Automated urinalysis with expert review for incidental identification of atypical urothelial cells: An anticipated bladder carcinoma diagnosis.

BACKGROUND: The most common diagnostic technique for the detection of malignant/atypical urothelial cells (m/AUC) is urinary cytology (Ucytol). Urinary sediment (Used) examination, often prescribed for asymptomatic, healthy subjects, can incidentally identify suspicious/AUC (s/AUC) in routine daily practice. METHODS: Unstained, unfixed and uncentrifuged urine samples were analyzed with an automated intelligent microscopy (AIM) system. From January 2010, any incidental identification of s/AUC through expert revision of unclassified cell images was reported, according to internal protocols, as an additional note on the patients' laboratory report. Patients' symptomatology or previous history was unknown to the pathologist. All referrals from January 2010 to December 2014, with the s/AUC note, were reviewed and cross-referenced with Ucytol and histology data from a central database. RESULTS: Of the 162 patients identified with s/AUC (0.1/1000 samples), 84% (n=136) performed further Ucytol and/or histological examinations. The sensitivity of the identification of non-transitory AUC at Used was 87.5%. Positive histological examinations were 91.2% classified as high-grade urothelial carcinoma. CONCLUSIONS: Routine Used examination, with an AIM and expert revision, can identify the presence of AUC in a clinical laboratory setting, and for some subjects, may anticipate bladder carcinoma diagnosis.

Expression of p89(c-Mybex9b), an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells.

The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.

Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing cells via transcriptional repression of STAT 5 and Mcl-1.

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

c-Myb and its target Bmi1 are required for p190BCR/ABL leukemogenesis in mouse and human cells.

Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.