The etiology of paragangliomas (PGLs) is unclear, as recent data suggest low penetrance of SDHxmutations (1), and mice mutated in Sdhb/d are vulnerable to aberrant differentiation but do not develop PGLs (2-3). Studying prospectively- collected head and neck PGLs by electron microscopy (EM), we found cytomegalovirus-like particles in all tumors.To confirm CMV infection we analysed a series of over 140 PGL cases for SDHxmutational status and CMV infection. Germline point mutations and large deletion/rearrangements of the SDHxgenes were analysed by PCR, sequencing and MLPA. Blood and tumor DNAs were analysed for CMV sequences using PCR. Tumor tissues were analysed by rapid in situ hybridization (RISH) with a large CMV probe, EM, cryo-immuno EM, IF, IHC and WB for several CMV proteins. Viral transmissibility to HELF, MRC5, and HEK293T cells from PGL cells or PGL tissue extracts was tested with protocols modified from Rous (4) and Heine et al. (5). PGL-derived cells were treated with ganciclovir/valganciclovir and imatinib in vitroand in vivo. SDHxmutational analysis showed a mutation frequency of 34.3%. CMV-like particles were observed in all the 51 cases analysed by EM. The CMV reactive proteins IE, gB, pp65, vMIA and their partner cellular proteins (ZEB1, PDGFRA, viperin) were detected by IF, IHC, immuno-EM, and WB in all cases. In our attempts to amplify the viral DNA using CMV-specific primers, we detected a viral glycoprotein B sequence stretch in 68.2% and 82.3% of the tested PGL and matched blood samples respectively. Overall, 36/40 PGLs examined by RISH resulted positive. Exposure of HELF, HEK293T, and MRC5 cell lines to PGL cells or tissue extracts resulted in cytopathic effects and PCR positivity for CMV sequences. PGL-derived xenografts presented the same viral particles and proteins of the original tumors, while CMV sequences were retrievable from the blood of the xenografted mice. Ganciclovir and imatinib inhibited paraganglioma cell growth, downregulated the expression of CMV proteins and their host partners, and significantly prevented engraftment of paraganglioma cells. Overall, this suggests that infection with a CMV- like virus is common in PGLs. ACKNOWLEDGMENT: Work supported by AIRC Grant IG 16932. 1. Benn DE et al (2018) J Med Genet doi: 10.1136/jmedgenet-2018-105427. 2. Bayley JP et al (2009) PLoS One 4: e7987. 3. Ashtekar A et al (2017) Endocr Relat Cancer doi: 10.1530/ERC-17-0229. 4. Rous P (1911) J Exp Med doi:10.1084/jem.13.4.397.

Possible role of a CMV-like virus in paragangliomas

M. R. Pantalone
;
F. Verginelli;S. Perconti;D. L. Esposito;S. Vespa;VALENTINUZZI, SILVIA;DE FABRITIIS, SIMONE;TARANTINI, VITTORIA;S. Soliman;A. Ramassone;M. Di Marco;SERLUCA, MICHELE;A. Veronese;R. Visone;C. T. Paties;M. Sanna;R. Mariani- Costantini
2018

Abstract

The etiology of paragangliomas (PGLs) is unclear, as recent data suggest low penetrance of SDHxmutations (1), and mice mutated in Sdhb/d are vulnerable to aberrant differentiation but do not develop PGLs (2-3). Studying prospectively- collected head and neck PGLs by electron microscopy (EM), we found cytomegalovirus-like particles in all tumors.To confirm CMV infection we analysed a series of over 140 PGL cases for SDHxmutational status and CMV infection. Germline point mutations and large deletion/rearrangements of the SDHxgenes were analysed by PCR, sequencing and MLPA. Blood and tumor DNAs were analysed for CMV sequences using PCR. Tumor tissues were analysed by rapid in situ hybridization (RISH) with a large CMV probe, EM, cryo-immuno EM, IF, IHC and WB for several CMV proteins. Viral transmissibility to HELF, MRC5, and HEK293T cells from PGL cells or PGL tissue extracts was tested with protocols modified from Rous (4) and Heine et al. (5). PGL-derived cells were treated with ganciclovir/valganciclovir and imatinib in vitroand in vivo. SDHxmutational analysis showed a mutation frequency of 34.3%. CMV-like particles were observed in all the 51 cases analysed by EM. The CMV reactive proteins IE, gB, pp65, vMIA and their partner cellular proteins (ZEB1, PDGFRA, viperin) were detected by IF, IHC, immuno-EM, and WB in all cases. In our attempts to amplify the viral DNA using CMV-specific primers, we detected a viral glycoprotein B sequence stretch in 68.2% and 82.3% of the tested PGL and matched blood samples respectively. Overall, 36/40 PGLs examined by RISH resulted positive. Exposure of HELF, HEK293T, and MRC5 cell lines to PGL cells or tissue extracts resulted in cytopathic effects and PCR positivity for CMV sequences. PGL-derived xenografts presented the same viral particles and proteins of the original tumors, while CMV sequences were retrievable from the blood of the xenografted mice. Ganciclovir and imatinib inhibited paraganglioma cell growth, downregulated the expression of CMV proteins and their host partners, and significantly prevented engraftment of paraganglioma cells. Overall, this suggests that infection with a CMV- like virus is common in PGLs. ACKNOWLEDGMENT: Work supported by AIRC Grant IG 16932. 1. Benn DE et al (2018) J Med Genet doi: 10.1136/jmedgenet-2018-105427. 2. Bayley JP et al (2009) PLoS One 4: e7987. 3. Ashtekar A et al (2017) Endocr Relat Cancer doi: 10.1530/ERC-17-0229. 4. Rous P (1911) J Exp Med doi:10.1084/jem.13.4.397.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11564/711504
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