Lack of evolutionary convergence in multiple primary lung cancer suggests insufficient specificity of personalized therapy.

Multiple primary lung cancer (MPLC) is an increasingly prevalent subtype of lung cancer. According to recent genomic studies, the different lesions of a single MPLC patient exhibit functional similarities that may reflect evolutionary convergence. We perform whole-exome sequencing for a unique cohort of MPLC patients with multiple samples from each lesion found. Using our own and other relevant public data, evolutionary tree reconstruction reveals that cancer driver gene mutations occurred at the early trunk, indicating evolutionary contingency rather than adaptive convergence. Additionally, tumors from the same MPLC patient are as genetically diverse as those from different patients, while within-tumor genetic heterogeneity is significantly lower. Furthermore, the aberrant molecular functions enriched in mutated genes for a sample show a strong overlap with other samples from the same tumor, but not with samples from other tumors or other patients. Overall, there is no evidence of adaptive convergence during the evolution of MPLC. Most importantly, the similar between-tumor diversity and between-patient diversity suggest that personalized therapies may not adequately account for the genetic diversity among different tumors in an MPLC patient. To fully exploit the strategic value of precision medicine, targeted therapies should be designed and delivered on a per-lesion basis.

Learning curve of uniportal video-assisted thoracoscopic lobectomy: an analysis of the proficiency of 538 cases from a single centre.

OBJECTIVES: Uniportal video-assisted thoracoscopic surgery (UniVATS) is widely used as a minimally invasive thoracic operation. The goal of our study was to analyse the effect of long-term experience with the UniVATS lobectomy on the learning curve. METHODS: The learning curves were quantitatively evaluated by the unadjusted cumulative sum, and they were segmented using joinpoint linear regression analysis. The variables were compared between subgroups using trend analysis, and linear regression analysis was applied to correlate clinical characteristics at different stages of the learning curve with the duration of the operation. RESULTS: The learning curve for the UniVATS lobectomy can be divided into 3 phases of proficiency at ∼200-300 procedures, with a fourth phase as the number of procedures increases. The 1st-52nd, 52nd-156th, 156th-244th and 244th-538th procedures comprised the preliminary learning stage, preliminary proficiency stage, proficiency stage and advanced proficiency stage, respectively. Surgical outcomes and their variability between stages improved with increasing case numbers, with the most significant addition of an auxiliary operating port and conversions. In multivariable analysis, as stages progressed, influences other than surgical experience increased the operative time, with male and extensive pleural adhesions in the preliminary proficiency stage; male and incomplete pulmonary fissures in the proficiency stage; and male, extensive pleural adhesions and incomplete pulmonary fissures in the advanced proficiency stage. CONCLUSIONS: As the number of procedures increases, there may be 4 different proficiency stages in the UniVATS lobectomy learning curve. The surgeon enters the fourth stage at approximately the 244th procedure. Moreover, at stage 4, the perioperative indicators tend to stabilize, and influences other than surgical experience become more significant.

Near-infrared fluorescence imaging-guided lymphatic mapping in thoracic esophageal cancer surgery.

