In-depth analysis of alternative splicing landscape in multiple myeloma and potential role of dysregulated splicing factors.

Splicing changes are common in cancer and are associated with dysregulated splicing factors. Here, we analyzed RNA-seq data from 323 newly diagnosed multiple myeloma (MM) patients and described the alternative splicing (AS) landscape. We observed a large number of splicing pattern changes in MM cells compared to normal plasma cells (NPC). The most common events were alterations of mutually exclusive exons and exon skipping. Most of these events were observed in the absence of overall changes in gene expression and often impacted the coding potential of the alternatively spliced genes. To understand the molecular mechanisms driving frequent aberrant AS, we investigated 115 splicing factors (SFs) and associated them with the AS events in MM. We observed that ~40% of SFs were dysregulated in MM cells compared to NPC and found a significant enrichment of SRSF1, SRSF9, and PCB1 binding motifs around AS events. Importantly, SRSF1 overexpression was linked with shorter survival in two independent MM datasets and was correlated with the number of AS events, impacting tumor cell proliferation. Together with the observation that MM cells are vulnerable to splicing inhibition, our results may lay the foundation for developing new therapeutic strategies for MM. We have developed a web portal that allows custom alternative splicing event queries by using gene symbols and visualizes AS events in MM and subgroups. Our portals can be accessed at http://rconnect.dfci.harvard.edu/mmsplicing/ and https://rconnect.dfci.harvard.edu/mmleafcutter/ .

Robotic Resection of Pulmonary Epithelial Myoepithelial Carcinoma: A Case Report.

Background Pulmonary epithelial-myoepithelial carcinoma (P-EMC) is an extremely rare, well-differentiated, and malignant neoplasm originating from submucosal bronchial glands in the lung. EMCs arise mainly in the salivary glands. Case Description This case represents an asymptomatic 78-year-old male with a remote 75-pack-year history of smoking who presents with a solitary endobronchial lesion, which is suggestive of a primary lung EMC, detected on annual screening chest computed tomography (CT) scan. Conclusion A recent review of literature reveals less than 50 documented cases of the pulmonary subtype of this tumor worldwide. We are reporting a unique case of robot-assisted pulmonary lobectomy for a P-EMC.

Pediatric Autoimmune Ocular Myasthenia Gravis: Evaluation of Presentation and Treatment Outcomes in a Large Cohort.

BACKGROUND: In autoimmune myasthenia gravis (MG), autoantibodies target the neuromuscular junction. Ocular myasthenia gravis (OMG) is localized, affecting only extraocular and/or levator palpebrae muscles. OMG presents across all ages, varying in presentation, treatment modalities, and outcomes. Recently, there have been advances in MG/OMG treatment; their utilization and effectiveness are an important part of optimal disease management. METHODS: We completed a retrospective chart review of children aged 18 years or younger with a confirmed diagnosis of OMG presenting from 2002 to 2019. RESULTS: Forty-two patients were included with mean age at presentation of 8.5 years (2 to 18 years). Twenty-one patients (50%) had positive antibodies; 90% had acetylcholine receptor antibodies. Ten patients developed generalized symptoms with mean time to generalization of 13.6 months. Multiple logistic regression showed that older age of onset was a trend predictive factor (P = 0.054; odds ratio 1.17) for generalized disease. All patients were treated with pyridostigmine. Immunomodulating agents included steroids (15), mycophenolate mofetil (four), and intravenous immunoglobulin (one). Three patients underwent thymectomy. Twenty patients reached minimal manifestation status, and 12 achieved remission. Gender, race, and positive antibody status were not statistically significant predictors for advanced immunosuppressive therapy. CONCLUSIONS: We summarize one of the largest cohorts of pediatric patients with OMG who have undergone up-to-date diagnostic and therapeutic regimens. The predictors of outcome and treatment pathway for OMG patients suggested by this report may be further elucidated by future prospective studies.

Clinical exome sequencing in the diagnosis of pediatric neuromuscular disease.

