Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens.

The aims of this study were first, to systematically assess the inter-observer reproducibility of mean Epidermal Growth Factor Receptor (EGFR) gene copy number (MCN) in histological and cytological specimens from lung and non-lung cancers, second to compare the performance of this quantitative approach to the current Colorado criteria for the assessment of fluorescence in situ hybridization (FISH) positivity and third to develop a model to convert cytology into histology MCN. EGFR FISH analysis was performed on 170 histological and 153 cytological specimens. The MCN and Colorado criteria were assessed by two independent observers and agreement evaluated using Bland-Altman plots and kappa values. Conversion of cytology into histology MCN was tested on two randomized subgroups of specimens. Applying the Colorado criteria, agreement between observers was moderate with histology (k=0.5) and excellent with cytology (k=0.94). Positivity in histology versus cytology led to fair agreement (k=0.33). MCN was significantly greater in cytology compared to histology (p<0.001) with excellent inter-observer agreement in both sample types (r=0.99 and 0.89, respectively). The average difference in MCN between observers was -0.003 (95%CI: -0.05 to 0.05) and 0.008 (95%CI: -0.09 to 0.11) for cytology and histology, respectively. A reliable conversion model with an R(2)-value of 0.91 in the validation subgroup (p<0.001) was obtained. The MCN is a reproducible scoring method for evaluating EGFR FISH in samples from lung and non-lung cancers. This quantitative scoring system may constitute an alternative to current methods of gene copy number assessment and will further help to optimize patient selection for targeted therapies.

Unifocal Langerhans cell histiocytosis of the oral mucosa.

A 24-year-old man was admitted for a painful gingival ulcer. Histology and immunohistochemistry of a lesional biopsy revealed the diagnosis of Langerhans cell histiocytosis (LCH). To rule out multifocal disease, a complete staging was performed. There was no evidence of bony lesions or any other organ involvement. The diagnosis of LCH restricted to the oral mucosa was established. The complete oral lesion was ablated by CO(2) laser and subsequently treated topically with triamcinolone acetonide. The patient is still in remission after one year of follow-up. LCH confined to the oral mucosa is rare. It presents usually as an inflammatory or ulcerative lesion, easily leading to misinterpretation and delayed diagnosis. Patients with limited unifocal mucocutaneous disease, as in the present case, usually have an excellent prognosis. However, the oral lesion may represent an early sign of LCH, predating and progressing to an aggressive life-threatening multiorgan disease.

A cassette system to study embryonic stem cell differentiation by inducible RNA interference.

Although differentiation of pluripotent embryonic stem cells is restricted by a hierarchy of transcription factors, little is known about whether post-transcriptional mechanisms similarly regulate early embryoid differentiation. We developed a system where small hairpin (sh)RNAs can be induced in embryonic stem (ES) cells from a defined locus following integration by Flp recombinase-mediated DNA recombination. To verify the system, the key transcription factor Stat3, which maintains pluripotency, was downregulated by shRNA, and the expected morphological and biochemical markers of differentiation were observed. Induction of shRNA specific for the post-transcriptional regulator Brf1 (Zfp36L1) amplified the cardiac markers with strong stimulation of cardiomyocyte formation within embryoid bodies. These findings identify Brf1 as a novel potential regulator of cardiomyocyte formation and suggest that post-transcriptional mechanisms are of importance to early development and, possibly, to regenerative medicine. The inducible RNA interference system presented here should also allow assignment of function for candidate genes with suspected roles in ES cell development. Disclosure of potential conflicts of interest is found at the end of this article.

Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference.

HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover.

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.