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Purpose: Few guidelines exist for the perioperative management (PM) of neonates with surgical conditions (SC). This study examined the current neonatal PM in Italy., Methods: We invited 51 neonatal intensive care units with pediatric surgery in their institution to participate in a web-based survey. The themes included (1) the involvement of the neonatologist during the PM; (2) the spread of bedside surgery (BS); (3) the critical issues concerning the neonatal PM in operating rooms (OR) and the actions aimed at improving the PM., Results: Response rate was 82.4%. The neonatologist is involved during the intraoperative management in 42.9% of the responding centers (RC) and only when the surgery is performed at the patient's bedside in 50.0% of RCs. BS is reserved for extremely preterm (62.5%) or clinically unstable (57.5%) infants, and the main barrier to its implementation is the surgical-anesthesiology team's preference to perform surgery in a standard OR (77.5%). Care protocols for specific SC are available only in 42.9% of RCs., Conclusion: Some critical issues emerged from this survey: the neonatologist involvement in PM, the spread of BS, and the availability of specific care protocols need to be implemented to optimize the care of this fragile category of patients., (© 2024. The Author(s).)

Background: The European Reference Network for rare Inherited Congenital Anomalies, ERNICA, guidelines for gastroschisis cover perinatal period to help teams to improve care., Method: A systematic literature search including 136 publications was conducted. Research findings were assessed following the GRADE methodology. The evidence to decision framework was used to determine the strength and direction of recommendations., Results: The mode or timing of delivery do not impact neonatal mortality, risk of NEC or time on parenteral nutrition (PN). Intra or extra abdominal bowel dilatation predict complex gastroschisis and longer length of hospital stay but not increased perinatal mortality. Outcomes after Bianchi procedure and primary fascia closure under anesthesia are similar. Sutureless closure decreases the rate of surgical site infections and duration of ventilation compared to surgical closure. Silo-staged closure with or without intubation results in similar outcomes. Outcomes of complex gastroschisis (CG) undergoing early or delayed surgical repair are similar. Early enteral feeds starting within 14 days is associated with lower risk of surgical site infection., Recommendations: The panel suggests vaginal birth between 37 and 39 w in cases of uncomplicated gastroschisis. Bianchi's approach is an option in simple gastroschisis. Sutureless closure is suggested when general anesthesia can be avoided, sutured closure. If anesthesia is required. Silo treatment without ventilation and general anesthesia can be considered. In CG with atresia primary intestinal repair can be attempted if the condition of patient and intestine allows. Enteral feeds for simple gastroschisis should start within 14 days., (© 2024. The Author(s).)

Background: Tunneled central venous catheters (CVC) are crucial in the management of children affected by short bowel syndrome (SBS). This work aims to investigate the outcomes of tunneled CVC and to identify factors influencing their survival., Methods: All the children diagnosed with SBS and undergone a procedure of insertion of a tunneled CVC from 2010 to 2019 were included. Demographic data and surgical information about the procedures were collected. Regression models and Kaplan-Meier analysis were performed to estimate the survival., Results: Eighteen patients, eight males (44%), with a median length of residual bowel measuring 72 cm (IQR 50-102 cm), were enrolled. Thirty-nine Broviac CVCs were inserted with a mean number of 2.2 CVCs per patient and 13365 line-days. The overall incidence of complications was 3.2/1000 line-days, and the incidence of central line associated bloodstream infections (CLABSI) was 1.1/1000 line-days. No episode of catheter thrombosis was reported. The median survival was 269 days (IQR 82-1814 days). The survival was negatively influenced by a younger age at insertion ( R 2  = 0.29; p  < 0.001), 2.7 Fr diameter (median survival 76 days; p  < 0.001) and the occurrence of complications (median survival 169 days; p  = 0.002). The length of residual bowel was a mild risk factor for anticipated removal (OR 1.1; CI95 1.0-1.1; p  = 0.05)., Conclusion: CVC-related complications negatively influenced the survival of the line. An elder age at insertion together with a larger CVC diameter increased the survival of the line, while a shorter residual bowel was associated with an anticipated removal due to complications., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background : Many prognostic factors for CDH patients are described and validated in the current literature: the size of diaphragmatic defects, need for patch repair, pulmonary hypertension and left ventricular dysfunction are recognized as the most influencing outcomes. The aim of this study is to analyze the influence of these parameters in the outcome of CDH patients in our department and identify any further prognostic factors. Methods : An observational retrospective single-center study was conducted including all patients treated at our centre with posterolateral CDH between 01.01.1997 and 12.31.2019. The main outcomes evaluated were mortality and length of hospital stay. A univariate and multivariate analysis was performed. Results : We identified 140 patients with posterolateral CDH; 34.8% died before discharge. The overall median length of stay was 24 days. A univariate analysis confirmed that both outcomes are associated with the size of diaphragmatic defects, need for patch repair and presence of spleen-up ( p < 0.05). A multivariate analysis identified that the need for patch repair and maximum dopamine dose used for cardiac dysfunction are independent parameters associated with the length of stay only ( p < 0.001). Conclusions : In our series, the duration of hospitalization is longer for newborns with CDH treated with higher doses of dopamine for left ventricular dysfunction or needing patch repair in large diaphragmatic defects.

