Iya Znoyko

Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate

BACKGROUND Liver injury transforms hepatic stellate cells into myofibroblast (MFB)-like cells. With recovery from injury, MFBs undergo apoptosis, but it is unknown whether they can also revert to quiescence. AIM To determine whether human (h)MFBs become quiescent if cultured on a basement membrane-like substrate (Matrigel). METHODS hMFBs obtained from cirrhotic liver were re-cultured on plastic or Matrigel. Expression of genes of collagen metabolism was assayed before and after transforming growth factor beta (TGFbeta) and Oncostatin M (OSM) stimulation. RESULTS hMFBs had typical MFB-like morphology, with abundant alpha-smooth muscle actin (SMA) but no cytoplasmic lipid droplets. hMFBs re-cultured on Matrigel reverted to alphaSMA-negative, lipid droplet-positive quiescent morphology. alphaSMA, collagen alpha1(1) (COL1A1) and collagen alpha2(1) (COL1A2) messages were upregulated in hMFBs cultured on plastic, but suppressed by Matrigel. The opposite was true for metalloproteinase-1 mRNA. OSM but not TGFbeta reduced alphaSMA mRNA by 30% while TGFbeta but not OSM upregulated COL1A1 mRNA by 48%, in hMFBs on plastic. TGFbeta and OSM stimulated COL1A1 gene expression in Matrigel by 50 and 60%, respectively. CONCLUSIONS Matrigel culture de-activates hMFBs yet collagen gene expression still responds to fibrogenic cytokines. The responses of hMFB gene expression to TGFbeta and OSM, are regulated differently by the extracellular matrix.

Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The methods currently used for monitoring CLL and determining conditions for treatment are limited in their ability to predict disease progression, patient survival, and response to therapy. Although clonal diversity and the acquisition of new chromosomal abnormalities during the disease course (clonal evolution) have been associated with disease progression, their prognostic potential has been underappreciated because cytogenetic and fluorescence in situ hybridization (FISH) studies have a restricted ability to detect genomic abnormalities and clonal evolution. We hypothesized that whole genome analysis using high resolution single nucleotide polymorphism (SNP) microarrays would be useful to detect diversity and infer clonal evolution to offer prognostic information. In this study, we used the Infinium Omni1 BeadChip (Illumina, San Diego, CA) array for the analysis of genetic variation and percent mosaicism in 25 non-selected CLL patients to explore the prognostic value of the assessment of clonal diversity in patients with CLL. We calculated the percentage of mosaicism for each abnormality by applying a mathematical algorithm to the genotype frequency data and by manual determination using the Simulated DNA Copy Number (SiDCoN) tool, which was developed from a computer model of mosaicism. At least one genetic abnormality was identified in each case, and the SNP data was 98% concordant with FISH results. Clonal diversity, defined as the presence of two or more genetic abnormalities with differing percentages of mosaicism, was observed in 12 patients (48%), and the diversity correlated with the disease stage. Clonal diversity was present in most cases of advanced disease (Rai stages III and IV) or those with previous treatment, whereas 9 of 13 patients without detected clonal diversity were asymptomatic or clinically stable. In conclusion, SNP microarray studies with simultaneous evaluation of genomic alterations and mosaic distribution of clones can be used to assess apparent clonal evolution via analysis of clonal diversity. Since clonal evolution in CLL is strongly correlated with disease progression, whole genome SNP microarray analysis provides a new comprehensive and reliable prognostic tool for CLL patients.

