Diverse Phenotypic Manifestations in a Family with a Novel RYR2 E4107A Variant.

Cardiac ryanodine receptor (RyR2) gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Conversely, RyR2 loss-of-function mutations cause a new disease entity, termed calcium release deficiency syndrome (CRDS), which may include RYR2-related long QT syndrome (LQTS). Importantly, unlike CPVT, patients with CRDS do not always exhibit exercise- or epinephrine-induced ventricular arrhythmias, which precludes a diagnosis of CRDS. Here we report a boy and his father, who both experienced exercise-induced cardiac events and harbor the same RYR2 E4107A variant. In the boy, an exercise stress test (EST) and epinephrine provocation test (EPT) did not induce any ventricular arrhythmias. QTc was slightly prolonged (QTc: 474 ms), and an EPT induced QTc prolongation (QTc-baseline: 466 ms, peak: 532 ms, steady-state: 527 ms). In contrast, in his father, QTc was not prolonged (QTc: 417 ms), and neither an EST nor EPT induced QTc prolongation. However, an EST induced multifocal premature ventricular contraction (PVC) bigeminy and bidirectional PVC couplets. Thus, they exhibited distinct clinical phenotypes: the boy exhibited LQTS (or CRDS) phenotype, whereas his father exhibited CPVT phenotype. These findings suggest that, in addition to the altered RyR2 function, other unidentified factors, such as other genetic, epigenetic, and environmental factors, and aging, may be involved in the diverse phenotypic manifestations. Considering that a single RYR2 variant can cause both CPVT and LQTS (or CRDS) phenotypes, in cascade screening of patients with CPVT and CRDS, an EST and EPT are not sufficient and genetic analysis is required to identify individuals who are at increased risk for life-threatening arrhythmias.

Prediction of response to promising first-line chemotherapy in ovarian cancer patients with residual peritoneal tumors: practical biomarkers and robust multiplex models.

BACKGROUND: Platinum/taxane (TC) chemotherapy with debulking surgery stays the mainstay of the treatment in ovarian cancer patients with peritoneal metastasis, and recently its novel modality, intraperitoneal carboplatin with dose-dense paclitaxel (ddTCip), was shown to have greater therapeutic impact. Nevertheless, the response varies among patients and consequent recurrence, or relapse often occurs. Discovery of therapeutic response predictor to ddTCip and/or TC therapy is eagerly awaited to improve the treatment outcome. METHODS: Using datasets in 76 participants in our ddTCip study and published databases on patients received TC therapy, we first validated a total of 75 previously suggested markers, sought out more active biomarkers through the association analyses of genome-wide transcriptome and genotyping data with progression-free survival (PFS) and adverse events, and then developed multiplex statistical prediction models for PFS and toxicity by mainly using multiple regression analysis and the classification and regression tree (CART) algorithm. RESULTS: The association analyses revealed that SPINK1 could be a possible biomarker of ddTCip efficacy, while ABCB1 rs1045642 and ERCC1 rs11615 would be a predictor of hematologic toxicity and peripheral neuropathy, respectively. Multiple regression analyses and CART algorithm finally provided a potent efficacy prediction model using 5 gene expression data and robust multiplex toxicity prediction models-CART models using a total of 4 genotype combinations and multiple regression models using 15 polymorphisms on 12 genes. CONCLUSION: Biomarkers and multiplex models composed here could work well in the response prediction of ddTCip and/or TC therapy, which might contribute to realize optimal selection of the key therapy.

Hyperplasia of Arachnoid Trabecular Cells: A Hitherto Undescribed Lesion Observed in the Setting of Neurofibromatosis Type 1.

