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Background: Single-agent pembrolizumab represents the standard first-line option for metastatic non-small-cell lung cancer (NSCLC) patients with a PD-L1 (programmed death-ligand 1) expression of ≥ 50%., Methods: We conducted a multicenter retrospective study aimed at evaluating the clinicopathologic correlates of pembrolizumab effectiveness in patients with treatment-naïve NSCLC and a PD-L1 expression of ≥ 50%., Results: One thousand and twenty-six consecutive patients were included. The objective response rate (ORR) was 44.5% (95% CI 40.2-49.1), while the median progression free survival (PFS) and overall survival (OS) were 7.9 months (95% CI 6.9-9.5; 599 events) and 17.2 months (95% CI 15.3-22.3; 598 censored patients), respectively. ECOG-PS ≥ 2 (p < 0.0001) and bone metastases (p = 0.0003) were confirmed to be independent predictors of a worse ORR. Former smokers (p = 0.0002), but not current smokers (p = 0.0532) were confirmed to have a significantly prolonged PFS compared to never smokers at multivariate analysis. ECOG-PS (p < 0.0001), bone metastases (p < 0.0001) and liver metastases (p < 0.0001) were also confirmed to be independent predictors of a worse PFS. Previous palliative RT was significantly related to a shortened OS (p = 0.0104), while previous non-palliative RT was significantly related to a prolonged OS (p = 0.0033). Former smokers (p = 0.0131), but not current smokers (p = 0.3433) were confirmed to have a significantly prolonged OS compared to never smokers. ECOG-PS (p < 0.0001), bone metastases (p < 0.0001) and liver metastases (p < 0.0001) were also confirmed to be independent predictors of a shortened OS. A PD-L1 expression of ≥ 90%, as assessed by recursive partitioning, was associated with significantly higher ORR (p = 0.0204), and longer and OS (p = 0.0346) at multivariable analysis., Conclusion: Pembrolizumab was effective in a large cohort of NSCLC patients treated outside of clinical trials. Questions regarding the effectiveness in clinical subgroups, such as patients with poorer PS and with liver/bone metastases, still remain to be addressed. We confirmed that the absence of tobacco exposure, and the presence of bone and liver metastasis are associated with worse clinical outcomes to pembrolizumab. Increasing levels of PD-L1 expression may help identifying a subset of patients who derive a greater benefit from pembrolizumab monotherapy.

Introduction: Pemetrexed maintenance therapy (MT) after induction with platinum-based chemotherapy has recently become a common treatment strategy for advanced nonsquamous non-small-cell lung cancer (NSCLC). However, the benefits of MT should be weighed with consideration of the patients' perceptions and preferences. The aim of the present study was to evaluate patients' attitudes toward MT and to describe physicians' awareness of their patients' inclinations., Materials and Methods: We administered a 12-question anonymous survey and the Distress Thermometer Questionnaire to patients with advanced or recurrent nonsquamous NSCLC. The survey was also distributed to the referring physicians., Results: From December 2014 to July 2015, 92 patients and 37 physicians were enrolled. All 92 patients completed the questionnaire at T0 (before starting chemotherapy) and 56.5% also did so at T1 (after completion of induction). The physicians completed the survey only at T0. Most patients had a positive attitude toward MT at both T0 (78.9%) and T1 (86.5%), and 100% of the physicians thought their patients would be in favor of MT. The physicians believed that their patients' attitudes toward MT would decrease proportionally with the reduction in the magnitude of the overall survival increase and expected benefits. The decrease expected by the physicians was much greater than that reported by the patients. This was especially true for an overall survival increase as small as 1 month (51.9% of patients accepting MT vs. 13.5% supposed by physicians) or when the only treatment benefit was radiologic tumor stabilization (69.3% of patients accepting MT vs. 37.8% supposed by physicians)., Conclusion: NSCLC patients have a generally positive attitude toward MT, which is not directly proportional to the expected benefits and greater than the attitude expected by physicians., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Nitric oxide (NO), generated in skeletal muscle mostly by the neuronal NO synthases (nNOSμ), has profound effects on both mitochondrial bioenergetics and muscle development and function. The importance of NO for muscle repair emerges from the observation that nNOS signalling is defective in many genetically diverse skeletal muscle diseases in which muscle repair is dysregulated. How the effects of NO/nNOSμ on mitochondria impact on muscle function, however, has not been investigated yet., Methods: In this study we have examined the relationship between the NO system, mitochondrial structure/activity and skeletal muscle phenotype/growth/functions using a mouse model in which nNOSμ is absent. Also, NO-induced effects and the NO pathway were dissected in myogenic precursor cells., Results: We show that nNOSμ deficiency in mouse skeletal muscle leads to altered mitochondrial bioenergetics and network remodelling, and increased mitochondrial unfolded protein response (UPR(mt)) and autophagy. The absence of nNOSμ is also accompanied by an altered mitochondrial homeostasis in myogenic precursor cells with a decrease in the number of myonuclei per fibre and impaired muscle development at early stages of perinatal growth. No alterations were observed, however, in the overall resting muscle structure, apart from a reduced specific muscle mass and cross sectional areas of the myofibres. Investigating the molecular mechanisms we found that nNOSμ deficiency was associated with an inhibition of the Akt-mammalian target of rapamycin pathway. Concomitantly, the Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase 1 (Mul-1) axis was also dysregulated. In particular, inhibition of nNOS/NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent-protein kinases induced the transcriptional activity of FoxO3 and increased Mul-1 expression. nNOSμ deficiency was also accompanied by functional changes in muscle with reduced muscle force, decreased resistance to fatigue and increased degeneration/damage post-exercise., Conclusions: Our results indicate that nNOSμ/NO is required to regulate key homeostatic mechanisms in skeletal muscle, namely mitochondrial bioenergetics and network remodelling, UPR(mt) and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance.