Background: Pediatric robotic-assisted surgeries have increased in recent years; however, guidance documents are still lacking. This study aimed to develop evidence-based recommendations, or best practice statements when evidence is lacking or inadequate, to assist surgical teams internationally., Methods: A joint consensus taskforce of anesthesiologists and surgeons from the Italian Society of Pediatric and Neonatal Anesthesia and Intensive Care (SARNePI) and the Italian Society of Pediatric Surgery (SICP) have identified critical areas and reviewed the available evidence. The taskforce comprised 21 experts representing the fields of anesthesia (n = 11) and surgery (n = 10) from clinical centers performing pediatric robotic surgery in the Italian cities of Ancona, Bologna, Milan, Naples, Padua, Pavia, Perugia, Rome, Siena, and Verona. Between December 2020 and September 2021, three meetings, two Delphi rounds, and a final consensus conference took place., Results: During the first planning meeting, the panel agreed on the specific objectives, the definitions to apply, and precise methodology. The project was structured into three subtopics: (i) preoperative patient assessment and preparation; (ii) intraoperative management (surgical and anesthesiologic); and (iii) postoperative procedures. Within these phases, the panel agreed to address a total of 18 relevant areas, which spanned preoperative patient assessment and patient selection, anesthesiology, critical care medicine, respiratory care, prevention of postoperative nausea and vomiting, and pain management., Conclusion: Collaboration among surgeons and anesthesiologists will be increasingly important for achieving safe and effective RAS procedures. These recommendations will provide a review for those who already have relevant experience and should be particularly useful for those starting a new program., (© 2022. The Author(s).)