Central nervous system manifestations, a variety of benign and malignant tumors as well as non-neoplastic abnormalities, are found in over 70% of neurofibromatosis type 1 (NF1) patients. Herein, we report hitherto undescribed space-occupying lesions in the setting of NF1. We aimed to clarify their characteristics, especially whether they represent neoplastic or non-neoplastic (hyperplastic) lesions. All 3 cases were preoperatively assessed as non-neoplastic; 2 and 1 cases were suspected to be arachnoid cysts and dilation of subarachnoid space, respectively. However, all lesions were revealed to be whitish jelly-like masses by operation, and the histology composed of spindle cells resembling arachnoid trabecular cells with moderate cellularity and cellular uniformity gave an impression that these lesions may be neoplastic. In contrast, electron microscopic analysis showed that the characteristics of these cells were compatible with those of normal arachnoid trabecular cells. Furthermore, whole-exome sequencing and array comparative genomic hybridization did not show any obvious alterations suggestive of their neoplastic nature. DNA methylation analysis demonstrated that these lesions were epigenetically distinct not only from meningiomas but also from normal healthy meninges. In conclusion, considering the clinicopathologic aspects of the present lesions and the results of the molecular analysis that failed to suggest their neoplastic nature, they may represent previously unrecognized rare hyperplasia of arachnoid trabecular cells, which may be associated with NF1.

Survival advantage of locoregional and systemic therapy in oligometastatic breast cancer: an international retrospective cohort study (OLIGO-BC1).

BACKGROUND: An international retrospective cohort study was conducted to clarify the survival advantage of combination therapy with locoregional and systemic therapy (ST) in oligometastatic breast cancer (BC). METHODS: Patients with oligometastatic BC diagnosed from 2007 to 2012 were enrolled in center hospitals in China, Korea and Japan. It was defined as a low-volume metastatic disease at up to five sites and not necessarily in the same organ. Cases with brain, pleural, peritoneal and pericardial metastases were excluded. The primary endpoint was overall survival (OS) from the initial diagnosis of oligometastases. OS was summarized using the Kaplan-Meier method. A multivariable Cox regression model was used to estimate the hazard ratio (HR) for clinicopathological factors. RESULTS: Among 1,295 cases registered from February 2018 to May 2019, 932 remained for analysis after the exclusion of unavailable cases and locoregional recurrence. One metastatic site was found in 400 cases, 2 in 243, 3 in 130, 4 in 86 and 5 in 73. At the median follow-up of 4.5 years, 5-year OS was 54.7% and 39.7% for 321 cases in the combination therapy group and 611 cases in the ST group, respectively. An adjusted HR was 0.66 (95% confidence interval: 0.55, 0.79). Some types of ST without chemotherapy alone, younger age, ECOG performance status 0, early-stage BC, non-triple negative subtype, fewer metastatic sites and longer duration of surgery to relapse were significantly favorable prognostic factors. CONCLUSION: Combination therapy may be considered for longer survival under some conditions in oligometastatic BC.

Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.

Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.

Biomarker discovery for practice of precision medicine in hypopharyngeal cancer: a theranostic study on response prediction of the key therapeutic agents.

BACKGROUND: Hypopharyngeal cancer is a relatively rare malignancy with poor prognosis. Current chemotherapeutic algorithm is still far from personalized medicine, and the identification of the truly active therapeutic biomarkers and/or targets is eagerly awaited. METHODS: Venturing to focus on the conventional key chemotherapeutic drugs, we identified the most correlative genes (and/or proteins) with cellular sensitivity to docetaxel (TXT), cisplatin (CDDP) and 5-fluorouracil (5-FU) in the expression levels, through 3 steps approach: genome-wide screening, confirmation study on the quantified expression levels, and knock-down and transfection analyses of the candidates. The probable action pathways of selected genes were examined by Ingenuity Pathway Analysis using a large-scale database, The Cancer Genome Atlas. RESULTS: The first genome-wide screening study derived 16 highly correlative genes with cellular drug sensitivity in 15 cell lines (|R| > 0.8, P < 0.01 for CDDP and 5-FU; |R| > 0.5, P < 0.05 for TXT). Among 10 genes the observed correlations were confirmed in the quantified gene expression levels, and finally knock-down and transfection analyses provided 4 molecules as the most potent predictive markers-AGR2 (anterior gradient 2 homolog gene), and PDE4D (phosphodiesterase 4D, cAMP-specific gene) for TXT; NINJ2 (nerve Injury-induced protein 2); CDC25B (cell division cycle 25 homolog B gene) for 5-FU- in both gene and protein expression levels. Overexpression of AGR2, PDE4D signified worse response to TXT, and the repressed expression sensitized TXT activity. Contrary to the findings, in the other 2 molecules, NINJ2 and CDC25, there observed opposite relationship to cellular drug response to the relevant drugs. IPA raised the potential that each selected molecule functionally interacts with main action pathway (and/or targets) of the relevant drug such as tubulin ß chain genes for TXT, DNA replication pathway for CDDP, and DNA synthesis pathway and thymidylate synthetase gene for 5-FU. CONCLUSION: We newly propose 4 molecules -AGR2, PDE4D,NINJ2 and CDC25B) as the powerful exploratory markers for prediction of cellular response to 3 key chemotherapeutic drugs in hypopharyngeal cancers and also suggest their potentials to be the therapeutic targets, which could contribute to the development of precision medicine of the essential chemotherapy in hypopharyngeal patients. (339 words).

