Real-time tests of multiple genome alterations take the first steps into the clinic: a learning example

Molecular characterization is increasingly changing clinical practice, in both diagnosis and treatment. BRAF is a proto-oncogene that is mutated in ~2%-4% of lung cancers, but the incidence rises to 40%-45% among papillary thyroid cancers. Furthermore, BRAF is a promising target in lung cancer treatment. The present case study covers both the challenges of molecular differential diagnosis and the perspectives opened by targeted therapy by discussing the history of a 78-year-old female affected by a papillary histotype carcinoma with BRAF mutation associated with both thyroid and lung localizations. A differential diagnosis was possible as a consequence of a multidisciplinary approach including an in-depth molecular characterization. Based on this molecular feature, the patient was successfully treated with the BRAF inhibitor dabrafenib after the failure of treatment with standard regimen. To the best of our knowledge, this is the first published case of non-small-cell lung cancer with metastasis to thyroid and with BRAF V600E mutation

Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes

Nano-in-Nano Approach for Enzyme Immobilization Based on Block Copolymers

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability

Prognostic significance of KRAS mutation rate in metastatic colorectal cancer patients.

No abstract availabl

Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

none24noIntroduction: Activating mutations of K-Ras gene have a well-established role as predictors of resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) patients. Their prognostic value is controversial, and no data regarding the prognostic value of mutation rate, defined as the percentage of mutated alleles/ tumor sample, are available. We aimed to evaluate the prognostic value of K-Ras mutation rate in a homogenous cohort of mCRC patients receiving first-line doublet plus bevacizumab. Patients and Methods: This retrospective study enrolled 397 K-Ras mutant mCRC patients from 6 Italian centers, and 263 patients were fully evaluable for our analysis. K-Ras mutation rate was assessed by pyrosequencing. Patients with less than 60% of cancer cells in tumor tissue were excluded. No patients received anti-EGFR containing anticancer therapy, at any time. Median mutation rate was 40% and was adopted as cut-off. The primary and secondary endpoints were PFS and OS respectively. Results: At univariate analysis, K-Ras mutation rate higher than 40% was significantly associated with lower PFS (7.3 vs 9.1 months; P < 0.0001) and OS (21 vs 31 months; P = 0.004). A multivariate model adjusted for age at diagnosis, site of origin of tumor tissue (primary vs metastases), referral center, number of metastatic sites, and first-line chemotherapy backbone, showed that K-Ras mutation rate remained a significant predictor of PFS and OS in the whole population. Discussion: Our data demonstrate an association between K-Ras mutation rate and prognosis in mCRC patients treated with bevacizumab-containing first-line therapy. These data deserve to be verified in an independent validation set.openVincenzi B.; Cremolini C.; Sartore-Bianchi A.; Russo A.; Mannavola F.; Perrone G.; Pantano F.; Loupakis F.; Rossini D.; Ongaro E.; Bonazzina E.; Dell'Aquila E.; Imperatori M.; Zoccoli A.; Bronte G.; Maglio G.D.; Fontanini G.; Natoli C.; Falcone A.; Santini D.; Onetti-Muda A.; Siena S.; Tonini G.; Aprile G.Vincenzi, B.; Cremolini, C.; Sartore-Bianchi, A.; Russo, A.; Mannavola, F.; Perrone, G.; Pantano, F.; Loupakis, F.; Rossini, D.; Ongaro, E.; Bonazzina, E.; Dell'Aquila, E.; Imperatori, M.; Zoccoli, A.; Bronte, G.; Maglio, G. D.; Fontanini, G.; Natoli, C.; Falcone, A.; Santini, D.; Onetti-Muda, A.; Siena, S.; Tonini, G.; Aprile, G

Lower Lid Reconstruction Utilizing Auricular Conchal Chondral-Perichondral Tissue in Patients with Neoplastic Lesions

Purpose. To assess surgical outcomes of lower lid reconstruction surgery using auricular conchal tissue. Methods. This prospective study included 20 patients that underwent reconstructive lower lid surgery using autologous auricle chondral-perichondral graft tissue. Auricle tissue was used to provide adequate support and protection with similar conjunctiva tarsal structures on overlying soft tissues in patients with pathologic inferior lid tissue loss requiring reconstructive surgery. Biopsies with histopathology and cytology analysis were taken after 1 year. Cytology analysis using CK19 was used to confirm newly formed conjunctiva overlying the graft. Results. All patients showed no graft rejection. Surgical outcomes were generally good, with minimal or no ocular complications. 16 of 20 patients had excellent results, showing good lid symmetry and esthetics, minimal auricular discomfort, patient satisfaction and proper lid function. Surgical outcomes were highly dependent on proper post-op conjunctiva formation. All patients were positive for CK19, thus indicating proper conjunctiva tissue formation. Conclusions. Lower lid reconstruction surgery using auricular chondral-perichondral conchal tissue is a good alternative in patients with neoplastic lesions. Autologous chondral-perichondral tissue provides good functional and mechanical support in the reconstructed lid, thus reducing the risks of ectropion and corneal exposure and ensuring a protected ocular surface

The storm of NGS in NSCLC diagnostic-therapeutic pathway: How to sun the real clinical practice