Collagen binding α2β1 and α1β1 integrins play contrasting roles in regulation of Ets-1 expression in human liver myofibroblasts

Activation of hepatic stellate cells from quiescence to myofibroblast-like cells (MFBs) is a pivotal event in hepatic fibrogenesis. Plastic-cultured stellate cells (an established in vitro model of the activated phenotype) recultured on Matrigel™ revert to quiescence. In the present study we analyzed the molecular mechanism underlying this process, focusing on the effect of collagen receptors α2β1 and α1β1 integrin signaling on the expression of Ets-1 transcription factor and its target gene MMP1 in cultured human MFBs. Cells grown in 3-dimensional (3D) substrates (Matrigel™ or collagen type I gel) markedly upregulated Ets-1 and MMP1 messages, in comparison to cells cultured on plastic. A similar effect but less intense was mimicked by stimulation of α2β1 or blocking of α1β1 integrin in cells grown on plastic. We observed increased expression of MMP1 transcripts with parallel changes in MMP1 promoter activity, and in mRNA and protein levels of upstream transcription factors Ets-1 and c-Jun. Interference with α2β1 and α1β1 integrin function in cells cultured in a 3D collagen substrate resulted in an even greater effect. Morphologically, stimulation of α2β1 integrin resulted in formation of multicellular networks, probably by facilitation of cell migration. Thus, we report the novel observation that in cultured human MFBs reverting to quiescence, the expression of transcription factor Ets-1 and its downstream target MMP1 can be modulated by changes in the microenvironment, which are mediated, at least in part, by the balance between collagen receptor integrin α2β1 and α1β1 activities

A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.

Tetraploidy and 5q deletion in myelodysplastic syndrome: a case report.

Tetraploidy is a very rare cytogenetic abnormality in myelocytic malignancies, and its significance is unclear to date. We report here on a 68-year-old male diagnosed with myelodysplastic syndrome/refractory anemia with excess blasts (MDS/RAEB). Cytogenetic analysis of his bone marrow biopsy at initial clinical presentation and in subsequent studies revealed the presence of two abnormal clones, 92,XXYY and 92,XXYY,del(5)(q13q33). Interphase fluorescence in situ hybridization analysis of abnormal cells confirmed interstitial deletion in 5q, demonstrated predominance of the tetraploid clone and persistent presence of the tetraploid clone with 5q deletion. The patient was not responsive to Revlimid (lenalidomide) treatment, which is routinely used in patients with 5q- syndrome. However, a subsequent course of therapy with the methyl-transferase inhibitor decitabine resulted in clinical and cytogenetic remission. Our data suggest that the unique complex abnormality of tetraploidy and 5q deletion described here for the first time in MDS is characterized by distinct disease etiology, the mechanism of which could involve epigenetic inactivation of gene expression via methylation.

Chromothripsis in Two Patients With Renal Cell Carcinoma: A Case Series.

Chromothripsis has been described as the catastrophic shattering of a chromosomal region with inaccurate genomic reassembly. Although chromothripsis occurs in a number of other epithelial cell tumors, it has only rarely been reported in renal cell carcinoma (RCC). We describe 2 individuals in their sixth decade who presented to our institution with RCC and apparent chromothripsis identified by single nucleotide polymorphism whole genome microarray analysis. This result suggests that chromothripsis might be a more common mechanism leading to the development of RCC than previously known. Detection of chromothripsis in patients with RCC might, in part, dictate the prognosis and, ultimately, guide therapeutic decisions based on the typically poorer outcome and more advanced disease documented in other tumor types A specific treatment regimen for chromothripsisrelated tumors is not currently available. However, it is conceivable that molecularly targeted therapies might one day be used to exploit the underlying etiologies and subsequent effects of chromothripsis, because some of the innumerable cells and/or pathways affected might be more sensitive to particular drugs.

First Report of Prenatal Ascertainment of a Fetus With Homozygous Loss of the SOX10 Gene and Phenotypic Correlation by Autopsy Examination

The SOX10 gene plays a vital role in neural crest cell development and migration. Abnormalities in SOX10 are associated with Waardenburg syndrome Types II and IV, and these patients have recognizable clinical features. This case report highlights the first ever reported homozygous loss of function of the SOX10 gene in a human. This deletion is correlated using family history, prenatal ultrasound, microarray analysis of amniotic fluid, and ultimately, a medical autopsy examination to further elucidate phenotypic effects of this genetic variation. Incorporating the use of molecular pathology into the autopsy examination of fetuses with suspected congenital anomalies is vital for appropriate family counseling, and with the ability to use formalin-fixed and paraffin-embedded tissues, has become a practical approach in autopsy pathology.