Novel CACNA1C R511Q mutation, located in domain â -â ¡ linker, causes non-syndromic type-8 long QT syndrome.

BACKGROUND: Gain-of-function mutations in CACNA1C encoding Cav1.2 cause syndromic or non-syndromic type-8 long QT syndrome (LQTS) (sLQT8 or nsLQT8). The cytoplasmic domain (D)Ⅰ-Ⅱ linker in Cav1.2 plays a pivotal role in calcium channel inactivation, and mutations in this site have been associated with sLQT8 (such as Timothy syndrome) but not nsLQT8. OBJECTIVE: Since we identified a novel CACNA1C mutation, located in the DⅠ-Ⅱ linker, associated with nsLQTS, we sought to reveal its biophysical defects. METHODS: Target panel sequencing was employed in 24 genotype-negative nsLQTS probands (after Sanger sequencing) and three family members. Wild-type (WT) or R511Q Cav1.2 was transiently expressed in tsA201 cells, then whole-cell Ca2+ or Ba2+ currents (ICa or IBa) were recorded using whole-cell patch-clamp techniques. RESULTS: We identified two CACNA1C mutations, a previously reported R858H mutation and a novel R511Q mutation located in the DⅠ-Ⅱ linker. Four members of one nsLQTS family harbored the CACNA1C R511Q mutation. The current density and steady-state activation were comparable to those of WT-ICa. However, persistent currents in R511Q-ICa were significantly larger than those of WT-ICa (WT at +20 mV: 3.3±0.3%, R511Q: 10.8±0.8%, P<0.01). The steady-state inactivation of R511Q-ICa was weak in comparison to that of WT-ICa at higher prepulse potentials, resulting in increased window currents in R511Q-ICa. Slow component of inactivation of R511Q-ICa was significantly delayed compared to that of WT-ICa (WT-tau at +20 mV: 81.3±3.3 ms, R511Q-tau: 125.1±5.0 ms, P<0.01). Inactivation of R511Q-IBa was still slower than that of WT-IBa, indicating that voltage-dependent inactivation (VDI) of R511Q-ICa was predominantly delayed. CONCLUSIONS: Delayed VDI, increased persistent currents, and increased window currents of R511Q-ICa cause nsLQT8. Our data provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.

Double-layer omics analysis of castration- and X-ray-resistant prostate cancer cells.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P < 0.01). The Lethal dose 50 values of carbon ion beams were 0.9 Gy and 0.7 Gy, respectively (P = 0.09). On DNA mutation analysis, AR mutation was observed specifically in LNCaP-LA. From RNA-seq, 181 genes were identified as differentially expressed genes (DEGs; FDR <0.10, P < 0.00076) between LNCaP and LNCaP-LA. Function analysis suggested that cell death was suppressed in LNCaP-LA, and pathway analysis suggested that the NRF2-pathway involved in intracellular oxidative stress prevention was activated in LNCaP-LA. LNCaP-LA showed X-ray resistance compared to LNCaP and sensitivity to carbon ion beams. The AR mutation and the NRF2-pathway were suggested as causes of resistance.

International Retrospective Cohort Study of Conversion Therapy for Stage IV Gastric Cancer 1 (CONVO-GC-1).

