Volume 150, Issue 3, Pages 552-561 (September 2018) Somatic mutation profiling of vulvar cancer: Exploring therapeutic targets  Sebastian Zięba, Artur Kowalik, Kamil Zalewski, Natalia Rusetska, Krzysztof Goryca, Agata Piaścik, Marcin Misiek, Elwira Bakuła- Zalewska, Janusz Kopczyński, Kamil Kowalski, Jakub Radziszewski, Mariusz Bidziński, Stanisław Góźdź, Magdalena Kowalewska  Gynecologic Oncology  Volume 150, Issue 3, Pages 552-561 (September 2018) DOI: 10.1016/j.ygyno.2018.06.026 Copyright © 2018 The Authors Terms and Conditions

Fig. 1 Distribution of pathogenic mutations in hrHPV-positive (n = 52) and hrHPV-negative (n = 29) VSCC tumors. Each column corresponds to an individual tumor, while each row corresponds to the mutated gene. Tumor samples with no mutations detected are depicted in grey, missense mutations are highlighted in green, whereas black hits indicate nonsense mutations. The OncoPrinter tool from cBioPortal was used to generate this figure (http://www.cbioportal.org/oncoprinter.jsp). Gynecologic Oncology 2018 150, 552-561DOI: (10.1016/j.ygyno.2018.06.026) Copyright © 2018 The Authors Terms and Conditions

Fig. 2 Expression of total mTOR protein detected by IHC. A – HSIL sample, B – VSCC sample obtained from a patient with no progression, C - VSCC sample obtained from a patient experiencing disease progression, D – a sample of VSCC recurrence. Upper panel – 40× and lower panel – 10× magnification. Gynecologic Oncology 2018 150, 552-561DOI: (10.1016/j.ygyno.2018.06.026) Copyright © 2018 The Authors Terms and Conditions

Fig. 3 Percentage of mTOR-staining tumor cells (upper panel) and immune infiltrating cells (lower panel) in HSIL and VSCC samples. Asterisks indicate the significance as follow: **** for <0.0001, *** for 0.0001 to 0.001, ** for 0.001 to 0.01 and * for 0.01 to 0.05 p-values. Gynecologic Oncology 2018 150, 552-561DOI: (10.1016/j.ygyno.2018.06.026) Copyright © 2018 The Authors Terms and Conditions

Fig. 4 Effect of rapamycin (red), everolimus (blue) and AZD2014 (yellow) at 10 μM concentrations on viability of A431, CAL-39 and SW-954 cells. The results are expressed as a mean of three experiments performed in three technical replicates. Asterisks indicate the significance of difference of cells viability (%) compared to control cells (white) according to the following convention: **** for <0.0001, *** for 0.0001 to 0.001, ** for 0.001 to 0.01 and * for 0.01 to 0.05 p-values. Gynecologic Oncology 2018 150, 552-561DOI: (10.1016/j.ygyno.2018.06.026) Copyright © 2018 The Authors Terms and Conditions

Fig. 5 Signaling pathways implicated in VSCC carcinogenesis. PI3KCA/AKT/mTOR and MAPK cellular pathways may be activated by RTK and/or by the mutated downstream members of these signaling circuits. Contained within the cytoplasm, these signaling network members function either as oncogenic (blue) or tumor suppressor (green) proteins. Genes encoding some of these proteins are frequently mutated in VSCC tumors, resulting in mTORC1 activation. In addition to the RB and TP53 mutations, two main cell cycle regulators may be inactivated by viral proteins produced in HPV infected individuals. Abbreviations: AKT - AKT Serine/Threonine Kinase 1; CDK4/6 - Cyclin Dependent Kinase 4/Cyclin Dependent Kinase 6; CDKN2A - Cyclin Dependent Kinase Inhibitor 2A; MAPK - Mitogen-Activated Protein Kinase; mTOR - Mechanistic Target Of Rapamycin Kinase; PI3KCA - Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha; PTEN - Phosphatase And Tensin Homolog; FBWX7 - F-Box And WD Repeat Domain Containing 7; RAS – N-, K- and H-RAS; RB - RB Transcriptional Corepressor; RTK - receptor tyrosine kinases. Gynecologic Oncology 2018 150, 552-561DOI: (10.1016/j.ygyno.2018.06.026) Copyright © 2018 The Authors Terms and Conditions