PD-1 inhibitor plus oncolytic vaccinia virus is a safe and effective treatment option for metastatic renal cell carcinoma.

BACKGROUND: Although a combination of immune checkpoint inhibitors (ICIs) is recommended as the first line treatment option for metastatic renal cell carcinoma (mRCC), several immune-related adverse events (irAEs) occur, especially hepatitis. We explored the therapeutic benefits and safety profile of combining oncolytic vaccinia virus, JX-594, with a programmed cell death protein-1 (PD-1) inhibitor. METHODS: We used early-stage and advanced-stage orthotopic murine mRCC models developed by our group. PD-1 inhibitor monotherapy or a PD-1 inhibitor combined with either JX-594 or a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor were systemically injected through the peritoneum. An immunofluorescence analysis was performed to analyze the tumor immune microenvironment (TIME). irAEs were assessed in terms of hepatitis. RESULTS: In the early-stage mRCC model mice, the combination of JX-594 and a PD-1 inhibitor significantly decreased the primary tumor size and number of lung nodules, compared with the ICI combination, but the JX-594 and PD-1 inhibitor combination and ICI combination did not differ significantly in the advanced-stage mRCC model mice. The JX-594 and PD-1 inhibitor combination induced tumor-suppressing TIME changes in both the early- and advanced-stage mRCC models. Furthermore, mice treated with the ICI combination had significantly greater hepatic injuries than those treated with the JX-594 and PD-1 inhibitor combination which was evaluated in early-stage mRCC model. CONCLUSIONS: The JX-594 and PD-1 inhibitor combination effectively reduced primary tumors and the metastatic burden, similar to ICI combination therapy, through dynamic remodeling of the TIME. Furthermore, hepatitis was significantly decreased in the JX-594 and PD-1 inhibitor combination group, suggesting the potential benefit of that combination for reducing ICI-induced toxicity.

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

sRAGE attenuates angiotensin II-induced cardiomyocyte hypertrophy by inhibiting RAGE-NFκB-NLRP3 activation.

OBJECTIVE AND DESIGN: The receptor for advanced glycation endproducts (RAGE) is an innate immunity receptor that has been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, the possibility that RAGE-mediated signaling is involved in angiotensin II (Ang II)-induced cardiac left ventricular hypertrophy has yet to be investigated. We therefore determined whether RAGE has a role in regulating pathological cardiac hypertrophy. MATERIALS AND SUBJECTS: Protein abundance was estimated using Western blotting and intracellular ROS level and phospho-p65 were detected using fluorescence microscopy. Enzyme-linked immunosorbent assay was used to detect HMGB1 and IL-1ß. All in vitro experiments were performed using H9C2 cells. TREATMENTS: To induce cardiomyocyte hypertrophy, 300 nM Ang II was treated for 48 h and 2 µg/ml sRAGE was treated 1 h prior to addition of Ang II. RESULTS: sRAGE attenuated Ang II-induced cardiomyocyte hypertrophy by downregulating RAGE and angiotensin II type 1 receptor expression. Secretion levels of high motility group box 1 and interleukin-1ß, estimated from a cell culture medium, were significantly reduced by sRAGE. Activated PKCs and ERK1/2, important signals in left ventricular hypertrophy (LVH) development, were downregulated by sRAGE treatment. Furthermore, we found that nuclear factor-κB and NOD-like receptor protein 3 (NLRP3) were associated with RAGE-mediated cardiomyocyte hypertrophy. CONCLUSIONS: In the context of these results, we conclude that RAGE induces cardiac hypertrophy through the activation of the PKCs-ERK1/2 and NF-κB-NLRP3-IL1ß signaling pathway, and suggest that RAGE-NLRP3 may be an important mediator of Ang II-induced cardiomyocyte hypertrophy. In addition, we determined that inhibition of RAGE activation with soluble RAGE (sRAGE) has a protective effect on Ang II-induced cardiomyocyte hypertrophy.

Discovery and characterization of a novel 7-aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibitor.

We describe a novel 7-aminopyrazolo[1,5-a]pyrimidine (7-APP) derivative as a potent hepatitis C virus (HCV) inhibitor. A series of 7-APPs was synthesized and evaluated for inhibitory activity against HCV in different cell culture systems. The synthesis and preliminary structure-activity relationship study of 7-APP are reported.

Gene Expression Analysis of Aggressive Adult Xp11.2 Translocation Renal Cell Carcinoma at Clinical Stage T1N0M0 to Identify Potential Prognostic and Therapeutic Biomarkers.

Xp11.2 translocation renal cell carcinoma (tRCC), involving transcription factor E3 (TFE3) gene fusions, is a rare and aggressive RCC variant when present in adults and has been recently recognized as a unique entity in RCC. Biomarkers and treatment guidelines do not exist for patients with aggressive Xp11.2 tRCC. The aim was to identify and evaluate therapeutic biomarkers for aggressive Xp11.2 tRCC. RNA sequencing was performed using formalin-fixed, paraffin-embedded tissues from 11 adult patients with clinical T1N0M0 Xp11.2 tRCC, including three patients with aggressive characteristics (recurrence or cancer-specific death after nephrectomy). Thirty genes were differentially expressed between the aggressive and non-aggressive groups, even after adjustment, and were associated with KEGG pathways related to the aggressiveness of Xp11.2 tRCC. PIK3R2, involved in various KEGG pathways, including the PI3K/AKT/mTOR pathway, was overexpressed in the Xp11.2 tRCC cell lines UOK120 and UOK146. The PI3K pathway inhibitor LY294002 showed a significant therapeutic benefit. This study provides the first candidate biomarker, PIK3R2, for aggressive clinical T1N0M0 Xp11.2 tRCC. Furthermore, this study is the first to recommend a targeted drug, LY294002, for aggressive Xp11.2 tRCC based on the molecular pathophysiology.

Systemic Injection of Oncolytic Vaccinia Virus Suppresses Primary Tumor Growth and Lung Metastasis in Metastatic Renal Cell Carcinoma by Remodeling Tumor Microenvironment.

Immune checkpoint inhibitors and tyrosine kinase inhibitors are the first-line treatment for metastatic renal cell carcinoma (mRCC), but their benefits are limited to specific patient subsets. Here, we aimed to evaluate the therapeutic efficacy of JX-594 (pexastimogene devacirepvec, Pexa-vec) monotherapy by systemic injection in comparison with sunitinib monotherapy in metastatic orthotopic RCC murine models. Two highly metastatic orthotopic RCC models were developed to compare the treatment efficacy in the International Metastatic RCC Database Consortium favorable-risk and intermediate- or poor-risk groups. JX-594 was systemically injected through the peritoneum, whereas sunitinib was orally administered. Post-treatment, tumor microenvironment (TME) remodeling was determined using immunofluorescence analysis. Systemic JX-594 monotherapy injection demonstrated therapeutic benefit in both early- and advanced-stage mRCC models. Sunitinib monotherapy significantly reduced the primary tumor burden and number of lung metastases in the early-stage, but not in the advanced-stage mRCC model. Systemic JX-594 delivery remodeled the primary TME and lung metastatic sites by increasing tumor-infiltrating CD4/8+ T cells and dendritic cells. Systemic JX-594 monotherapy demonstrated significantly better therapeutic outcomes compared with sunitinib monotherapy in both early- and advanced-stage mRCCs by converting cold tumors into hot tumors. Sunitinib monotherapy effectively suppressed primary tumor growth and lung metastasis in early-stage mRCC.

Development of a highly pulmonary metastatic orthotopic renal cell carcinoma murine model.

The incidence of renal cell carcinoma (RCC) is high, and its outcomes remain poor. Mortality is attributable largely to metastatic disease and a dearth of effective therapeutic interventions. The lungs are the most common metastatic site. To elucidate the biological mechanisms underlying pulmonary metastasis and identify superior therapeutic strategies, we developed a novel and clinically relevant murine RCC model exhibiting enhanced pulmonary metastasis. Mice underwent intrarenal implantation using luciferase-expressing Renca, a murine renal adenocarcinoma cell line. Primary renal tumor progression and development of metastatic lung lesions were monitored in live mice using bioluminescent imaging, followed by post-mortem organ assessment. Cells were isolated from pulmonary metastases for reimplantation, followed by repeat monitoring and assessment. This process was repeated once more for a total of two in vivo passages to select for pulmonary metastatic Renca cell subpopulations. However, a single round of in vivo selection was sufficient to produce a near-maximally metastatic subpopulation. Relative to Renca cell-implanted mice, subpopulation-implanted mice exhibited shorter implantation-metastasis intervals (5 days), shorter implantation-moribundity intervals (sacrificed at 18.6±2.9 versus 22.3±1.1 days), a higher number of metastatic lung lesions at 23 days (183.9±39.0 versus 172.6±38.2) and poorer survival. Implantation of cells derived from the second round of in vivo selection produced no further significant differences in the above metrics. This model consistently and efficiently recapitulates RCC pulmonary metastasis while allowing in vivo monitoring of tumor progression, thereby facilitating elucidation of the molecular mechanisms underlying pulmonary metastasis and evaluation of therapeutic modalities.

The DEAD/DEAH Box Helicase, DDX11, Is Essential for the Survival of Advanced Clear Cell Renal Cell Carcinoma and Is a Determinant of PARP Inhibitor Sensitivity.

Genes associated with the DEAD-box helicase DDX11 are significant biomarkers of aggressive renal cell carcinoma (RCC), but their molecular function is poorly understood. We analyzed the molecular pathways through which DDX11 is involved in RCC cell survival and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity. Immunohistochemistry and immunoblotting determined DDX11 expression in normal kidney tissues, benign renal tumors, and RCC tissues and cell lines. Quantitative polymerase chain reaction validated the downregulation of DDX11 in response to transfection with DDX11-specific small interfering RNA. Proliferation analysis and apoptosis assays were performed to determine the impact of DDX11 knockdown on RCC cells, and the relevant effects of sunitinib, olaparib, and sunitinib plus olaparib were evaluated. DDX11 was upregulated in high-grade, advanced RCC compared to low-grade, localized RCC, and DDX11 was not expressed in normal kidney tissues or benign renal tumors. DDX11 knockdown resulted in the inhibition of RCC cell proliferation, segregation defects, and rapid apoptosis. DDX11-deficient RCC cells exhibited significantly increased sensitivity to olaparib compared to sunitinib alone or sunitinib plus olaparib combination treatments. Moreover, DDX11 could determine PARP inhibitor sensitivity in RCC. DDX11 could serve as a novel therapeutic biomarker for RCC patients who are refractory to conventional targeted therapies and immunotherapies.

AXIN2 and SNAIL expression predict the risk of recurrence in cutaneous squamous cell carcinoma after Mohs micrographic surgery.

Recurrence is a common complication observed during cutaneous squamous cell carcinoma (cSCC) treatment; however, biomarkers for predicting recurrence in cSCC remain unknown. The present study aimed to investigate the predictive value of axis inhibition protein 2 (AXIN2) and SNAIL expression in cSCC recurrence. AXIN2 and SNAIL expression was evaluated using immunohistochemistry in 111 cSCC tissue samples obtained from 18 patients who presented recurrence (recurrence interval, 1-91 months) and 93 patients who did not experience recurrence following Mohs micrographic surgery (MMS) during the follow-up period (156 months). Nomogram construction was performed using patients' clinicopathological characteristics and AXIN2 and SNAIL protein expression. The results demonstrated that high AXIN2 (histoscore >100) and SNAIL (histoscore >100) expression was detected in 35 and 44 cSCC tissues, respectively. Furthermore, the expression levels of AXIN2 and SNAIL were significantly associated in patients with cSCC (P=0.001). AXIN2 and SNAIL expression levels were significantly associated with tumor size (P=0.021 and P=0.044, respectively) and recurrence of cSCC (P=0.017 and P=0.042, respectively). In addition, the results of the Kaplan-Meier curve analysis revealed that recurrence-free survival was significantly associated with tumor size (P=0.025), differentiation status (P<0.001), AXIN2 expression (P=0.001) and SNAIL expression (P=0.001). Furthermore, the results of the multivariate analysis demonstrated that age (P=0.043), AXIN2 expression (P=0.001) and SNAIL expression (P=0.045) were independent risk factors for cSCC recurrence in the present cohort. A nomogram for predicting the 1-, 2-, 3-, and 5-year recurrence-free survival was developed for patients with cSCC by including independent risk factors with a concordance index of 0.75. The results suggested that high AXIN2 and SNAIL expression may be considered as potential risk factors for cSCC recurrence. This nomogram may therefore be useful to assess the probability of recurrence in patients with cSCC following MMS.

Reliable screening and classification of phosphodiesterase type 5 inhibitors in dietary supplements using gas chromatography / mass spectrometry combined with specific common ions.

Illegal dietary supplements adulterated with phosphodiesterase type 5 inhibitors (PDE-5i) are increasingly widely distributed through internet markets and underground routes. For this reason, it demands development of reliable screening methods to determine a wide range of PDE-5i drugs in various types of dietary supplements. Herein, we developed a screening method using gas chromatography-mass spectrometry (GC-MS) for simultaneous detection of 53 PDE-5i drugs in supplements. Common formulations (such as capsule, powder, pill, and tablet) of supplements with complicated matrices were treated by simple liquid-liquid extraction and trimethylsilyl (TMS) derivatization. With the aid of TMS derivatization, 53 PDE-5i drugs could be successfully separated and detected within 15 min, using a short microbore GC column (15 m). Moreover, owing to enhanced detection sensitivity and selectivity of PDE-5i TMS derivatives, 0.5 mg of sample was sufficient to screen and confirm targeted PDE-5i drugs. In this study, specific common ions according to structural characteristics of PDE-5i drugs were found under the electron ionization (EI) of their TMS derivatives. These specific common fragments could reflect the common pharmacophores for 4 classes of PDE-5i drugs (sildenafil, other sildenafil, vardenafil, and tadalafil analogues). Based on characteristic EI fragment ions, extracted common ion chromatograms (ECICs) and discriminant analysis (DA) were effectively used for reliable screening and classification of various types of PDE-5i drugs. Specific ECICs and DA using characteristic EI fragments here will aid in identification of newly emerging PDE-5i counterfeits in supplements. This study will be helpful to supervise illegal adulteration of PDE-5i drugs in dietary supplements to protect public health and consumer safety.

Radiological assessment of effectiveness of soluble RAGE in attenuating Angiotensin II-induced LVH mouse model using in vivo 9.4T MRI.

We investigated the effectiveness of soluble Receptor for Advanced Glycation Endproducts (sRAGE) in attenuating angiotensin II (AngII)-induced left ventricular hypertrophy (LVH) using in vivo 9.4T cine-magnetic resonance imaging (CINE-MRI). Mice were divided into four groups: AngII (n = 9), saline (n = 10), sRAGE (n = 10), and AngII + sRAGE (n = 10). CINE-MRI was performed in each group after administration of the AngII or sRAGE, and CINE-MR images were analyzed to obtain parameters indicating cardiac anatomical and functional changes including end-diastolic and end-systolic blood volume, end-diastolic and end-systolic myocardial volume, ejection fraction, end-diastolic and end-systolic myocardial mass, and LV wall thickness. LVH observed in AngII group was significantly attenuated by sRAGE. These trends were also observed in histological analysis, demonstrating that cardiac function tracking using in vivo and real-time 9.4T MR imaging provides valuable information about the cardiac remodeling induced by AngII and sRAGE in an AngII-induced LV hypertrophy mice model.

Variation of the 3' telomeric overhang lengths in human cells.

The telomeric G-rich 3' overhang is important for the maintenance of chromosomal integrity by stabilizing T-loop structure in which the 3' overhang invades the double-stranded telomeric DNA. However, the 3' overhang length has not been examined in different human cell lines, and its regulatory mechanism has not been revealed. In this study, we examined overhang length in 56 human cancerous cell lines and five normal cell lines, originated from various tissues. In cancer cells, relative overhang length existed in a wide range from 23% to 308% and showed no significant association with tissue types although short overhang was noted in brain, cervix, and colorectal cells. Normal cells exhibited overhangs in the range from 92% to 202%, which were relatively longer than those seen in cancer cells (p = 0.002). The overhang length was positively correlated with telomere length (p < 0.001), and showed no correlation with mRNA levels of hTERT, a catalytic protein of telomerase, POT1, an overhang binding protein and TPP1, a POT1 interacting protein. This study demonstrates a broad distribution of overhang length in human cells, suggesting a dynamic regulation of 3' overhang length. The overhang length seems to be closely associated with telomere length and might be regulated by multiple mechanisms.

Interpretation of digital breast tomosynthesis: preliminary study on comparison with picture archiving and communication system (PACS) and dedicated workstation.

OBJECTIVE: To compare the diagnostic accuracy and efficiency in the interpretation of digital breast tomosynthesis (DBT) images using a picture archiving and communication system (PACS) and a dedicated workstation. METHODS: 97 DBT images obtained for screening or diagnostic purposes were stored in both a workstation and a PACS and evaluated in combination with digital mammography by three independent radiologists retrospectively. Breast Imaging-Reporting and Data System final assessments and likelihood of malignancy (%) were assigned and the interpretation time when using the workstation and PACS was recorded. Receiver operating characteristic curve analysis, sensitivities and specificities were compared with histopathological examination and follow-up data as a reference standard. RESULTS: Area under the receiver operating characteristic curve values for cancer detection (0.839 vs 0.815, p = 0.6375) and sensitivity (81.8% vs 75.8%, p = 0.2188) showed no statistically significant differences between the workstation and PACS. However, specificity was significantly higher when analysing on the workstation than when using PACS (83.7% vs 76.9%, p = 0.009). When evaluating DBT images using PACS, only one case was deemed necessary to be reanalysed using the workstation. The mean time to interpret DBT images on PACS (1.68 min/case) was significantly longer than that to interpret on the workstation (1.35 min/case) (p < 0.0001). CONCLUSION: Interpretation of DBT images using PACS showed comparable diagnostic performance to a dedicated workstation, even though it required a longer reading time. Advances in knowledge: Interpretation of DBT images using PACS is an alternative to evaluate the images when a dedicated workstation is not available.