Objective: The anesthetic management of fetal operative procedures (FOP) is a highly debated topic. Literature on fetal pain perception and response to external stimuli is rapidly expanding. Nonetheless, there is no consensus on the fetal consciousness nor on the instruments to measure pain levels. As a result, no guidelines or clinical recommendations on anesthesia modality during FOP are available. This systematic literature review aimed to collect the available knowledge on the most common fetal interventions, and summarize the reported outcomes for each anesthetic approach. Additional aim was to provide an overall evaluation of the most commonly used anesthetic agents., Methods: Two systematic literature searches were performed in Embase, Medline, Web of Science Core Collection and Cochrane Central Register of Controlled Trials up to December 2021. To best cover the available evidence, one literature search was mostly focused on fetal surgical procedures; while anesthesia during FOP was the main target for the second search. The following fetal procedures were included: fetal transfusion, laser ablation of placental anastomosis, twin-reversed arterial perfusion treatment, fetoscopic endoluminal tracheal occlusion, thoraco-amniotic shunt, vesico-amniotic shunt, myelomeningocele repair, resection of sacrococcygeal teratoma, ligation of amniotic bands, balloon valvuloplasty/septoplasty, ex-utero intrapartum treatment, and ovarian cyst resection/aspiration. Yielded articles were screened against the same inclusion criteria. Studies reporting anesthesia details and procedures' outcomes were considered. Descriptive statistical analysis was performed and findings were reported in a narrative manner., Results: The literature searches yielded 1,679 articles, with 429 being selected for full-text evaluation. A total of 168 articles were included. Overall, no significant differences were found among procedures performed under maternal anesthesia or maternal-fetal anesthesia. Procedures requiring invasive fetal manipulation resulted to be more effective when performed under maternal anesthesia only. Based on the available data, a wide range of anesthetic agents are currently deployed and no consistency has been found neither between centers nor procedures., Conclusions: This systematic review shows great variance in the anesthetic management during FOP. Further studies, systematically reporting intraoperative fetal monitoring and fetal hormonal responses to external stimuli, are necessary to identify the best anesthetic approach. Additional investigations on pain pathways and fetal pain perception are advisable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Duci, Pulvirenti, Fascetti Leon, Capolupo, Veronese, Gamba and Tognon.)

Low-middle income countries (LMICs) are currently experiencing an important population growth, leading to a substantial raise in the number of children living in those areas. As a consequence, the existing gap between the need for surgical and anesthetic care and the available therapeutic options will increase. To overcome this, an improvement in the available expertise, infrastructures, and supplies will be mandatory. The implementation of educational and training programs for local healthcare providers should be a top priority. Alongside, the population's awareness on the necessity to seek for medical care should be deployed, together with an eased access to health facilities. Based on the existing literature and our 20-years' experience in humanitarian missions, our article aims to investigate the status of pediatric surgery in LMICs, and the role of western aids in the implementation of this ever-increasing field of expertise., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pulvirenti, Gortan, Cumba, Gamba and Tognon.)

Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual's leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML., (© 2022. The Author(s).)

Objectives: One lung ventilation (OLV) is the preferred ventilation technique for thoracoscopy as it provides a better exposure of the operative field and grants the protection of the healthy lung. Preoperative evaluation of lung exclusion is necessary and different methods are available. In recent years lung ultrasound (US) gained popularity and its use for monitoring the endotracheal tube position is widely reported. The existing evidence on adults addresses lung US as effective, yet only few data are available in children. Therefore, we present our experience with lung US as verification method for pediatric OLV. Methods: All patients undergoing OLV for video-assisted thoracoscopic surgery from January 2019 to May 2021 and for whom lung exclusion was confirmed through lung US were involved. Lung exclusion was considered effective when absence of lung motion and presence of lung pulse were encountered. When lung US did not match these criteria, repositioning of the endobronchial device followed by US verification was performed. When lung US met the exclusion criteria surgery was started and direct thoracoscopic observation was used to verify lung exclusion. Results: A total of 20 patients, accounting for 22 procedures, were involved. Absence of lung motion and presence of lung pulse were assessed in the operative-side lung for all patients. Lung exclusion was confirmed through thoracoscopy. Postoperative lung US proved the reappearance of lung motion in the previously excluded lung. Conclusions: In our center experience lung US resulted to be a safe, effective, and time-saving verification method for OLV. Further studies are needed to define its sensitivity and specificity.