The increasing number of approved drugs along with next generation sequencing (NGS) technologies look out as potential revolution of biomolecular characterization of non-small-cell lung cancer (NSCLC). Nevertheless, several aspects impact on success rate of NGS in clinical practice: a multidisciplinary approach and thorough knowledge of strengths and limits of each technologic diagnostic tool are required. Crucial preliminary step is the selection of the best available sample before testing, aware of clinical condition and setting of disease. Genomic data should be than integrated in the clinical context and matched with available therapeutic options; Molecular Tumor Boards (MTB) are worldwide emerging interdisciplinary groups implemented to transfer the impact of precision medicine in clinical practice. In order to guarantee equity in treatment, these considerations should find their application widely and rapidly. Aim of this review is offering an overview of emerging biomarkers, relative upcoming targeted drugs, and new diagnostic chances with an authors' perspective about a real-life diagnostic-therapeutic algorithm useful for daily clinical practice

A 16q deletion involving FOXF1 enhancer is associated to pulmonary capillary hemangiomatosis

BACKGROUND: Pulmonary capillary hemangiomatosis (PCH) is an uncommon pulmonary disorder, with variable clinical features depending on which lung structure is affected, and it is usually linked to pulmonary arterial hypertension. Congenital PCH has been very rarely described and, so far, the only causative gene identified is EIF2AK4, which encodes for a translation initiation factor. However, not all PCH cases might carry a mutation in this gene. CASE PRESENTATION: We report the clinical and cytogenetic characterization of a patient (male, newborn, first child of healthy non-consanguineous parents) died after three days of life with severe neonatal pulmonary hypertension, due to diffuse capillary hemangiomatosis diagnosed post mortem. Conventional karyotyping, Microarray-Based Comparative Genomic Hydridization (CGHa) and quantitative PCR were performed. CGHa revealed a heterozygous chromosome 16q23.3q24.1 interstitial deletion, spanning about 2.6 Mb and involving a FOXF1 gene enhancer. Quantitative PCR showed that the proband’s deletion was de novo. Microsatellite analysis demonstrate that the deletion occurred in the maternal chromosome 16. CONCLUSION: FOXF1 loss of function mutation have been so far identified in alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), a lung disease different from PCH. Our data suggest the hypothesis that disruption of the FOXF1 gene enhancer could be a genetic determinant of PCH. Moreover, our findings support the idea that FOXF1 is a paternally imprinted gene. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12881-015-0241-7) contains supplementary material, which is available to authorized users

Η επίδραση ενός προγράμματος άσκησης παράλληλα με θεραπεία ενζυμικής υποκατάστασης σε ασθενείς με νόσο Pompe

Η ασθένεια Pompe είναι μια σπάνια αυτοσωμική διαταραχή η οποία χαρακτηρίζεται από την έλλειψη της όξινης α-γλυκοσιδάσης, που έχει σαν αποτέλεσμα τη συσσώρευση γλυκογόνου στα λισοσωμάτια. Η όψιμη μορφή της ασθένειας χαρακτηρίζεται από προοδευτική δυσλειτουργία των σκελετικών και αναπνευστικών μυών. Σε συνδυασμό με την πρόσφατα προταθείσα θεραπεία ενζυμικής υποκατάστασης, έχουν προταθεί θεραπείες όπως η δίαιτα πλούσια σε πρωτεΐνες και η άσκηση, παρόλο που λίγα είναι γνωστά για την αποτελεσματικότητα στη φυσική κατάσταση των ασθενών. Ο σκοπός της παρούσας εργασίας ήταν να διερευνήσει την επίδραση της άσκησης στη μυϊκή δύναμη και τη σύσταση του σώματος σε πέντε (5) ασθενείς με Pompe όψιμης έναρξης, οι οποίοι συμμετείχαν σε θεραπεία ενζυμικής υποκατάστασης. Όλοι οι ασθενείς ακολούθησαν ένα πρόγραμμα 20 εβδομάδων αεροβικής άσκησης και ασκήσεων με αντιστάσεις, υπό την επίβλεψη ειδικού. Πριν και μετά την προπονητική περίοδο η σύσταση του σώματος μετρήθηκε με τη μέθοδος απορροφησιομετρίας διπλοενεργειακής δέσμης ακτινών X ενώ η ισομετρική μυϊκή δύναμη μετρήθηκε με ένα ειδικό δυναμόμετρο. Η λειτουργική ικανότητα μετρήθηκε με τη δοκιμασία 6 λεπτών περπάτημα. Μετά την προπονητική περίοδο παρατηρήθηκε μία στατιστικώς σημαντική αύξηση στη μυϊκή δύναμη (15-50% στα διάφορα μέρη του σώματος, ρ < 0.05) και στην απόσταση που περπάτησαν στα 6 λεπτά (203.8±177 m πριν vs. 248.2±184 m μετά, ρ< 0.01), ενώ η συνολική μάζα σώματος και η μάζα των κάτω άκρων δεν άλλαξε σημαντικά. Αυτά τα αποτελέσματα υποδηλώνουν ότι η άσκηση έχει ένα θετικό αποτέλεσμα στη μυϊκή δύναμη και τη λειτουργική ικανότητα σε ασθενείς με Pompe όψιμης έναρξης που λαμβάνουν θεραπεία ενζυμικής αποκατάστασης