Comparison of immunohistochemical markers that can be used for targeted therapy between microsatellite stable and instable endometrial cancers, together with morphological and clinical parameters

Aim: The purpose of our study was to evaluate the differences in tumor microenvironments of microsatellite instabile and stable endometrioid type endometrial carcinomas by using CD3, CD4, CD8, CD45, PD-L1 antibodies. It was aimed to investigate the prognostic effect of tumor immune microenvironment together with histomorphological features which may help predicting response to immunotherapy. Materials and methods: The study group consisted of 108 patients who were diagnosed as endometrioid type endometrial carcinoma at Ankara University Medical School, Pathology Department between January 2020 – December 2022 and met the inclusion criterias. In 51 of these, MMR system deficiency was demonstrated by immunohistochemistry (MMRd group). 57 had no MMR protein loss with IHC (MMRp group). Among MMRd group, the cases showing microsatellite instability by PCR-MSI test or NGS were determined as "MSI/MMRd" group (n=35). Remaining 16 cases that did not show MSI by molecular analyses, and 57 MMRp cases together constituted the "MSS/MMRp" group (n=73). Archival HE, MSH2, MSH6, PMS2, MLH1, ER, PR, p53 stained sections of all cases were reevaluated for FIGO grade, stage, prognostic group, presence and extent of lymphovascular invasion, lymph node metastasis, depth of myometrial invasion, presence and extent of MELF type invasion, presence and extent of squamous and mucinous differentiation, presence and extent of necrosis, comedonecrosis, psammomatous calcification, tumor localisation, hormone receptor status, MMR proteins and p53 expressions. Six tissue-microarray (TMA) blocks were constructed in which each case was represented by at least 2 (max 5) cores of 1 mm size. CD3, CD4, CD8, CD45, PD-L1 stained TMA sections were scanned and evaluated on digital images. CD3, CD4, CD8, CD45 positive lymphocyte densities were calculated for both intratumoral and stromal compartments as follows: number of positive cells/mm2 . For PD-L1, both density and combined positive scores were calculated for neoplastic cells. For stromal PD-L1 evaluation, percentage of tumor stroma that was covered by PD-L1 positive stromal mononuclear inflammatory cells were estimated. Two different categorisations were used for both stromal and tumoral PD-L1 staining by using %1 and %25 cut-offs (negative, <1%, 1-25%, >25%) followed by "negative" and "positive" groups. Statistical analyses was performed using SPSS 23.0 (IBM, Armank, NY, USA), p<0.05 was considered statistically significant. 4 Results: The MSI/MMRd group showed more cases with advanced stage, high histological grade, larger tumor size than the MSS/MMRp group. LVI, tumor necrosis and MELF type invasion was more frequent and extensive in the MSI/MMRd group. Lymph node metastasis, comedonecrosis and psammomatous calcification were more common in the MSI/MMRd. Intratumoral CD3 and CD8 positive lymphocyte density, total CD8 positive lymphocyte density, PD-L1 density in stromal lymphocytes and tumor cells were significantly higher in the MSI/MMRd. In cases with extensive MELF, an increase in the PD-L1 staining in stromal lymphocytes was observed. Although there was no difference in terms of survival, recurrence or metastasis between MSI/MMRd and MSS/MMRp cases; clinicopathological features like older age, LVI, lymph node metastasis, high grade, advanced stage, psammoma bodies, extensive necrosis, deep myometrial invasion were found to be associated with decreased survival. Of these, LVI, extensive necrosis and deep myometrial invasion were also associated with recurrence/metastasis. Multivariate Cox analysis using LVI, extent of necrosis, FIGO grade and myometrial invasion showed that FIGO grade and extent of necrosis were independent prognostic factors for survival. In addition, survival was significantly decreased in the group that PD-L1 was totally negative in neoplastic cells. Moreover, increase in intratumoral CD8 positive lymphocyte density was associated with prolonged survival time in the MSI/MMRd group. While, increase in stromal CD4 positive lymphocyte density was associated with decreased survival in the MSS/MMRp. Conclusions: Although poor prognostic morphological findings were more common in the MSI/MMRd group, there was no difference in terms of survival, recurrence or metastasis between MSI/MMRd and MSS/MMRp cases. The reason of this is thought to be related with the differences in tumor microenvironment among tumor groups. Increased intratumoral CD8 positive lymphocyte density being correlated with better survival as shown in our study which can be considered as a proof of this argument. There are conflicting results in the literature regarding the prognostic significance of PD-L1 expression of neoplastic cells and stromal lymphocytes. In our study, increased PD-L1 expression on tumor cells was found to be associated with prolonged survival, which in fact may be related to the correlation between PDL1 and CD8 positive cells among all compartments. Anahtar Kelimeler: Endometrial carcinoma, microsatellite instability, tumor microenvironment, prognosis, immun checkpoint.