Inherited predisposition to myeloid malignancies is more common than previously appreciated. We analyzed the whole-exome sequencing data of paired leukemia and skin biopsy samples from 391 adult patients from the Beat AML 1.0 consortium. Using the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines for variant interpretation, we curated 1547 unique variants from 228 genes. The pathogenic/likely pathogenic (P/LP) germline variants were identified in 53 acute myeloid leukemia (AML) patients (13.6%) in 34 genes, including 6.39% (25/391) of patients harboring P/LP variants in genes considered clinically actionable (tier 1). 41.5% of the 53 patients with P/LP variants were in genes associated with the DNA damage response. The most frequently mutated genes were CHEK2 (8 patients) and DDX41 (7 patients). Pathogenic germline variants were also found in new candidate genes (DNAH5, DNAH9, DNMT3A, and SUZ12). No strong correlation was found between the germline mutational rate and age of AML onset. Among 49 patients who have a reported history of at least one family member affected with hematological malignancies, 6 patients harbored known P/LP germline variants and the remaining patients had at least one variant of uncertain significance, suggesting a need for further functional validation studies. Using CHEK2 as an example, we show that three-dimensional protein modeling can be one of the effective methodologies to prioritize variants of unknown significance for functional studies. Further, we evaluated an in silico approach that applies ACMG curation in an automated manner using the tool for assessment and (TAPES) prioritization in exome studies, which can minimize manual curation time for variants. Overall, our findings suggest a need to comprehensively understand the predisposition potential of many germline variants in order to enable closer monitoring for disease management and treatment interventions for affected patients and families., (© 2022 by The American Society of Hematology.)

Extraperitoneal approach is sometimes recommended for kidney transplantation (KT) in children weighting <15 kg. We hypothesized that this approach might be as successful as in patients with normal weight. Data of all consecutive KTs performed between 2013 and 2019 were retrospectively reviewed. Early outcomes and surgical complications were compared between children weighing ≤15 kg (low-weight (LW) group) and those weighing >15 kg (Normal-weight (NW) group). All the 108 KTs were performed through an extraperitoneal approach. The LW group included 31 patients (mean age 3.5 ± 1.4 years), whose mean weight was 11.1 ± 2.0 kg. In the LW group,-a primary graft nonfunction (PNGF) occurred in one patient (3.2%), surgical complications occurred in nine (29%), with four venous thrombosis. In the NW group, PNGF occurred in one case (1.3%), delayed graft function (DGF) in eight (10%), surgical complications in 11 (14%) with only one case of venous thrombosis. In both groups, no need for patch during wound closure and no wound dehiscence were reported. The extraperitoneal approach can be effectively used in LW children. No differences were observed in the overall complication rate (P = 0.10), except for the occurrence of venous thrombosis (P = 0.02). This might be related to patients' characteristics of the LW group., (© 2021 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

Pediatric abdominal surgery is constantly evolving, alongside the advent of new surgical technologies. A combined use of new tools and traditional surgical approaches can be useful in the management of complex cases, allowing less invasive procedures and sometimes even avoiding multiple interventions. This combination of techniques has implications even from the anesthetic point of view, especially in post-operative pain control. Thereby, tertiary level centres, including highly-specialized professionals and advanced equipment, can maximize the effectiveness of treatments to improve the final outcomes. Our paper aims to present some possible combinations of techniques recently used at our institution to provide a one-session, minimally invasive treatment within different areas of abdominal surgery.

Background: Little is known about the surgical conditions affecting the pediatric population in low-income countries. In this article we describe the epidemiology of pediatric surgical diseases observed in Mutoyi hospital, a first-level hospital in Burundi. Methods and Findings: We retrospectively reviewed the records of all children (0-14 years) admitted to the Surgery ward from January 2017 to December 2017. We also reviewed the records of all the patients admitted to the Neonatology ward in 2017 and among them we selected the ones in which a surgical diagnosis was present. Five hundred twenty-eight children were admitted to the surgical ward during the study period. The most common conditions requiring hospitalization were abscesses (29.09%), fractures (13.59%), osteomyelitis (9.76%), burns (5.40%) and head injuries (4.36%). The average length of stay was 16 days. Fifty-six newborns were admitted to the Neonatology ward for a surgical condition; 29% of them had an abscess. Conclusions: Conditions requiring surgical care are frequent in Burundian children and have a completely different spectrum from the western ones. This is due on one side to an under-diagnosis of certain conditions caused by the lack of diagnostic tools and on the other to the living conditions of the population. This difference should lead to intervention plans tailored on the actual necessities of the country and not on the western ones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gortan, Caravaggi, Brooks, Butoyi, Bambara, Nkurunziza, Mulemangabo, Nzeyimana, Harakaneza, Nshimirimana, Tognon, Gamba, Parigi and Dalla Gasperina.)