BACKGROUND: The diagnosis of uncommon pediatric neuromuscular disease (NMD) is challenging due to genetic and phenotypic heterogeneity, yet is important to guide treatment, prognosis, and recurrence risk. Patients with diagnostically challenging presentations typically undergo extensive testing with variable molecular diagnostic yield. Given the advancement in next generation sequencing (NGS), we investigated the value of clinical whole exome sequencing (ES) in uncommon pediatric NMD. METHODS: A retrospective cohort study of 106 pediatric NMD patients with a combination of ES, chromosomal microarray (CMA), and candidate gene testing was completed at a large tertiary referral center. RESULTS: A molecular diagnosis was achieved in 37/79 (46%) patients with ES, 4/44 (9%) patients with CMA, and 15/74 (20%) patients with candidate gene testing. In 2/79 (3%) patients, a dual molecular diagnosis explaining the neuromuscular disease process was identified. A total of 42 patients (53%) who received ES remained without a molecular diagnosis at the conclusion of the study. CONCLUSIONS: Due to NGS, molecular diagnostic yield of rare neurological diseases is at an all-time high. We show that ES has a higher diagnostic rate compared to other genetic tests in a complex pediatric neuromuscular disease cohort and should be considered early in the diagnostic journey for select NMD patients with challenging presentations in which a clinical diagnosis is not evident.

Hospital Readmissions for Hyperparathyroidism After Bariatric Surgery in the United States: A National Database Review.

INTRODUCTION: The incidence and significance of hyperparathyroidism in patients after bariatric surgery have been established to some degree; however, the impact it has on the national healthcare system has not. We sought to assess the risk of readmission and related comorbidities in this patient population. METHODS: The Healthcare Cost and Utilization Project Nationwide Readmission Database was queried for all patients who underwent Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Multivariate logistic regression analysis was conducted to identify factors associated with readmission for hyperparathyroidism. RESULTS: A total of 915,792 patients between 2010 and 2015 were queried; 43.2% had undergone SG and 56.8% had RYGB. A total of 589 patients were readmitted for hyperparathyroidism; 80.8% were female and 68% had a Charlson comorbidity index ≥ 2. Factors associated with readmission were as follows: age 45-64 years (odds ratio [OR] 1.42, p=0.001), Medicare (OR 3.01, p<0.001) or Medicaid (OR 2.61, p<0.001) insurance status, lower median household income, renal failure (OR 17.14, p<0.001), hypertension (OR 2.89, p<0.001), and deficiency anemia (OR 2.62, p<0.01). CONCLUSIONS: Parathyroid axis monitoring may provide benefits to predictably high-risk patients. Appropriate surveillance may decrease the impact of bariatric hyperparathyroidism readmission on the U.S. healthcare system.

YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Dual PAK4-NAMPT Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldenström Macroglobulinemia.

PURPOSE: p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. EXPERIMENTAL DESIGN: We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. RESULTS: We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. CONCLUSIONS: These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

Non-overlapping Control of Transcriptome by Promoter- and Super-Enhancer-Associated Dependencies in Multiple Myeloma.

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy.

Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma.

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific 'MYC target gene signatures' and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.

Thirty-day readmissions following parathyroidectomy: Evidence from the National Readmissions Database, 2013-2014.

PURPOSE: Parathyroidectomy is one of the most common procedures performed in the United States, and are increasingly being performed safely in the outpatient setting. However, complications from surgery can be life-threatening, and thus an understanding of who may be at risk is essential. We analyzed and compared the risk factors for patients readmitted within 30 days following inpatient parathyroidectomy for primary or secondary hyperparathyroidism. MATERIALS AND METHODS: We reviewed the National Readmissions Database from 2013 to 2014 for patients who received inpatient parathyroidectomy for primary or secondary hyperparathyroidism. The primary outcome was non-elective readmission within 30 days. Multivariate logistic regression was used to analyze risk factor odds ratios for readmission. RESULTS: 7171 patients underwent inpatient parathyroidectomies in 2013 and 2014. 59.89% of parathyroidectomies were performed for primary hyperparathyroidism, with a 5.6% readmission rate. Most common causes of readmission were septicemia (13.69%), hypocalcemia (12.86%), heart failure (10.79%) and renal failure (9.54%). Having Medicare (OR: 1.71, CI:1.14-2.59, p = .01), Medicaid (OR: 3.24, CI: 2.03-5.17, p < .001), and self-paying (OR: 2.43, CI: 1.11-5.32, p = .02), were associated with increased odds of readmission for those with primary hyperparathyroidism. 21.99% of parathyroidectomies were performed for secondary hyperparathyroidism, with a 19.4% readmission rate. Most common causes of readmission were hypocalcemia (22.88%), hungry bone syndrome (14.38%), electrolyte disorders (13.73%), and renal failure (11.11%). CONCLUSION: Patients with secondary hyperparathyroidism are older, poorer and have more comorbidities than patients with primary hyperparathyroidism, and are more likely to be readmitted within 30 days of parathyroidectomy.

Pediatric laparoscopic appendectomy, risk factors, and costs associated with nationwide readmissions.