Aim: Much attention has been paid to conversion therapy for stage IV gastric cancer, however, its operative comorbidities and survival benefit have not yet been clarified. CONVO-GC-1, an international retrospective cohort study, was designed to investigate the role of conversion surgery in Japan, Korea, and China. Methods: The rate of operative complications was the primary endpoint and the overall survival (OS), according to the four-category criteria previously published (Gastric Cancer:19; 2016), was analyzed as the secondary endpoint. Results: A total of 1206 patients underwent surgery after chemotherapy with curative intent. Operative complications were observed in 290 (24.0%) patients in all grades, including pancreatic fistula and surgical site infection. The median survival time (MST) of all resected patients was 36.7 mo (M) and those of R0, R1, and R2 resection were 56.6 M, 25.8 M, and 21.7 M, respectively. Moreover, the MST of R0 patients were 47.8 M, 116.7 M, 44.8 M in categories 1, 2, and 3, respectively, and not reached in category 4. Interestingly, the MST of P1 patients was as favorable as that of P0CY1 patients if R0 resection was achieved. The MST of patients with liver metastasis was also favorable regardless of the number of lesions, and the MST of patients with para-aortic lymph node (LN) No 16a1/b2 metastasis was not inferior to that of patients with para-aortic LN No 16a2/b1 metastasis. Conclusion: Conversion therapy for stage IV gastric cancer is safe and could be a new therapeutic strategy to improve the survival of patients, especially those with R0 resection.

Fibroblast-derived prolargin is a tumor suppressor in hepatocellular carcinoma.

Cancer-associated fibroblasts (CAF) are important constituents of the tumor microenvironment (TME) and are major drivers of tumorigenesis. Yet, therapies aiming at eliminating CAF have failed to cure patients. This setback has raised questions regarding whether CAF exclusively favour cancer progression, or if they may also assume tumor-suppressor functions. In the present study, we used proteomics and single cell RNA-sequencing analysis to examine the CAF landscape in hepatocellular carcinoma (HCC). We thereby unveil three major CAF populations in HCC, one of which specifically expressing the prolargin protein. This CAF subpopulation (further termed as CAF_Port) shared a strong transcriptomic signature with portal liver fibroblasts. We further show that CAF_Port deposit prolargin in the TME and that its levels are lower in tumors as compared to the peritumoral region. Mechanistically, aggressive cancer cells degraded prolargin using matrix metalloprotease activity. Survival analysis of 188 patients revealed that high prolargin protein levels correlate with good patient outcome (HR = 0.37; p = 0.01). In vivo, co-injection of cancer cells with fibroblasts silenced for prolargin, led to faster tumor development (5-fold; p = 0.01), mainly due to stronger angiogenesis. Using protein-protein interaction study and structural modelling, we further demonstrate that prolargin binds and inhibits the activity of several pro-agiogenic proteins, including hepatocyte and fibroblast growth factors. In conclusion, prolargin is angiogenesis modulator and CAF-derived tumor suppressor in HCC. Stabilizing prolargin levels in the CAF_Port subpopulation may revert their tumor-antagonizing properties, warranting exploration in further pre-clinical studies.

AKT3 Is a Novel Regulator of Cancer-Associated Fibroblasts in Head and Neck Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in tumor progression by promoting epithelial-to-mesenchymal transition, angiogenesis, and immunosuppression. In the present study, we sought to identify the key regulators of the pro-tumoral functions of CAFs in head and neck squamous cell carcinoma (HNSCC). mRNA expression data obtained from The Cancer Genome Atlas revealed that CAF-specific mRNA expression correlated with genes that relate to an immunosuppressive microenvironment in a HNSCC cohort. RNA sequencing of CAFs and normal fibroblasts isolated from HNSCC specimens identified 1127 differentially expressed genes (DEGs) and several upregulated pathways in CAFs. Among the 1127 DEGs, we identified 13 immune function-related genes and focused on AKT3 as a potential regulator of CAFs. The targeted depletion of AKT3 in CAFs revealed that AKT3 promotes their myofibroblastic phenotype. AKT3-transduced CAFs exhibited downregulated the expression of immunosuppressive cytokine genes, impairing T-cell suppression and pro-tumoral macrophage induction. The immunohistochemistry of 72 HNSCC patients showed that AKT3 expression in CAFs positively correlated with tumor infiltration by CAFs, tumor-associated macrophages, dendritic cells, and T cells. Moreover, AKT3 expression in CAFs was an independent prognostic factor for overall survival. In conclusion, AKT3 is a potential target for cancer therapy that inhibits the pro-tumoral function of CAFs and reverses CAF-mediated immunosuppression.