Neuroblastoma (NB) is the most common extracranial, solid, pediatric malignancy and, despite the constant progress of treatment and development of innovative therapies, remains a complex, challenging disease causing major morbidity and mortality in children. There is significant variability in the management of neuroblastoma, partially due to the heterogeneity of the clinical and biological behavior, and partially secondary to the different approaches between treating institutions. Anesthesia takes an integral part in the multidisciplinary care of patients with NB, from diagnosis to surgery and pain control. This paper aims to review and discuss the critical steps of the perioperative and operative management of children undergoing surgery for neuroblastoma. Anesthesia and analgesia largely depend on tumor location, surgical approach, and extension of the surgical dissection. Attention should be paid to the physio-pathological changes on cardiovascular, gastrointestinal, and immune systems induced by the tumor or by chemotherapy. At the time of surgery meticulous patient preparation needs to be carried out to optimize intraoperative monitoring and minimize the risk of complications. The cross-sectional role of anesthesia in cancer care requires effective communication between all members of the multidisciplinary team.

Introduction: Our aim is to compare thoracoscopy to thoracotomy in the treatment of congenital lung malformations (CLM) in children., Materials and Methods: We report a retrospective monocentric cohort study. Patients treated at our Center for CLM (1991-2020) were divided in two groups: patients treated with video-assisted thoracoscopic surgery (VATS) and open thoracotomy (OT). Characteristics of the two groups were compared through statistical analysis (GraphPad Prism7). A p value less than .05 was considered statistically significant., Results: One hundred six patients were included: 58 in VATS group, 48 in OT group. Prenatal diagnosis was possible in 73.6%. The most frequent surgical procedures were lobectomy (43.4%) and sequestrectomy (22.6%). All VATS patients underwent lung exclusion, mostly by endobronchial blocker (69%). Mean operative time was 146.1 min (±52.04 SD) in VATS and 159.2 (±46.53 SD) in OT (p = .1973). Conversion to OT was necessary in 20.6% of VATS patients, but decreased in the last 5 years (6.2%). There were not any intraoperative complication. Respectively in VATS and OT group, length of stay (LOS) was 4.5 days ± 3.6 SD versus 7.7 ± 3.4 SD (p < .0001), chest tube duration 2.8 days ± 3.4 SD versus 3.7 ± 2.4 SD (p < .0001), antibiotic treatment duration 3.7 days ± 4.7 SD versus 5 ± 2.6 SD (p = .1196). Postoperative complications were described in 22.6%. The commonest histological diagnosis (40.6%) was congenital pulmonary airway malformation., Conclusion: VATS resulted a feasible, effective and safe technique. Operative time and postoperative complications were similar in VATS and OT groups. VATS conversion rate decreased in time. VATS had a statistically significant shorter LOS and chest tube duration., (© 2020 Wiley Periodicals LLC.)