Assessment of the treatment response of spinal meningiomas after radiosurgery focusing on serial MRI findings.

PURPOSE: This study evaluated the response of spinal meningiomas to treatment, by monitoring changes in magnetic resonance imaging (MRI) findings after stereotactic radiosurgery (SRS). MATERIALS AND METHODS: Serial follow-up MRIs of 11 patients with spinal meningiomas who underwent SRS were retrospectively reviewed. Changes in tumor volume, T2 signal intensity (T2SI), and contrast enhancement were evaluated. RESULTS: The mean MRI follow-up period was 46.9 months (range 13-108 months). The local tumor control rate was 100 % in overall tumor volume, although boost SRS was performed for marginal recurrence in case 8 and rapid decompression in case 5. Seven tumors showed decreased T2SI. Each of the remaining four tumors showed variable T2SIs. In most tumors, the enhancement patterns did not change. Two of three patients with en plaque type meningiomas showed increased intramedullary T2SI, thought to be due to compressive myelopathy and peritumoral edema. CONCLUSIONS: SRS resulted in successful local tumor control in all patients, although the follow-up period was not long. Changes in T2SI and contrast enhancement patterns of the tumors were evaluated on serial MRI. In addition, close follow-up with MRI is desirable to monitor intramedullary signal changes in cases of spinal meningiomas with a wider contact area with the spinal cord.

Dovitinib (TKI258), a multi-target angiokinase inhibitor, is effective regardless of KRAS or BRAF mutation status in colorectal cancer.

INTRODUCTION: We aimed to determine whether KRAS and BRAF mutant colorectal cancer (CRC) cells exhibit distinct sensitivities to the multi-target angiokinase inhibitor, TKI258 (dovitinib). MATERIALS AND METHODS: We screened 10 CRC cell lines by using receptor tyrosine kinase (RTK) array to identify activated RTKs. MTT assays, anchorage-independent colony-formation assays, and immunoblotting assays were performed to evaluate the in vitro anti-tumor effects of TKI258. In vivo efficacy study followed by pharmacodynamic evaluation was done. RESULTS: Fibroblast Growth Factor Receptor 1 (FGFR1) and FGFR3 were among the most highly activated RTKs in CRC cell lines. In in vitro assays, the BRAF mutant HT-29 cells were more resistant to the TKI258 than the KRAS mutant LoVo cells. However, in xenograft assays, TKI258 equally delayed the growth of tumors induced by both cell lines. TUNEL assays showed that the apoptotic index was unchanged following TKI258 treatment, but staining for Ki-67 and CD31 was substantially reduced in both xenografts, implying an anti-angiogenic effect of the drug. TKI258 treatment was effective in delaying CRC tumor growth in vivo regardless of the KRAS and BRAF mutation status. CONCLUSIONS: Our results identify FGFRs as potential targets in CRC treatment and suggest that combined targeting of multiple RTKs with TKI258 might serve as a novel approach to improve outcome of patients with CRC.

Subtelomeric DNA methylation and telomere length in human cancer cells.

Subtelomeric epigenetic modifications are known to be associated with telomere length. We examined subtelomeric DNA methylation at seven sites for five chromosomes by methylation-specific PCR (MSP) and two sites for two chromosomes by bisulfite genomic sequencing (BGS) in 20 human cancer cell lines and subsequently analyzed their association with telomere length. Full-methylation (55/140) was more frequently found compared to un-methylation (35/140) (p=0.01). Subtelomeric-methylation patterns varied from region to region; full-methylation and un-methylation were dominant at one of 9q sites (20/20) and 9p (18/20), respectively. MSP and BGS data exhibited no apparent correlation between methylation status and telomere length. In addition, Hep3B subclones that possessed different telomere lengths exhibited no changes in methylation status according to telomeres. In summary, subtelomeres might form distinct chromatin structures from region to region and effect of subtelomeric DNA methylation on telomere regulation might be little.