Immunohistochemical detection of PD-L1 among diverse human neoplasms in a reference laboratory: observations based upon 62,896 cases.

Targeting of the PD1/PD-L1 immune checkpoint pathway has rapidly gained acceptance as a therapeutic strategy for a growing number of malignancies. Testing for expression of PD-L1 in tumor cells and immune cells has been used as a companion or complementary test for drugs targeting the PD1/PD-L1 pathway. We evaluated the results of PD-L1 testing in a large reference lab cohort. Using Food and Drug Administration-approved methods and interpretive instructions for each individual test, 62,896 cases were evaluated for PD-L1 using antibody clone 22C3, 28-8, SP142, or SP263. Case data analyzed included test results and information on tumor location and clinical history. No clinical outcome information was available and no attempt was made to correlate PD-L1 results with any other tests performed. The following numbers of cases were evaluated: 22C3 with tumor proportion score [n = 52585], 22C3 with combined positive score [n = 2631], 28-8 [n = 4191], SP142 [n = 850], and SP263 [n = 70]. In 22C3/tumor proportion score cases, the general results were as follows: negative 33.1% (n = 17,405), (low) expression 33.9% (n = 17,822), and high expression 29.5% (n = 15,486). In cases identified as metastatic, the results were as follows: negative 35.9% (n = 1411), (low) expression 30.8% (n = 1211), and high expression 30.7% (n = 1208). We found broad ranges of expression in tumor types with increasing positivity, as adenocarcinomas were reported as poorly differentiated, whereas squamous cell carcinomas showed more positivity as tumors were described as well-differentiated. The results of many individual tumor types were evaluated and showed, in general, high levels of positive expression. Practical challenges and observations of PD-L1 stain results and interpretation are also discussed.

A real-world, comparative study of FDA-approved diagnostic assays PD-L1 IHC 28-8 and 22C3 in lung cancer and other malignancies.

AIMS: At the time of analysis, two widely used, drug-specific, tumour-cell programmed death ligand 1 (PD-L1) assays were approved by the US Food and Drug Administration for anti-PD-1 therapies: the Dako PD-L1 immunohistochemistry (IHC) 28-8 pharmDx assay and the Dako PD-L1 IHC 22C3 pharmDx assay. Given that the majority of current PD-L1 testing in US clinical practice is performed at commercial reference laboratories, we aimed to evaluate the concordance of the 28-8 and 22C3 assays in a real-world setting. METHODS: Matched PD-L1 IHC 28-8 and 22C3 results from routine assessment were obtained from 1930 patients, including 412 confirmed to have lung cancer, submitted from hospitals in over 38 US states/territories. Biopsies were stained, reviewed and scored by trained/certified pathologists at a single cancer reference laboratory between 2015 and 2017. Rate of concordance between assay findings was assessed by Bland-Altman analysis; overall per cent agreement (OPA), positive per cent agreement and negative per cent agreement; and Cohen's kappa. RESULTS: PD-L1 IHC 28-8 and 22C3 displayed strong correlation across all samples and in samples with a confirmed lung cancer diagnosis irrespective of biopsy site. The OPA was 97%-98% for all samples, depending on the expression level defining PD-L1 positivity. In the Bland-Altman analysis, the mean difference in percentage of tumour cells positively stained for PD-L1 between the paired assay findings was -0.80% for all samples and -0.93% in samples with a confirmed lung cancer diagnosis. CONCLUSIONS: These data, in conjunction with recent findings, support the analytical concordance of the PD-L1 IHC 28-8 and 22C3 assays for assessing per cent tumour-cell membrane PD-L1 expression.

Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification.

PURPOSE: While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. PATIENTS AND METHODS: Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. RESULTS: Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. CONCLUSION: The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer.

BACKGROUND: The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer. METHODS: In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing. RESULTS: Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. CONCLUSIONS: This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification.

Biomarkers.

Biomarkers are characteristics objectively measured and evaluated as indicators of: normal biologic processes, pathogenic processes, or pharmacologic response(s) to a therapeutic intervention. In environmental research and risk assessment, biomarkers are frequently referred to as indicators of human or environmental hazards. Discovering and implementing new biomarkers for toxicity caused by exposure to a chemical either from a therapeutic intervention or accidentally through the environment continues to be pursued through the use of animal models to predict potential human effects, from human studies (clinical or epidemiologic) or biobanked human samples, or the combination of all such approaches. The key to discovering or inferring biomarkers through computational means involves the identification or prediction of the molecular target(s) of the chemical(s) and the association of these targets with perturbed biological pathways. Two examples are given in this chapter: (1) inferring potential human biomarkers from animal toxicogenomics data, and (2) the identification of protein targets through computational means and associating these in one example with potential drug interactions and in another case with increasing the risk of developing certain human diseases.

Translational biology approach to identify causative factors for rare toxicities in humans and animals.

