Ursula R. Kees

Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate?

BackgroundThe use of microarray technology to assess gene expression levels is now widespread in biology. The validation of microarray results using independent mRNA quantitation techniques remains a desirable element of any microarray experiment. To facilitate the comparison of microarray expression data between laboratories it is essential that validation methodologies be critically examined. We have assessed the correlation between expression scores obtained for 48 human genes using oligonucleotide microarrays and the expression levels for the same genes measured by quantitative real-time RT-PCR (qRT-PCR).ResultsCorrelations with qRT-PCR data were obtained using microarray data that were processed using robust multi-array analysis (RMA) and the MAS 5.0 algorithm. Our results indicate that when identical transcripts are targeted by the two methods, correlations between qRT-PCR and microarray data are generally strong (r = 0.89). However, we observed poor correlations between qRT-PCR and RMA or MAS 5.0 normalized microarray data for 13% or 16% of genes, respectively.ConclusionThese results highlight the complementarity of oligonucleotide microarray and qRT-PCR technologies for validation of gene expression measurements, while emphasizing the continuing requirement for caution in interpreting gene expression data.

BRD–NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3–NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4–NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3–NUT and BRD4–NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4–NUT and BRD3–NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD–NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3–NUT and BRD4–NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD–NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia.

Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.

Intrathoracic carcinoma in an 11-year-old- girl showing a translocation t(15;19)

We report here a rare case of intrathoracic undifferentiated carcinoma in an 11-year-old girl. The biopsy tissue stained with antibodies against cytokeratin and carcino-embryonic antigen. The same staining was obtained using a cell line, PER-403, which was derived from the tumor. DNA from PER-403 cells was tested for the presence of Epstein-Barr virus genes, yielding a negative result. The cytogenetic analysis found a translocation t(15;19) (p12;q13), which has not previously been described in a carcinoma.

Increased Frequency of Aberrations in the p53/MDM2/p14ARF Pathway in Neuroblastoma Cell Lines Established at Relapse

p53 mutations have been reported in cell lines derived from relapsed neuroblastoma tumors. We hypothesize that functional inactivation of p53 by mutation or other mechanisms is common in relapsed neuroblastoma and can contribute to chemoresistance. Our aim was to determine the frequency of p53 mutations, p14(ARF) methylation, or deletion and MDM2 amplification in 23 neuroblastoma cell lines (6 derived at diagnosis and 17 derived at relapse). One cell line was p53 mutant (BE2c) and two cell lines were deleted for p14(ARF) (LAN-6 and SHEP). Two cell lines were methylated for p14(ARF) (GIMEN and PER-108), one of which had low levels of p14(ARF) mRNA expression which increased following demethylation with 5-aza-2/deoxycytidine treatment (GIMEN), and four cell lines were confirmed to be MDM2-amplified. All these cell lines were derived from neuroblastomas at relapse. Inactivation of the p53 pathway was observed in 9 out of 17 neuroblastoma cell lines (53%) established at relapse and in none of the cell lines established from pretreatment tumors. If these data are confirmed in neuroblastoma tumors, this suggests that p53-independent therapy and reactivation of inactive p53 approaches would be useful in the management of relapsed neuroblastoma.

Expression of HOX11 in childhood T-lineage acute lymphoblastic leukaemia can occur in the absence of cytogenetic aberration at 10q24 : a study from the Children's Cancer Group (CCG)

Clonal genetic aberrations in tumour cells provide critical information for the development of new diagnostic and therapeutic strategies for patients. In paediatric T-cell acute lymphoblastic leukaemia (T-ALL) chromosomal translocations are present in 30–35% of cases. HOX11 and the closely related HOX11L2 genes play a key role in T-ALL. HOX11 is aberrantly activated by either of the two chromosomal translocations, t(7;10) and t(10;14). In this study, HOX11 expression levels were measured by real-time quantitative reverse-transcriptase polymerase chain reaction. We show that leukaemic blasts from 15/76 (19.7%) paediatric T-ALL patients expressed the HOX11 gene at high level and 22/76 (28.9%) at low level, yet the reported frequency for chromosomal rearrangement of 10q24 is 4–7%. Direct cytogenetic analysis revealed that only 2/16 specimens that showed HOX11 expression exhibited abnor-malities at 10q24. These results confirm and extend our previously published findings, and implicate mechanisms other than gross chromosomal translocations for the deregulation of HOX11. Analysis of clinical outcome for the whole study group showed a trend for better outcome for patients with leukaemic blasts expressing HOX11 at high level. A statistically significant difference in clinical outcome was found in a subgroup of 20 patients treated for high-risk disease on CCG-1901 from the Childrens Cancer Group, where HOX11 expression in leukaemic blasts conferred a prognostic advantage (P=0.01).

