The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.

Ten subjects were assessed by 11 examiners (three physical therapy students, three physical therapists experienced in rehabilitation, and five physical therapists experienced in orthopedics) to determine intrarater and interrater agreement for an assessment procedure involving palpation and observation of iliac crest heights. The results of two separate trials in a single session showed that both groups of experienced physical therapists had slightly higher intrarater and interrater agreement than student physical therapists. Findings suggest that a need exists to improve the reliability of this commonly used physical therapy assessment procedure.

No abstract

e12070 Background: In triple negative breast cancer (TNBC), anti-PD-L1 is associated with gains in overall survival, however only in tumors with > 1% PD-L1+ ICs. Locoregional cytokine therapy is being evaluated as a method of priming and redirecting ICs to tumors, which may increase I-O benefit particularly in PD-L1 negative tumors. We report a sensitive method of quantifying I-O-related changes in PD-L1 and ICs using mIHC and single-cell hierarchical regression, which controls for within-tumor and across-patient PD-L1/IC heterogeneity. Methods: Pre-treatment and resection tissues from a phase Ib trial of locoregional cytokines (IRX-2, n = 16 subjects) in ESBC were analyzed. IRX-2 contains immunostimulatory cytokines (GM-CSF, IL-2, IFN-α, INF-γ, and IL-12) and was injected in peri-areolar tissue, which communicates directly with tumor-draining lymphatics. Specimens were analyzed for 1) H&E stromal TILs score; 2) clinical PD-L1 IC expression (Ventana SP142, 2 blinded pathologists); and 3) mIHC (PerkinElmer Vectra). InForm software was used to obtain geospatial outputs of tumor cells (CK+) and ICs (CD3, CD8, CD163) across multiple regions of interest (mean:18; range: 9-32). Mixed-effects modeling was used to evaluate for changes in PD-L1, ICs, and IC/tumor distance metrics. Results: PD-L1 and IC quantity by mIHC was highly concordant with clinical PD-L1 IC classification (JT test = 211, p < .001) and TIL score (r = 0.77). Cytokine therapy was associated with higher PD-L1 IC classification in 9/13 tumors, and increased PD-L1 ICs by mIHC in 14/15 (+337%, 95% CI: 120,769%). After adjusting for heterogeneity, cytokine therapy increased CD3+CD8+ ICs (+173%, CI: 93,287%) and CD3+CD8- ICs (+120%, CI: 21,301%). Therapy was also associated with increased effector-helper T-cell clustering (nearest-neighbor distance, -40%, CI: -29,-50%) and effector cell tumor localization (stromal-tumor interface CD8+ density +90%, Cl: 74, 305%). Conclusions: Our method is concordant with clinical PD-L1 and sTILs scores, and can additionally quantify IC and IC distances. This assay may allow for comparative I-O assessments with increased resolution, and will be used in a randomized phase II trial of pembrolizumab, chemo +/- IRX-2 in stage II/III TNBC.