AKT3 is a key regulator of head and neck squamous cell carcinoma.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays a vital role in cell proliferation, apoptosis, metabolism, and angiogenesis in various human cancers, including head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to clarify the role of AKT, which is a major downstream effector of the PI3K-AKT-mTOR pathway, in HNSCC. We first investigated the mRNA expression of AKT isoforms using RNA-sequencing data from The Cancer Genome Atlas database. We observed a specific elevation of AKT3 expression in HNSCC tissues when compared with that in normal tissues. Furthermore, AKT3 expression correlated with genes related to the immunosuppressive microenvironment more than the other AKT isoforms and PIK3CA. Accordingly, we focused on AKT3 and performed a knockdown approach using an HNSCC cell line. AKT3 knockdown cells exhibited impaired proliferation, a shift in the cell cycle from G2/M to G1/G0 phase, an increase in apoptotic cells, and downregulation of gene expression related to immunosuppression, as well as the knockdown of its upstream regulator PIK3CA. We also performed immunohistochemistry for both AKT3 and PIK3CA using surgical specimens from 72 patients with HNSCC. AKT3 expression in tumor cells correlated with immune cell infiltration and unfavorable prognosis when compared with PIK3CA. These findings suggested that AKT3 expression is a potential biomarker for predicting the immunoreactivity and prognosis of HNSCC. Furthermore, the isoform-specific inhibition of AKT3 could be developed as a novel cancer therapy that efficiently suppresses the PI3K-AKT-mTOR pathway.

Ependymoma-like tumor with mesenchymal differentiation harboring C11orf95-NCOA1/2 or -RELA fusion: A hitherto unclassified tumor related to ependymoma.

Recurrent fusion genes involving C11orf95, C11orf95-RELA, have been identified only in supratentorial ependymomas among primary CNS tumors. Here, we report hitherto histopathologically unclassifiable high-grade tumors, under the tentative label of "ependymoma-like tumors with mesenchymal differentiation (ELTMDs)," harboring C11orf95-NCOA1/2 or -RELA fusion. We examined the clinicopathological and molecular features in five cases of ELTMDs. Except for one adult case (50 years old), all cases were in children ranging from 1 to 2.5 years old. All patients presented with a mass lesion in the cerebral hemisphere. Histologically, all cases demonstrated a similar histology with a mixture of components. The major components were embryonal-appearing components forming well-delineated tumor cell nests composed of small uniform cells with high proliferative activity, and spindle-cell mesenchymal components with a low- to high-grade sarcoma-like appearance. The embryonal-appearing components exhibited minimal ependymal differentiation including a characteristic EMA positivity and tubular structures, but histologically did not fit with ependymoma because they lacked perivascular pseudorosettes, a histological hallmark of ependymoma, formed well-delineated nests, and had diffuse and strong staining for CAM5.2. Molecular analysis identified C11orf95-NCOA1, -NCOA2, and -RELA in two, one, and two cases, respectively. t-distributed stochastic neighbor embedding analysis of DNA methylation data from two cases with C11orf95-NCOA1 or -NCOA2 and a reference set of 380 CNS tumors revealed that these two cases were clustered together and were distinct from all subgroups of ependymomas. In conclusion, although ELTMDs exhibited morphological and genetic associations with supratentorial ependymoma with C11orf95-RELA, they cannot be regarded as ependymoma. Further analyses of more cases are needed to clarify their differences and similarities.

Multiple arrhythmic and cardiomyopathic phenotypes associated with an SCN5A A735E mutation.