Promoter demethylation accompanies reactivation of the HOX11 proto-oncogene in leukemia

Despite considerable work on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes in lymphoid tumors. The HOX11 proto‐oncogene is frequently activated in T‐cell acute lymphoblastic leukemia (T‐ALL). HOX11 activation can occur in the absence of translocation of the gene to the T‐cell receptor locus (Salvati et al., 1995 ), implying that activation mechanisms must be involved other than the juxtaposition of the gene to adjacent enhancing sequences. We tested whether the methylation status of the proximal promoter was correlated with expression status in T‐ALL and found that, in all cases, expression of HOX11 in T‐ALL was associated with extensive demethylation of the proximal HOX11 promoter, regardless of whether or not translocation was involved. In contrast, cells that did not express HOX11 showed a more methylated pattern of CpG residues in the proximal promoter. Methylation of this sequence in vitro was sufficient to silence the proximal promoter. We propose a model in which the selection of leukemia clones via a pathway involving HOX11 expression requires the demethylation of its promoter as a prerequisite for additional gene activation mechanisms.

The gene expression signature of relapse in paediatric acute lymphoblastic leukaemia: implications for mechanisms of therapy failure

Despite significant improvements in the treatment of childhood acute lymphoblastic leukaemia (ALL), the prognosis for relapsing patients remains poor. The aim of this study was to generate a transcriptional profile of relapsed ALL to increase our understanding of the mechanisms involved in therapy failure. RNA was extracted from 11 pairs of cryopreserved pre‐B ALL bone marrow specimens taken from the same patients at diagnosis and relapse, and analysed using HG‐U133A microarrays. Relapse specimens overexpressed genes that are involved with cell growth and proliferation, in keeping with their aggressive phenotype. When tested in 72 independent specimens of pre‐B ALL and T‐ALL, the identified genes could successfully differentiate between diagnosis and relapse in either lineage, indicating the existence of relapse mechanisms common to both. These genes have functions relevant for oncogenesis, drug resistance and metastasis, but are not related to classical multidrug‐resistance pathways. Increased expression of the top‐ranked gene (BSG) at diagnosis was significantly associated with adverse outcome. Several chromosomal loci, including 19p13, were identified as potential hotspots for aberrant gene expression in relapsed ALL. Our results provide evidence for a link between drug resistance and the microenvironment that has previously only been considered in the context of solid tumour biology.

Germ-line and somatic DICER1 mutations in pineoblastoma

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.

Altered glucose metabolism in childhood pre-B acute lymphoblastic leukaemia.

The cells of solid tumours are known to have an altered metabolism, with high rates of glucose uptake and glycolysis, which results in the excessive production of lactate. To date there has been no definitive research documenting metabolic changes in acute lymphoblastic leukaemia (ALL) cells. In order to investigate whether ALL cells have an altered metabolism, we initially compared the transcriptional profiles of 22 specimens from paediatric patients diagnosed with ALL to five CD34+ specimens isolated from bone marrow, which was verified in an independent cohort of 101 specimens. Profiling revealed the upregulation of genes facilitating glycolysis in the ALL specimens compared to the CD34+ specimens, while those involved in the tricarboxylic acid cycle were downregulated. Functional studies supported the microarray findings threefold: (1) higher expression of the glucose transport protein glucose transporter 1 in ALL compared to CD34+ specimens, (2) the excessive production of lactate in ALL cell lines and (3) sensitivity of ALL cell lines to the glycolysis inhibitor 2-deoxy-D-glucose. While metabolic alterations have been well documented in solid tumours, this is the first study to provide direct evidence for the existence of metabolic changes in the leukaemic cells of ALL patients. The finding offers new options for targeted therapy for ALL patients.