A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.

3-M syndrome is an autosomal recessive primordial growth disorder characterized by small birth size and post-natal growth restriction associated with a spectrum of minor anomalies (including a triangular-shaped face, flat cheeks, full lips, short chest and prominent fleshy heels). Unlike many other primordial short stature syndromes, intelligence is normal and there is no other major system involvement, indicating that 3-M is predominantly a growth-related condition. From an endocrine perspective, serum GH levels are usually normal and IGF-I normal or low, while growth response to rhGH therapy is variable but typically poor. All these features suggest a degree of resistance in the GH-IGF axis. To date, mutations in three genes CUL7, OBSL1 and CCDC8 have been shown to cause 3-M. CUL7 acts an ubiquitin ligase and is known to interact with p53, cyclin D-1 and the growth factor signalling molecule IRS-1, the link with the latter may contribute to the GH-IGF resistance. OBSL1 is a putative cytoskeletal adaptor that interacts with and stabilizes CUL7. CCDC8 is the newest member of the pathway and interacts with OBSL1 and, like CUL7, associates with p53, acting as a co-factor in p53-medicated apoptosis. 3-M patients without a mutation have also been identified, indicating the involvement of additional genes in the pathway. Potentially damaging sequence variants in CUL7 and OBSL1 have been identified in idiopathic short stature (ISS), including those born small with failure of catch-up growth, signifying that the 3-M pathway could play a wider role in disordered growth.

Correction to: British Journal of Cancer (2008) 98, 1403–1414. doi:10.1038/sj.bjc.6604316 During correction of this article, the affiliations of the authors were changed, therefore causing an error in the footnote attributing credit for the work involved in the paper. The correct affiliations are shown above and the corrected footnote should read ‘This work is attributed to institutions 1–3 and 5 above’.

Fanconi anemia (FA) is an inherited disease with congenital abnormalities and an extreme risk of acute myeloid leukemia (AML). Genetic events occurring during malignant transformation in FA and the biology of FA‐associated AML are poorly understood, but are often preceded by the development of chromosomal aberrations involving 3q26‐29 in bone marrow of FA patients. We report here the molecular cytogenetic characterization of FA‐derived AML cell lines SB1685CB and SB1690CB by conventional and array comparative genomic hybridization, fluorescence in situ hybridization, and SKY. We identified gains of a 3.7 MB chromosomal region on 3q26.2‐26.31, which preceded transformation to overt leukemia. This region harbors the oncogenic transcription factor EVI1. A third FA‐derived cell line, FA‐AML1, carried a translocation with ectopic localization of 3q26 including EVI1 . Rearrangements of 3q, which are rare in childhood AML, commonly result in overexpression of EVI1, which determines specific gene expression patterns and confers poor prognosis. We detected overexpression of EVI1 in all three FA‐derived AML. Our results suggest a link between the FA defect, chromosomal aberrations involving 3q and overexpression of EVI1 . We hypothesize that constitutional or acquired FA defects might be a common factor for the development of 3q abnormalities in AML. In addition, cryptic imbalances as detected here might account for overexpression of EVI1 in AML without overt 3q26 rearrangements. © 2007 Wiley‐Liss, Inc.

Fanconi anemia (FA) is an inherited disease with congenital abnormalities and an extreme risk of acute myeloid leukemia (AML). Genetic events occurring during malignant transformation in FA and the biology of FA-associated AML are poorly understood, but are often preceded by the development of chromosomal aberrations involving 3q26-29 in bone marrow of FA patients. We report here the molecular cytogenetic characterization of FA-derived AML cell lines SB1685CB and SB1690CB by conventional and array comparative genomic hybridization, fluorescence in situ hybridization, and SKY. We identified gains of a 3.7 MB chromosomal region on 3q26.2-26.31, which preceded transformation to overt leukemia. This region harbors the oncogenic transcription factor EVII. A third FA-derived cell line, EA-AMLI, carried a translocation with ectopic localization of 3q26 including EVII. Rearrangements of 3q, which are rare in childhood AML, commonly result in overexpression of EVII, which determines specific gene expression patterns and confers poor prognosis. We detected overexpression of EVII in all three FA-derived AML. Our results suggest a link between the FA defect, chromosomal aberrations involving 3q and overexpression of EVII. We hypothesize that constitutional or acquired FA defects might be a common factor for the development of 3q abnormalities in AML. In addition, cryptic imbalances as detected here might account for overexpression of EVII in AML without overt 3q26 rearrangements. (c) 2007 Wiley-Liss, Inc.

The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.

Background: Used alone, the MAS5.0 algorithm for generating expression summaries has been criticized for high False Positive rates resulting from exaggerated variance at low intensities.Results: Here we show, with replicated cell line data, that, when used alongside detection calls, MAS5 can be both selective and sensitive. A set of differentially expressed transcripts were identified that were found to be changing by MAS5, but unchanging by RMA and GCRMA. Subsequent analysis by real time PCR confirmed these changes. In addition, with the Latin square datasets often used to assess expression summary algorithms, filtered MAS5.0 was found to have performance approaching that of its peers.Conclusion: When used alongside detection calls, MAS5 is a sensitive and selective algorithm for identifying differentially expressed genes.

10074 Background: The genetic determinants of survival and metastasis in soft tissue sarcoma (STS) are poorly understood and progress in this field has been limited by the rare nature of STS and the need for fresh/frozen tissue (FT) for gene microarray analyses. Our objective was to determine whether valid gene microarray data can be obtained from archival formalin-fixed paraffin-embedded tumours (FFPET) in order to retrospectively identify prognostic STS gene signatures and potential molecular targets from patients with known clinical outcome data. Methods: Total RNA was extracted from macrodissected viable tumour tissue for 34 FFPET (14 liposarcomas, 11 leiomyosarcomas and 9 synovial sarcomas) and two paired FFPET and FT primary leiomyosarcomas (modified Optimum kit, Ambion, for FFPET RNA extraction and Trizol, Invitrogen, for FT RNA extraction). One-cycle labelled cRNA was hybridised to Affymetrix U133 Plus 2 microarrays, and strict QC protocols used to identify 19 arrays for further analysis using R and BioConductor. Log rank regression and Kaplan-Meier plots of disease-specific survival were performed to identify genes predictive of survival and/or metastasis. Results: Similar fold changes in gene expression were obtained for paired cRNA samples, although the number of genes called present was lower for cRNA from FFPET. Five hundred genes (selected from FFPET cRNA arrays) were discriminatory for survival (p=0.0006 to 0.0088) across all STS subtypes and correctly assigned 17/19 cases by leave-one-out cross validation. Many are known tumour prognostic genes, significant for survival, metastasis, invasion, angiogenesis and apoptosis. Twenty-five novel and known candidate genes (with 3–10 fold differential expression) were selected for validation on 19 test and 60 independent cases by RT-PCR for relative gene expression and immunohistochemistry for protein detection. Conclusions: We have shown that prognostic information can be derived from archival FFPET, permitting the identification of candidate prognostic genes and therapeutic targets in STS, and opening the way for studies in other tumours where FT is unavailable. No significant financial relationships to disclose.