Context: The circumcision is the most frequent procedure in paediatric surgery worldwide, performed for medical and ritual purposes. In developing countries, because of the difficult accessibility to healthcare, even a common procedure could be unsafe., Aims: The aim of the article is to compare the perioperative and anaesthesiological management of circumcision in children between two Italian and two sub-Saharan African hospitals., Materials and Methods: Medical records of paediatric circumcision from January 2014 to December 2016 have been reviewed. The involved hospitals were: Padua (Italy), Ferrara (Italy), Sao José em Bor (Guinea Bissau) and Yaoundé (Cameroun)., Results: In Padua, 77 circumcisions were performed, 19 of these (24.6%) were ritual. In 75 children (97.4%), locoregional anaesthesia (LRA) together with sedation was used; only one complication (1.3%) occurred. In Ferrara, 200 interventions were done, 140 (70%) ritual; general anaesthesia was administered to 183 (93.5%) patients. There were five complications (2.5%). In Bissau, 53 procedures were performed, 21 (39.6%) ritual; in 34 children (64.1%), LRA with sedation was preferred. Two complications (3.8%) were reported. In Yaoundé, 60 children were circumcised, 15 (25%) for ritual purposes; in 51 (85%), only LRA was performed; there was only one (1.7%) complication. In the African hospital, no post-operative analgesia was administered., Conclusion: Despite the different anaesthesiological techniques, the study shows no difference in rate of complications for the in-hospital setting. Training of the local medical team in pain management and post-operative care should be emphasised., Competing Interests: None

The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.

Background: The preoperative assessment involves the process of evaluating the patient's clinical condition, which is intended to define the physical status classification, eligibility for anesthesia and the risks associated with it, thus providing elements to select the most appropriate and individualized anesthetic plan. The aim of this recommendation was provide a framework reference for the preoperative evaluation assessment of pediatric patients undergoing elective surgery or diagnostic/therapeutic procedures., Methods: We obtained evidence concerning pediatric preoperative evaluation from a systematic search of the electronic databases MEDLINE and Embase between January 1998 and February 2012. We used the format developed by the Italian Center for Evaluation of the Effectiveness of Health Care's scoring system for assessing the level of evidence and strength of recommendations., Results: We produce a set of consensus guidelines on the preoperative assessment and on the request for preoperative tests. A review of the existing literature supporting these recommendations is provided. In reaching consensus, emphasis was placed on the level of evidence, clinical relevance and the risk/benefit ratio., Conclusion: Preoperative evaluation is mandatory before any diagnostic or therapeutic procedure that requires the use of anesthesia or sedation. The systematic prescription of complementary tests in children should be abandoned, and replaced by a selective and rational prescription, based on the patient history and clinical examination performed during the preoperative evaluation.

The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non-small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1(G2032R) mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3β is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3β. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.

