Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers.

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm2 tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

Specificity and functional analysis of the pH-responsive element within renal glutaminase mRNA.

The specificity and the functional significance of the binding of a specific cytosolic protein to a direct repeat of an eight-base AU sequence within the 3'-nontranslated region of the glutaminase (GA) mRNA were characterized. Competition experiments established that the protein that binds to this sequence is not an AUUUA binding protein. When expressed in LLC-PK(1)-F(+) cells, the half-life of a beta-globin reporter construct, betaG-phosphoenolpyruvate carboxykinase, was only slightly affected (1.3-fold) by growth in acidic (pH 6.9, 10 mM HCO(-)(3)) vs. normal (pH 7.4, 25 mM HCO(-)(3)) medium. However, insertion of short segments of GA mRNA containing the direct repeat or a single eight-base AU sequence was sufficient to impart a fivefold pH-responsive stabilization to the chimeric mRNA. Furthermore, site-directed mutation of the direct repeat of the 8-base AU sequence in a betaG-GA mRNA, which contains 956 bases of the 3'-nontranslated region of the GA mRNA, completely abolished the pH-responsive stabilization of the wild-type betaG-GA mRNA. Thus either the direct repeat or a single eight-base AU sequence is both sufficient and necessary to create a functional pH-response element.

Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA.

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.