Human bone marrow mesenchymal stem cell-derived exosomes loaded with gemcitabine inhibit pancreatic cancer cell proliferation by enhancing apoptosis

BACKGROUND Pancreatic cancer remains one of the most lethal malignancies,and has limited effective treatment.Gemcitabine(GEM),a chemotherapeutic agent,is commonly used for clinical treatment of pancreatic cancer,but it has characteristics of low drug delivery efficiency and significant side effects.The study tested the hypothesis that human bone marrow mesenchymal stem cell(MSC)-derived exosomes loaded with GEM(Exo-GEM)would have a higher cytotoxicity against human pancreatic cancer cells by enhancing their apoptosis.AIM To investigate the cytotoxicity of MSC-derived Exo-GEM against pancreatic cancer cells in vitro.METHODS Exosomes were isolated from MSCs and characterized by transmission electron microscopy and nanoparticle tracking analysis.Exo-GEM through electroporation,sonication,or incubation,and the loading efficiency was evaluated.The cytotoxicity of Exo-GEM or GEM alone against human pancreatic cancer Panc-1 and MiaPaca-2 cells was assessed by MTT and flow cytometry assays.RESULTS The isolated exosomes had an average size of 76.7 nm.The encapsulation efficacy and loading efficiency of GEM by electroporation and sonication were similar and significantly better than incubation.The cytotoxicity of Exo-GEM against pancreatic cancer cells was stronger than free GEM and treatment with 0.02μM Exo-GEM significantly reduced the viability of both Panc-1 and MiaPaca-2 cells.Moreover,Exo-GEM enhanced the frequency of GEMinduced apoptosis in both cell lines.CONCLUSION Human bone marrow MSC-derived Exo-GEM have a potent cytotoxicity against human pancreatic cancer cells by enhancing their apoptosis,offering a promising drug delivery system for improving therapeutic outcomes.

Efficacy and safety of gemcitabine plus S-1 vs. gemcitabine plus nab-paclitaxel in treatment-na?ve advanced pancreatic ductal adenocarcinoma

Objective:Gemcitabine plus nab-paclitaxel(GnP)is the standard first-line therapy for advanced pancreatic ductal adenocarcinoma(PDAC).S-1,an oral fluoropyrimidine derivative,as compared with gemcitabine,is non-inferior in terms of overall survival(OS)and is associated with lower hematologic toxicity.Accordingly,S-1 is a convenient oral alternative treatment for advanced PDAC.This study was aimed at comparing the efficacy and safety of gemcitabine plus S-1(GS)vs.GnP as first-line chemotherapy for advanced PDAC.Methods:Patients with advanced PDAC who received first-line GS or GnP at the Peking Union Medical College Hospital between March 2011 and November 2022 were evaluated.Results:A total of 300 patients were assessed,of whom 84 received GS and 216 received GnP.The chemotherapy completion rate was higher with GS than GnP(50.0%vs.30.3%,P=0.0028).The objective response rate(ORR)was slightly higher(14.3%vs.9.7%,P=0.35),and the median OS was significantly longer(17.9 months vs.13.3 months,P=0.0078),in the GS group than the GnP group.However,the median progression-free survival(PFS)did not significantly differ between groups.Leukopenia risk was significantly lower in the GS group than the GnP group(14.9%vs.28.1%,P=0.049).Conclusions:As first-line chemotherapy for advanced PDAC,the GS regimen led to a significantly longer OS than the GnP regimen.The PFS,ORR,and incidence of severe adverse events were comparable between the GS and GnP groups.

Discussion on gemcitabine combined with targeted drugs in the treatment of pancreatic cancer

Pancreatic cancer is a malignant tumor with poor prognosis.The treatment of pancreatic cancer depends on the tumor stage and type,and includes local treatment(surgery,radiotherapy and ablation intervention)and systemic therapy(chemotherapy,targeted therapy and immunotherapy).We read with great interest the review“Effective combinations of anti-cancer and targeted drugs for pancreatic cancer treatment”published on World J Gastroenterol and intended to share some of our perspectives in pancreatic cancer treatment.This review presents the therapeutic effects of the combination of gemcitabine and targeted drugs,which gives us a deeper insight into the combination treatments for pancreatic cancer.

