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Figure S1. Correlation of RACGAP1 Immunohistochemical Staining and mRNA Expression; Figure S2. Survivin Expression in CS99 Treated With YM155

Table S4. Short Tandem Repeat Profiling of Primary Cell Line DNA and Patient Tumor Tissue DNA.

Supplemental Figure and Table Legends

Purpose: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets.Experimental Design: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line–derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA).Results: Rac GTPase–activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease.Conclusions: The RACGAP1–STAT3–survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676–86. ©2016 AACR.

Background Mutations in the NMDA receptor are known to disrupt glutamatergic signaling crucial for early neurodevelopment, often leading to severe global developmental delay/intellectual disability, epileptic encephalopathy, and cerebral palsy phenotypes. Both seizures and movement disorders can be highly treatment-refractory. Results We describe a targeted ABA n = 1 patient trial with intrathecal MgSO4, rationally designed based on the electrophysiologic properties of this gain of function mutation in the GRIN1 NMDA subunit. Conclusion Although the invasive nature of the trial necessitated a short-term, non-randomized, unblinded intervention, multimodal neurophysiologic monitoring indicated benefit, providing class II evidence in support of intrathecal MgSO4 for select forms of GRIN disorders.

Opsoclonus-myoclonus-ataxia syndrome is a rare neuroimmunologic disorder typically presenting in previously healthy infants and toddlers. It is characterized by a clinical triad of (1) erratic saccadic intrusions; (2) myoclonus and/or ataxia; (3) behavioral features, typified by developmental plateauing, irritability and insomnia. About half of cases are associated with an underlying neuroblastoma and diagnostic imaging is essential once OMAS is suspected. A thorough workup, including serum, urine, and cerebrospinal fluid studies is critical to identify underlying biomarkers of OMAS itself or neuroblastoma. Historically, many children had relatively poor long-term outcomes, with residual neurologic and/or neuropsychiatry sequelae typical. More recent concepts have emphasized combined immunotherapy regimens that offer hope for better outcomes in children with this remarkable, challenging disease.

BackgroundNormative childhood motor network resting-state fMRI effective connectivity is undefined, yet necessary for translatable dynamic resting-state network informed treatments in pediatric movement disorders.MethodCross-spectral dynamic causal modelling of resting-state fMRI was investigated in 19 neurotypically developing 5-7-year-old children. Fully connected six-node motor network models were created for each hemisphere including primary motor cortex, striatum, subthalamic nucleus, globus pallidus internus, thalamus, and contralateral cerebellum. Parametric Empirical Bayes with exhaustive Bayesian model reduction and Bayesian modeling averaging were used to create a group model for each hemisphere; Purdue Pegboard Test (PPBT) scores for relevant hand motor behavior were also entered as a covariate at the group level to determine the brain-behavior relationship.ResultsOverall, the resting-state functional MRI effective connectivity of motor cortico-basal ganglia-cerebellar networks was similar across hemispheres, with greater connectivity in the left hemisphere. The motor network effective connectivity relationships between the nodes were consistent and robust across subjects. Additionally, the PPBT score for each hand was positively correlated with the thalamus to contralateral cerebellum connection.DiscussionThe normative effective connectivity from resting-state functional MRI in children largely reflect the direction of inter-nodal signal predicted by other prior modalities and was consistent and robust across subjects, with differences from these prior task-dependent modalities that likely reflect the motor rest-action state during acquisition. Effective connectivity of the motor network was correlated with motor behavior, indicating effective connectivity brain-behavior relationship has physiological meaning in the normally developing. Thus, it may be helpful for future studies in children with movement disorders, wherein comparison to normative effective connectivity will be critical for network-targeted intervention.Impact StatementThis is the first study to use pediatric resting-state functional MRI to create a normative effective connectivity model of the motor network and to also show correlation with behavior, which may have therapeutic implications for children with movement disorders.

ObjectiveTo determine whether mutations reported for ZDHHC15 can cause mixed neurodevelopmental disorders, we performed both functional studies on variant pathogenicity and ZDHHC15 function in animal models.MethodsWe examined protein function of 4 identified variants in ZDHHC15 in a yeast complementation assay and locomotor defects of loss-of-function genotypes in a Drosophila model.ResultsAlthough we assessed multiple patient variants, only 1 (p.H158R) affected protein function. We report a patient with a diagnosis of hypotonic cerebral palsy, autism, epilepsy, and intellectual disability associated with this bona fide damaging X-linked variant. Features include tall forehead with mild brachycephaly, down-slanting palpebral fissures, large ears, long face, facial muscle hypotonia, high-arched palate with dental crowding, and arachnodactyly. The patient had mild diminished cerebral volume, with left-sided T2/FLAIR hyperintense periatrial ovoid lesion. We found that loss-of-function mutations in orthologs of this gene cause flight and coordinated movement defects in Drosophila.ConclusionsOur findings support a functional expansion of this gene to a role in motor dysfunction. Although ZDHHC15 mutations represent a rare cause of neurodevelopmental disability, candidate variants need to be carefully assessed before pathogenicity can be determined.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

Parental worries regarding a child’s movement are relatively common in pediatric neurological practice, particularly when one considers concerns about early development, unusual or paroxysmal movements during the preschool years, and the broad spectrum of movement patterns and mannerisms that typical children display. In differentiating benign from concerning movements, phenomenology (the appearance of the movement) is key. Although a good history is crucial, the importance of being able to observe the movement in question cannot be stressed enough, and video clips are invaluable in effectively diagnosing movements of concern.

Enteroviruses frequently result in respiratory and gastrointestinal illness; however, multiple subtypes, including poliovirus, can cause severe neurologic disease. Recent biennial increases (i.e., 2014, 2016, and 2018) in cases of non-polio acute flaccid paralysis have led to speculations that other enteroviruses, specifically enterovirus D68 (EV-D68), are emerging to fill the niche that was left from poliovirus eradication. A cluster of 11 suspect cases of pediatric acute flaccid myelitis (AFM) was identified in 2016 in Phoenix, AZ. Multiple genomic analyses identified the presence of EV-D68 in the majority of clinical AFM cases. Beyond limited detection of herpesvirus, no other likely etiologies were found in the cluster. These findings strengthen the likelihood that EV-D68 is a cause of AFM and show that the rapid molecular assays developed for this study are useful for investigations of AFM and EV-D68., Enteroviruses are a common cause of respiratory and gastrointestinal illness, and multiple subtypes, including poliovirus, can cause neurologic disease. In recent years, enterovirus D68 (EV-D68) has been associated with serious neurologic illnesses, including acute flaccid myelitis (AFM), frequently preceded by respiratory disease. A cluster of 11 suspect cases of pediatric AFM was identified in September 2016 in Phoenix, AZ. To determine if these cases were associated with EV-D68, we performed multiple genomic analyses of nasopharyngeal (NP) swabs and cerebrospinal fluid (CSF) material from the patients, including real-time PCR and amplicon sequencing targeting the EV-D68 VP1 gene and unbiased microbiome and metagenomic sequencing. Four of the 11 patients were classified as confirmed cases of AFM, and an additional case was classified as probable AFM. Real-time PCR and amplicon sequencing detected EV-D68 virus RNA in the three AFM patients from which NP swabs were collected, as well as in a fourth patient diagnosed with acute disseminated encephalomyelitis, a disease that commonly follows bacterial or viral infections, including enterovirus. No other obvious etiological causes for AFM were identified by 16S or RNA and DNA metagenomic sequencing in these cases, strengthening the likelihood that EV-D68 is an etiological factor. Herpes simplex viral DNA was detected in the CSF of the fourth case of AFM and in one additional suspect case from the cluster. Multiple genomic techniques, such as those described here, can be used to diagnose patients with suspected EV-D68 respiratory illness, to aid in AFM diagnosis, and for future EV-D68 surveillance and epidemiology.

Study Objective To evaluate the ability of the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) surgical risk calculator to predict discharge to postacute care and perioperative complications in gynecologic oncology patients undergoing minimally invasive surgery (MIS). Design A retrospective chart review (Canadian Task Force classification II-1). Setting A university hospital. Patients All patients undergoing MIS on the gynecologic oncology service from January 1, 2009, to December 30, 2013. Interventions Surgical procedures were reviewed, and appropriate Common Procedural Terminology codes were assigned. Twenty-one preoperative risk factors were abstracted from the chart and entered into the ACS NSQIP surgical risk calculator. The predicted risk of discharge to postacute care and 8 additional postoperative complications were calculated and recorded. Actual postoperative complications were abstracted from the medical record. The association between the calculated risk and the actual outcome was determined using logistic regression. The ability of the calculator to accurately predict a particular event was assessed using the c-statistic and Brier score. Measurements and Main Results Of the 876 patients reviewed, a majority underwent hysterectomy (71.6%), with almost half of those patients undergoing additional cancer staging procedures (34.8%). Although the calculator was a poor predictor of postoperative complications, it was a strong predictor for discharge to postacute care (c-statistic = 0.91, Brier score = 0.02) with an odds ratio of 2.31 (95% confidence interval, 1.65–3.25; p

A majority of cervical cancers are caused by human papillomavirus (HPV); however, HPV-negative cervical cancers exist and, though rare, are more aggressive. No prior reports examine HPV-negative cancer of the cervix in a female pseudohermaphrodite with congenital adrenal hyperplasia (CAH). This is a case of a 78-year-old phenotypic male with hypospadias and absent testicles who presented with urinary retention and urosepsis. He was diagnosed with a pelvic mass on imaging and with a female mosaic karyotype (45,X/47,XXX/46 XX). He was taken to the operating room and found to have a rare form of HPV-negative cervical cancer: gastric-type adenocarcinoma (GAS). This study examines the presentation, management, and outcome of a GAS cervical cancer in a patient with a known lack of HPV exposure secondary to the unique anatomy of female pseudohermaphrodism.