BACKGROUND: SCN5A mutations are associated with multiple arrhythmic and cardiomyopathic phenotypes including Brugada syndrome (BrS), sinus node dysfunction (SND), atrioventricular block, supraventricular tachyarrhythmias (SVTs), long QT syndrome (LQTS), dilated cardiomyopathy and left ventricular noncompaction. Several single SCN5A mutations have been associated with overlap of some of these phenotypes, but never with overlap of all the phenotypes. OBJECTIVE: We encountered two pedigrees with multiple arrhythmic phenotypes with or without cardiomyopathic phenotypes, and sought to identify a responsible mutation and reveal its functional abnormalities. METHODS: Target panel sequencing of 72 genes, including inherited arrhythmia syndromes- and cardiomyopathies-related genes, was employed in two probands. Cascade screening was performed by Saner sequencing. Wild-type or identified mutant SCN5A were expressed in tsA201 cells, and whole-cell sodium currents (INa) were recorded using patch-clamp techniques. RESULTS: We identified an SCN5A A735E mutation in these probands, but did not identify any other mutations. All eight mutation carriers exhibited at least one of the arrhythmic phenotypes. Two patients exhibited multiple arrhythmic phenotypes: one (15-year-old girl) exhibited BrS, SND, and exercise and epinephrine-induced QT prolongation, the other (4-year-old boy) exhibited BrS, SND, and SVTs. Another one (30-year-old male) exhibited all arrhythmic and cardiomyopathic phenotypes, except for LQTS. One male suddenly died at age 22. Functional analysis revealed that the mutant did not produce functional INa. CONCLUSIONS: A non-functional SCN5A A735E mutation could be associated with multiple arrhythmic and cardiomyopathic phenotypes, although there remains a possibility that other unidentified factors may be involved in the phenotypic variability of the mutation carriers.

Reduced current density, partially rescued by mexiletine, and depolarizing shift in activation of SCN5A W374G channels as a cause of severe form of Brugada syndrome.

BACKGROUND: SCN5A-related Brugada syndrome (BrS) can be caused by multiple mechanisms including trafficking defects and altered channel gating properties. Most SCN5A mutations at pore region cause trafficking defects, and some of them can be rescued by mexiletine (MEX). OBJECTIVE: We recently encountered symptomatic siblings with BrS and sought to identify a responsible mutation and reveal its biophysical defects. METHODS: Target panel sequencing was performed. Wild-type (WT) or identified mutant SCN5A was transfected into tsA201 cells. After incubation of transfected cells with or without 0.1 mM MEX for 24-36 hr, whole-cell sodium currents (INa ) were recorded using patch-clamp techniques. RESULTS: The proband was 29-year-old male who experienced cardiopulmonary arrest. Later, his 36-year-old sister, who had been suffering from recurrent episodes of syncope since 12 years, was diagnosed with BrS. An SCN5A W374G mutation, located at pore region of domain 1 (D1 pore), was identified in both. The peak density of W374G-INa was markedly reduced (WT: 521 ± 38 pA/pF, W374G: 60 ± 10 pA/pF, p < .01), and steady-state activation (SSA) was shifted to depolarizing potentials compared with WT-INa (V1/2 -WT: -39.1 ± 0.8 mV, W374G: -30.9 ± 1.1 mV, p < .01). Incubation of W374G-transfected cells with MEX (W374G-MEX) increased INa density, but it was still reduced compared with WT-INa (W374G-MEX: 174 ± 19 pA/pF, p < .01 versus W374G, p < .01 versus WT). The SSA of W374G-MEX-INa was comparable to W374G-INa (V1/2 -W374G-MEX: -31.6 ± 0.7 mV, P = NS). CONCLUSIONS: Reduced current density, possibly due to a trafficking defect, and depolarizing shift in activation of SCN5A W374G are underlying biophysical defects in this severe form of BrS. Trafficking defects of SCN5A mutations at D1 pore may be commonly rescued by MEX.

Distinctive roles of syntaxin binding protein 4 and its action target, TP63, in lung squamous cell carcinoma: a theranostic study for the precision medicine.