PURPOSE: Identifying the lymphatic drainage pathway is important for accurate lymph node (LN) dissection in esophageal cancer (EC). This study aimed to assess lymphatic drainage mapping in thoracic EC using near-infrared fluorescent (NIRF) imaging with indocyanine green (ICG) and identify its feasibility for intraoperative LN drainage visualization and dissection. METHODS: From November 2019 to August 2020, esophagectomy was performed using intraoperative NIRF navigation with ICG injected into the esophageal submucosa by endoscopy. All LNs were divided into four groups according to the NIRF status and presence of metastasis: NIRF+LN+, NIRF+LN-, NIRF-LN+, and NIRF-LN-. RESULTS: Regional LNs were detected in all 84 enrolled patients with thoracic EC. A total of 2164 LNs were removed, and the mean number of dissected LNs was 25.68 ± 12.00. NIRF+ LNs were observed in all patients and distributed at 19 LN stations, which formed lymphatic drainage maps. The top five LN stations of NIRF+ probability in upper thoracic EC were No. 7, 106ecR, 107, 1, and 106recL; in middle thoracic EC, they were No. 107, 7, 110, 1, and 105; and in lower thoracic EC, they were No. 107, 7, 110, 106recR, and 1. There were no cases of ICG-related adverse events or chylothorax. The 30-day mortality rate was 0%. Major complications included anastomotic fistula (7.14%), pneumonia (4.76%), pleural effusion (13.10%), atelectasis (3.75%), hoarseness (8.33%), and arrhythmia (4.76%). CONCLUSION: Regional LN mapping of thoracic EC was performed using ICG/NIRF imaging, which showed different preferred LN drainage stations in various anatomical locations of the thoracic esophagus. ICG/NIRF imaging is feasible for intraoperative LN drainage visualization and dissection. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT04173676 ( http://www. CLINICALTRIALS: gov/ ).

Effect of single-port inflatable mediastinoscopy simultaneous laparoscopic-assisted radical esophagectomy on respiration and circulation.

BACKGROUND AND PURPOSE: We previously developed a new surgical method, namely, single-port inflatable mediastinoscopy simultaneous laparoscopic-assisted radical esophagectomy. The purpose of this study was to evaluate the effect of carbon dioxide inflation on respiration and circulation using this approach. METHODS: From April 2018 to October 2020, 105 patients underwent this novel surgical approach. The changes in respiratory and circulatory functions were reported when the mediastinal pressure and pneumoperitoneum pressure were 10 and 12 mmHg, respectively. Data on blood loss, operative time, and postoperative complications were also collected. RESULTS: 104 patients completed the operation successfully, except for 1 patient who was converted to thoracotomy because of intraoperative injury. During the operation, respectively, the heart rate, mean arterial pressure, central venous pressure, peak airway pressure, end-expiratory partial pressure of carbon dioxide and partial pressure of carbon dioxide increased in an admissibility range. The pH and oxygenation index decreased 1 h after inflation, but these values were all within a safe and acceptable range and restored to the baseline level after CO2 elimination. Postoperative complications included anastomotic fistula (8.6%), pleural effusion that needed to be treated (8.6%), chylothorax (0.9%), pneumonia (7.6%), arrhythmia (3.8%) and postoperative hoarseness (18.2%). There were no cases of perioperative death. CONCLUSIONS: When the inflation pressure in the mediastinum and abdomen was 10 mmHg and 12 mmHg, respectively, the inflation of carbon dioxide from single-port inflatable mediastinoscopy simultaneous laparoscopic-assisted radical esophagectomy did not cause serious changes in respiratory and circulatory function or increase perioperative complications.

Lymphadenectomy Along Bilateral Recurrent Laryngeal Nerves Under Single-Incision Mediastinoscopy.

This report proposes an approach for lymphadenectomy along bilateral recurrent laryngeal nerves (RLNs) under mediastinoscopy through a single left-neck incision. After pneumomediastinum is established, esophagectomy is begun over the aortic arch to the level of the lower edge of the left main bronchus, and lymphadenectomy along the left RLN is also accomplished. At the level of the lower edge of the right subclavian artery, between the trachea and the esophagus, the instruments can gain access to the right RLN. The lymphadenectomy may be performed up to 2 cm above the upper edge of the right subclavian artery.

Mediastinal lymphadenectomy under laparoscopic assisted single-port inflatable mediastinoscopy through left neck approach.

