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. 2020 Jun 16;10:9705. doi: 10.1038/s41598-020-66257-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Quantification of PD-1 and PD-L1 in FIHC images. (A) Expression of PD-1+ at the lamina propria (LP) and PD-L1 + (LP and epithelium) calculated as MFI × area in progressing (green bars) versus control (grey bars) (*P < 0.05, ** P < 0.01). MFI of PD-L1 was calculated at the epithelium (B) and lamina propria (C) and PD-1 at the lamina propria (D). Expression of PD-L1 was calculated as the product of MFI and average positive area at the epithelium (E) and lamina propria (F) and PD-1 at the lamina propria (G). A total of 5 images per sample were analyzed, and the average of the results is shown in the bar graph - progressing (green bars) versus control (grey bars)(*P < 0.05, ** P < 0.01). (H) Differences in expression of PD-L1 (MFI × Area) between matched premalignant and OSCC patients. One case has 2 previous biopsies and “a” represents the most recent biopsy (1 year) and “b” represents the earlier biopsy (2 year). There are no significant differences between the groups (paired t-tests between OSCC and premalignant cases, P > 0.4) (I) Bland-Altman analysis, difference versus average, between two examiners (bias of 289 ± 1446 and a 95% limit of the agreement between −2546 and +3124). (J) Linear regression of the scores of the two examiners (slope of 0.8615 ± 0.02436 and R² = 0.8742).