Citations

Abstract Multiple myeloma (MM) is a malignant disease of plasma cells that accumulate in the bone marrow. The survival mechanisms of myeloma cells are still not fully understood. There are several cytokines that are known to support the survival and growth of myeloma cells, such as IL-6, IGF-I, TNF-a, and IL-1β. Recent evidence has also shown that the TNF family member, B lymphocyte stimulator (BLyS), is critical for normal B cell development and homeostasis, as well as for survival of malignant B cells, including MM. However, it should be noted that the precise mechanisms by which BLyS promotes the survival of MM cells remain uncertain. Our group has already reported that MM cell lines and primary myeloma cells can express BLyS and its receptors. Thus, exogenous BLyS augmented the survival of MM cells, including the IL-6 dependent MM cell line, KAS-6/1. To better study the mechanism underlying BLyS-mediated survival of myeloma cells, we sought to establish a KAS-6/1 cell line variant which was solely dependent on exogenous BLyS for growth and survival. We have previously reported that KAS-6/1 cells do not appear to express autocrine IL-6, and therefore are highly dependent on exogenous IL-6 for survival. To establish a BLyS-dependent KAS-6/1 variant, cells were cultured long-term in RPMI containing 0.5% BSA or 1% FCS in the absence of exogenous IL-6, and in the presence or absence of exogenous BLyS. KAS-6/1 cells cultured in the absence of cytokines were non-viable after two weeks of culture, whereas KAS-6/1 cells cultured with BLyS alone remained viable and proliferated, albeit at a lower rate than parental KAS-6/1 cells maintained with IL-6. We next focused on identifying the mechanisms of BLyS-dependent survival of the KAS-6/1/BLyS (KAS-6/B) variant cell line. Of interest, KAS-6/B cells placed in short term culture exhibited cytokine independent survival and proliferation and this was completely inhibited using a neutralizing IL-6 antibody. These results therefore suggested that the mechanism underlying BLyS-mediated survival and proliferation of the KAS-6/B cells is induction of an autocrine IL-6 pathway. This notion is further supported by the ability of BLyS to induce IL-6 expression at the transcriptional level, and at the protein level as revealed by an IL-6 specific ELISA. Consistent with the concept that BLyS induced autocrine IL-6 expression in the KAS-6/B cells, we also observed that STAT-3 was constitutively activated in these cells. In addition, we tested the ability of BLyS-conditioned media obtained from KAS-6/B cells to stimulate the proliferation of a different IL-6 dependent MM cell line, JMW. Whereas BLyS alone failed to stimulate JMW cell proliferation, BLyS-conditioned media obtained from the KAS-6/B cells stimulated proliferation, and in a manner that was inhibited by a neutralizing IL-6 antibody. Finally, BLyS stimulation of primary patient MM cells similarly resulted in increased IL-6 expression. IL-6 is known to play a major role in the malignant progression of MM by regulating the growth and survival of tumor cells. IL-6 levels often increase during disease progression, and it has been demonstrated that IL-6 may derive from the tumor cells themselves or from other cell types in the bone marrow. Because of this, it is important to fully understand what signals are capable of inducing IL-6 expression in this disease. In summary, our results demonstrate for the first time the ability of BLyS to maintain myeloma cell survival via the induction of an autocrine IL-6 pathway. Our results underscore the importance of IL-6 in this disease, and further demonstrate the complexity of the deregulated cytokine network in this disease.

Abstract HACS1 is a SH3 (Src homology 3) and SAM (sterile alpha motif) domain containing adaptor protein that is expressed in activated B and dendritic cells, is up-regulated by Interleukin 4 in the process of B cell activation and likely serves to dampen the immune response. To elucidate the function of HACS1, we generated HACS1 gene knockout mice by deletion of the SH3 and SAM domains. HACS1−/− mice were viable and fertile and had normal bone marrow B cell development and normal splenic T and B cell populations. However, adult HACS1−/− mice had increased numbers of peritoneal B1 cells (IgM+CD5+). Upon LPS plus BCR or TCR stimulations, splenic cells from HACS1−/− mice demonstrated an upregulation of activation markers with increased intensity of CD23 expression or increased population of CD69 positive cells. Purified B220+ splenic B cells from HACS1−/− mice showed increased cell proliferation upon BCR stimulation. Both T helper type 1 (Th1) and T helper type 2 (Th2) humoral responses were enhanced in HACS1−/− mice upon immunization with T cell-dependent antigen NP-KLH, which resulted in increased production of interferon-gamma (IFN-γ), anti-NP IgG2a and IL-4, as well as anti-NP IgG1 and IgE. Upon immunization with T cell-independent antigens such as TNP-LPS and TNP-Ficoll, HACS1−/− mice had increased production of anti-TNP IgM and IgG3 as compared to normal controls. The in vitro maturation of bone marrow-derived dentritic cells from HACS1−/− mice was similar to wild-type mice but HACS1−/− dentritic cells showed increased IL-12 production upon stimulation with anti-CD40 or LPS. There was no significant difference in antigen uptake by cultured dentritic cells from non-immunized wild-type or knockout mice. However, in immunized mice, an increase in antigen uptake in HACS1−/− dentritic cells was observed. We further demonstrate that the HACS1−/− B cells had increased tyrosine phosphorylation in the resting state. As deletion of the SH3 & SAM domains of HACS1 appears to generate a hypersensitive immune response, our results collectively support that HACS1 negatively regulates adaptive immunity.