Minimally invasive esophagectomy (MIE) has been developed for decades. However, conventional MIE requires transthoracic surgery, which can increase the risk of many perioperative cardiopulmonary complications. Therefore, mediastinoscopy-assisted transhiatal esophagectomy has been proposed, but the traditional surgical methods have shortcomings, such as unclear vision, especially during the dissection of mediastinal lymph nodes (LNs). A new approach for mediastinal lymphadenectomy under single-port inflatable mediastinoscopy with one left-neck incision is proposed. There are three difficulties in this procedure. (I) LNs along the left recurrent laryngeal nerve (RLN). After establishing pneumomediastinum, esophagectomy is performed over the aortic arch to the level of the lower edge of the left main bronchus, and lymphadenectomy along the left RLN is also accomplished during this process. (II) LNs along the right RLN. At the level of the lower edge of the right subclavian artery (RSA), between the trachea and the esophagus, instruments are used to access the right RLN. Lymphadenectomy of up to 2 cm can be accomplished at the upper edge of the RSA. (III) Subcarinal LNs. Between the trachea and esophagus, the left and right main bronchi are exposed along the trailing edge of the trachea down to the carina, and lymphadenectomy can be performed here. The surgical procedure described here in detail is the first mediastinal lymphadenectomy under mediastinoscopy with one single left-neck incision.

Glutathione Peroxidase 1 Promotes NSCLC Resistance to Cisplatin via ROS-Induced Activation of PI3K/AKT Pathway.

PURPOSE: Reactive oxygen species (ROS)-induced cytotoxicity is an important mechanism by which cisplatin kills tumor cells. Glutathione peroxidase family (GPXs) is an important member of antioxidant system which metabolizes intracellular ROS and maintains homeostasis of cells. Altered expressions of GPXs enzymes, especially GPX1, have been described in a variety of human cancers. However, their functional roles in cisplatin-based chemoresistance in human malignancies including non-small cell lung cancer have never been explored. METHODS: A panel of NSCLC cell lines were selected for this study. GPX1 expression was detected using quantitative RT-PCR and Western blot. Cisplatin-induced cell killing was analyzed by CCK8 assay. Intracellular ROS levels were detected by fluorescence-based flow cytometry analysis. In vitro overexpression and knockdown of GPX1 expression were performed using GPX1 expression vector and siRNA approaches. Protein levels of PTEN, NF-κB, BCL2, Bax, and phosphorylated AKT were detected with western blot analysis using specific antibodies. RESULTS: GPX1 expression was upregulated in a subset of NSCLC cell lines resistant to cisplatin treatment. Expression vector-mediated forced overexpression of GPX1 significantly increased cisplatin resistance in NSCLC cell lines, whereas RNA inference-mediated downregulation of GPX1 could restore sensitivity to cisplatin. Overexpression of GPX1 significantly suppressed elevation of intracellular ROS and activation of AKT pathway when NSCLC cell lines were exposed to different concentrations of cisplatin. Activation of the AKT pathway inhibited proapoptotic cascade and subsequently led to cisplatin resistance in NSCLC cells. Inhibition of NF-κB by its chemical inhibitor BAY can significantly downregulate GPX1 expression and restore the cisplatin sensitivity of the cell lines resistant to cisplatin. CONCLUSIONS: Our findings suggested that overexpression of GPX1 is a novel molecular mechanism for cisplatin-based chemoresistance in NSCLC. GPX1 overexpression blocks cisplatin-induced ROS intracellular accumulation, activates PI3K-AKT pathway by increased AKT phosphorylation, and further leads to cisplatin resistance in NSCLC cells. Inhibition of NF-κB signaling may be an alternative approach for restoring cisplatin sensitivity for NSCLC cells resistant to cisplatin-based chemotherapy.

Morin attenuates cigarette smoke-induced lung inflammation through inhibition of PI3K/AKT/NF-κB signaling pathway.

