High concordance of a closed-system, RT-qPCR breast cancer assay for HER2 mRNA, compared to clinically determined immunohistochemistry, fluorescence in situ hybridization, and quantitative immunofluorescence.

Historically, mRNA measurements have been tested on several commercially available platforms, but none have gained broad acceptance for assessment of HER2. An mRNA measurement, as a continuous value, has the potential for use in adjudication of the equivocal category. Here we use a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay in a closed, single-use cartridge, automated system. Multiple cores (1 mm in diameter) were retrospectively collected from 80 formalin-fixed paraffin-embedded (FFPE) tissue blocks with invasive breast cancer seen by Yale Pathology Labs between 1998 and 2011. Tissue cores were processed with a FFPE lysis kit to create lysates that were tested with the automated RT-qPCR assay. Results for IHC and FISH were extracted from the pathology reports and quantitative immunofluorescence (QIF) for each case was measured as previously described. Quality control testing showed that the GX platform RT-qPCR shows no case to case cross contamination on material from routine histology practices. Concordance between RT-qPCR and IHC/FISH was 91.25% (sensitivity=0.87; specificity=0.94; PPV=0.89; NPV=0.92) using a pre-defined delta Ct cut-off (dCt≥-1) for HER2. Concordance (OPA) between RT-qPCR and QIF was 94% (sensitivity=0.90; specificity=0.96; PPV=0.93; NPV=0.94) using dCt≥-1 and a previously defined cut-point for positivity by QIF. In conclusion, the closed system RT-qPCR assay shows >90% concordance with the ASCO/CAP HER2 IHC/FISH scoring. Additionally, the RT-qPCR assay is highly concordant (94%) with the continuous variable HER2 QIF assay, and may better reflect the true continuum of HER2 receptor status in invasive breast cancer. These initial results suggest that fast, closed system molecular assays may have future value for the adjudication of the ASCO/CAP HER2 equivocal category or possibly routine usage in time constrained or low resource settings.

Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer.

PD-L1 is expressed in a percentage of lung cancer patients and those patients show increased likelihood of response to PD-1 axis therapies. However, the methods and assays for the assessment of PD-L1 using immunohistochemistry are variable and PD-L1 expression appears to be highly heterogeneous. Here, we examine assay heterogeneity parameters toward the goal of determining variability of sampling and the variability due to pathologist-based reading of the immunohistochemistry slide. SP142, a rabbit monoclonal antibody, was used to detect PD-L1 by both chromogenic immunohistochemistry and quantitative immunofluorescence using a laboratory-derived test. Five pathologists scored the percentage of PD-L1 positivity in tumor- and stromal-immune cells of 35 resected non-small cell lung cancer cases, each represented on three separate blocks. An intraclass correlation coefficient of 94% agreement was seen among the pathologists for the assessment of PD-L1 in tumor cells, but only 27% agreement was seen in stromal/immune cell PD-L1 expression. The block-to-block reproducibility of each pathologist's score was 94% for tumor cells and 75% among stromal/immune cells. Lin's concordance correlation coefficient between pathologists' readings and the mean immunofluorescence score among blocks was 94% in tumor and 68% in stroma. Pathologists were highly concordant for PD-L1 tumor scoring, but not for stromal/immune cell scoring. Pathologist scores and immunofluorescence scores were concordant for tumor tissue, but not for stromal/immune cells. PD-L1 expression was similar among all the three blocks from each tumor, indicating that staining of one block is enough to represent the entire tumor and that the spatial distribution of heterogeneity of expression of PD-L1 is within the area represented in a single block. Future studies are needed to determine the minimum representative tumor area for PD-L1 assessment for response to therapy.