Abstract Bortezomib and related proteasome inhibitors have profound pre-clinical and clinical activity in Multiple Myeloma (MM). However, the critical molecular target(s) modulated by proteosome inhibition remains unknown. The overall response rate to bortezomib in relapsed and refractory MM patients is consistently 35%–50%, suggesting specific molecular targets or resistance mechanisms are inherent in patients. None of the known effects of bortezomib has been shown to correlate with clinical responses in patients or to explain the unique sensitivity of MM (as opposed to other malignancies) to these reagents. Therefore, in this study, we have conducted a small interfering RNA (siRNA) screen of the druggable genome in human myeloma cells to identify genes that modulate sensitivity to bortezomib. KMS11 human myeloma cells were reverse transfected with 13,984 synthetic short interfering RNAs (siRNA) against each of 6992 druggable targets (2 siRNA/gene with a single-siRNA per well/384 well format). At 24 hours post transfection, bortezomib was added at IC10, 30, 60 and 90. Cell viability was measured by ATP-dependent luminescence assay 72 hours post drug treatment. The resulting data from primary screening was evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >99% global transfection efficiency, <0.25 CV values across the dataset with minimal plate-to-plate and set-to-set variation observed. From analysis of the primary screening data, a core panel of 320 (4.6% of druggable targets in library) bortezomib sensitizers were identified. 187 (58%) of these were further confirmed in a secondary screening experiment by multiple (2 to 4) independent siRNA. Among these 187 genes (2.6% of the genes investigated), some known targets, such as proteasome subunits are evident. Other genes belonging to the same functional gene families (e.g. TNF receptor family, Ras family, kinases, oncogenes and transporter proteins) or functioning within the same network of signal transduction (e.g. NF-kB, Wnt and estrogen receptor pathways) are evident. Our primary screen also identified genes that attenuate bortezomib-induced cytotoxicity. Members in this group include genes implicated in the negative regulation of cell proliferation and survival, such as NF-kB inhibitors, JNK pathway activators, tumor suppressors, apoptosis inducers and phosphatases. These studies enable dissection of the molecular mechanism of bortezomib in myeloma cells and may serve to point to biomarkers for response prediction and novel chemotherapeutic combination therapies.

Medical education exists in the service of patients and communities and must continually calibrate its focus to ensure the achievement of these goals. To close gaps in U.S. health outcomes, medical education is steadily evolving to better prepare providers with the knowledge and skills to lead patient- and systems-level improvements. Systems-related competencies, including high-value care, quality improvement, population health, informatics, and systems thinking, are needed to achieve this but are often curricular islands in medical education, dependent on local context, and have lacked a unifying framework. The third pillar of medical education—health systems science (HSS)—complements the basic and clinical sciences and integrates the full range of systems-related competencies. Despite the movement toward HSS, there remains uncertainty and significant inconsistency in the application of HSS concepts and nomenclature within health care and medical education. In this Article, the authors (1) explore the historical context of several key systems-related competency areas; (2) describe HSS and highlight a schema crosswalk between HSS and systems-related national competency recommendations, accreditation standards, national and local curricula, educator recommendations, and textbooks; and (3) articulate 6 rationales for the use and integration of a broad HSS framework within medical education. These rationales include: (1) ensuring core competencies are not marginalized, (2) accounting for related and integrated competencies in curricular design, (3) providing the foundation for comprehensive assessments and evaluations, (4) providing a clear learning pathway for the undergraduate–graduate–workforce continuum, (5) facilitating a shift toward a national standard, and (6) catalyzing a new professional identity as systems citizens. Continued movement toward a cohesive framework will better align the clinical and educational missions by cultivating the next generation of systems-minded health care professionals.