BACKGROUND: Lung squamous cell carcinoma (LSCC) remains a challenging disease to treat, and further improvements in prognosis are dependent upon the identification of LSCC-specific therapeutic biomarkers and/or targets. We previously found that Syntaxin Binding Protein 4 (STXBP4) plays a crucial role in lesion growth and, therefore, clinical outcomes in LSCC patients through regulation of tumor protein p63 (TP63) ubiquitination. METHODS: To clarify the impact of STXBP4 and TP63 for LSCC therapeutics, we assessed relevance of these proteins to outcome of 144 LSCC patients and examined whether its action pathway is distinct from those of currently used drugs in in vitro experiments including RNA-seq analysis through comparison with the other putative exploratory targets and/or markers. RESULTS: Kaplan-Meier analysis revealed that, along with vascular endothelial growth factor receptor 2 (VEGFR2), STXBP4 expression signified a worse prognosis in LSCC patients, both in terms of overall survival (OS, p = 0.002) and disease-free survival (DFS, p = 0.041). These prognostic impacts of STXBP4 were confirmed in univariate Cox regression analysis, but not in the multivariate analysis. Whereas, TP63 (ΔNp63) closely related to OS (p = 0.013), and shown to be an independent prognostic factor for poor OS in the multivariate analysis (p = 0.0324). The action pathway of STXBP4 on suppression of TP63 (ΔNp63) was unique: Ingenuity pathway analysis using the knowledge database and our RNA-seq analysis in human LSCC cell lines indicated that 35 pathways were activated or inactivated in association with STXBP4, but the action pathway of STXBP4 was distinct from those of other current drug targets: STXBP4, TP63 and KDR (VEGFR2 gene) formed a cluster independent from other target genes of tumor protein p53 (TP53), tubulin beta 3 (TUBB3), stathmin 1 (STMN1) and cluster of differentiation 274 (CD274: programmed cell death 1 ligand 1, PD-L1). STXBP4 itself appeared not to be a potent predictive marker of individual drug response, but we found that TP63, main action target of STXBP4, might be involved in drug resistance mechanisms of LSCC. CONCLUSION: STXBP4 and the action target, TP63, could afford a key to the development of precision medicine for LSCC patients.

Novel Cardiocerebral Channelopathy Associated with a KCND3 V392I Mutation.

While a KCND3 V392I mutation uniquely displays a mixed electrophysiological phenotype of Kv4.3, only limited clinical information on the mutation carriers is available. We report two teenage siblings exhibiting both cardiac (early repolarization syndrome and paroxysmal atrial fibrillation) and cerebral phenotypes (epilepsy and intellectual disability), in whom we identified the KCND3 V392I mutation. We propose a link between the KCND3 mutation with a mixed electrophysiological phenotype and cardiocerebral phenotypes, which may be defined as a novel cardiocerebral channelopathy.

Biophysical defects of an SCN5A V1667I mutation associated with epinephrine-induced marked QT prolongation.

BACKGROUND: The epinephrine infusion test (EIT) typically induces marked QT prolongation in LQT1, but not LQT3, while the efficacy of ß-blocker therapy is established in LQT1, but not LQT3. We encountered an LQT3 family, with an SCN5A V1667I mutation, that exhibited epinephrine-induced marked QT prolongation. METHODS: Wild-type (WT) or V1667I-SCN5A was transiently expressed into tsA-201 cells, and whole-cell sodium currents (INa ) were recorded using patch-clamp techniques. To mimic the effects of epinephrine, INa was recorded after the application of protein kinase A (PKA) activator, 8-CPT-cAMP (200 µM), for 10 minutes. RESULTS: The peak density of V1667I-INa was significantly larger than WT-INa (WT: 469 ± 48 pA/pF, n = 20; V1667I: 690 ± 62 pA/pF, n = 19, P < .01). The steady-state activation (SSA) and fast inactivation rate of V1667I-INa were comparable to WT-INa . V1667I-INa displayed a significant depolarizing shift in steady-state inactivation (SSI) in comparison to WT-INa (V1/2 -WT: -88.1 ± 0.8 mV, n = 17; V1667I: -82.5 ± 1.1 mV, n = 17, P < .01), which increases window currents. Tetrodotoxin (30 µM)-sensitive persistent V1667I-INa was comparable to WT-INa . However, the ramp pulse protocol (RPP) displayed an increased hump in V1667I-INa in comparison to WT-INa . Although 8-CPT-cAMP shifted SSA to hyperpolarizing potentials in WT-INa and V1667I-INa to the same extent, it shifted SSI to hyperpolarizing potentials much less in V1667I-INa than in WT-INa (V1/2 -WT: -92.7 ± 1.3 mV, n = 6; V1667I: -85.3 ± 1.6 mV, n = 6, P < .01). Concordantly, the RPP displayed an increased hump in V1667I-INa , but not in WT-INa . CONCLUSIONS: We demonstrated an increase of V1667I-INa by PKA activation, which may provide a rationale for the efficacy of ß-blocker therapy in some cases of LQT3.