Over the last 10 years, the systemic treatment of advanced non-small-cell lung cancer has progressively moved away from the 'one-size-fits-all' approach to histological subtyping. Currently, there is a progressive implementation of targeted therapies based on specific molecular characteristics such as the EGF receptor sensitizing mutations and the anaplastic lymphoma kinase rearrangements. Despite the availability of effective agents against these abnormalities, acquired resistance is still a major issue. A new generation of tyrosine kinase inhibitors for EGF receptor and anaplastic lymphoma kinase targeting acquired resistance mechanisms have been recently investigated. Several promising tyrosine kinase inhibitors that hit other targets are also in clinical development, including: rat sarcoma gene/MEK, BRAF1, PIK3A, c-mesenchymal-epithelial transition, c-ros oncogene 1, rearranged during transfection, human EGFR 2, FGFR, VEGFR, PDGFR and discoidin death receptor 2. Furthermore, new advances in immunology have been achieved through the discovery of vaccines and immune checkpoint pathways such as the cytotoxic T-lymphocyte-associated antigen-4, programmed cell death protein 1 and its ligands.

The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Most of patients with newly diagnosed non-small cell lung cancer (NSCLC) present with locally advanced or metastatic disease. In this setting the goal of treatment is to prolong survival and to control disease- and treatment-related symptoms. Currently systemic cytotoxic chemotherapy remains the first-line treatment for most patients with stage IV NSCLC, but preferred treatments are now defined by histology and based on the presence of specific molecular abnormalities. In first-line the combination of platinum plus pemetrexed with or without bevacizumab is a reasonable choice in patients with non-squamous NSCLC. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) as first-line therapy are the recommended for patients with EGFR-sensitizing mutations. A small-molecule TKI of anaplastic lymphoma kinase (ALK), crizotinib, showed pronounced clinical activity in the treatment of patients with NSCLC positive for EML4-ALK and it has rapidly entered into daily clinical practice. Currently no agents are specifically approved for the treatment of squamous cell carcinoma of the lung. Second-line treatments include docetaxel, pemetrexed, or erlotinib as single agents. There is a growing evidence that cytotoxics are better than EGFR-TKIs in EGFR wild-type patients. In the setting of the third line, the only approved agent is erlotinib. In elderly patients with good performance status (PS), doublet chemotherapy including platinum should not be excluded, especially for those patients 70-75 years of age without comorbidities. The better selection of patients, the identification of specific predictive biomarkers, a reasonable sequencing of all active and available treatments, including targeted therapies and cytotoxic, may significantly contribute to extend the natural history of stage IV NSCLC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Physical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis. These autophagy-mediated metabolic improvements are thought to be largely dependent on the activation of the metabolic sensor PRKAA1/AMPK. However, it is unknown whether these important benefits stem from systemic adaptations or are due solely to alterations in skeletal muscle metabolism. To address this we utilized inducible, muscle-specific, atg7 knockout mice that we have recently generated. Our findings indicate that acute inhibition of autophagy in skeletal muscle just prior to exercise does not have an impact on physical performance, PRKAA1 activation, or glucose homeostasis. However, we reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. This effect appears to be gender specific affecting primarily females. We also establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondrial quality control during damaging physical activity.

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63(-/-) mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.

In early stage non-small cell lung cancer (NSCLC) large randomized trials have demonstrated that in patients with radically resected disease adjuvant chemotherapy improves 5-year survival rates. However, a customization of systemic treatment is needed to avoid treatments in patients cured by surgery alone or to justify the use of adjuvant chemotherapy in high risk patients, including those in stage IA. Recently, the possibility of identifying prognostic and predictive factors related to the genetic signatures of the tumor that could affect adjuvant and neo-adjuvant treatment choices for resectable non-small cell lung cancer (NSCLC) has been of interest. This review summarizes the current status and future opportunities for clinical application of genotyping and genomic tests in early NSCLC.

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3β activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin; affected muscles are characterized by continuous bouts of muscle degeneration, eventually leading to the exhaustion of the endogenous satellite cell pool. At present, only palliative treatments are available, although several gene and cell therapy-based approaches are being studied. In this study we proposed to overcome the limitations hampering intramuscular cell injection by using a biomaterial-based strategy. In particular, we used a three-dimensional (3D) collagen porous scaffold to deliver myogenic precursor cells (MPCs) in vivo in the mdx murine model of DMD. MPCs, derived from single fibres of wild-type donors, were expanded in vitro, seeded onto collagen scaffolds and implanted into the tibialis anterior muscles of normal and mdx mice. As a control, cells were delivered via direct intramuscular cell injection in the contralateral muscles. Scaffold-delivered MPCs displayed lower apoptosis and higher proliferation than injected cells; in terms of dystrophin restoration, collagen scaffolds yielded better results than direct injections. Importantly, time-course experiments indicated that the scaffolds acted as a cell reservoir, although cell migration was mostly contained within 400 µm from the scaffold-host tissue interface., (Copyright © 2010 John Wiley & Sons, Ltd.)

The size of skeletal muscle cells is precisely regulated by intracellular signaling networks that determine the balance between overall rates of protein synthesis and degradation. Myofiber growth and protein synthesis are stimulated by the IGF-1/Akt/mammalian target of rapamycin (mTOR) pathway. In this study, we show that the transcription factor JunB is also a major determinant of whether adult muscles grow or atrophy. We found that in atrophying myotubes, JunB is excluded from the nucleus and that decreasing JunB expression by RNA interference in adult muscles causes atrophy. Furthermore, JunB overexpression induces hypertrophy without affecting satellite cell proliferation and stimulated protein synthesis independently of the Akt/mTOR pathway. When JunB is transfected into denervated muscles, fiber atrophy is prevented. JunB blocks FoxO3 binding to atrogin-1 and MuRF-1 promoters and thus reduces protein breakdown. Therefore, JunB is important not only in dividing populations but also in adult muscle, where it is required for the maintenance of muscle size and can induce rapid hypertrophy and block atrophy.

Background and Aims: Newborn hearing screening and early intervention for congenital hearing loss have created a need for tools assessing the hearing development of very young children. A multidisciplinary evaluation of children's development is now becoming standard in clinical practice, though not many reliable diagnostic instruments exist. For this reason, the LittlEARS Auditory Questionnaire (LEAQ) was created to assess the auditory skills of a growing population of infants and toddlers who receive hearing instruments. The LEAQ relies on parent report, which has been shown to be a reliable way of assessing child development. Results with this tool in a group of children who received very early cochlear implantation are presented., Methods: The LEAQ is the first module of the LittlEARS comprehensive test battery for children under the age of two who have normal hearing (NH), cochlear implants (CIs) or hearing aids (HAs). The LEAQ is a parent questionnaire comprised of 35 "yes/no" questions which can be completed by parents in less than 10 min. Sixty-three children who received unilateral CIs at a young age were assessed longitudinally and their performance was compared to that of a NH group., Results: All CI children reached the maximum possible score on the LEAQ on average by 22 months of hearing age, i.e. 38 months of chronological age. In comparison, the NH group reached the maximum score by 24 months of age demonstrating that auditory skills of CI children often develop quicker than those of NH children. In the two comparison groups of children aged (a) younger and older than 12 months, and (b) between 6-9 and 21-24 months at first fitting, the early implanted children reached the highest scores faster than the later implanted children. Furthermore, three children with additional needs were tested. They showed slower growth over time but also received benefits from early implantation., Conclusions: The LEAQ is a quick and effective tool for assessing auditory skills of very young children with or without hearing loss. In our study, the auditory skills of children with CI progressed very quickly after implantation and were comparable with those of NH peers., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The production of engineered three-dimensional (3D) skeletal muscle grafts holds promise for treatment of several diseases. An important factor in the development of such approach involves the capability of preserving myogenicity and regenerative potential during ex vivo culturing. We have previously shown that electrical stimulation of myogenic cells grown in monolayer could improve the differentiation process. Here we investigated the effect of exogenous electrical field, specifically designed to mimic part of the neuronal activity, on muscle precursor cells (MPCs) cultured within 3D collagen scaffolds. Our data showed that electric stimulation did not affect cell viability and increased by 65.6% the release rate of NO(x), an early molecular activator of satellite cells in vivo. NO(x) release rate was decreased by an inhibitor of NO synthase, both in stimulated and non-stimulated cultures, confirming the endocrine origin of the measured NO(x). Importantly, electrical stimulation also increased the expression of two myogenic markers, MyoD and desmin. We also carried out some preliminary experiments aimed at determining the biocompatibility of our seeded collagen scaffolds, implanting them in the tibialis anterior muscles of syngeneic mice. Ten days after transplantation, we could observe the formation of new myofibers both inside the scaffold and at the scaffold/muscle interface. Altogether, our findings indicate that electrical stimulation could be a new strategy for the effective 3D expansion of muscle precursor cells in vitro without losing myogenic potential and that 3D collagen matrices could be a promising tool for delivering myogenic cells in recipient muscles.

Background: Adjuvant treatment for rectal cancer is still controversial. This study reports on overall survival and disease-free survival, toxicity, downstaging, and surgical morbidity in rectal cancer patients who received combined chemoradiation therapy followed by curative surgery., Methods: Between 1993 and 1998, 51 patients (31 males and 20 females; median age, 60 years; range, 33-73 years) underwent chemoradiation therapy followed by radical surgery for middle and lower rectal adenocarcinoma. Criteria for giving preoperative radiotherapy (total 45 Gy in 25 fractions of 1.8 Gy/day for 5 weeks) and chemotherapy (5-fluorouracil 350 mg/m2/day and leucovorin 10 mg/m2/day, bolus on days 1-5 and 29-33) were an age younger than 75 years; an Eastern Cooperative Oncology Group performance status score of 0 to 2; and clinical preoperative stage II-III. Forty-three low anterior and eight abdominoperineal resections were performed. Median follow-up time was 29 (range, 3-63) months., Results: Although grade 3 to 4 toxicity occurred in 14 cases (27.4%), all patients completed the planned adjuvant therapy. At pathology, a complete response was found in eight (15.7%) cases. Of the remaining 43 cases, 22 were stage I, 12 were stage II, and 9 were stage III. Five-year actuarial disease-free survival and overall survival rates were 86.4% and 85.5%, respectively. Whereas no local recurrences were found, 4 patients had distant metastases. Three patients died (1 of cancer-related causes), 45 are alive and disease free, and 3 are alive with disease., Conclusions: The combined preoperative chemoradiation approach used by us seems to improve the disease-free survival and overall survival of selected patients with rectal cancer. However, a longer follow-up time is required to confirm these preliminary results.