Loss-of-function Mutations K11E or E271K Lead to Novel Tumor Suppression, Implicate Nucleolar Helicase DDX24 Oncogenicity.

Purpose: Mutations (K11E or E271K) of DEAD-box RNA helicase 24 (DDX24) were related to multi-organ venous lymphatic malformation syndrome (MOVLD). However, the relationship between these mutations and DDX24-function still remains unknown. Understanding whether K11E and E271K cause "loss-of-function" or "gain-of-function" for DDX24 is significant for related diseases. DDX24 was reported to be related to tumors closely, thus this study aims to explore how K11E and E271K affect DDX24-function in tumor proliferation. Methods: Cell lines stably expressing wild-type DDX24, K11E-DDX24, E271K-DDX24, along with vector only based on Chinese hamster ovary cells (CHO) and Balb/c tumor-bearing mice models were constructed. Then immunofluorescence staining, proliferation assay and colony formation assay in vitro and 18F-FDG PET/CT-scan were performed. Finally, the tumor tissues were collected to perform transcriptome sequencing to predict the potential mechanism. Results: Contrasted with CHO-WT-DDX24, CHO-K11E-DDX24 or CHO-E271K-DDX24 showed a decreased number of nucleoli, a slower proliferation rate and a lower colony formation rate significantly. Moreover, mice, inoculated with CHO-K11E-DDX24 or CHO-E271K-DDX24 cells, showed lower tumor formation rate, slower tumor growth rate, better prognosis, reduced standard uptake value and Ki of glucose in subcutaneous tumors. Sequencing indicated CHO-K11E-DDX24 or CHO-E271K-DDX24 caused increasing expression of TNF or chemokines and alteration in immune-related signal pathways. Conclusion: K11E or E271K mutation could lead to "loss-of-function" of DDX24 in cell proliferation and tumor bearing mice, which may be acted by non-specific immune killing to inhibit tumor growth.

Preclinical Evaluation of [64Cu]NOTA-CP01 as a PET Imaging Agent for Metastatic Esophageal Squamous Cell Carcinoma.

Targeting metastatic esophageal squamous cell carcinoma (ESCC) has been a challenge in clinical practice. Emerging evidence demonstrates that C-X-C chemokine receptor 4 (CXCR4) highly expresses in ESCC and plays a pivotal role in the process of tumor metastasis. We developed a copper-64 (t1/2 = 12.7 h, 19% beta+) labeling route of NOTA-CP01 derived from LY2510924, a cyclopeptide-based CXCR4 potent antagonist, in an attempt to noninvasively visualize CXCR4 expression in metastatic ESCC. Precursor NOTA-CP01 was designed by modifying the C-terminus of LY2510925 with bis-t-butyl NOTA via a butane-1,4-diamine linker. The radiolabeling process was finished within 15 min with high radiochemical yield (>95%), radiochemical purity (>99%), and specific activity (10.5-21 GBq/µmol) (non-decay-corrected). The in vitro solubility and stability tests revealed that [64Cu]NOTA-CP01 had a high water solubility (log P = -3.44 ± 0.12, n = 5) and high stability in saline and fetal bovine serum. [64Cu]NOTA-CP01 exhibited CXCR4-specific binding with a nanomolar affinity (IC50 = 1.61 ± 0.96 nM, Kd = 0.272 ± 0.14 nM) similar to that of the parental LY2510924. The in vitro cell uptake assay indicated that the [64Cu]NOTA-CP01-selective accumulation in EC109 cells was CXCR4-specific. Molecular docking of the CXCR4/NOTA-CP01 complex suggested that the Lys, Arg, and NOTA of this ligand have a strong polar interaction with the key residues of CXCR4, which explains the tight affinity of [64Cu]NOTA-CP01 for CXCR4. To test the target engagement in vivo, prolonged-time positron emission computed tomography (PET) imaging was performed at 0.5, 4, 6, 8, 12, 16, and 24 h postinjection of [64Cu]NOTA-CP01 to the EC109 tumor-bearing mice. The EC109 tumors were most visible with high contrast to the contralateral background at 6 h postinjection. The tracer revealed receptor-specific tumor accumulation, which was illustrated by effective blocking via coinjection with a blocking dose of LY2510924. Quantification analysis of the prolonged-time images showed that there was obvious radioactivity accumulation in the tumor (1.27 ± 0.19%ID/g) with the best tumor-to-blood ratio (4.79 ± 0.06) and tumor-to-muscle ratio (15.44 ± 2.94) at 6 h postinjection of the probe. The immunofluorescence and immunohistochemistry confirmed the positive expression of CXCR4 in the EC109 tumor and ESCC and metastatic lymph nodes of patients, respectively. We concluded that [64Cu]NOTA-CP01 possessed a very high target engagement for CXCR4-positive ESCC and could be a potential candidate in the clinical detection of metastatic ESCC.

