The Immune Landscape of Undifferentiated Pleomorphic Sarcoma

INTRODUCTION: Undifferentiated pleomorphic sarcoma (UPS) can be associated with a relatively dense immune infiltration. Immune checkpoint inhibitors (anti-PD1, anti-PDL1, and anti-CTLA4) are effective in 20% of UPS patients. We characterize the immune microenvironment of UPS and its association with oncologic outcomes. MATERIAL AND METHODS: Surgically resected UPS samples were stained by immunohistochemistry (IHC) for the following: tumor-associated immune cells (CD3, CD8, CD163, CD20), immune checkpoints (stimulatory: OX40, ICOS; inhibitory: PD-L1, LAG3, IDO1, PD1), and the adenosine pathway (CD73, CD39). Sections were reviewed for the presence of lymphoid aggregates (LA). Clinical data were retrospectively obtained for all samples. The Wilcoxon rank-sum and Kruskal-Wallis tests were used to compare distributions. Correlations between biomarkers were measured by Spearman correlation. Univariate and multivariate Cox models were used to identify biomarkers associated with overall survival (OS) and disease-free survival (DFS). Unsupervised clustering was performed, and Kaplan-Meier curves and log-rank tests used for comparison of OS and DFS between immune clusters. RESULTS: Samples analyzed (n=105) included 46 primary tumors, 34 local recurrences, and 25 metastases. LA were found in 23% (n=10/43), 17% (n=4/24), and 30% (n=7/23) of primary, recurrent, and metastatic samples, respectively. In primary UPS, CD73 expression was significantly higher after preoperative radiation therapy (p=0.009). CD39 expression was significantly correlated with PD1 expression (primary: p=0.002, recurrent: p=0.004, metastatic: p=0.001), PD-L1 expression (primary: p=0.009), and CD3+ cell densities (primary: p=0.016, recurrent: p=0.043, metastatic: p=0.028). In recurrent tumors, there was a strong correlation between CD39 and CD73 (p=0.015), and both were also correlated with CD163+ cell densities (CD39 p=0.013; CD73 p\u3c0.001). In multivariate analyses, higher densities of CD3+ and CD8+ cells (Cox Hazard Ratio [HR]=0.33; p=0.010) were independently associated with OS (CD3+, HR=0.19, p\u3c0.001; CD8+, HR= 0.33, p=0.010) and DFS (CD3+, HR=0.34, p=0.018; CD8+, HR=0.34, p= 0.014). Unsupervised clustering of IHC values revealed three immunologically distinct clusters: immune high, intermediate, and low. In primary tumors, these clusters were significantly associated with OS (log-rank p\u3c0.0001) and DFS (p\u3c0.001). CONCLUSION: We identified three immunologically distinct clusters of UPS Associated with OS and DFS. Our data support further investigations of combination anti-PD-1/PD-L1 and adenosine pathway inhibitors in UPS

The coronal pulp cavity index: A forensic tool for age determination in adults

Background: Various biochemical and histological methods are available for human age determination which are invasive and may require extraction of teeth. The present study aims to assess the accuracy of age estimation from tooth-coronal index (TCI) of known age and sex individuals and to present a noninvasive method for age estimation. Materials and Methods: This retrospective study comprised 88 patients, which included 54 males and 34 females. An orthopantomogram of these individuals were taken, and premolars and molars in the same were evaluated. The height of the crown (coronal height [CH]) and the height of the coronal pulp cavity (coronal pulp cavity height [CPCH]) was digitally measured on the computer screen. The TCI given by Ikeda et al. in 1985 (TCI = [CPCH × 100]/CH.) was computed on each tooth and regressed on real age of the sample. The mean, median, range, and standard deviation of the computed index were calculated. The correlation between the actual age and the estimated age was calculated using t-test. P < 0.05 was considered significant. Results: Results revealed that there is a significant correlation between the TCI with age. Increase in TCI observed with age; however, it showed no significant sex difference. Conclusion: TCI is a precise, noninvasive and easily used reliable biomarker for age estimation and is applicable to both living and dead individuals

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach