Study of chromatin remodeling genes implicates SMARCA4 as a putative player in oncogenesis in neuroblastoma.

In neuroblastoma (NB), genetic alterations in chromatin remodeling (CRGs) and epigenetic modifier genes (EMGs) have been described. We sought to determine their frequency and clinical impact. Whole exome (WES)/whole genome sequencing (WGS) data and targeted sequencing (TSCA®) of exonic regions of 33 CRGs/EMGs were analyzed in tumor samples from 283 NB patients, with constitutional material available for 55 patients. The frequency of CRG/EMG variations in NB cases was then compared to the Genome Aggregation Database (gnomAD). The sequencing revealed SNVs/small InDels or focal CNAs of CRGs/EMGs in 20% (56/283) of all cases, occurring at a somatic level in 4 (7.2%), at a germline level in 12 (22%) cases, whereas for the remaining cases, only tumor material could be analyzed. The most frequently altered genes were ATRX (5%), SMARCA4 (2.5%), MLL3 (2.5%) and ARID1B (2.5%). Double events (SNVs/small InDels/CNAs associated with LOH) were observed in SMARCA4 (n = 3), ATRX (n = 1) and PBRM1 (n = 1). Among the 60 variations, 24 (8.4%) targeted domains of functional importance for chromatin remodeling or highly conserved domains but of unknown function. Variations in SMARCA4 and ATRX occurred more frequently in the NB as compared to the gnomAD control cohort (OR = 4.49, 95%CI: 1.63-9.97, p = 0.038; OR 3.44, 95%CI: 1.46-6.91, p = 0.043, respectively). Cases with CRG/EMG variations showed a poorer overall survival compared to cases without variations. Genetic variations of CRGs/EMGs with likely functional impact were observed in 8.4% (24/283) of NB. Our case-control approach suggests a role of SMARCA4 as a player of NB oncogenesis.

QuantumClone: clonal assessment of functional mutations in cancer based on a genotype-aware method for clonal reconstruction.

Motivation: In cancer, clonal evolution is assessed based on information coming from single nucleotide variants and copy number alterations. Nonetheless, existing methods often fail to accurately combine information from both sources to truthfully reconstruct clonal populations in a given tumor sample or in a set of tumor samples coming from the same patient. Moreover, previously published methods detect clones from a single set of variants. As a result, compromises have to be done between stringent variant filtering [reducing dispersion in variant allele frequency estimates (VAFs)] and using all biologically relevant variants. Results: We present a framework for defining cancer clones using most reliable variants of high depth of coverage and assigning functional mutations to the detected clones. The key element of our framework is QuantumClone, a method for variant clustering into clones based on VAFs, genotypes of corresponding regions and information about tumor purity. We validated QuantumClone and our framework on simulated data. We then applied our framework to whole genome sequencing data for 19 neuroblastoma trios each including constitutional, diagnosis and relapse samples. We confirmed an enrichment of damaging variants within such pathways as MAPK (mitogen-activated protein kinases), neuritogenesis, epithelial-mesenchymal transition, cell survival and DNA repair. Most pathways had more damaging variants in the expanding clones compared to shrinking ones, which can be explained by the increased total number of variants between these two populations. Functional mutational rate varied for ancestral clones and clones shrinking or expanding upon treatment, suggesting changes in clone selection mechanisms at different time points of tumor evolution. Availability and implementation: Source code and binaries of the QuantumClone R package are freely available for download at https://CRAN.R-project.org/package=QuantumClone. Contact: gudrun.schleiermacher@curie.fr or valentina.boeva@inserm.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Sequential Analysis of cfDNA Reveals Clonal Evolution in Patients with Neuroblastoma Receiving ALK-Targeted Therapy.

PURPOSE: The study of cell-free DNA (cfDNA) enables sequential analysis of tumor cell-specific genetic alterations in patients with neuroblastoma. EXPERIMENTAL DESIGN: Eighteen patients with relapsing neuroblastoma having received lorlatinib, a third-generation ALK inhibitor, were identified (SACHA national registry and/or in the institution). cfDNA was analyzed at relapse for nine patients and sequentially for five patients (blood/bone marrow plasma) by performing whole-genome sequencing library construction followed by ALK-targeted ddPCR of the hotspot mutations [F1174L, R1275Q, and I1170N; variant allele fraction (VAF) detection limit 0.1%] and whole-exome sequencing (WES) to evaluate disease burden and clonal evolution, following comparison with tumor/germline WES. RESULTS: Overall response rate to lorlatinib was 33% (CI, 13%-59%), with response observed in 6/10 cases without versus 0/8 cases with MYCN amplification (MNA). ALK VAFs correlated with the overall clinical disease status, with a VAF < 0.1% in clinical remission, versus higher VAFs (>30%) at progression. Importantly, sequential ALK ddPCR detected relapse earlier than clinical imaging. cfDNA WES revealed new SNVs, not seen in the primary tumor, in all instances of disease progression after lorlatinib treatment, indicating clonal evolution, including alterations in genes linked to tumor aggressivity (TP53) or novel targets (EGFR). Gene pathway analysis revealed an enrichment for genes targeting cell differentiation in emerging clones, and cell adhesion in persistent clones. Evidence of clonal hematopoiesis could be observed in follow-up samples. CONCLUSIONS: We demonstrate the clinical utility of combining ALK cfDNA ddPCR for disease monitoring and cfDNA WES for the study of clonal evolution and resistance mechanisms in patients with neuroblastoma receiving ALK-targeted therapy.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma.

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS: We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS: We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS: Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations.

The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

Whole-Exome Sequencing of Cell-Free DNA Reveals Temporo-spatial Heterogeneity and Identifies Treatment-Resistant Clones in Neuroblastoma.

Purpose: Neuroblastoma displays important clinical and genetic heterogeneity, with emergence of new mutations at tumor progression.Experimental Design: To study clonal evolution during treatment and follow-up, an innovative method based on circulating cell-free DNA (cfDNA) analysis by whole-exome sequencing (WES) paired with target sequencing was realized in sequential liquid biopsy samples of 19 neuroblastoma patients.Results: WES of the primary tumor and cfDNA at diagnosis showed overlap of single-nucleotide variants (SNV) and copy number alterations, with 41% and 93% of all detected alterations common to the primary neuroblastoma and cfDNA. CfDNA WES at a second time point indicated a mean of 22 new SNVs for patients with progressive disease. Relapse-specific alterations included genes of the MAPK pathway and targeted the protein kinase A signaling pathway. Deep coverage target sequencing of intermediate time points during treatment and follow-up identified distinct subclones. For 17 seemingly relapse-specific SNVs detected by cfDNA WES at relapse but not tumor or cfDNA WES at diagnosis, deep coverage target sequencing detected these alterations in minor subclones, with relapse-emerging SNVs targeting genes of neuritogenesis and cell cycle. Furthermore a persisting, resistant clone with concomitant disappearance of other clones was identified by a mutation in the ubiquitin protein ligase HERC2Conclusions: Modelization of mutated allele fractions in cfDNA indicated distinct patterns of clonal evolution, with either a minor, treatment-resistant clone expanding to a major clone at relapse, or minor clones collaborating toward tumor progression. Identification of treatment-resistant clones will enable development of more efficient treatment strategies. Clin Cancer Res; 24(4); 939-49. ©2017 AACR.

Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse.

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ~85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression.

Genomic Copy Number Profiling Using Circulating Free Tumor DNA Highlights Heterogeneity in Neuroblastoma.

PURPOSE: The tumor genomic copy number profile is of prognostic significance in neuroblastoma patients. We have studied the genomic copy number profile of cell-free DNA (cfDNA) and compared this with primary tumor arrayCGH (aCGH) at diagnosis. EXPERIMENTAL DESIGN: In 70 patients, cfDNA genomic copy number profiling was performed using the OncoScan platform. The profiles were classified according to the overall pattern, including numerical chromosome alterations (NCA), segmental chromosome alterations (SCA), and MYCN amplification (MNA). RESULTS: Interpretable and dynamic cfDNA profiles were obtained in 66 of 70 and 52 of 70 cases, respectively. An overall identical genomic profile between tumor aCGH and cfDNA was observed in 47 cases (3 NCAs, 22 SCAs, 22 MNAs). In one case, cfDNA showed an additional SCA not detected by tumor aCGH. In 4 of 8 cases with a silent tumor aCGH profile, cfDNA analysis revealed a dynamic profile (3 SCAs, 1 NCA). In 14 cases, cfDNA analysis did not reveal any copy number changes. A total of 378 breakpoints common to the primary tumor and cfDNA of any given patient were identified, 27 breakpoints were seen by tumor aCGH, and 54 breakpoints were seen in cfDNA only, including two cases with interstitial IGFR1 gains and two alterations targeting TERT CONCLUSIONS: These results demonstrate the feasibility of cfDNA copy number profiling in neuroblastoma patients, with a concordance of the overall genomic profile in aCGH and cfDNA dynamic cases of 97% and a sensitivity of 77%, respectively. Furthermore, neuroblastoma heterogeneity is highlighted, suggesting that cfDNA might reflect genetic alterations of more aggressive cell clones. Clin Cancer Res; 22(22); 5564-73. ©2016 AACRSee related commentary by Janku and Kurzrock, p. 5400.

Detection of tumor ALK status in neuroblastoma patients using peripheral blood.

New protocols based on ALK-targeted therapy by crizotinib or other ALK-targeting molecules have opened for the treatment of patients with neuroblastoma (NB) if their tumors showed mutation and/or amplification of the ALK gene. However, tumor samples are not always available for analysis of ALK mutational status in particular at relapse. Here, we evaluated the ALK mutational status of NB samples by analysis of circulating DNA, using the droplet digital PCR (ddPCR) system. ddPCR assays was developed for the detection of ALK mutations at F1174 and R1275 hotspots found in NB tumors and was applied for the analysis of circulating DNA obtained from 200 µL of serum or plasma samples collected from 114 patients with NB. The mutations F1174L (exon 23 position 3520, T>C and position 3522, C>A) and the mutation R1275Q (exon 25 position 3824, G>A) were detected in circulating DNA. The sensitivity of our test was 100%, 85%, and 92%, respectively, and the specificity was 100%, 91%, and 98%, respectively. In conclusion, the assay that we have developed offers a reliable, noninvasive blood test to assess ALK mutational status at F1174 and R1275 hotspots and should help clinicians to identify patients showing an ALK mutation in particular when no tumor tissue is available.

Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis.

PURPOSE: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing. EXPERIMENTAL DESIGN: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage. RESULTS: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%-40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%-73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed. CONCLUSIONS: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy.

Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations.

The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Fission yeast Rad52 phosphorylation restrains error prone recombination pathways.

Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

Stress activated protein kinase pathway modulates homologous recombination in fission yeast.

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.

Comparação da cifose torácica entre o paciente com doença pulmonar obstrutiva crônica e o indivíduo saudável pelo método flexicurva / Comparación de la cifosis torácica entre el paciente con enfermedad pulmonar obstructiva crónica y el individuo sano por el método flexicurva / Thoracic kyphosis comparison between a patient with chronic obstructive pulmonary disease and a healthy individual by flexicurve method

RESUMO A doença pulmonar obstrutiva crônica (DPOC) é caracterizada pela obstrução do fluxo aéreo, aprisionamento de ar e pela hiperinsuflação pulmonar. Esses fatores fisiopatológicos podem comprometer a mobilidade diafragmática, causar deformidades na caixa torácica e consequentemente aumentar o ângulo da curvatura torácica. Comparamos o ângulo da curvatura torácica entre pacientes com DPOC e indivíduos saudáveis pelo método flexicurva. Participaram do estudo 37 pacientes com DPOC e 37 indivíduos saudáveis. Todos os indivíduos realizaram as seguintes avaliações: antropometria, espirometria e mensuração do ângulo da curvatura torácica. Os dados foram analisados e tratados com análise descritiva como média e desvio-padrão. O teste de Shapiro-Wilk foi utilizado para verificar a normalidade dos dados. O teste t de Student foi utilizado para comparar o ângulo da curvatura torácica dos pacientes portadores de DPOC com os indivíduos saudáveis. O nível de significância adotado foi de 5%. A média de idade do grupo com DPOC foi de 65,70±7,91 anos, o índice de massa corporal (IMC), 26,73±5,34kg/m2, e VEF1 (% previsto), 50,65±19,08, apresentando grau de obstrução moderada. Os indivíduos saudáveis apresentaram em média 62,49±7,27 anos, IMC de 26,97±3,55kg/m² e VEF1 (% previsto) de 94,05±9,44. Não houve diferença significante entre os pacientes com DPOC e os indivíduos saudáveis no ângulo da curvatura torácica: 56,67±11,31 e 55,42±9,61 graus, respectivamente (p=0,61). O método flexicurva mostrou-se uma ferramenta útil e prática para avaliar a cifose torácica, identificando que não houve diferença entre as curvaturas torácicas dos pacientes com DPOC com obstrução moderada e dos indivíduos saudáveis.

RESUMEN La enfermedad pulmonar obstructiva crónica (EPOC) se caracteriza por la obstrucción del flujo de aire, aprisionamiento de aire e por la hiperinsuflación pulmonar. Estos factores fisiopatológicos pueden comprometer la movilidad diafragmática, causar deformidades en la caja torácica y consecuentemente aumentar el ángulo de la curvatura torácica. Se comparó el ángulo de la curvatura torácica entre pacientes con EPOC y de individuos sanos mediante el método flexicurva. Participaron del estudio 37 pacientes con EPOC y 37 individuos sanos. Todos los individuos realizaron las siguientes evaluaciones: antropometría, espirometría y medición del ángulo de la curvatura torácica. Los datos fueron analizados y tratados con análisis descriptivo como media y desviación estándar. Se utilizó la prueba de Shapiro-Wilk para comprobar la normalidad de los datos, y la prueba t de Student para comparar el ángulo de la curvatura torácica de los pacientes portadores de EPOC con los individuos sanos. El nivel de significancia adoptado fue del 5%. La edad promedio del grupo con EPOC fue 65,70±7,91 años, el índice de masa corporal (IMC), 26,73±5,34kg/m2, y VEF1 (% previsto), 50,65±19,08, presentando grado de obstrucción moderada. Los individuos sanos presentaron un promedio de 62,49±7,27 años, IMC de 26,97±3,55kg/m² y VEF1 (% previsto) de 94,05±9,44. No hubo diferencia significante entre los pacientes con EPOC y los pacientes sanos en el ángulo de la curvatura torácica: 56,67±11,31 y 55,42±9,61 grados, respectivamente (p=0,61). El método flexicurva ha mostrado ser una herramienta útil y práctica para evaluar la cifosis torácica, identificando que no hubo diferencia entre las curvaturas torácicas de los pacientes con EPOC con obstrucción moderada y de los individuos sanos.

ABSTRACT Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction, air entrapment and pulmonary hyperinflation. These pathophysiological factors can compromise the diaphragmatic mobility, causing deformities in the thoracic cavity and consequently increasing the angle of the thoracic curvature. We compared the angle of the thoracic curvature between COPD patients and healthy individuals by the flexicurve method. Thirty-seven patients with COPD and 37 healthy individuals participated in the study. All subjects performed the following evaluations: anthropometry, spirometry, and measurement of the thoracic curvature angle. The data were analyzed and treated with descriptive analysis such as mean and standard deviation. The Shapiro-Wilk test was used to verify the normality of the data. The Student's t-test was used to compare the thoracic curvature angle of patients with COPD with healthy individuals. The significance level adopted was 5%. The mean age of the COPD group was 65.70±7.91 years, body mass index (BMI) of 26.73±5.34kg/m2, and FEV1(expected %) of 50.65±19.08, showing moderate obstruction degree. Healthy individuals showed an average of 7.27±62.49 years, BMI of 26.97±3.55kg/m² and FEV1(expected %) of 94.05±0 9.44. We did not observe any significant difference between patients with COPD and healthy individuals in the thoracic curvature angle: 56.67±11.31 and 55.42±9.61 degrees, respectively (p=0.61). The flexicurve method proved to be a useful and practical tool for assessing the thoracic kyphosis, and it also identified no difference between the thoracic curvature of COPD patients with moderate obstruction and of healthy individuals.

Innovative strategies in the public administration: report of the experience at the School of Dentistry (Institute of Science and Technology) - Universidade Estadual Paulista “Julio de MesquitaFillho” – UNESP, Campus of São José dos Campos / Estratégias inovadoras na administração pública: relato de experiência da faculdade de odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP Campus de São José dos Campos

This ARTICLE aims to show an organization changing process, which is being performed in a public university, School of Dentistry of São José dos Campos – UNESP. A Strategic Planning was developed to promote such change, first using the usual model of this tool. Then the strategies were aligned with the expectations of a Balanced Scorecard engaged with the targets of a public organization (student success on the market, learning and growth, structure to work and leadership). To work the strategic review and the creation of renewal strategies a method from Matrix of Four Actions was used, aiming to identify values to add to the institution by diagnoses previewed on this tool, which makes easier the study of the fact that will be raised, eliminated, reduced and created. All this process was established along with the leadership development through trainings, individual and group coaching and the tracking of critical points of the strategic areas of this University Teaching Institute

O artigo tem por objetivo apresentar o processo de mudança organizacional que está em fase de implantação na instituição pública do ensino superior, Faculdade de Odontologia de São José dos Campos – UNESP, Campus de São José dos Campos. Para promover esta mudança foi desenvolvido o Planejamento Estratégico, inicialmente utilizando se o modelo tradicional desta ferramenta. Posteriormente as estratégias foram alinhadas às perspectivas de um Balanced Scorecard adaptado às necessidades dos objetivos estratégicos desta organização pública (sucesso do aluno no mercado, aprendizado e crescimento, infraestrutura de trabalho e governança institucional). Para promover a revisão estratégica e a criação de estratégias de inovação foi utilizada a metodologia da Matriz das Quatro Ações, visando identificar valores a agregar à instituição por meio do diagnóstico previsto nesta ferramenta, que facilita o levantamento dos fatores a serem elevados, eliminados, reduzidos e criados. Todo esse processo foi estabelecido conjuntamente com o desenvolvimento das lideranças de cada setor da instituição, por meio de treinamentos, sessões coaching individual e coletivo e mapeamento dos processos críticos das áreas estratégicas desta IES (Instituição de Ensino Superior).