Moderate-to-strong expression of FGFR3 and TP53 alterations in a subpopulation of choroid plexus tumors.

Deregulation of fibroblast growth factor receptor (FGFR) signaling is tightly associated with numerous human malignancies, including cancer. Indeed, FGFR inhibitors are being tested as anti-tumor drugs in clinical trials. Among gliomas, FGFR3 fusions occur in IDH wild-type diffuse gliomas leading to high FGFR3 protein expression and both, FGFR3 and FGFR1, show elevated expression in aggressive ependymomas. The aim of this study was to uncover the expression of FGFR1 and FGFR3 proteins in choroid plexus tumors and to further characterize FGFR-related as well as other genetic alterations in FGFR3 expressing tumors. Expression levels of FGFR1 and FGFR3 were detected in 15 choroid plexus tumor tissues using immunohistochemistry of tissue microarrays and 6 samples were subjected to whole mount FGFR3 staining. Targeted sequencing was used for deeper molecular analysis of two FGFR3 positive cases. Moderate expression of FGFR1 or FGFR3 was evidenced in one third of the studied choroid plexus tumors. Targeted sequencing of a choroid plexus carcinoma and an atypical choroid plexus papilloma, both with moderate-to-strong FGFR3 expression, revealed lack of protein-altering mutations or fusions in FGFR1 or FGFR3, but TP53 was altered in both tumors. FGFR3 and FGFR1 proteins are expressed in a subpopulation of choroid plexus tumors. Further studies using larger cohorts of patients will allow identification of the clinicopathological implications of FGFR1 and FGFR3 expression in choroid plexus tumors.

A High-throughput workflow for CRISPR/Cas9 mediated combinatorial promoter replacements and phenotype characterization in yeast.

Due to the rapidly increasing sequence information on gene variants generated by evolution and our improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling towards the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome. Here we describe a CRISPR/Cas9 mediated high-throughput workflow for combinatorial and multiplexed replacement of native promoters with synthetic promoters and the following high-throughput phenotype characterization in the yeast Saccharomyces cerevisiae. The workflow is demonstrated with three central metabolic genes, ZWF1, PGI1 and TKL1 encoding a glucose-6-phosphate dehydrogenase, phosphoglucose isomerase and transketolase, respectively. The synthetic promoter donor DNA libraries were generated by PCR and transformed to yeast cells. A 50% efficiency was achieved for simultaneous replacement at three individual loci using short 60-bp flanking homology sequences in the donor promoters. Phenotypic strain characterization was validated and demonstrated using liquid handling automation and 150 µl cultivation volume in 96-well plate format. The established workflow offers a robust platform for automated engineering and improvement of yeast strains.

Clinical association analysis of ependymomas and pilocytic astrocytomas reveals elevated FGFR3 and FGFR1 expression in aggressive ependymomas.

BACKGROUND: Fibroblast growth factor receptors (FGFRs) are well-known proto-oncogenes in several human malignancies and are currently therapeutically targeted in clinical trials. Among glioma subtypes, activating FGFR1 alterations have been observed in a subpopulation of pilocytic astrocytomas while FGFR3 fusions occur in IDH wild-type diffuse gliomas, resulting in high FGFR3 protein expression. The purpose of this study was to associate FGFR1 and FGFR3 protein levels with clinical features and genetic alterations in ependymoma and pilocytic astrocytoma. METHODS: FGFR1 and FGFR3 expression levels were detected in ependymoma and pilocytic astrocytoma tissues using immunohistochemistry. Selected cases were further analyzed using targeted sequencing. RESULTS: Expression of both FGFR1 and FGFR3 varied within all tumor types. In ependymomas, increased FGFR3 or FGFR1 expression was associated with high tumor grade, cerebral location, young patient age, and poor prognosis. Moderate-to-strong expression of FGFR1 and/or FGFR3 was observed in 76% of cerebral ependymomas. Cases with moderate-to-strong expression of both proteins had poor clinical prognosis. In pilocytic astrocytomas, moderate-to-strong FGFR3 expression was detected predominantly in non-pediatric patients. Targeted sequencing of 12 tumors found no protein-altering mutations or fusions in FGFR1 or FGFR3. CONCLUSIONS: Elevated FGFR3 and FGFR1 protein expression is common in aggressive ependymomas but likely not driven by genetic alterations. Further studies are warranted to evaluate whether ependymoma patients with high FGFR3 and/or FGFR1 expression could benefit from treatment with FGFR inhibitor based therapeutic approaches currently under evaluation in clinical trials.

Strong FGFR3 staining is a marker for FGFR3 fusions in diffuse gliomas.

BACKGROUND: Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled-coil protein 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibition. As high FGFR3 expression has been detected in fusion-positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level as well as its potential for indicating FGFR3 fusions. METHODS: We performed FGFR3 immunohistochemistry on tissue microarrays containing 676 grades II-IV astrocytomas and 116 grades II-III oligodendroglial tumor specimens. Fifty-one cases were further analyzed using targeted sequencing. RESULTS: Moderate to strong FGFR3 staining was detected in gliomas of all grades, was more common in females, and was associated with poor survival in diffuse astrocytomas. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 15 strongly stained cases, whereas no fusions were found in 36 negatively to moderately stained cases. Fusion-positive cases were predominantly female and negative for IDH and EGFR/PDGFRA/MET alterations. These and moderately stained cases show lower MIB-1 proliferation index than negatively to weakly stained cases. Furthermore, stronger FGFR3 expression was commonly observed in malignant tissue regions of lower cellularity in fusion-negative cases. Importantly, subregional negative FGFR3 staining was also observed in a few fusion-positive cases. CONCLUSIONS: Strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a clinically applicable predictive marker for treatment regimens based on FGFR inhibitors.

A Model of the Spatio-temporal Dynamics of Drosophila Eye Disc Development.

Patterning and growth are linked during early development and have to be tightly controlled to result in a functional tissue or organ. During the development of the Drosophila eye, this linkage is particularly clear: the growth of the eye primordium mainly results from proliferating cells ahead of the morphogenetic furrow (MF), a moving signaling wave that sweeps across the tissue from the posterior to the anterior side, that induces proliferating cells anterior to it to differentiate and become cell cycle quiescent in its wake. Therefore, final eye disc size depends on the proliferation rate of undifferentiated cells and on the speed with which the MF sweeps across the eye disc. We developed a spatio-temporal model of the growing eye disc based on the regulatory interactions controlled by the signals Decapentaplegic (Dpp), Hedgehog (Hh) and the transcription factor Homothorax (Hth) and explored how the signaling patterns affect the movement of the MF and impact on eye disc growth. We used published and new quantitative data to parameterize the model. In particular, two crucial parameter values, the degradation rate of Hth and the diffusion coefficient of Hh, were measured. The model is able to reproduce the linear movement of the MF and the termination of growth of the primordium. We further show that the model can explain several mutant phenotypes, but fails to reproduce the previously observed scaling of the Dpp gradient in the anterior compartment.