recurrences exhibited an increase of beclin-1, which co-localized with cyclin D1 in the cytoplasm. Our results suggest therapy-induced d...
To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter m... more To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). We assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. Clustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). The EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.
Anaplastic giant cell ependymoma (AGCE) is a very rare neoplasm. Its cytological features, helpfu... more Anaplastic giant cell ependymoma (AGCE) is a very rare neoplasm. Its cytological features, helpful for the intraoperative diagnosis, have been reported only once. AGCE is characterized by giant cells with intranuclear inclusions, besides other findings, observable in ependymal neoplasms, such as intracytoplasmic vacuoles, epithelial and glial features of the tumor cells and ependymal pseudorosettes. These findings can be detected also in intraoperative squash smear. Herein we describe a pineal AGCE, highlighting the cytological and histological correlations and underlining some useful diagnostic clues of this unusual entity.
Beclin 1 is is an autophagy gene, the role of which as a tumour suppressor has recently been reco... more Beclin 1 is is an autophagy gene, the role of which as a tumour suppressor has recently been recognized in a few studies. We examined the expression of Beclin 1 protein in 212 primary human brain tumours, including 97 high-grade glial tumours, 29 low-grade glial tumours, 4 grade III meningiomas, 19 grade II meningiomas, 52 grade I meningiomas, and 11 medulloblastomas. In 94 cases, including 56 glial tumours, 35 meningiomas, and 3 medulloblastomas we also assessed Beclin 1 mRNA expression by real-time RT-PCR. In most high-grade astrocytic, ependymal neoplasms and atypical meningiomas we found a decrease of cytoplasmic protein expression that was, instead, high in the majority of low-grade tumours and in medulloblastomas. The expression level of Beclin 1 mRNA was significantly lower in glioblastomas than in grade II (p=0.04) and grade I (p=0.01) astrocytomas; in grade III than in grade I astrocytomas (p=0.01); in grade II than in grade I meningiomas (p=0.03); and in all glial tumours when compared to all meningiomas (p<0.0001). Cytoplasmic expression is thought to be linked to the functional protein. Our observations are in line with studies that demonstrated decreased expression of Beclin 1 in human breast, ovarian, prostate and ovarian cancer and furtherly support its involvement also in brain tumours, which had previously been demonstrated in a few experimental studies, both in spontaneous and in therapy-induced autophagy. Furthermore, our study suggests possible differences of Beclin 1 involvement and its role among the different histotypes of brain neoplasms. Further studies are needed to highlight Beclin 1 function in tumour suppression and/or in tumour survival through autophagy and other related programmed cell death processes in brain tumours.
Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical ... more Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.
Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was desig... more Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.
Glioblastoma (GB) recurrences are rarely removed, therefore, tissue modifications induced by radi... more Glioblastoma (GB) recurrences are rarely removed, therefore, tissue modifications induced by radiotherapy, and temozolomide chemotherapy are scarcely known. Nuclear cyclin D1 is associated with GB progression and resistance to therapy. We previously found that the expression of autophagic protein beclin-1 is a major determinant of prognosis in GB. In 31 patients with primary GB and their recurrences, we investigated the protein expression of cyclin D1 and beclin-1, before and after radiotherapy-temozolomide therapy by immunohistochemistry. Most (20/31) primary GBs were negative for nuclear cyclin D1, and highly expressed beclin-1. In their recurrences, cytoplasmic cyclin D1 positivity was observable, which co-localized with beclin-1. Eleven primary GBs instead exhibited low beclin-1 expression and were positive for nuclear cyclin D1; three of their recurrences exhibited an increase of beclin-1, which co-localized with cyclin D1 in the cytoplasm. Our results suggest therapy-induced d...
To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter m... more To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). We assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. Clustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). The EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.
Anaplastic giant cell ependymoma (AGCE) is a very rare neoplasm. Its cytological features, helpfu... more Anaplastic giant cell ependymoma (AGCE) is a very rare neoplasm. Its cytological features, helpful for the intraoperative diagnosis, have been reported only once. AGCE is characterized by giant cells with intranuclear inclusions, besides other findings, observable in ependymal neoplasms, such as intracytoplasmic vacuoles, epithelial and glial features of the tumor cells and ependymal pseudorosettes. These findings can be detected also in intraoperative squash smear. Herein we describe a pineal AGCE, highlighting the cytological and histological correlations and underlining some useful diagnostic clues of this unusual entity.
Beclin 1 is is an autophagy gene, the role of which as a tumour suppressor has recently been reco... more Beclin 1 is is an autophagy gene, the role of which as a tumour suppressor has recently been recognized in a few studies. We examined the expression of Beclin 1 protein in 212 primary human brain tumours, including 97 high-grade glial tumours, 29 low-grade glial tumours, 4 grade III meningiomas, 19 grade II meningiomas, 52 grade I meningiomas, and 11 medulloblastomas. In 94 cases, including 56 glial tumours, 35 meningiomas, and 3 medulloblastomas we also assessed Beclin 1 mRNA expression by real-time RT-PCR. In most high-grade astrocytic, ependymal neoplasms and atypical meningiomas we found a decrease of cytoplasmic protein expression that was, instead, high in the majority of low-grade tumours and in medulloblastomas. The expression level of Beclin 1 mRNA was significantly lower in glioblastomas than in grade II (p=0.04) and grade I (p=0.01) astrocytomas; in grade III than in grade I astrocytomas (p=0.01); in grade II than in grade I meningiomas (p=0.03); and in all glial tumours when compared to all meningiomas (p<0.0001). Cytoplasmic expression is thought to be linked to the functional protein. Our observations are in line with studies that demonstrated decreased expression of Beclin 1 in human breast, ovarian, prostate and ovarian cancer and furtherly support its involvement also in brain tumours, which had previously been demonstrated in a few experimental studies, both in spontaneous and in therapy-induced autophagy. Furthermore, our study suggests possible differences of Beclin 1 involvement and its role among the different histotypes of brain neoplasms. Further studies are needed to highlight Beclin 1 function in tumour suppression and/or in tumour survival through autophagy and other related programmed cell death processes in brain tumours.
Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical ... more Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.
Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was desig... more Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.
Uploads
Papers by Marzia Toscano