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Background: A 15-gene expression profile test has been clinically validated and is widely utilized in newly diagnosed uveal melanoma (UM) patients to assess metastatic potential of the tumor. As most patients are treated with eye-sparing radiotherapy, there is limited tumor tissue available for testing, and technical reliability and success of prognostic testing are critical. This study assessed the analytical performance of the 15-gene expression test for UM and the correlation of molecular class with pathologic characteristics., Methods: Inter-assay, intra-assay, inter-instrument/operator, and inter-site experiments were conducted, and concordance of the 15-gene expression profile test results and associated discriminant scores for matched tumor samples were evaluated. Technical success was determined from de-identified clinical reports from January 2010 - May 2016. Pathologic characteristics of enucleated tumors were correlated with molecular class results., Results: Inter-assay concordance on 16 samples run on 3 consecutive days was 100%, and matched discriminant scores were strongly correlated (R 2  = 0.9944). Inter-assay concordance of 46 samples assayed within a one year period was 100%, with an R 2 value of 0.9747 for the discriminant scores. Intra-assay concordance of 12 samples run concurrently in duplicates was 100%; discriminant score correlation yielded an R 2 of 0.9934. Concordance between two sites assessing the same tumors was 100% with an R 2 of 0.9818 between discriminant scores. Inter-operator/instrument concordance was 96% for Class 1/2 calls and 90% for Class 1A/1B calls, and the discriminant scores had a correlation R 2 of 0.9636. Technical success was 96.3% on 5516 samples tested since 2010. Increased largest basal diameter and thickness were significantly associated with Class 1B and Class 2 vs. Class 1A signatures., Conclusions: These results show that the 15-gene expression profile test for UM has robust, reproducible performance characteristics. The technical success rate during clinical testing remains as high as first reported during validation. As molecular testing becomes more prevalent for supporting precision medicine efforts, high technical success and reliability are key characteristics when testing such limited and precious samples. The performance of the 15-gene expression profile test in this study should provide confidence to physicians who use the test's molecular classification to inform patient management decisions.

Background: Hepatitis C virus (HCV) translation is initiated in a cap-independent manner by an internal ribosome entry site (IRES) located within the 5' untranslated region (5'UTR). Sequence changes in this region could affect translation efficiency and presumably viral replication., Aim: To determine translation efficiency of 5'UTR variants developing during post-transfusion hepatitis C in two immunocompetent subjects and in two immunosuppressed liver recipients with recurrent HCV., Methods: Sequential samples were screened for 5'UTR changes by single-strand conformation polymorphism followed by cloning and sequencing whenever band pattern suggested sequence changes. 5'UTR variants were tested for IRES activity using a bicistronic dual luciferase expression plasmid transfected into HepG2 and Huh7 cell-lines., Results: In the transfused patients, translation efficiency of 5'UTR variants from early post-transfusion samples was 5.1- to 13.7-fold higher than that of predominant variants found in late follow-up samples. Post-transplant variants in the other two patients had 2.6- to 5.9-fold higher translation efficiency than those present only in pretransplant samples., Conclusion: In the immunocompetent host there may be selection of low translation efficiency HCV variants over the course of infection. However, in immunosuppressed subjects the opposite seems to be true as low translation efficiency variants are superseded by high translation efficiency variants.

We have developed an in vitro system in which RNA polymerase I accurately transcribes Xenopus ribosomal RNA (rRNA) genes. Cloned Xenopus laevis ribosomal DNA (rDNA) is incubated in a homogenate of manually isolated Xenopus borealis oocyte nuclei. The resultant RNA is analyzed by an S1 nuclease mapping procedure specific for the initiation region of X. laevis rRNA. We show that transcription initiates in vitro on the cloned X. laevis rDNA at the in vivo initiation site. In addition, the dinucleotide ApG significantly stimulates transcription in vitro by acting as a sequence-specific primer. The ApG stimulation is dependent on the template concentration and independent of the ribonucleoside triphosphate levels suggesting that the primer circumvents or augments an initiation-specific factor. We have also investigated transcription of Xenopus rDNA in a mouse cell extract. This heterologous extract can catalyze accurate initiation at a low level. However, under different ionic conditions, this extract transcribes X. laevis rDNA approximately 100-fold more efficiently, but synthesis initiates 4 nucleotides upstream from the in vivo initiation site. There is strong sequence conservation between this start site, the in vivo X. laevis start site, and the in vivo mouse start site suggesting that these sequences are important in RNA polymerase I initiation and that species specificity for RNA polymerase I transcription may not be as great as suggested earlier.

Transcription of ribosomal RNA genes by RNA polymerase I is generally accepted as being highly species specific, a conclusion based on numerous reports that rRNA genes of one species are not transcribed by factors of even closely related species. It thus was striking to find that cloned rDNA from the frog Xenopus laevis is specifically transcribed in extracts prepared from mouse cells. The data in this paper demonstrate that this heterologous transcription is due to a normal initiation process and not to a fortuitous event. Transcription of Xenopus rDNA in the mouse cell extract is directed by the same large promoter (residue-141 to +6) that is utilized to promote the synthesis of frog rRNA in homologous Xenopus systems. Moreover, the same factors of the mouse cell extract that transcribe the homologous mouse rDNA also catalyze transcription from the X. laevis rDNA promoter. We conclude that polymerase I transcriptional machinery does not evolve as rapidly as prior studies would suggest.