AbstractProper cell‐cycle progression is essential for the self‐renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination‐based cell‐cycle indicator (FUCCI) has allowed the dual‐color visualization of the G1 and S/G2/M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage‐specific FUCCI reporters have not yet been developed to analyze complex tissue‐specific cell‐cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell‐cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell‐cycle‐dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac‐specific TNNT2‐FUCCI reporter for lineage‐specific cell‐cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell‐cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.

AbstractImportanceThe impact of tumour thickness on radiation complications following plaque radiotherapy for uveal melanoma in the anti‐vascular endothelial growth factor (VEGF) era remains unknown.BackgroundTo evaluate treatment outcomes following plaque radiotherapy and prophylactic intravitreal bevacizumab for uveal melanoma based on initial tumour thickness.DesignThis was a retrospective, interventional case series.ParticipantsPatients with uveal melanoma were included in this study.MethodsA review of medical records was conducted of patients with uveal melanoma treated with plaque radiotherapy and prophylactic intravitreal bevacizumab from 7 July 2000 to 2 November 2018.Main outcomes measuresRadiation‐related outcomes of cystoid macular oedema (CME), radiation maculopathy, papillopathy, retinopathy, iris neovascularization (NVI) and neovascular glaucoma (NVG) were compared based on tumour thickness (small [<3.0 mm] vs medium [3.1‐8.0 mm] vs large [>8.0 mm]).ResultsOf 1131 eyes, 341 (30%) had small, 633 (56%) medium and 157 (14%) large melanoma. Comparison (small vs medium vs large) at 4 years following radiotherapy revealed large melanoma with greater Kaplan‐Meier estimated risk of CME (37% vs 37% vs 63%, P < .001), earlier onset of CME (33 vs 26 vs 19 months, P < .001) and greater development of NVI (<1% vs 2% vs 13%, P < .001) and NVG (1% vs 2% vs 12%, P < .001). Radiation‐induced maculopathy, papillopathy and retinopathy were not associated with tumour thickness.Conclusions and relevanceCompared with small and medium uveal melanoma, large uveal melanoma demonstrated greater 48‐month risk for CME, shorter time to CME onset and greater development of NVI and NVG following plaque radiotherapy and prophylactic intravitreal bevacizumab.

Objectives We compared risk-adjusted short- and long-term outcomes between standard pancreaticoduodenectomy (SPD) and a pylorus-preserving pancreaticoduodenectomy (PPPD). Methods The National Cancer Database was queried for the years 2004 to 2014 to identify patients with adenocarcinoma of the pancreatic head undergoing SPD and PPD. Margin status, lymph node yield, length of stay (LOS), 30- and 90-day mortality, and overall survival were compared. Results A total of 11,172 patients were identified, of whom 9332 (83.5%) underwent SPD and 1840 (16.5%) PPPD. There was no difference in patient age, sex, stage, tumor grade, radiation treatment, and chemotherapy treatment between the 2 groups. Total number of regional lymph nodes was examined, and surgical margin status and overall survival were also comparable. However, patients undergoing PPPD had a shorter LOS (11.3 vs 12.3 days, P < 0.001), lower 30-day mortality (2.5% vs 3.7%, P = 0.02), and 90-day mortality (5.5% vs 6.9%, P = 0.03). On multivariate analyses, patients undergoing SPD were at higher risk for 30-day mortality compared with PPPD (odds ratio, 1.51; 95% confidence interval, 1.07–2.13). Conclusions Standard pancreaticoduodenectomy and PPPD are oncologically equivalent, yet PPPD is associated with a reduction in postoperative mortality and shorter LOS.

BackgroundPretreatment determination of renal cell carcinoma aggressiveness may help to guide clinical decision‐making.PurposeTo evaluate the efficacy of residual convolutional neural network using routine MRI in differentiating low‐grade (grade I–II) from high‐grade (grade III–IV) in stage I and II renal cell carcinoma.Study TypeRetrospective.PopulationIn all, 376 patients with 430 renal cell carcinoma lesions from 2008–2019 in a multicenter cohort were acquired. The 353 Fuhrman‐graded renal cell carcinomas were divided into a training, validation, and test set with a 7:2:1 split. The 77 WHO/ISUP graded renal cell carcinomas were used as a separate WHO/ISUP test set.Field Strength/Sequence1.5T and 3.0T/T2‐weighted and T1 contrast‐enhanced sequences.AssessmentThe accuracy, sensitivity, and specificity of the final model were assessed. The receiver operating characteristic (ROC) curve and precision‐recall curve were plotted to measure the performance of the binary classifier. A confusion matrix was drawn to show the true positive, true negative, false positive, and false negative of the model.Statistical TestsMann–Whitney U‐test for continuous data and the chi‐square test or Fisher's exact test for categorical data were used to compare the difference of clinicopathologic characteristics between the low‐ and high‐grade groups. The adjusted Wald method was used to calculate the 95% confidence interval (CI) of accuracy, sensitivity, and specificity.ResultsThe final deep‐learning model achieved a test accuracy of 0.88 (95% CI: 0.73–0.96), sensitivity of 0.89 (95% CI: 0.74–0.96), and specificity of 0.88 (95% CI: 0.73–0.96) in the Fuhrman test set and a test accuracy of 0.83 (95% CI: 0.73–0.90), sensitivity of 0.92 (95% CI: 0.84–0.97), and specificity of 0.78 (95% CI: 0.68–0.86) in the WHO/ISUP test set.Data ConclusionDeep learning can noninvasively predict the histological grade of stage I and II renal cell carcinoma using conventional MRI in a multiinstitutional dataset with high accuracy.Level of Evidence3Technical Efficacy Stage2

AbstractBackgroundBody mass index (BMI) and diabetes are established risk factors for colorectal cancer (CRC), likely through perturbations in metabolic traits (e.g. insulin resistance and glucose homeostasis). Identification of interactions between variation in genes and these metabolic risk factors may identify novel biologic insights into CRC etiology.MethodsTo improve statistical power and interpretation for gene‐environment interaction (G × E) testing, we tested genetic variants that regulate expression of a gene together for interaction with BMI (kg/m2) and diabetes on CRC risk among 26 017 cases and 20 692 controls. Each variant was weighted based on PrediXcan analysis of gene expression data from colon tissue generated in the Genotype‐Tissue Expression Project for all genes with heritability ≥1%. We used a mixed‐effects model to jointly measure the G × E interaction in a gene by partitioning the interactions into the predicted gene expression levels (fixed effects), and residual G × E effects (random effects). G × BMI analyses were stratified by sex as BMI‐CRC associations differ by sex. We used false discovery rates to account for multiple comparisons and reported all results with FDR <0.2.ResultsAmong 4839 genes tested, genetically predicted expressions of FOXA1 (P = 3.15 × 10−5), PSMC5 (P = 4.51 × 10−4) and CD33 (P = 2.71 × 10−4) modified the association of BMI on CRC risk for men; KIAA0753 (P = 2.29 × 10−5) and SCN1B (P = 2.76 × 10−4) modified the association of BMI on CRC risk for women; and PTPN2 modified the association between diabetes and CRC risk in both sexes (P = 2.31 × 10−5).ConclusionsAggregating G × E interactions and incorporating functional information, we discovered novel genes that may interact with BMI and diabetes on CRC risk.

OBJECTIVE The nature of the volume-outcome relationship in cases with severe traumatic brain injury (TBI) remains unclear, with considerable interhospital variation in patient outcomes. The objective of this study was to understand the state of the volume-outcome relationship at different levels of trauma centers in the United States. METHODS The authors queried the National Trauma Data Bank for the years 2007–2014 for patients with severe TBI. Case volumes for each level of trauma center organized into quintiles (Q1–Q5) served as the primary predictor. Analyzed outcomes included in-hospital mortality, total hospital length of stay (LOS), and intensive care unit (ICU) stay. Multivariable regression models were performed for in-hospital mortality, overall complications, and total hospital and ICU LOSs to adjust for possible confounders. The analysis was stratified by level designation of the trauma center. Statistical significance was established at p < 0.001 to avoid a type I error due to a large sample size. RESULTS A total of 122,445 patients were included. Adjusted analysis did not demonstrate a significant relationship between increasing hospital volume of severe TBI cases and in-hospital mortality, complications, and nonhome hospital discharge disposition among level I–IV trauma centers. However, among level II trauma centers, hospital LOS was longer for the highest volume quintile (adjusted mean difference [MD] for Q5: 2.83 days, 95% CI 1.40–4.26 days, p < 0.001, reference = Q1). For level III and IV trauma centers, both hospital LOS and ICU LOS were longer for the highest volume quintile (adjusted MD for Q5: LOS 4.6 days, 95% CI 2.3–7.0 days, p < 0.001; ICU LOS 3.2 days, 95% CI 1.6–4.8 days, p < 0.001). CONCLUSIONS Higher volumes of severe TBI cases at a lower level of trauma center may be associated with a longer LOS. These results may assist policymakers with target interventions for resource allocation and point to the need for careful prehospital decision-making in patients with severe TBI.