The molecular landscape and microenvironment of salivary duct carcinoma reveal new therapeutic opportunities.

Purpose: Salivary duct carcinoma (SDC) is a rare and aggressive salivary gland cancer subtype with poor prognosis. The mutational landscape of SDC has already been the object of several studies, however little is known regarding the functional genomics and the tumor microenvironment despite their importance in oncology. Our investigation aimed at describing both the functional genomics of SDC and the SDC microenvironment, along with their clinical relevance. Methods: RNA-sequencing (24 tumors), proteomics (17 tumors), immunohistochemistry (22 tumors), and multiplexed immunofluorescence (3 tumors) data were obtained from three different patient cohorts and analyzed by digital imaging and bioinformatics. Adjacent non-tumoral tissue from patients in two cohorts were used in transcriptomic and proteomic analyses. Results: Transcriptomic and proteomic data revealed the importance of Notch, TGF-ß, and interferon-γ signaling for all SDCs. We confirmed an overall strong desmoplastic reaction by measuring α-SMA abundance, the level of which was associated with recurrence-free survival (RFS). Two distinct immune phenotypes were observed: immune-poor SDCs (36%) and immune-infiltrated SDCs (64%). Advanced bioinformatics analysis of the transcriptomic data suggested 72 ligand-receptor interactions occurred in the microenvironment and correlated with the immune phenotype. Among these interactions, three immune checkpoints were validated by immunofluorescence, including CTLA-4/DC86 and TIM-3/galectin-9 interactions, previously unidentified in SDC. Immunofluorescence analysis also confirmed an important immunosuppressive role of macrophages and NK cells, also supported by the transcriptomic data. Conclusions: Together our data significantly increase the understanding of SDC biology and open new perspectives for SDC tumor treatment. Before applying immunotherapy, patient stratification according to the immune infiltrate should be taken into account. Immune-infiltrated SDC could benefit from immune checkpoint-targeting therapy, with novel options such as anti-CTLA-4. Macrophages or NK cells could also be targeted. The dense stroma, i.e., fibroblasts or hyaluronic acid, may also be the focus for immune-poor SDC therapies, e.g. in combination with Notch or TGF-ß inhibitors, or molecules targeting SDC mutations.

A phase 2 study of intraperitoneal carboplatin plus intravenous dose-dense paclitaxel in front-line treatment of suboptimal residual ovarian cancer.

BACKGROUND: We evaluated the efficacy of intraperitoneal (IP) carboplatin in combination with dose-dense paclitaxel (ddTCip) for suboptimal residual ovarian cancer. METHODS: This was a phase 2 study to evaluate ddTCip. Patients with stage II-IV ovarian carcinoma, who underwent primary cytoreductive surgery and had radiologically evaluable disease after surgery, were eligible to participate in this study. IP carboplatin (AUC = 6) was administered on day 1, and intravenous paclitaxel (80 mg/m2) was administered on days 1, 8 and 15. The primary endpoint was response rate. Secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. Interval- debulking surgery followed by the same regimen was allowed when indicated. RESULTS: A total of 117 patients were considered eligible for this study prior to surgery and temporarily registered. Of the 117 patients, 76 patients met the inclusion criteria and were enrolled in this study. Fifty-nine (83.1%) patients had objective clinical responses. Median PFS and OS were 18.3 and 55.5 months, respectively. Sixty-four (84.2%) patients had grade 3/4 neutropenia, 43 (56.5%) patients had anaemia and 17 (22.4%) patients had thrombocytopenia. Port-related adverse events occurred in nine (11.8%) patients. CONCLUSIONS: Front-line chemotherapy with ddTCip therapy appears safe and effective, even for patients with suboptimal residual ovarian cancer. TRIAL REGISTRATION: UMIN Clinical Trials Registry (ID: UMIN000001713) on February 16th, 2009.