Cigarette smoke (CS) is a major factor that leads to lung inflammation. The prevalence of CS-induced lung injury has continuously increased worldwide. Morin exists in a large member of plants and fruits that has been reported to have antioxidant and anti-inflammatory properties. In the present study, we tested the mechanism and protective effects of morin on CS-induced lung inflammation in mice. The mice were exposed to CS for 2 h twice a day for 4 weeks. Morin (10, 20, and 40 mg/kg) was treated to mice through oral gavage 1 h before CS administration. 24 h after the last CS exposure, the mice were euthanized. The lung tissues were collected and the pathological changes, wet/dry ratio, MPO activity, MDA levels, and P13K/ATK/NF-κB signaling pathway expression were detected. The bronchial alveolar lavage fluid (BALF) was obtained and the levels of inflammatory cells and inflammatory cytokines were measured. The results showed that morin treatment significantly inhibited lung pathological changes, wet/dry ratio, MPO activity, and MDA level. The levels of total cells, neutrophils, macrophages, as well as the production of inflammatory cytokines in the BALF induced by CS were also suppressed by morin. Further research showed that morin dramatically suppressed the activation of P13K/ATK/NF-κB singling pathway induced by CS. This study highlights the protective effects of morin on CS-induced lung inflammation, which may, at least part, be mediated through inhibiting P13K/ATK/NF-κB signaling pathway. These finding demonstrated that morin could be a potential drug for CS-induced lung injury.

Left- and right-sided video-assisted thoracoscopic thymectomy exhibit similar effects on myasthenia gravis.

BACKGROUND: Unilateral video-assisted thoracoscopic (VATS) thymectomy features less operative trauma, improved cosmesis, and similar efficiency compared with transsternal (TS) thymectomy for treatment of patients with myasthenia gravis (MG). Unilateral VATS thymectomy can be easily performed from either side of the thorax, because thymus is located in the middle of mediastinum. Nevertheless, the side that provides better outcomes remains controversial. This study presents our experience on treatments for MG and reveals the differences between the unilateral VATS thymectomy performed on each side. METHODS: Eighty-one consecutive patients with MG who underwent TS or VATS thymectomy on either side between January 2003 and December 2012 were enrolled in the study. Clinicopathologic data and surgical outcomes were retrospectively analyzed and compared among different surgical approaches. RESULTS: TS thymectomy was administered in 50 patients, whereas unilateral VATS approaches were performed on the remaining 31 patients, 15 on the left side and 16 on the right side. The VATS group exhibited a significantly shorter surgery duration (P<0.001), less intraoperative blood loss (P=0.009), shorter postoperative hospital stay (P=0.025), smaller thoracic drainage volume (P=0.033), shorter thoracic drainage duration (P=0.006), and less postoperative complications (P<0.001) compared with the TS group. However, disease remission rates did not significantly differ among the groups (P=0.988). The left-sided group exhibited considerably longer thoracic drainage duration than the right-sided group (P=0.041). Moreover, surgical time (P=0.736), intraoperative blood loss (P=0.281), postoperative hospital stay (P=0.599), thoracic drainage volume (P=0.571), postoperative complications (P=0.742) and therapeutic effect (P=1.000) did not significantly differ among the groups. Multivariate analysis revealed that the ocular type of MG is the only independent factor for clinical remission (P=0.002). CONCLUSIONS: Unilateral VATS thymectomy can reduce surgical risks and shorten hospitalization duration without threatening the therapeutic effect. This technique can be safely and effectively performed by experienced surgeons in either side of the thorax.

1,25-Dihydroxyvitamin D3 alleviates salivary adenoid cystic carcinoma progression by suppressing GPX1 expression through the NF-κB pathway.

1,25-Dihydroxyvitamin D3 (1,25D3) is the active form of vitamin D with antineoplastic effects. The glutathione peroxidase-1 (GPX1) gene is associated with tumour progression. The present study aimed to explore the role of GPX1 in 1,25D3-mediated progression of salivary adenoid cystic carcinoma (SACC). Downregulating GPX1 expression inhibited SACC cell proliferation, chemoresistance, motility, and uPA secretion, but promoted apoptosis via the NF-κB pathway. Pre-processing 1,25D3 inhibited expression of NF-κB/GPX1/uPA, which subsequently suppressed cell motility and cisplatin-resistance in ACC-2 cells. In conclusion, 1,25D3 works as a modifier of NF-κB/GPX1/uPA expression, inhibiting cisplatin-resistance and cell invasive ability of SACC cells. The present study comprehensively elucidated the potential mechanism underlying the effects of vitamin D on chemoresistance and invasive potential in SACC.

