Case report: targeted sequencing improves the diagnosis of multiple synchronous lung cancers.

Background: The ability to distinguish satellite nodules, multiple primary lung cancers (MPLCs), and intrapulmonary metastases (IPM) is crucial for prognosis and treatment. The traditional diagnostic criteria for MPLC/IPM including the Martini and Melamed (MM) criteria and the comprehensive histologic assessment (CHA) criteria, mainly relies on histological comparison between multiple lesions. However, many challenges remain in distinguishing them in clinical practice. Case Description: We herein present a report of 3 lung adenocarcinoma cases who presented with 2 lesions, with improved diagnosis based on targeted sequencing covering driver genes. Based on histopathological features, patient 1 (P1) was classified as MPLC, whereas patients 2 and 3 (P2, P3) were classified as satellite nodules. However, targeted sequencing revealed the clonality status of these lesions and improved their diagnosis. The result of the molecular testing indicated that P1 is IPM and the other two patients (P2, P3) should be diagnosed with MPLC. Conclusions: Different lesions in the same case had different driver mutations, suggesting that the 2 lesions were driven by different molecular events. Therefore, targeted sequencing containing driver genes should be used for the diagnosis of multiple synchronous lung cancers. A limitation of this report is the short follow up period, and long-term outcomes of the patients require further follow up.

Minimally Invasive Versus Open McKeown for Patients with Esophageal Cancer: A Retrospective Study.

INTRODUCTION: McKeown minimally invasive esophagectomy (McKeown-MIE) offers advantages in short-term outcomes compared with McKeown open esophagectomy (McKeown-OE); however, debate as to whether MIE is equivalent or better than OE regarding survival outcomes is ongoing. OBJECTIVE: The aim of this study was to compare long-term survival between McKeown-MIE and McKeown-OE in a large cohort of esophageal cancer (EC) patients. METHODS: We used a prospective database (independently managed by LinkDoc company) of the Thoracic Surgery Department at Henan Cancer Hospital and included patients who underwent McKeown-MIE and McKeown-OE for EC from 1 January 2015 to 6 January 2018. The perioperative data and overall survival (OS) rate in the two groups were retrospectively compared. RESULTS: We included 502 patients who underwent McKeown-MIE (n = 306) or McKeown-OE (n = 196) for EC. The median age in the total patient population was 63 years. All baseline characteristics were well-balanced between the two groups. There was a significantly shorter mean operative time (269.76 min vs. 321.14 min, p < 0.001) in the OE group. The 30-day and in-hospital mortality rates were 0, and there was no difference in 90-day mortality (p = 0.053) between the groups. The postoperative stay was shorter in the MIE group and was 14 days and 18 days in the MIE and OE groups, respectively (p < 0.001). The OS at 60 months was 58.8% and 41.6% in the MIE and OE groups, respectively (p < 0.001) [hazard ratio 1.783, 95% confidence interval 1.347-2.359]. CONCLUSIONS: These results showed that McKeown-MIE was associated with better long-term survival than McKeown-OE for patients with resectable EC.

SMYD3 confers cisplatin chemoresistance of NSCLC cells in an ANKHD1-dependent manner.

BACKGROUND: Cisplatin (DDP) remains the backbone of chemotherapy for non-small cell lung cancer (NSCLC), yet its clinical efficacy is limited by DDP resistance. We aim to investigate the role of the SET and MYND domain-containing protein 3 (SMYD3) in DDP resistance of NSCLC. METHODS: Expression pattern of SMYD3 was determined in NSCLC tissues using qRT-PCR, which also validated its correlation with NSCLC clinicopathological stages. Impacts of SMYD3 on DDP resistance were evaluated by knocking down SMYD3 in DDP-resistant cells and overexpressing it in DDP-sensitive cells, and assessed for several phenotypes: IC50 by MTT, long-term proliferation by colony formation, apoptosis and cell-cycle distribution by flow cytometry. The interaction between Ankyrin Repeat and KH Domain Containing 1 (ANKHD1) and SMYD3 was examined by co-immunoprecipitation and immunofluorescence. The transcriptional regulation of SMYD3 on cyclin-dependent kinase 2 (CDK2) promoter regions was confirmed using chromatin-immunoprecipitation. The in vivo experiments using DDP-resistant cells with altered SMYD3 and ANKHD1 expression were further performed to verify the SMYD3/ANKHD1 axis. RESULTS: Highly expressed SMYD3 was observed in NSCLC tissues or cells, acted as a sensitive indicator for NSCLC, correlated with higher TNM stages or resistant to DDP treatment, and shorter overall survival. The promotion of SMYD3 on DDP resistance requires co-regulator, ANKHD1. CDK2 was identified as a downstream effector. In vivo, SMYD3 knockdown inhibited the growth of DDP-resistant NSCLC cells, which was abolished by ANKHD1 overexpression. CONCLUSIONS: SMYD3 confers NSCLC cells chemoresistance to DDP in an ANKHD1-dependent manner, providing novel therapeutic targets to overcome DDP resistance in NSCLC .

