SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer.

BACKGROUND Mutational analysis of the KRAS gene has recently been established as a complementary in vitro diagnostic tool for the identification of patients with colorectal cancer who will not benefit from anti-epidermal growth factor receptor (EGFR) therapies. Assessment of the mutation status of KRAS might also be of potential relevance in other EGFR-overexpressing tumors, such as those occurring in breast cancer. Although KRAS is mutated in only a minor fraction of breast tumors (5%), about 60% of the basal-like subtype express EGFR and, therefore could be targeted by EGFR inhibitors. We aimed to study the mutation frequency of KRAS in that subtype of breast tumors to provide a molecular basis for the evaluation of anti-EGFR therapies. METHODS Total, genomic DNA was obtained from a group of 35 formalin-fixed paraffin-embedded, triple-negative breast tumor samples. Among these, 77.1% (27/35) were defined as basal-like by immunostaining specific for the established surrogate markers cytokeratin (CK) 5/6 and/or EGFR. KRAS mutational status was determined in the purified DNA samples by Real Time (RT)-PCR using primers specific for the detection of wild-type KRAS or the following seven oncogenic somatic mutations: Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val and Gly13Asp. RESULTS We found no evidence of KRAS oncogenic mutations in all analyzed tumors. CONCLUSIONS This study indicates that KRAS mutations are very infrequent in triple-negative breast tumors and that EGFR inhibitors may be of potential benefit in the treatment of basal-like breast tumors, which overexpress EGFR in about 60% of all cases.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Combined vemurafenib and cobimetinib in BRAF-mutated melanoma.

BACKGROUND The combined inhibition of BRAF and MEK is hypothesized to improve clinical outcomes in patients with melanoma by preventing or delaying the onset of resistance observed with BRAF inhibitors alone. This randomized phase 3 study evaluated the combination of the BRAF inhibitor vemurafenib and the MEK inhibitor cobimetinib. METHODS We randomly assigned 495 patients with previously untreated unresectable locally advanced or metastatic BRAF V600 mutation-positive melanoma to receive vemurafenib and cobimetinib (combination group) or vemurafenib and placebo (control group). The primary end point was investigator-assessed progression-free survival. RESULTS The median progression-free survival was 9.9 months in the combination group and 6.2 months in the control group (hazard ratio for death or disease progression, 0.51; 95% confidence interval [CI], 0.39 to 0.68; P<0.001). The rate of complete or partial response in the combination group was 68%, as compared with 45% in the control group (P<0.001), including rates of complete response of 10% in the combination group and 4% in the control group. Progression-free survival as assessed by independent review was similar to investigator-assessed progression-free survival. Interim analyses of overall survival showed 9-month survival rates of 81% (95% CI, 75 to 87) in the combination group and 73% (95% CI, 65 to 80) in the control group. Vemurafenib and cobimetinib was associated with a nonsignificantly higher incidence of adverse events of grade 3 or higher, as compared with vemurafenib and placebo (65% vs. 59%), and there was no significant difference in the rate of study-drug discontinuation. The number of secondary cutaneous cancers decreased with the combination therapy. CONCLUSIONS The addition of cobimetinib to vemurafenib was associated with a significant improvement in progression-free survival among patients with BRAF V600-mutated metastatic melanoma, at the cost of some increase in toxicity. (Funded by F. Hoffmann-La Roche/Genentech; coBRIM ClinicalTrials.gov number, NCT01689519.).

Combined vemurafenib and cobimetinib in BRAF-mutated melanoma.