BACKGROUND: Previous studies of readmission after pediatric laparoscopic appendectomy have been limited to individual hospitals or noncompeting public pediatric hospitals. The purpose of this study was to evaluate the risk factors and costs associated with nonelective, 30-d readmissions in pediatric patients nationwide across public and private hospitals. MATERIALS AND METHODS: The Nationwide Readmission Database for 2013 was queried for all patients under the age of 18 y with a diagnosis of acute appendicitis undergoing laparoscopic appendectomy. Using multivariate logistic regression with 26 different variables, the odds ratios (ORs) for nonelective readmissions within 30 d were determined. The costs of readmission were calculated as well as the most common diagnoses on readmission. RESULTS: In 2013, there were 12,730 patients under the age of 18 y undergoing laparoscopic appendectomy, and 3.4% were readmitted within 30 d. The overall mean age was 11.6 ± 3.8 y, and the mean age of the readmitted patients was 10.7 ± 4.0 whereas the mean age of patients not readmitted was 11.6 ± 3.8 (P < 0.01, 95% CI: 0.54-1.26). The total cost of readmissions was $3,645,502 with a weighted nationwide estimated cost of $10,351,690. The mean readmission cost was $8304 ± 7864. The most common diagnosis group on readmission was postoperative, posttraumatic, other device infections (36.0%), whereas the most common principal diagnosis was other postoperative infection (38.5%) and the most common secondary diagnosis was peritoneal abscess (11.9%). CONCLUSIONS: Readmission within 30 d after laparoscopic appendectomy in pediatric patients represents a significant resource burden. This study elucidates the patient characteristics that predispose these patients to readmission. Efforts to reduce these readmissions should be focused around preventing infections in patients with these predisposing risk factors.

Acute abdominal pain secondary to chilaiditi syndrome.

Chilaiditi syndrome is a rare condition occurring in 0.025% to 0.28% of the population. In these patients, the colon is displaced and caught between the liver and the right hemidiaphragm. Patients' symptoms can range from asymptomatic to acute intermittent bowel obstruction. Diagnosis is best achieved with CT imaging. Identification of Chilaiditi syndrome is clinically significant as it can lead to many significant complications such as volvulus, perforation, and bowel obstruction. If the patient is symptomatic, treatment is usually conservative. Surgery is rarely indicated with indications including ischemia and failure of resolution with conservative management.

Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.

The ß-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and ß-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of ß-tropomyosin-related myopathies through the identification of a novel ß-tropomyosin mutation in two clinical contexts not previously associated with ß-tropomyosin. The first clinical phenotype is core-rod myopathy, with a ß-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of ß-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric ß-tropomyosin, a change predicted to alter protein-protein binding between ß-tropomyosin and other molecules and to disturb head-to-tail polymerization of ß-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del ß-tropomyosin in the developing zebrafish. p.K7del ß-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail ß-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel ß-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with ß-tropomyosin and pathogenic data from the first animal model of ß-tropomyosin-related myopathies.

Anisotropic regulation of Ankrd2 gene expression in skeletal muscle by mechanical stretch.

The diaphragm muscles in vivo are subjected to mechanical forces both in the direction of the muscle fibers and in the direction transverse to the fibers. However, the effect of directional mechanical forces in skeletal muscle gene regulation is completely unknown. Here, we identified that stretch in the longitudinal and transverse directions to the diaphragm muscle fibers up-regulated Ankrd2 gene expression by two distinct signaling pathways in wild-type (WT) and mdm, a mouse model of muscular dystrophy with early-onset of progressive muscle-wasting. Stretch in the longitudinal direction activated both NF-kappaB and AP-1 transcription factors, whereas stretch in the transverse direction activated only AP-1 transcription factor. Interestingly, longitudinal stretch activated Ankrd2 promoter only by NF-kappaB, whereas transverse stretch activated Ankrd2 promoter by AP-1. Moreover, we found that longitudinal stretch activated Akt, which up-regulated Ankrd2 expression through NF-kappaB. However, transverse stretch activated Ras-GTP, Raf-1, and Erk1/2 proteins, which up-regulated Ankrd2 expression through AP-1. Surprisingly, the stretch-activated NF-kappaB and AP-1 signaling pathways was not involved in Ankrd2 regulation at the basal level, which was high in the mdm mouse diaphragm. Taken together, our data show the anisotropic regulation of Ankrd2 gene expression in the diaphragm muscles of WT and mdm mice via two distinct mechanosensitive signaling pathways.