Cancer cells are often characterized by high proliferation rates, a consequence of increased mitotic signaling coupled with unchecked cellular growth. We recently demonstrated that vascular endothelial cells unexpectedly express ferlins, a family of muscle-specific proteins capable of regulating the fusion of lipid patches to the plasma membrane, and that these highly regulated membrane fusion events are essential to endothelial cell proliferation and homeostasis. Here, we show that human and mouse breast cancer cell lines also express myoferlin at various levels, and that the processes of transformation, epithelial-mesenchymal transition, and metastasis do not appear to have any effect on myoferlin expression in vitro. In vivo, we observed that solid mouse and human carcinoma tissues also express high levels of myoferlin protein. Loss-of-function studies performed in mice revealed that myoferlin gene knockdown can attenuate cancer cell proliferation in vitro and decrease tumor burden, and that accelerated tumor cell growth appears to rely on intact myoferlin-dependent membrane repair and signaling under exponential growth conditions. To our knowledge, these data provide the first evidence of myoferlin expression in solid human and mouse tumors. We have thus identified a novel membrane repair process that likely helps sustain the high growth rates characteristic of tumors, and we suggest that interfering with normal myoferlin expression and/or membrane repair and remodeling may provide therapeutically relevant antiproliferative effects., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Infantile fibrosarcoma (IFS; also known as cellular congenital mesoblastic nephroma, CMN, when in the kidney) is a rare, undifferentiated tumour often characterized by the ETV6-NTRK3 fusion transcript. Our goal was to identify downstream pathways, diagnostic markers and potential therapeutic targets for IFS/CMN. Global gene expression, reverse-phase protein array and ETV6-NTRK3 fusion analyses were performed on 14 IFS/CMN and compared with 41 other paediatric renal tumours. These analyses confirm significant receptor tyrosine kinase (RTK) activation, with evidence of PI3-Akt, MAPK and SRC activation. In particular, GAB2 docking protein, STAT5-pTyr-694, STAT3-pSer-729 and YAP-pSer-127 were elevated, and TAZ-pSer-89 was decreased. This provides mRNA and proteomic evidence that GAB2, STAT activation and phosphorylation of the Hippo pathway transcription co-activators YAP and TAZ contribute to the RTK signal transduction in IFS/CMN. All IFS/CMN tumours displayed a distinctive gene expression pattern that may be diagnostically useful. Unexpectedly, abundant ETV6-NTRK3 transcript copies were present in only 7/14 IFS, with very low copy number in 3/14. An additional 4/14 were negative by RT-PCR and absence of ETV6-NTRK3 was confirmed by FISH for both ETV6 and NTRK3. Therefore, molecular mechanisms other than ETV6-NTRK3 fusion are responsible for the development of some IFS/CMNs and the absence of ETV6-NTRK3 fusion products should not exclude IFS/CMN as a diagnosis., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Hyperproliferation induced by various oncogenic proteins, including activated Ras, is the most prominent and well characterized feature of cancerous cells. This property has been exploited in the development of the most successful anti-cancer treatments to target rapidly dividing cells. Here we argue that hyperproliferation may in fact be detrimental to survival during particular stages of cancer progression such as dissemination from primary tumor and establishing metastatic outgrowth. Our recent work has demonstrated that elevation of YB-1 protein levels, which is frequently observed in human cancers, is associated with reduced proliferation rates in disseminated mesenchymal-like breast carcinoma cells. In breast cancer cell lines with activated Ras-MAPK signaling, YB-1 inhibited cellular proliferation, while inducing an epithelial-to-mesenchymal transition (EMT). The underlying mechanism involves YB-1-mediated translational repression of pro-growth transcripts and activation of the messages encoding EMT-associated proteins, many of which are also known to inhibit proliferation. In addition to the lack of epithelial polarity, increased mobility and invasiveness, YB-1-overexpressing cells displayed a remarkable ability to shut down proliferation and survive in anchorage-independent conditions. These findings support the view that while an increase in proliferation is important for the initiation and maintenance of primary tumors, growth inhibition could ultimately be crucial for survival of carcinoma cells in the circulation and secondary organs, thereby leading to the development of a more malignant phenotype.

Increased expression of the transcription/translation regulatory protein Y-box binding protein-1 (YB-1) is associated with cancer aggressiveness, particularly in breast carcinoma. Here we establish that YB-1 levels are elevated in invasive breast cancer cells and correlate with reduced expression of E-cadherin and poor patient survival. Enforced expression of YB-1 in noninvasive breast epithelial cells induced an epithelial-mesenchymal transition (EMT) accompanied by enhanced metastatic potential and reduced proliferation rates. YB-1 directly activates cap-independent translation of messenger RNAs encoding Snail1 and other transcription factors implicated in downregulation of epithelial and growth-related genes and activation of mesenchymal genes. Hence, translational regulation by YB-1 is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, likely acting together to promote metastatic spread.

An emerging theme in cancer biology is that although some malignancies occur through the sequential acquisition of different genetic alterations, certain dominantly acting oncoproteins such as those associated with chromosomal translocations have multiple functions and do not require additional mutations for cell transformation. The ETV6-NTRK3 (EN) chimeric tyrosine kinase, a potent oncoprotein expressed in tumors derived from multiple cell lineages, functions as a constitutively active protein tyrosine kinase. Here, we show that EN suppresses TGF-beta signaling by directly binding to the type II TGF-beta receptor, thereby preventing it from interacting with the type I TGF-beta receptor. This activity requires a functional EN protein tyrosine kinase, and type II TGF-beta receptor appears to be a direct target of EN. Our findings provide evidence for a previously undescribed mechanism by which oncogenic tyrosine kinases can block TGF-beta tumor suppressor activity.