Milošević, Dragana

Abstract Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

BACKGROUND There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters. METHODS miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA. RESULTS miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability. CONCLUSIONS Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.

Abstract Background: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. Methods: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. Results: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100–10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell–derived neoplasms. Conclusions: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

ABSTRACT The trimeric fusion (F) glycoproteins of morbilliviruses are activated by furin cleavage of the precursor F 0 into the F 1 and F 2 subunits. Here we show that an additional membrane-proximal cleavage occurs and modulates F protein function. We initially observed that the ectodomain of approximately one in three measles virus (MV) F proteins is cleaved proximal to the membrane. Processing occurs after cleavage activation of the precursor F 0 into the F 1 and F 2 subunits, producing F 1a and F 1b fragments that are incorporated in viral particles. We also detected the F 1b fragment, including the transmembrane domain and cytoplasmic tail, in cells expressing the canine distemper virus (CDV) or mumps virus F protein. Six membrane-proximal amino acids are necessary for efficient CDV F 1a/b cleavage. These six amino acids can be exchanged with the corresponding MV F protein residues of different sequence without compromising function. Thus, structural elements of different sequence are functionally exchangeable. Finally, we showed that the alteration of a block of membrane-proximal amino acids results in diminished fusion activity in the context of a recombinant CDV. We envisage that selective loss of the membrane anchor in the external subunits of circularly arranged F protein trimers may disengage them from pulling the membrane centrifugally, thereby facilitating fusion pore formation.