A phase III study on neoadjuvant chemotherapy versus neoadjuvant toripalimab plus chemotherapy for locally advanced esophageal squamous cell carcinoma: Henan Cancer Hospital Thoracic Oncology Group 1909 (HCHTOG1909).

BACKGROUND: Neoadjuvant chemotherapy (NAC) and neoadjuvant chemoradiotherapy (NACR) are the standard treatments for esophageal squamous cell carcinoma (ESCC). However, the 5-year overall survival (OS) rate is still far from satisfactory. In recent years, immune checkpoint inhibitors (ICIs) have shown promising results in the treatment of ESCC. More than 20 phase II clinical trials have been launched to explore combinations of ICIs in the neoadjuvant setting for ESCC. Based on our phase II clinical trial, a two-arm phase III trial was launched in Henan Cancer Hospital. ICIs combined with NAC may usher in a new era and may benefit locally advanced, resectable ESCC patients. METHODS: A two-arm phase III trial was launched in April 2020 in Henan Cancer Hospital. Patient recruitment will be completed within 18 months. The primary endpoint is event-free survival (EFS). The secondary endpoints include pathologic complete response (pCR), disease-free survival (DFS) rate, overall response rate (ORR), R0 resection rate, major pathologic response (MPR), adverse events (AEs), complication rate and quality of life (QOL). A biobank of pretreatment, resected tumor tissue and paired blood samples will be built for translational research in the future. DISCUSSION: This RCT directly compares NAC with neoadjuvant toripalimab plus chemotherapy in terms of EFS for locally advanced ESCC. The results may usher in a new era of resectable ESCC treatment. TRIAL REGISTRATION: NCT04280822 (https://www.clinicaltrials.gov/ct2/show/NCT04280822). Registered title: "A Phase III, Randomized Controlled Study of Neo-adjuvant Toripalimab (JS001) in Combination with Chemotherapy versus Neo-adjuvant Chemotherapy for Resectable Esophageal Squamous Cell Carcinoma". Version 1.0/Nov. 21, 2019.

Neoadjuvant chemotherapy versus neoadjuvant chemoradiotherapy for locally advanced oesophageal squamous cell carcinoma: a single-Centre, open-label, randomized, controlled, clinical trial (HCHTOG1903).

BACKGROUND: Neoadjuvant therapy plus oesophagectomy has been accepted as the standard treatment for patients with potentially curable locally advanced oesophageal cancer. No completed randomized controlled trial (RCT) has directly compared neoadjuvant chemotherapy and neoadjuvant chemoradiation in patients with oesophageal squamous cell carcinoma (ESCC). The aim of the current RCT is to investigate the impact of neoadjuvant chemotherapy plus surgery and neoadjuvant chemoradiotherapy plus surgery on overall survival for patients with resectable locally advanced ESCC. METHODS: This open label, single-centre, phase III RCT randomized patients (cT2-T4aN + M0 and cT3-4aN0M0) in a 1:1 fashion to receive either the CROSS regimen (paclitaxel 50 mg/m2; carboplatin (area under the curve = 2), q1w, 5 cycles; and concurrent radiotherapy, 41.4 Gy/23 F, over 5 weeks) or neoadjuvant chemotherapy (paclitaxel 175 mg/m2; and cisplatin 75 mg/m2, q21d, 2 cycles). Assuming a 12% 5-year overall survival difference in favour of the CROSS regimen, 80% power with a two-sided alpha level of 0.05 and a 5% dropout each year for an estimated 3 years enrolment, the power calculation requires 456 patients to be recruited (228 in each group). The primary endpoint is 5-year overall survival, with a minimum 5-year follow-up. The secondary endpoints include 5-year disease-free survival, toxicity, pathological complete response rate, postoperative complications, postoperative mortality and quality of life. A biobank of pre-treatment and resected tumour tissue will be built for translational research in the future. DISCUSSION: This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies. TRIAL REGISTRATION: NCT04138212, date of registration: October 24, 2019.

HIPK3 Promotes Growth and Metastasis of Esophageal Squamous Cell Carcinoma via Regulation of miR-599/c-MYC Axis.

