Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

<p>Abstract</p> <p>Background</p> <p>Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23.</p> <p>Conclusions</p> <p>Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future.</p

Polycomb repressor complex 1 member, BMI1 contributes to urothelial tumorigenesis through p16-independent mechanisms

Urothelial carcinoma (UC) causes significant morbidity and remains the most expensive cancer to treat due to the need for repeated resections and life-long monitoring for patients with non-muscle-invasive bladder cancer (NMIBC). Novel therapeutics and stratification approaches are needed to improve the outlook for both NMIBC and muscle-invasive bladder cancer (MIBC). We investigated the expression and effects of B Lymphoma Mo-MLV Insertion Region 1 (BMI1) in UC. BMI1 was found to be over-expressed in most UC cell lines and primary tumors by quantitative-real-time-PCR and immunohistochemistry. In contrast to some previous reports, no association with tumor stage or grade was observed in 2 independent tumor panels. Furthermore, up-regulation of BMI1 was detected in premalignant bladder lesions, suggesting a role early in tumorigenesis. BMI1 is not located within a common region of genomic amplification in UC. The CDKN2A locus (which encodes the p16 tumor suppressor gene) is a transcriptional target of BMI1 in some cellular contexts. In UC cell lines and primary tissues, no correlation between BMI1 and p16 expression was observed. Retroviral-mediated over-expression of BMI1 immortalized normal human urothelial cells (NHUC) in vitro and was associated with induction of telomerase activity, bypass of senescence and repression of differentiation. The effects of BMI1 on gene expression were identified by expression microarray analysis of NHUC-BMI1. MetacoreTM analysis of the gene expression profile implicated downstream effects of BMI1 on α4/β1 integrin-mediated adhesion, cytoskeleton remodelling and CREB1-mediated transcription

Functional and molecular characterisation of mammary side population cells

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2–0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology

Telomerase reverse transcriptase and telomeric-repeat binding factor protein 1 as regulators of telomerase activity in pancreatic cancer cells

Telomerase adds hexameric repeats of 5′-TTAGGG-3′ termed telomeres to ends of chromosomal DNA. This enzyme has been implicated in cellular immortalization and cellular senescence. Recently, a number of relevant genes have been cloned, including these encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein-1 (TEP1). Also important are genes encoding human telomeric-repeat binding factor protein (TRF) 1 and 2. To clarify mechanisms regulating telomerase activity, we studied telomerase activity, the telomeric restriction fragment (TRF) length and gene expression of these telomerase components and the telomeric-repeat binding factor proteins in sequential observation following X-irradiation of cultured pancreatic cancer cells. We previously reported that PANC-1 cells are better able to tolerate thermal stress, antineoplastic drugs, and exposure to tumour necrosis factor than MIAPaCa-2 cells. MIAPaCa-2 and PANC-1 cells were exposed to X-irradiation, their telomerase activity was increased at 2 days and then decreased gradually. Of the three telomerase components, only hTERT mRNA expression showed parallel changes. TRF length was stable just before and after X-irradiation. Among binding factor proteins, TRF1 mRNA showed reciprocal changes possibly directed toward maintaining a stable telomere length. In this study, our results demonstrate that not only hTERT but also TRF1 are important regulator of telomerase activity. © 2001 Cancer Research Campaign http://www.bjcancer.co

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer

Telomerase reactivation is a hallmark of human carcinogenesis. Increased telomerase activity may result from gene amplification and/or overexpression. This study evaluates the prognostic value of hTERT gene amplification and mRNA overexpression in 144 resectable non-small-cell lung cancer (NSCLC) specimens. The hTERT gene copy number was assessed by quantitative polymerase chain reaction (qPCR) on laser-capture microdissected tumour cells of 81 tumours, and by fluorescence in situ hybridisation (FISH) on a subset of 59 tumours. hTERT mRNA level was determined by reverse transcription (RT)–qPCR in 130 tumours. In total, 57% of (46 out of 81) primary NSCLC specimens demonstrated hTERT amplification, which was significantly more common (P<0.001) in adenocarcinoma (30 out of 40) than in squamous cell carcinoma (13 out of 37). The hTERT mRNA overexpression was noted in 74% (94 out of 130) of tumours; it was more frequent in squamous cell than in adenocarcinoma (87 vs 68%, P=0.03). Overexpression was significantly associated with amplification (P=0.03), especially in adenocarcinoma. The hTERT gene amplification was prognostic for shorter recurrence-free survival (hazard ratio=2.16, P=0.03). These data indicate that gene amplification is an important mechanism for hTERT overexpression in lung adenocarcinoma and is an independent poor prognostic marker for disease-free survival in NSCLC

Psychology Knowledge Revision Campaign: Assessing Student Learning through a Classroom and Laboratory based Research Project

Commonsense beliefs are barriers to learning for all sciences but Psychology is especially vulnerable as people have emotionally held beliefs about human behavior. One known method for knowledge revision is to provide learners with refutation information about misconceptions. Refutation-style texts have been considered a viable strategy for changing psychological misconceptions in classroom-based and laboratory studies. The current project aims to integrate teaching with refutation-style information to the preceding laboratory work. Eighteen participants enrolled in psychology courses were administered a pre-test of 20 true/false statements; half the statements were psychology facts and the other were psychology misconceptions (e.g. “men and women communicate in different ways”). Next the participants viewed 10 posters, with each poster being presented by a psychology student as part of their own research course. Posters contained information and graphics that refuted the misconception in an attempt to revise knowledge. Poster presentations resembled the type of information teachers would present in a classroom. After viewing all 10 posters the participants took a post-test in the same true/false format but this time the participant was instructed to explain why he/she chose the answer they did. Seven to ten days later, participants took the post-test again to assess their long-term retention. Data revealed that the refutation-style presentations were successful in revising knowledge for psychology misconceptions. Participants answered more of the psychology misconceptions correct on the post-test survey than the pre-test and this knowledge persisted on the long-term post text. In fact, nearly all participants scored perfectly on the post tests