Genome-wide RNA splicing (with gene expression) can be used to discover variations that drive specific diseases and / or change the susceptibility in individuals to drug responses including tissue specific toxicities. Evidence linking causative SNPs to individual splicing differences between individuals is emerging and this may lead to a better understanding of susceptibilities related to rare drug-induced toxicities. The development of more sensitive genomics tools is expected to further the study of variations in molecular phenotype from alternative splicing of pre-mRNA. This report highlights a genomics platform developed to measure splicing changes that occur in response to drug exposures, and therefore is applicable for the study of drug-induced toxicity. The platform is applicable for humans, all toxicology species, and specialized model systems. For efficiency, multiple samples can be combined into a single sequencing run and individual sequences can be separated via informatics. Biobanked specimens from clinical trials, toxicology studies, from commercial sources, and/or from public 'omics' data resources such as in NCBI are the only sample or non-sample data requirements.

Functional consequences of mTOR inhibition.

TOR (target of rapamycin) is a serine-threonine protein kinase that is conserved across a diverse range of species from fungi to mammals. The signaling pathway that is anchored by TOR is also conserved across species. In mammals, mTOR integrates growth factor, amino acid, nutrient and energy sensing signals, and thus plays a major role in cell growth and proliferation, protein synthesis and autophagy. As a result of the pivotal role of mTOR in signaling, the aberrant regulation of mTOR has been implicated in several disease processes, including cancer, diabetes, ocular diseases and neurodegenerative disorders, as well as in lifespan extension. More recently, rapamycin (sirolimus) analogs that antagonize the mTOR signaling pathway have been approved for the treatment of several cancers. This review describes some recent advances in the understanding of mTOR signaling, with an emphasis on the functional consequences of mTOR inhibition and therapeutic intervention strategies.

Interactions between traditional Chinese medicines and Western therapeutics.

Traditional Chinese medicine (TCM) is a holistic approach to health that attempts to bring the body, mind and spirit into harmony. TCM is an essential part of the healthcare system in several Asian countries, and is considered a complementary or alternative medical system in most Western countries. An integration of the traditional Chinese and Western systems of medicine has begun in multiple medical centers internationally, and there is increasing evidence that several herbs and combinations of herbs used in TCM impart important pharmacological effects. The number of databases and compilations of herbs, herbal formulations, phytochemical constituents and molecular targets is increasing, primarily because of the widespread use of TCM in combination with Western drugs. The continued popularity of herbal remedies worldwide suggests that evidence-based research in this field, as well as information regarding the potential efficacy and safety of phytochemical constituents in herbs and TCM formulations, are essential, particularly when TCM is used in combination with other drugs. Herb-drug interactions are similar to drug-drug interactions in terms of their effects on ADME properties. Improvements in the knowledge of the molecular targets and metabolic pathways, as well as of the synergistic and inhibitory effects associated with important phytochemicals from herbs and herbal formulations, will lead to the development of rational approaches for the safe combination of healthcare systems from different cultures.

Profiling human phosphodiesterase genes and splice isoforms.

A mere 21 human phosphodiesterase (PDE) genes are responsible for modulating cellular levels of cAMP and cGMP in response to stimuli. Considering the importance of cAMP and cGMP to disparate physiological functions including visual response, smooth muscle relaxation, platelet aggregation, immune response, and cardiac contractibility, perhaps the 200 or more splice isoforms of PDE genes also play a major functional role. We profiled the human PDEs across 25 tissue samples using splice sensitive oligonucleotide microarrays containing probes for exons and exon-exon junctions. Our results suggest that PDEs exhibit tissue-specific differences in expression, as demonstrated by the high expression of PDE4B in skeletal muscle. At the splice variant level, the majority of PDE genes--notably 1A, 1C, 2A, 4C, 4D, 5A, 7A, 8A, 8B, 9A, 10A, and 11A--exhibited tissue-specific splicing with potential functional implications for PDE biology. This work validates expression of many EST transcripts, and confirms and expands on published findings based on PCR and cloning, illuminating some of the complexity of cAMP and cGMP processing.

The mouse kinome: discovery and comparative genomics of all mouse protein kinases.

We have determined the full protein kinase (PK) complement (kinome) of mouse. This set of 540 genes includes many novel kinases and corrections or extensions to >150 published sequences. The mouse has orthologs for 510 of the 518 human PKs. Nonorthologous kinases arise only by retrotransposition and gene decay. Orthologous kinase pairs vary in sequence conservation along their length, creating a map of functionally important regions for every kinase pair. Many species-specific sequence inserts exist and are frequently alternatively spliced, allowing for the creation of evolutionary lineage-specific functions. Ninety-seven kinase pseudogenes were found, all distinct from the 107 human kinase pseudogenes. Chromosomal mapping links 163 kinases to mutant phenotypes and unlocks the use of mouse genetics to determine functions of orthologous human kinases.

Evolution of protein kinase signaling from yeast to man.

Protein phosphorylation controls many cellular processes, especially those involved in intercellular communication and coordination of complex functions. To explore the evolution of protein phosphorylation, we compared the protein kinase complements ('kinomes') of budding yeast, worm and fly, with known human kinases. We classify kinases into putative orthologous groups with conserved functions and discuss kinase families and pathways that are unique, expanded or lost in each lineage. Fly and human share several kinase families involved in immunity, neurobiology, cell cycle and morphogenesis that are absent from worm, suggesting that these functions might have evolved after the divergence of nematodes from the main metazoan lineage.