Methylation of neurofilament light polypeptide promoter is associated with cell invasion and metastasis in NSCLC.

The role of NEFL in NSCLC remains largely unknown. Immunohistochemistry was performed to investigate the expression of NEFL in 108 lung cancer specimens. NEFL expression was associated with decreased lymph node metastases and favorable prognosis. Furthermore, real-time PCR and Western blot were used to investigate the expression of the NEFL gene in NSCLC cell lines. Subsequently, lentivirus-mediated RNA interference and overexpression were used to demonstrate that knocked-down of NEFL enhanced the invasion and migration of A549 and H460 NSCLC cells, whereas NEFL overexpression resulted in a suppression of the invasion and migration of GLC-82 and L78 cells in vitro. In addition, bisulfite sequence PCR assay demonstrated that NEFL downregulation was associated with promoter methylation, and NEFL expression was restored after treatment with 5-Aza-dC. Finally, we demonstrated that NEFL inhibited the NF-κB pathway, thereby suppressing the expression of uPA and decreasing NSCLC invasiveness and migration. Our studies suggest that NEFL methylation is a novel mechanism for NSCLC invasion and metastasis and that NEFL may represent a candidate biomarker for recurrence and survival in patients with NSCLC.

DNA promoter hypermethylation contributes to down-regulation of galactocerebrosidase gene in lung and head and neck cancers.

Galactocerebrosidase (GALC) is a lysosomal enzyme responsible for glycosphingolipids degradation byproducts of which are important for synthesis of apoptosis mediator ceramide. Reduced expression of GALC has been identified in human malignancies; however, molecular mechanisms underlying down-regulation of GALC expression in cancer remain unknown. We performed methylation and expression analysis on GALC gene in a panel of head and neck cancer (HNC) and lung cancer cell lines, attempting to understand the regulation of GALC in human cancer. QRT-PCR and western blot analysis were performed to detect the expression of GALC in HNC. Bisulfite DNA sequencing and real-time qMSP were used to detect the methylation of GALC in HNC and lung cancer cell lines. 5aza-dC treatment assay was used to analysis the functional effect of GALC methylation on GALC expression in HNC. Reduction or complete absence of GALC expression was observed in more than a half of the tested HNC cell lines (8/14). 7 out of 8 cell lines with down-regulated expression harbored heavy CpG island methylation, while all cell lines with abundant expression of the gene contained no methylation. Hypermethylation was also found in primary HNC tumor tissues and lung cancer cell lines whereas absent in normal oral mucosa tissues. Demethylating treatment demonstrated that 5aza-dC significantly restored GALC expression in cell lines with methylated promoter while showed no effect on cell lines without promoter hypermethylation. Our findings for the first time demonstrated that promoter hypermethylation contributed to down-regulation of GALC Gene, implicating epigenetic inactivation of GALC may play a role in tumorigenesis of cancer.

The expression of MACC1 and its role in the proliferation and apoptosis of salivary adenoid cystic carcinoma.