Indicators and prediction models for the severity of Covid-19.

OBJECTIVES: Coronavirus disease 2019 (Covid-19) is outbreaking globally. We aimed to analyse the clinical characteristics, cardiac injury, electrocardiogram and computed tomography (CT) features of patients confirmed Covid-19 and explored the prediction models for the severity of Covid-19. METHODS: A retrospective and single-centre study enrolled 98 laboratory-confirmed Covid-19 patients. Clinical data, electrocardiogram and CT features were collected and analysed using Statistical Package for the Social Sciences software. RESULTS: There were 46 males and 52 females, with a median age of 44 years, categorised into three groups, including mild, moderate and severe/critical Covid-19. The rate of abnormal electrocardiograms in severe/critical group (79%) was significantly higher than that in the mild group (17%) (P = .027), which (r = 0.392, P = .005) positively related to the severity of Covid-19 (OR: 5.71, 95% CI: 0.45-3.04, P = .008). Age older than 60 years old, comorbidities, whether had symptoms on admission, fatigue, CT features, laboratory test results such as platelet count, lymphocyte cell count, eosinophil cell count, CD3+ cell count, CD4+ cell count, CD8+ cell count, the ratio of albumin/globulin decreased and D-dimer, C-reactive protein (CRP), B-type natriuretic peptide (BNP), cardiac troponin I (cTnI) elevated were the risk factors for the increased severity of Covid-19. The logistic model, adjusted by age, lobular involvement score and lymphocyte cell count, could be applied for assessing the severity of Covid-19 (AUC, 0.903; Sensitivity, 90.9%; Specificity, 78.1%). CONCLUSIONS: Age >60 years old, chronic comorbidities, lymphocytopoenia and lobular involvement score were associated with the Covid-19 severity. The inflammation induced by Covid-19 caused myocardial injury with elevated BNP and cTnI level and abnormal electrocardiograms.

Early Therapeutic Vaccination Prediction of Hepatocellular Carcinoma via Imaging OX40-Mediated Tumor Infiltrating Lymphocytes.

The overall prognosis for hepatocellular carcinoma (HCC) patients is poor but immunotherapeutic strategies may represent a novel and effective tool for HCC. However, the prediction of the early response for the immunotherapeutic effect of HCC remains a big challenge. We developed a novel near-infrared fluorescence (NIRF) probe (IRDye800-AbOX40) for OX40-targeted imaging. The H22 dual-tumor-bearing mice models were established and treated with CpG ODN intratumoral vaccination. Sixteen hours after vaccination, the mice were injected with the probe via the tail vein and conducted with NIRF imaging. The uptake of this probe in HCC tumors was greatly increased as early as 40 h post vaccination and reached a plateau between 54 and 112 h, while the untreated tumors showed a lower uptake, which was further confirmed by ex vivo imaging and flow cytometry. Immunofluorescence staining identified the colocalization of CD3 and OX40 in the tumor microenvironment. Moreover, immunohistochemistry analysis showed that OX40 expression level on tumor infiltrating lymphocytes (TILs) was associated with the fluorescence signal of the H22 tumors. IRDye800-AbOX40 could be used as a specific NIRF probe for noninvasive imaging of OX40 expression on TILs, which may aid in predicting the early response to immunotherapy of HCC.

Co-delivery of dimeric camptothecin and chlorin e6 via polypeptide-based micelles for chemo-photodynamic synergistic therapy.

BACKGROUND: The integration of photodynamic therapy with a chemical drug-delivery system has displayed great potential in enhancing anticancer therapy. However, the solubility and non-specific biodistribution of both chemotherapeutic agents and photosensitizers continue to pose challenges that hinder their clinical applications. METHOD: A polypeptide-based nanoscale drug delivery system was fabricated to address the prementioned issues. An amphiphilic polymer was formed by conjugating the photosensitizer chlorin e6 (Ce6) onto a polypeptide poly-(L-lysine)-b-polyphenylalanine (PKF) for encapsulating the model drug dimeric camptothecin (DCPT), and the nanoparticles (PCD) with high drug loading efficiency were further modified with acid-sensitive polyethylene glycol (PEG) to yield the drug delivery sytem (PPCD). RESULTS: The DCPT and Ce6 encapsulation efficiency were analyzed as 99% and 73.5%, respectively. In phosphate-buffered saline (PBS) solution at a pH of 7.4, the PEG shell improved the stability of micelles and shielded their positive charge while in the acidic tumor microenvironment, the pH-sensitive PEG layer was removed to expose the cationic nanoparticles, thus facilitating the cellular uptake of PPCD micelles. Benefiting from the enhanced cellular internalization, the amount of intracellular reactive oxygen species (ROS) treated with PCD and PPCD micelles were obviously increased. Furthermore, the enhanced anti-cancer efficacy prompted by PPCD micelles was validated through cellular and animal study. CONCLUSION: This study presents a promising method to promote the solubility and biodistribution of both chemotherapeutic agent and photosensitizer, thereby facilitating the further application of chemo-photodynamic cancer therapy.

Comparative Effectiveness of MRI, 4D-CT and Ultrasonography in Patients with Secondary Hyperparathyroidism.

Objective: Accurate preoperative localization of abnormal parathyroid glands is crucial for successful surgical management of secondary hyperparathyroidism (SHPT). This study was conducted to compare the effectiveness of preoperative MRI, 4D-CT, and ultrasonography (US) in localizing parathyroid lesions in patients with SHPT. Methods: We performed a retrospective review of prospectively collected data from a tertiary-care hospital and identified 52 patients who received preoperative MRI and/or 4D-CT and/or US and/or 99mTc-MIBI and subsequently underwent surgery for SHPT between May 2013 and March 2020. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each imaging modality to accurately detect enlarged parathyroid glands were determined using histopathology as the criterion standard with confirmation using the postoperative biochemical response. Results: A total of 198 lesions were identified intraoperatively among the 52 patients included in this investigation. MRI outperformed 4D-CT and US in terms of sensitivity (P < 0.01), specificity (P = 0.455), PPV (P = 0.753), and NPV (P = 0.185). The sensitivity and specificity for MRI, 4D-CT, and US were 90.91%, 88.95%, and 66.23% and 58.33%, 63.64%, and 50.00%, respectively. The PPV of combined MRI and 4D-CT (96.52%) was the highest among the combined 2 modalities. The smallest diameter of the parathyroid gland precisely localized by MRI was 8×3 mm, 5×5 mm by 4D-CT, and 5×3 mm by US. Conclusion: MRI has superior diagnostic performance compared with other modalities as a first-line imaging study for patients undergoing renal hyperparathyroidism, especially for ectopic or small parathyroid lesions. We suggest performing US first for diagnosis and then MRI to make a precise localization, and MRI proved to be very helpful in achieving a high success rate in the surgical treatment of renal hyperparathyroidism in our own experience.

Quantitative immunohistochemistry (IHC) analysis of biomarker combinations for human esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous carcinoma (ESCC) is one of the most common cancers in developing countries. However, currently there are no specific biomarkers for ESCC. This study evaluated the expression of proliferating cell nuclear antigen (PCNA), tumor suppressor protein p53, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) as biomarkers for ESCC. METHODS: This study included 60 clinical cases (30 ESCC and 30 non-ESCC cases that were confirmed pathologically). The expression of PCNA, p53, EGFR, and VEGF were investigated using a quantitative computerized immunohistochemistry (IHC) method. The expression level of each protein was indicated by a H-score from the quantitative analysis. Receiver operating characteristic curve (ROC) and area under curve (AUC) analyses were performed. The sensitivity and specificity of each individual protein and combinations of the proteins were calculated. RESULTS: The H-score analysis indicated that expressions of EGFR, PCNA, and VEGF were statistically significantly higher in ESCC than non-ESCC patients; however, p53 was not. The panels of combinations of these proteins were more sensitive than that of any single protein. In the triplicate combination, the AUC prediction probability increased to 0.86, while the single protein AUC prediction probabilities were 0.74 (EGFR), 0.80 (PCNA), and 0.70 (VEGF). CONCLUSIONS: The high expression of PCNA, EGFR, and VEGF suggests that they are potential biomarkers for ESCC. The combination of these biomarkers may provide targets for molecular therapy and molecular imaging.

Metformin and oxyphotodynamic therapy as a novel treatment approach for triple-negative breast cancer.