BACKGROUND/AIMS: this experimental design was based on HIPK3 to explore the pathogenesis of ESCC. METHODS: RT-qPCR was used to detect the expression of CircHIPK3 and miR-599 in ESCC tissues and cell lines.CCK-8, colony formation, flow cytometry and transwell assay were used to detect the effects of CircHIPK3 and miR-599 on tumor cell proliferation, apoptosis and migration and invasion. Target gene prediction and screening, luciferase reporter assays were used to validate downstream target genes of CircHIPK3 and miR-599.mRNA and protein expression of c-MYC were detected by RT-qPCR and Western blotting. The tumor changes in mice were detected by in vivo experiments in nude mice. RESULTS: HIPK3 was highly expressed in ESCC tissues and cell lines. In addition, HIPK3 expression levels were associated with advanced TNM stage, lymph node metastasis and tumor size. Moreover, HIPK3 was significantly promoted cell proliferation and migration of ESCC cells. In addition, HIPK3 was able to inhibit miRNA-599 expression and up-regulate the expression level of c-MYC. Finally, the results of in vivo animal models confirmed that HIPK3 promoted ESCC progression by modulating the miR-599/c-MYC axis. CONCLUSION: HIPK3 can regulate the proliferation of esophageal squamous cell carcinoma cells by regulating miR-599/c-MYC axis, thereby inhibiting the occurrence and development of esophageal squamous cell carcinoma.

Silencing lncRNA AGAP2-AS1 Upregulates miR-195-5p to Repress Migration and Invasion of EC Cells via the Decrease of FOSL1 Expression.

The interaction of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs has been implicated in various types of cancers, including esophageal cancer (EC). The current study aimed to investigate the role of AGAP2-AS1/miR-195-5p/Fos-like antigen-1 (FOSL1) in EC progression. The expression of AGAP2-AS1, miR-195-5p, and FOSL1 in tumor tissues isolated from EC patients and EC cell lines was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the results of which illustrated that AGAP2-AS1 and FOSL1 were increased while miR-195-5p was reduced in EC. Next, the ectopic expression, knockdown, and reporter assay experiments were all employed to elucidate the mechanism of AGAP2-AS1/miR-195-5p/FOSL1 in the processes of EC cell proliferation, cell cycle, apoptosis, invasion, and migration as well as tumor growth. Knockdown of AGAP2-AS1 or overexpression of miR-195-5p reduced EC cell proliferation, migration, and invasion, blocked cell cycle entry, and elevated apoptosis. FOSL1 was found to be specifically targeted by miR-195-5p. AGAP2-AS1 was observed to upregulate FOSL1 by binding to miR-195-5p. Silencing of AGAP2-AS1 was observed to restrain the development of EC both in vitro and in vivo through upregulating miR-195-5p and downregulating FOSL1. Taken together, AGAP2-AS1 knockdown exercises suppressive effects on the development of EC through miR-195-5p-dependent downregulation of FOSL1. Therefore, targeting AGAP2-AS1 could be a future direction to develop a novel molecule-targeted therapeutic strategy for EC.

FoxO3a inhibiting expression of EPS8 to prevent progression of NSCLC: A new negative loop of EGFR signaling.

BACKGROUND: The resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) is a major challenge in the treatment of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms behind resistance is therefore an important issue. Here we assessed the role of EGFR pathway substrate 8 (EPS8) and Forkhead box O 3a (FoxO3a) as potentially valuable targets in the resistance of NSCLC . METHODS: The expression levels of EPS8 and FoxO3a in patients with NSCLC (n = 75) were examined by immunohistochemistry staining, while in cells were detected by qPCR and western blot. The effects of EPS8 and FoxO3a on resistance, migration and invasion, cell cycle arrest were detected by MTT, transwell and flow cytometry, respectively. Chromatin immunoprecipitation and luciferase reporter assays were performed to determine the mechanisms of EPS8 expression and FoxO3a regulation. FINDINGS: We observed that the expression of EPS8 inversely correlated with FoxO3a in NSCLC cell lines and NSCLC patients. FoxO3a levels were significantly decreased in tumor tissues compared with para-carcinoma tissues, while EPS8 is opposite. Besides, they play reverse roles in the resistance to gefitinib, the migration and invasion abilities, the cell cycle arrest in vitro and the tumor growth in vivo. Mechanistically, FoxO3a inhibits EPS8 levels by directly binding its gene promoter and they form a negative loop in EGFR pathway. INTERPRETATION: Targeting FoxO3a and EPS8 in EGFR signaling pathway prevents the progression of NSCLC, which implied that the negative loop they formed could served as a therapeutic target for overcoming resistance in NSCLC. FUNDS: National Natural Science Foundation of China, Science and Technology Project of Henan, Outstanding Young Talent Research Fund of Zhengzhou University and the National Scholarship Fund.

Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy.

Colorectal cancer (CRC) is the third highest mortality cancer in the United States and frequently metastasizes to liver and lung. Smad2 is a key element downstream of the TGF-ß signaling pathway to regulate cancer metastasis by promoting epithelial to mesenchymal transition and maintaining the cancer stem cell (CSC) phenotype. In this study, we show that hsa-miR-140-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in CRC cell lines decreases Smad2 expression levels, leading decreased cell invasion and proliferation, and increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy. We have systematically identified targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished tumor formation and metastasis in vivo. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues. Higher hsa-miR-140 expression is significantly correlated with better survival in stage III and IV colorectal cancer patients.The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in CRC progression and metastasis, and may have potential as a novel therapeutic molecule to treat CRC.

Comparison of the analgesic effects of cryoanalgesia vs. parecoxib for lung cancer patients after lobectomy.

This study was designed to compare the analgesic effects of cryoanalgesia and parecoxib in lung cancer patients after lobectomy. A total of 178 lung cancer patients awaiting large-sized lobectomy were enrolled in the study. The patients were randomly divided into Group A (intercostal nerve cryoanalgesia) and Group B (parecoxib). The analgesic and adverse effects were compared between the two groups. The pain score of Group A was significantly lower than that of Group B (P < 0.05). The patients in Group A used significantly less morphine than those in Group B (P < 0.05). There were also significantly fewer complications in Group A than in Group B (P < 0.05). Cryoanalgesia of the intercostal nerves can be considered an economical, safe and simple technique for the long-term management of post-lobectomy pain.

Berberine induces apoptosis and DNA damage in MG­63 human osteosarcoma cells.

Berberine, an isoquinoline alkaloid extracted from the dry root of Coptidis Rhizoma, has been found to exhibit marked anticancer effects on a panel of established cancer cells. Among the human osteosarcoma lines treated, MG­63 cells were found to be the most sensitive. The present study investigated the potential genotoxic effect of berberine on MG­63 human osteosarcoma cells. The effect of berberine on cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5­diphenyltetrazolium bromide assay and cell apoptosis was analyzed by flow cytometry and a DNA ladder assay. γH2AX focus formation was used to detect DNA damage in MG-63 cells. Berberine induced a significant increase in apoptosis in MG-63 cells in a concentration- and time-dependent manner, as determined by DNA fragmentation analysis and flow cytometry. Furthermore, berberine induced significant concentration- and time-dependent increases in DNA damage compared with that in the negative control. In conclusion, these observations indicated that berberine induced apoptosis and DNA damage in MG­63 cells.

Transforming growth factor-1 promotes the transcriptional activation of plasminogen activator inhibitor type 1 in carcinoma-associated fibroblasts.

Carcinoma-associated fibroblasts (CAFs) play a pivotal role in promoting the growth, invasion and metastasis of tumor cells. However, to date little is known about the oncogenic mechanisms of CAFs. This study aimed to identify the microenvironmental factors involved in tumor development and progression directed by CAFs in liver metastases. Tissue samples collected from 20 patients with colorectal liver metastases were used in this study. Histological and morphological characterization of the samples was performed using hybridization and immunohistological assays. The mRNA expression of α-smooth muscle actin (α-SMA) was measured by northern blotting. The expression of plasminogen activator inhibitor type 1 (PAI-1) was measured by enzyme-linked immunosorbent assay (ELISA). As a result, co-expression of Thy-1 (CD90) and α-SMA was identified in CAFs, while normal liver samples were negative for α-SMA and Thy-1. Compared with epidermal growth factor (EGF) and tumor necrosis factor (TNF) incubation, the expression of α-SMA increased significantly following transforming growth factor-1 (TGF-1) incubation (P<0.05), while platelet-derived growth factor (PDGF) caused a significant suppression of α-SMA expression (P<0.05). PAI-1 expression was significantly lower in unstimulated fibroblasts compared to TGF-1-treated fibroblasts (P<0.01). The levels of PAI-1 transcription were significantly higher in CAFs from the patient samples compared with the healthy controls. Taken together, our findings suggest that CAFs may be important in migration, matrix degradation, invasion and angiogenesis of tumors, and TGF-1 may promote the activation of PAI-1 transcription in CAFs.