BACKGROUND: The objective of this study was to investigate the relationship between metastasis-associated in colon cancer-1 and patient clinical characteristics. We also examined the role of metastasis-associated in colon cancer-1 in the proliferation and apoptosis in adenoid cystic carcinoma. MATERIAL AND METHODS: Metastasis-associated in colon cancer-1 expression was analysed in 65 paraffin-embedded tissue specimens of salivary adenoid cystic carcinoma and 25 adjacent non-cancerous tissues by immunohistochemistry (IHC). We used RNA interference technology to silence metastasis-associated in colon cancer-1 expression in ACCM cells. Cell Counting Kit-8 tests, transwell experiments and flow cytometry were used to test the proliferation, cisplatin resistance, migration, invasion and apoptosis of ACCM cells. RESULTS: Metastasis-associated in colon cancer-1 nuclear and cytoplasmic expression in salivary adenoid cystic carcinoma tissue was higher than in the adjacent normal salivary tissue. The expression level was closely associated with tumour histological grading, perineural invasion and surrounding tumour invasion. The downregulation of metastasis-associated in colon cancer-1 expression inhibited proliferation and induced apoptosis in ACCM cells. The knock-down of metastasis-associated in colon cancer-1 expression had no effect on migration, invasion and chemoresistance. CONCLUSIONS: Metastasis-associated in colon cancer-1 may have an important role in tumour development in adenoid cystic carcinoma. Metastasis-associated in colon cancer-1 is a potential biomarker for adenoid cystic carcinoma.

Downregulation of MACC1 inhibits invasion, migration and proliferation, attenuates cisplatin resistance and induces apoptosis in tongue squamous cell carcinoma.

The aim of the present study was to investigate the expression and function of metastasis-associated in colon cancer 1 (MACC1) in tongue squamous cell carcinoma (TSCC) and its relationship with the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147). Levels of MACC1 and EMMPRIN expression were analyzed in 65 paraffin­embedded tissue specimens of TSCC and in the adjacent non-cancerous tissues using immunohistochemistry (IHC). MACC1 expression was highly associated with lymphatic metastasis and EMMPRIN expression. Overexpression of MACC1 was significantly correlated with poor overall patient survival. A small interfering RNA (siRNA) was delivered into TSCCA cells to downregulate MACC1 expression. The CCK-8 assay showed that TSCCA cell proliferation was markedly reduced and that cisplatin resistance was attenuated. The suppression of MACC1 promoted the apoptosis of the TSCCA cell line. A Transwell experiment demonstrated that the migration and invasion abilities of the TSCCA cells were extremely downregulated. An ELISA experiment and western blotting revealed that the secretion of urokinase-type plasminogen activator system (uPA) in the supernatant of the culture medium and uPA expression were significantly reduced. Experiments revealed that the secretion of metalloproteinase 2 (MMP2) in the supernatant of the culture medium and MMP2 expression were not affected. MACC1 may serve as a novel biomarker and therapeutic target for TSCC.

High GPX1 expression promotes esophageal squamous cell carcinoma invasion, migration, proliferation and cisplatin-resistance but can be reduced by vitamin D.

Esophageal cancer is one of the most common cancers worldwide. Despite recent progress in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The glutathione peroxidase 1 (GPX1) gene located on chromosome 3p21.3 is associated with the cancer of several organs. According to available information, GPX1, a gene downstream of NF-κB, is considered to exert adverse effects on tumour progression and enhance malignancy in some cancers but has not been reported in esophageal cancer. It is also reported that vitamin D (Vit. D), a widely used drug in the clinical setting, could suppress GPX1 expression through the NF-κB pathway. Thus, it is speculated that Vit. D could reduce malignancy in esophageal cancer by altering the NF-κB pathway. In this study, we confirmed our speculation by finding that Vit. D, through the inhibition of GPX1, decreased the migratory, invasive and proliferative capabilities, as well as cisplatin resistance, in esophageal cancer cells. Furthermore, when invasion and migration were reduced in the GPX1-inhibited cells, the expression of urokinase type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP2) was also suppressed correspondingly. Therefore, we believe that, in esophageal cancer cells, the expression of GPX1 can promote invasion, migration, proliferation and cisplatin resistance, and Vit. D can reduce the associated malignancy through the NF-κB pathway. The Vit. D- and NF-κB-mediated decrease in GPX1 expression resulted in a decrease in MMP2- and uPA-mediated invasion and migration.