Background Biocidal products are mixtures of one or more active substances (a.s.) and a broad range of formulation additives. There is regulatory guidance currently under development that will specify how the combined effects of the a.s. and any relevant formulation additives shall be considered in the environmental risk assessment of biocidal products. The default option is a component-based approach (CBA) by which the toxicity of the product is predicted from the toxicity of ‘relevant’ components using concentration addition. Hence, unequivocal and practicable criteria are required for identifying the ‘relevant’ components to ensure protectiveness of the CBA, while avoiding unnecessary workload resulting from including by default components that do not significantly contribute to the product toxicity. The present study evaluated a set of different criteria for identifying ‘relevant’ components using confidential information on the composition of 21 wood preservative products. Theoretical approaches were complemented by experimentally testing the aquatic toxicity of seven selected products. Results For three of the seven tested products, the toxicity was underestimated for the most sensitive endpoint (green algae) by more than factor 2 if only the a.s. were considered in the CBA. This illustrated the necessity of including at least some additives along with the a.s. Considering additives that were deemed ‘relevant’ by the tentatively established criteria reduced the underestimation of toxicity for two of the three products. A lack of data for one specific additive was identified as the most likely reason for the remaining toxicity underestimation of the third product. In three other products, toxicity was overestimated by more than factor 2, while prediction and observation fitted well for the seventh product. Considering all additives in the prediction increased only the degree of overestimation. Conclusions Supported by theoretical calculations and experimental verifications, the present study developed criteria for the identification of CBA-relevant components in a biocidal product. These criteria are based on existing criteria stated in the regulation for classification, labelling and packaging of substances. The CBA was found sufficiently protective and reliable for the tested products when applying the here recommended criteria. The lack of available aquatic toxicity data for some of the identified relevant components was the main reason for underestimation of product toxicity.

Background Peripheral diagnostics for Alzheimer’s disease (AD) continue to be developed. Diagnostics capable of detecting AD before the onset of symptoms are particularly desirable, and, given the fact that early detection is imperative for alleviating long-term symptoms of the disease, methods which enable detection in the earliest stages are urgently needed. Saliva testing is non-invasive, and saliva is easy to acquire. A simple, non-invasive saliva test can potentially be used as an adjunct to diagnose AD during its earliest stages. Methods Salivary levels of beta amyloid 42 (Aβ42) were quantitated with enzyme-linked immunosorbent–type assays. Fifteen AD patients (7 men, mean age 77.8 ± 1.8 years, mean Mini-Mental State Examination [MMSE] score 19.0 ± 1.3) and 7 normal controls (2 men, mean age 60.4 ± 4.7 years, mean MMSE 29.0 ± 0.4) were enrolled. Results Salivary Aβ42 levels were significantly higher in AD patients than in controls (51.7 ± 1.6 pg/mL for AD and 21.1 ± 0.3 pg/mL for controls, p

Purpose: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets. Experimental Design: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line–derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA). Results: Rac GTPase–activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease. Conclusions: The RACGAP1–STAT3–survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676–86. ©2016 AACR.

e22009 Background: Uterine carcinosarcomas, also known as malignant mixed mullerian tumors (MMMT), are rare and highly aggressive neoplasms that carry a poor prognosis even when diagnosed at an early stage. A better understanding of the molecular mechanisms underlying the development of uterine carcinosarcoma may lead to specific targeted therapies for these refractory tumors. Methods: Using a next generation sequencing approach and bioinformatic analysis, we compared the whole transcriptomes of 6 histologically normal endometrial tissues and 6 uterine carcinosarcoma tissues from patients. Quantitative real-time PCR and immunoblotting were done to validate the differentially expressed genes. Lentiviral transfection with short hairpin RNAs was performed to achieve targeted gene silencing in a carcinosarcoma cell line. p53 and survivin levels were monitored by immunoblotting. Results: We identified 27 dramatically differentially expressed genes between normal endometrial tissues and uterine carcinosarcoma tissues from patients. We confirmed the differential expression of these genes between normal tissues and uterine carcinosarcoma tissues by qRT-PCR. Of these, we found that RACGAP1 is significantly overexpressed in all of the uterine carcinosarcoma tissues at the mRNA level and at the protein level, compared to normal endometrial tissues. Silencing of RACGAP1 by shRNA in a carcinosarcoma cell line remarkably reduces cancer cell proliferation and leads to p53 upregulation and survivin downregulation. Conclusions: Our data demonstrate that RACGAP1 may represent a novel biomarker and therapeutic target for uterine carcinosarcomas.

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