BACKGROUND The combined inhibition of BRAF and MEK is hypothesized to improve clinical outcomes in patients with melanoma by preventing or delaying the onset of resistance observed with BRAF inhibitors alone. This randomized phase 3 study evaluated the combination of the BRAF inhibitor vemurafenib and the MEK inhibitor cobimetinib. METHODS We randomly assigned 495 patients with previously untreated unresectable locally advanced or metastatic BRAF V600 mutation-positive melanoma to receive vemurafenib and cobimetinib (combination group) or vemurafenib and placebo (control group). The primary end point was investigator-assessed progression-free survival. RESULTS The median progression-free survival was 9.9 months in the combination group and 6.2 months in the control group (hazard ratio for death or disease progression, 0.51; 95% confidence interval [CI], 0.39 to 0.68; P<0.001). The rate of complete or partial response in the combination group was 68%, as compared with 45% in the control group (P<0.001), including rates of complete response of 10% in the combination group and 4% in the control group. Progression-free survival as assessed by independent review was similar to investigator-assessed progression-free survival. Interim analyses of overall survival showed 9-month survival rates of 81% (95% CI, 75 to 87) in the combination group and 73% (95% CI, 65 to 80) in the control group. Vemurafenib and cobimetinib was associated with a nonsignificantly higher incidence of adverse events of grade 3 or higher, as compared with vemurafenib and placebo (65% vs. 59%), and there was no significant difference in the rate of study-drug discontinuation. The number of secondary cutaneous cancers decreased with the combination therapy. CONCLUSIONS The addition of cobimetinib to vemurafenib was associated with a significant improvement in progression-free survival among patients with BRAF V600-mutated metastatic melanoma, at the cost of some increase in toxicity. (Funded by F. Hoffmann-La Roche/Genentech; coBRIM ClinicalTrials.gov number, NCT01689519.).

Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.

Beta-catenin-mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP-mediated inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is beta-catenin independent. In contrast to earlier reports, these data exclude an essential role for beta-catenin during hematopoiesis and lymphopoiesis.

Thyroid carcinoma with papillary and squamous features: report of a case with histogenetic considerations.

We present a case of an 82-year-old female with a painless left latero-cervical swelling, which increased in size over the course of 6 months, compressing adjacent organs. The histopathological examination, following dissection of the left thyroid lobe and ipsilateral cervical lymph nodes, yielded two intermingled morphologically distinct histotypes that included conventional papillary thyroid carcinoma (PTC) and poorly differentiated squamous cell carcinoma (SCC) with cystic features. The clinical presentation, the immunophenotype, and the genotype, especially of the malignant squamous component with partial expression of TTF1, marked expression of p63 and mutation of BRAF, were consistent with the diagnosis of a papillary thyroid carcinoma with squamous component. The possibility of a squamous cell carcinoma of unknown origin metastasizing to a primary papillary thyroid carcinoma cannot be completely ruled out. This particular presentation of thyroid carcinoma carries a poor prognosis in 20% of cases, with high recurrence rates and distant metastasis.

The ubiquitin-like protein LC3 regulates the Rho-GEF activity of AKAP-Lbc.

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis.

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1β in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence-associated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1β in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3- and caspase-1-independent IL-1β release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.

Diagnosis of Birt-Hogg-Dube syndrome in a patient with spontaneous pneumothorax

Birt-Hogg-Dube syndrome refers to a dermatologic syndrome, consisting of small papular skins lesion distributed on the scalp, forehead, face and neck, which is autosomal dominantly inherited. Subsequently patients may develop concomitant renal and thoracic pathology. We report the case of a patient with Birt-Hogg-Dube syndrome diagnosed after spontaneous pneumothorax.

Les biomarqueurs prédictifs dans le cancer colorectal [Predictive biomarkers in colorectal cancer]

While for many years the diagnosis and therapy of colon cancer did not change drastically, recently new drugs (irinotecan and oxaliplatin, used in adjuvant or neo-adjuvant approaches) and even more recently the introduction of therapies targeting the epidermal growth factor receptor (EGFR) through the monoclonal antibodies cetuximab and panitumumab, are revolutionizing the field. The finding that only patients with a tumor with a wild type (non mutated) KRAS gene respond to anti-EGFR therapy has also affected the way pathologists address colorectal cancer. Molecular analysis of the KRAS gene has become almost a routine in a very short period of time. Pathologists will have to be prepared for a new era: from standard morphology based diagnostic procedures to the prediction of response to therapy using molecular tools.

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

Wingless activity in the precursor cells specifies neuronal migratory behavior in the Drosophila nerve cord.

Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p