Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters

Use of the 22C3 anti–PD-L1 antibody to determine PD-L1 expression in multiple automated immunohistochemistry platforms

<div><p>Background</p><p>For non–small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers.</p><p>Methods</p><p>We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA (Ventana Medical Systems, Inc.), and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens.</p><p>Results</p><p>Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform.</p><p>Conclusion</p><p>Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner.</p></div

PD-L1 staining patterns on NSCLC specimens using the 22C3 antibody concentrate on the VENTANA BenchMark ULTRA platform (LDT) compared with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>(<i>A</i>) The PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform; (<i>B</i>) optimised LDT (CC1 64 minutes, 22C3 primary antibody dilution 1:50, OptiView amplification 12 minutes); (<i>C</i>) CC1 32 minutes, primary antibody dilution 1:50, OptiView amplification 12 minutes; (<i>D</i>) CC1 64 minutes, primary antibody dilution 1:50, OptiView amplification 4 minutes; (<i>E</i>) CC1 32 minutes, primary antibody dilution 1:100, OptiView amplification 12 minutes; (<i>F</i>) CC1 64 minutes, primary antibody dilution 1:100, OptiView amplification 12 minutes. Original magnification 5×. Inserts, original magnification 40×. PD-L1, programmed death ligand 1; NSCLC, non–small cell lung cancer; ASL48, Autostainer Link 48; LDT, laboratory-developed test; IHC, immunohistochemistry.</p

PD-L1 staining patterns on NSCLC specimens using the 22C3 antibody concentrate on the Dako ASL48 platform (LDT) compared with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>(<i>A</i>) The PD-L1 22C3 pharmDx kit on the Dako ASL48 platform; (<i>B</i>) optimised LDT (primary antibody dilution 1:50, 30-minute incubation); (<i>C</i>) LDT using primary antibody dilution 1:100, 30-minute incubation; (<i>D</i>) primary antibody dilution 1:200, 30-minute incubation; (<i>E</i>) primary antibody dilution 1:100, 60-minute incubation; (<i>F</i>) primary antibody dilution 1:100, 120-minute incubation. Original magnification 5×. Inserts, original magnification 40×. PD-L1, programmed death ligand 1; NSCLC, non–small cell lung cancer; ASL48, Autostainer Link 48; LDT, laboratory-developed test; IHC, immunohistochemistry.</p