81 Inclusion of PD-L1-expressing tumor cells in the combined positive score algorithm yields superior identification of positive specimens around diagnostic cut-offs across multiple indications

BackgroundPD-L1 IHC 22C3 pharmDx uses Tumor Proportion Score (TPS) and Combined Positive Score (CPS) scoring algorithms for the immunohistochemical (IHC) evaluation of PD-L1 protein expression in human cancer tissues; both algorithms include PD-L1 staining tumor cells (TC) in scoring and CPS also includes scoring of PD-L1 staining mononuclear inflammatory cells to aid in the identification of patients for treatment with pembrolizumab (KEYTRUDA®) using indication-specific diagnostic cut-offs. This study evaluated contribution of TC in determining specimen diagnostic status based on the CPS scoring algorithm by looking into four tumor indications approved for use with KEYTRUDA®: gastric or gastroesophageal junction (GEJ) adenocarcinoma (GC/GEJ), urothelial carcinoma (UC), head and neck squamous cell carcinoma (HNSCC), and esophageal squamous cell carcinoma (ESCC). Detection of specimens expressing PD-L1 is significantly dependent on the PD-L1 staining TC component.MethodsA retrospective analysis was done looking at Dako’s internal tumor bank of the mentioned indications that were all stained with PD-L1 IHC 22C3 pharmDx and scored using the TPS, CPS and Quantitative Immune Cell Density (QID) methods described in figure 1. Statistical analysis encompassed looking at the scores generated that met the following criteria: CPS>0, TPS>0 and CPS≠TPS and then evaluating the percentage of those samples that changed from positive to negative diagnostic status upon removal of the TC component from the scoring.ResultsA noticeable downward trend was observed in all four indications in the total number of positives with the removal of the TC component. Table 1 aptly captures this by showing the number of specimens for each indication that had changed from positive to negative around each indication’s diagnostic cut-off(s). The three indications that showed the highest percentages of diagnostic status change were HNSCC (CPS ≥20) with a remarkable 83.3% (130) followed by UC (CPS ≥10) at 46.3% (57) and ESCC (CPS ≥10) at 36.6% (45) of the specimens reclassified as negative.Abstract 81 Figure 1PD-L1 Scoring AlgorithmsThe TPS algorithm (a) is defined as the number of PD-L1 staining tumor cells divided by the total number of viable TC, multiplied by 100. The CPS algorithm (b) includes TC and IC and is defined as the number of PD-L1 staining cells (TC, lymphocytes and macrophages) divided by the total number of viable TC, multiplied by 100. In addition to TPS and CPS, QID (c) was also calculated to quantify the contribution from PD-L1 expressing IC, QID is defined as the CPS minus the TPS.Abstract 81 Table 1Agilent Tumor Bank CPS and QIDConclusionsPD-L1 IHC 22C3 pharmDx (Dako, USA) stains both TC and immune cells. Removal of the PD-L1 staining TC from the CPS algorithm reduces the number of specimens scored as positive for each indication’s respective diagnostic cut-off(s). Scoring only IC reduces the number of specimens scored as positive for each indication’s respective cutoff.

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OBJECTIVES/SPECIFIC AIMS: (1) Correlate PBX1 mRNA expression as measured by RNAScope in situ hybridization, at an RNA number/cell measurement, Versus by RT-qPCR by the ddCt method. (2) Validate PBX1 mRNA expression in a second independent cohort of neuroblastoma tumor samples, and correlate with patient outcomes. We expect that PBX1 expression will correlate whether detected by RNAScope or by RT-qPCR. This work has the promise of validating a novel biomarker of disease severity, and for clinical translation as the RNAscope technology has been CLIA-certified for clinical use for other genes. METHODS/STUDY POPULATION: Primary neuroblastoma tumor samples were acquired through the Children’s Oncology Group Tumor Bank, The Cooperative Human Tissue Network Tumor bank, and the Westmeade Tumor Bank (Westmeade, Australia), with patient outcomes annotated but sequestered until experiments are completed. RT-qPCR is performed using 1 μg total RNA isolated from each sample by Nucleospin RNA kit (Clontech), reverse transcribed by SuperScript VILO (ThermoFisher Scientific) and amplified using KiCqSTART SYBR Green qPCR mix (Sigma Aldrich). RNAScope was performed on sections of fresh frozen tumor, in triplicate, per manufacturer protocol (ACDBio) using company-designed probes. Statistical analyses performed using GraphPad Prism5. RESULTS/ANTICIPATED RESULTS: PBX1 mRNA expression as measured RNAScope correlated well with matched RT-qPCR values, with most PBX1 transcripts identified within the malignant cells and not in tumor stroma. Correlation with patient outcomes is ongoing (expected to be available at the time of presentation), but as the RNAScope values correlate with R>0.9 with RT-qPCR values, we expect good correlation with outcomes in our primary data set and matching validation set. DISCUSSION/SIGNIFICANCE OF IMPACT: PBX1 mRNA expression is an accurate prognostic biomarker of outcome in low and intermediate-risk neuroblastoma, and testing on an additional validation set is planned based on thresholds established by RNAScope. RNAScope is a method readily translatable to clinical use and its inclusion in future clinical trials will be further studied. It provides an additional benefit that concomitant immunohistochemistry can also be performed. Analysis of high-risk neuroblastomas for responsiveness to retinoic acid based on PBX1 expression is planned.

Development of a Prototype Immunohistochemistry Assay to Measure Programmed Death Ligand-1 Expression in Tumor Tissue

Context.— With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation. Objective.— To develop a prototype immunohistochemistry assay using the anti–PD-L1 antibody clone 22C3. Design.— The assay was developed and optimized using commercially available reagents and archival tumor-bank tissue. Results.— The optimized immunohistochemistry method had high precision and reproducibility. Using the prototype assay in 142 non–small cell lung cancer and 79 melanoma archival tumor-bank tissue samples, PD-L1 staining was observed at the plasma membrane of nucleated tumor and nontumor cells and, in some cases, as a distinct lichenoid pattern at the tumor-stroma border. Using a preliminary scoring method, 56% (80 of 142) of non–small cell lung cancer and 53% (42 of 79) of melanoma samples were defined as PD-L1+ based on a modified H-score of 1 or more or the presence of a distinctive staining pattern at the tumor-stroma interface. Conclusions.— The immunohistochemistry assay using the anti–PD-L1 antibody 22C3 merits further investigation in clinical trials and prevalence assessments to further understand the prognostic and predictive value of PD-L1 expression in cancer.