Intrapatient Tumor Heterogeneity in IHC Interpretation Using PD-L1 IHC 22C3 pharmDx.

Tumor heterogeneity may impact immunohistochemical (IHC) interpretation, thus potentially affecting decision making by treating oncologists for cancer patient management. Programmed cell death ligand-1 (PD-L1) IHC 22C3 pharmDx is a companion diagnostic used as an aid in identifying patient eligibility for treatment with pembrolizumab (KEYTRUDA). This study aims to investigate tumor heterogeneity impact on IHC staining when evaluating PD-L1 expression using PD-L1 IHC 22C3 pharmDx. The effect of tumor heterogeneity was evaluated based on the PD-L1 diagnostic status of PD-L1 IHC 22C3 pharmDx stained tumor tissue sections at relevant diagnostic cutoffs for non-small cell lung carcinoma, gastric or gastroesophageal junction adenocarcinoma, urothelial carcinoma, head and neck squamous cell carcinoma, esophageal cancer and triple negative breast cancer. Overall agreement for the PD-L1 diagnostic status was assessed for each tumor type within a given specimen block (Intra-Block), between specimen blocks from the same surgical resection (Intra-Case), and between intrapatient primary and metastatic specimens. Intrablock and intracase point estimates were above 75%, and primary versus metastatic point estimates were above 50%. The results suggest that PD-L1 expression is consistent across cut sections through a minimum of 150 µm within a tissue block and between blocks from the same surgical resection and is significantly maintained across primary and metastatic blocks from the same patient despite changes to the tissue microenvironment. These data provide confidence for histopathologists and oncologists that evaluation of PD-L1 expression at clinically relevant cutoffs is reproducible among different assessments (or samplings) of a single tumor specimen.

Aspergillus nidulans CkiA is an essential casein kinase I required for delivery of amino acid transporters to the plasma membrane.

Type I casein kinases are highly conserved among Eukaryotes. Of the two Aspergillus nidulans casein kinases I, CkiA is related to the δ/ε mammalian kinases and to Saccharomyces cerevisiae Hrr25p. CkiA is essential. Three recessive ckiA mutations leading to single residue substitutions, and downregulation using a repressible promoter, result in partial loss-of-function, which leads to a pleiotropic defect in amino acid utilization and resistance to toxic amino acid analogues. These phenotypes correlate with miss-routing of the YAT plasma membrane transporters AgtA (glutamate) and PrnB (proline) to the vacuole under conditions that, in the wild type, result in their delivery to the plasma membrane. Miss-routing to the vacuole and subsequent transporter degradation results in a major deficiency in the uptake of the corresponding amino acids that underlies the inability of the mutant strains to catabolize them. Our findings may have important implications for understanding how CkiA, Hrr25p and other fungal orthologues regulate the directionality of transport at the ER-Golgi interface.

Targets of DNA-binding proteins in bacterial promoter regions present enhanced probabilities for spontaneous thermal openings.

We mapped promoter regions of double-stranded DNA with respect to the probabilities of appearance of relatively large bubble openings exclusively due to thermal fluctuations at physiological temperatures. We analyzed five well-studied promoter regions of procaryotic type and found a spatial correlation between the binding sites of transcription factors and the position of peaks in the probability pattern of large thermal openings. Other distinct peaks of the calculated patterns correlate with potential binding sites of DNA-binding proteins. These results suggest that a DNA molecule would more frequently expose the bases that participate in contacts with proteins, which would probably enhance the probability of the latter to reach their targets. It also stands for using this method as a means to analyze DNA sequences based on their intrinsic thermal properties.

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans.

The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2-like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.

AgtA, the dicarboxylic amino acid transporter of Aspergillus nidulans, is concertedly down-regulated by exquisite sensitivity to nitrogen metabolite repression and ammonium-elicited endocytosis.

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

Multiple rescue factors within a Wolbachia strain.

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow "imprints" the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod(+) resc(+), mod(-) resc(+), mod(-) resc(-), and mod(+) resc(-). A so-called "suicide" mod(+) resc(-) strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod(+) resc(-) strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types.