Volume 145, Issue 3, Pages 446-452 (June 2017) Characterization of immune regulatory molecules B7-H4 and PD-L1 in low and high grade endometrial tumors  Amy Bregar, Amit Deshpande, Chris Grange, Tong Zi, Jennifer Stall, Heather Hirsch, Jason Reeves, Sriram Sathyanarayanan, Whitfield B. Growdon, Bo R. Rueda  Gynecologic Oncology  Volume 145, Issue 3, Pages 446-452 (June 2017) DOI: 10.1016/j.ygyno.2017.03.006 Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Immunohistochemistry for clinical detection of microsatellite instability. These panels depict representative images of the protein levels detected by immunohistochemistry for the mismatch repair proteins MLH1 (a), PMS2 (b), MSH2 (c) and MSH6 (d) which were assessed on all low grade endometrioid endometrial carcinomas in our cohort (n=40). No specimens were found to harbor loss of MSH2 or MSH6, however 13 tumors in the cohort had simultaneous loss of expression of MLH1 and PMS2 (Panel A) and were therefore classified as MSI high. All images were 10× magnification. Gynecologic Oncology 2017 145, 446-452DOI: (10.1016/j.ygyno.2017.03.006) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 (A) PD-L1 Scoring Criteria. These panels represent 3 individual tumor samples and demonstrate the range of PD-L1 protein levels detected utilizing two criteria to assign positivity (criteria A and B). Criteria A utilized a cutoff level of positive staining on >1% of carcinoma cells. Criteria B is more stringent and utilized a cutoff level of positive staining on >5% of carcinoma cells. The upper panels are 10× and the lower panels are 1× magnification (B) Correlation of expression with MSI status showed a significantly greater presence of PD-L1+ cells in MSI-high subsets of endometrial tumors utilizing both criteria A (p<0.03) and criteria B (p<0.005). Gynecologic Oncology 2017 145, 446-452DOI: (10.1016/j.ygyno.2017.03.006) Copyright © 2017 The Authors Terms and Conditions

Fig. 3 B7-H4 Scoring Criteria. These panels represent 4 individual tumor samples and demonstrate the range of B7-H4 protein levels detected. An intensity score and a % carcinoma stained proportion was assigned for each sample and an H-Score was generated by multiplying the two indexes. The upper panels are 5× magnification while the lower panels are 1 x magnification. Gynecologic Oncology 2017 145, 446-452DOI: (10.1016/j.ygyno.2017.03.006) Copyright © 2017 The Authors Terms and Conditions

Fig. 4 Co-expression of PD-L1 (criteria A) and B7-H4 across EnCa histologic subtypes. In contrast to low grade endometrial carcinomas, high grade endometrial carcinomas harbored a 50% co-expression of both PD-L1 (criteria A) and B7-H4 (p<0.01). This effect was primarily due to high grade endometrioid and carcinosarcoma subtypes and was statistically identical to the low grade endometrial subset with MSI. Notably, the uterine serous carcinomas demonstrated a low level of co-expression. Gynecologic Oncology 2017 145, 446-452DOI: (10.1016/j.ygyno.2017.03.006) Copyright © 2017 The Authors Terms and Conditions

Fig. 5 RNA expression profiling analysis of CD274 and VTCN1 from the Cancer Genome Atlas (TCGA). Panel A depicts the 4 molecular subgroups defined in the TCGA investigation of 383 endometrial carcinomas and demonstrates that the POLE and MSI subsets have the greatest proportion of lymphocyte infiltration based on a validated Th1 signature that is utilized as a surrogate marker. We observed higher levels of expression of the CD274, the gene coding PD-L1, in the Th1 top 25% quartile in each of the molecular subgroups. In addition to T cell infiltration, the greatest mutational burden was observed in the POLE and MSI subsets of endometrial tumors. Panel B examines the POLE and MSI subsets of tumors and plots the Th1 signature against expression of CD274 and VTCN1 finding a significant linear correlation with CD274 and Th1 index expression (r2=0.56), while there was consistent expression of VTCN1 across various levels of Th1 index expression (r2=0.004). These data suggest the message for PD-L1 is more dependent upon histologic tumor variant and degree of infiltrating lymphocytes, while the message for B7-H4 is not associated with T cell infiltration. Gynecologic Oncology 2017 145, 446-452DOI: (10.1016/j.ygyno.2017.03.006) Copyright © 2017 The Authors Terms and Conditions