BACKGROUND: Treatment for triple-negative breast cancer (TNBC) remains a significant challenge due to a lack of targeted therapies. While photodynamic therapy (PDT) has been utilized as a treatment approach for several types of cancer, oxyphotodynamic therapy (OPDT) is a novel method that improves treatment efficacy by increasing local oxygen concentration. Metformin (MET) has been demonstrated utility as an anti-tumor agent by acting through the adenosine monophosphate-activated protein kinase (AMPK) pathway. We hypothesized that MET in combination with heme, a byproduct of 5-aminolevulinic acid (ALA), may increase cytotoxicity for cancer treatment. This study aimed to investigate the synergistic effect of MET and ALA with PDT or OPDT on TNBC tumorigenic cells. METHODS: The treatment efficacy and phototoxicity of PDT or OPDT were determined using a cell viability assay. PDT/OPDT experiments were carried out in nine groups based on different combinations and concentrations of ALA and/or MET. To calculate the synergistic effect by compuSyn soft for different groups, cells were incubated with ALA and/or MET at the following concentrations (0, 0.25, 0.5,1, 2, 4, 8, 16, 24, and 32 mM). The fluorescence of ALA-induced protoporphyrin IX (PpIX) and MitoTracker Green were observed under a confocal microscope. RESULTS: The optimized therapeutic concentration ratio of ALA and MET was determined to be 1:1. The inhibition of cancer growth (IC50) for each group was 14.03, 10.62, 7.71, 18.27, 22.09, 23.96, 4.57, 10.20, and 8.18 mM, respectively. The combination index (CI) values (fa =0.5) of the last three combination groups (groups 7, 8, and 9) were 0.44, 1.70, and 1.47, respectively. PpIX fluorescence intensity of group 9 (ALA-MET-OPDT group) remained the highest among all groups, indicating an enhanced therapeutic effect. CONCLUSIONS: This study introduces OPDT as a novel anti-tumor therapy for TNBC. Furthermore, the combined use of ALA and MET had a synergistic anti-tumor effect in TNBC cells when combined with OPDT.

Quantitative radiomic model for predicting malignancy of small solid pulmonary nodules detected by low-dose CT screening.

BACKGROUND: It is a permanent challenge to differentiate small solid lung nodules. Massive data, extracted from medical image through radiomics analysis, may help early diagnosis of lung cancer. The aim of this study was to assess the usefulness of a quantitative radiomic model developed from baseline low-dose computed tomography (LDCT) screening for the purpose of predicting malignancy in small solid pulmonary nodules (SSPNs). METHODS: This retrospective study included malignant and benign SSPNs (6 to 15 mm) detected in baseline low-dose CT screening. The malignancy was confirmed pathologically, and benignity was confirmed by long term follow-up or pathological diagnosis. The non-contrast CT images were reconstructed with a lung kernel of a slice thickness of 1 mm and were processed to extract 385 quantitative radiomic features using Analysis-Kinetic software. A predictive model was established with the training set of 156 benign and 40 malignant nodules, and was tested with the validation set of 77 benign and 21 malignant nodules through the analysis of R square. The performance of the radiomic model in predicting malignancy was compared with that of the ACR Lung Imaging Reporting and Data System (ACR lung-RADS). RESULTS: In 294 cases of SSPNs, 61 lung cancers and 24 benign nodules were confirmed pathologically and the remaining 209 nodules were stable with long-term follow-up (4.1±0.9 years). Eleven non-redundant features, including 8 high-order texture features, were extracted from the training data set. The sensitivity and specificity of the prediction model in malignancy differentiation were 81.0% and 92.2% respectively. The accuracy was superior to ACR-lung RADS (89.8% vs. 76.5%). CONCLUSIONS: A radiomic model based on baseline low-dose CT screening for lung cancer can improve the accuracy in predicting malignancy of SSPNs.

Management of sphenoidal sinus lesions by septal-assisted approach: Surgical skills and advantages.

The aim of this study was to develop a less invasive trans-septal approach for the endoscopic management of sphenoid sinus lesions. We performed a septal-assisted surgical procedure for endoscopic sphenoidectomy in 38 patients with isolated or combined sphenoidal sinus lesions, including fungal balls, mucoceles, purulent cystic sphenoidal sinusitis, etc. The posterior portion of the nasal septum became flexible after removal of the vomer and the sphenoidal rostrum. The superior portion of the common meatus was expanded to accommodate the endoscope after the septum was repositioned contra-laterally. The lesions were individually managed through the enlarged ostiums while damage to the mucosa of the front sphenoidal wall was avoided. All the procedures were completed successfully without intraoperative complications, and the bony ostiums were identified easily and enlarged accurately. During the follow-up period of 16 weeks to 2 years, no re-atresia or restenosis was observed. The recurrence rate was 0. No postoperative complications were recorded. All the responses from the patients were satisfactory. It was concluded that endoscopic sphenoidectomy assisted by trans-septal approach is a feasible, safe, effective and minimally invasive approach for selected cases with unilateral or bilateral lesions in the sphenoid sinuses.