N-gamboyl Gemcitabine Inhibits Tumor Cells Proliferation and Migration

Gambogic acid(GA) is a natural substance with a good antitumor effect, but it is too lipophilic to be metabolized and excreted, thus accumulating in the body. Gemcitabine(GEM), one of the first-line antitumor drugs, has high hydrophilicity, which greatly shortens its half-life in vivo. We previously reported a compound named N-gamboyl gemcitabine(GAG), derived from the condensation of GEM and GA, whose hydrophilicity is better than GA and stability is better than GEM. Here, the antitumor performance of GAG was investigated for the first time by using several common tumor cell lines as tumor models. The results of in vitro study showed that GAG significantly inhibited the proliferation and migration of the tumor cells. The IC50 values of GAG for the tumor cells were lower than those of GEM and GA. The present study suggests that GAG has a promising potential to be developed into a broad-spectrum antitumor drug.

Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine

Background:The survival of pancreatic cancer cells,particularly cancer stem cells which are responsible for tumor relapse,depends on mitochondrial function.Mitochondrial transcription factor A(TFAM)is critical for the regulation of mitochondrial DNA and thus mitochondrial function.However,the possible involvement of TFAM in pancreatic cancer is unknown.Methods:Human samples were obtained from pancreatic cancers and their adjacent tissues;human pancreatic cell lines were cultured in RPMI1640 medium.TFAM expressions in pancreatic tissues and cultured cells were determined using immunohistochemistry,ELISA,and reverse transcription polymerase chain reaction(RT-PCR).The effect of TFAM on cell growth,migration,colony formation and apoptosis were evaluated.Mitochondrial biogenesis in pancreatic cancer and normal cells were examined.Results:The majority of pancreatic cancer tissues exhibited higher TFAM expression compared to the adjacent counterparts.Consistently,TFAM mRNA and protein levels were higher in pancreatic cancer cell lines than in immortalized normal pancreatic epithelial cells.There was no difference on TFAM level between gemcitabine-sensitive and resistant pancreatic cancer cells.Functional analysis demonstrated that TFAM overexpression activated pancreatic normal and tumor cells whereas TFAM inhibition effectively inhibited the growth of pancreatic cancer cells.TFAM inhibition enhanced gemcitabine’s cytotoxicity and suppressed growth,anchorage-independent colony formation and survival of gemcitabine-resistant pancreatic cancer cells.Mechanistic studies showed that TFAM inhibition resulted in remarkable mitochondrial dysfunction and energy crisis followed by oxidative stress.The basal mitochondrial biogenesis level correlated well with TFAM level in pancreatic cancer cells.Conclusions:TFAM played essential roles in pancreatic cancer via regulating mitochondrial functions which highlighted the therapeutic value of inhibiting TFAM to overcome gemcitabine resistance.

Nuclear PLD1 combined with NPM1 induces gemcitabine resistance through tumorigenic IL7R in pancreatic adenocarcinoma

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%.Of PDAC patients,15%-20%are eligible for radical surgery.Gemcitabine is an important chemotherapeutic agent for patients with PDAC;however,the efficacy of gemcitabine is limited due to resistance.Therefore,reducing gemcitabine resistance is essential for improving survival of patients with PDAC.Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC.Methods:We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment.Then,co-IP,ChIP,ChIP-seq,transcriptome sequencing,and qPCR were used to determine the specific mechanism by which phospholipase D1(PLD1)confers resistance to gemcitabine.Results:PLD1 combines with nucleophosmin 1(NPM1)and triggers NPM1 nuclear translocation,where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor(IL7R)expression.Upon interleukin 7(IL-7)binding,IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein,BCL-2,and induce gemcitabine resistance.The PLD1 inhibitor,Vu0155069,targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells.Conclusions:PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1,further promoting the downstream JAK1/STAT5/Bcl-2 pathway.Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity.

Gemcitabine-induced peripheral vascular disease and prolonged response in a patient with metastatic pancreatic adenocarcinoma:A case report

BACKGROUND Gemcitabine is an antimetabolite used in the treatment of pancreatic cancer.One of the side effects of gemcitabine is vascular toxicity.Here,we report the case of a patient treated with gemcitabine who had peripheral vascular disease concomi-tant with a prolonged antitumor response.CASE SUMMARY A 75-year-old man was diagnosed with locally recurrent pancreatic cancer.Partial response was achieved after 9 mo of gemcitabine.At the same time,the patient reported peripheral vascular disease without necrosis.Chemotherapy was suspended,and after one month the Positron Emission Tomography(PET)scan showed locoregional tumor recurrence.Gemcitabine was resumed and partial response was obtained,but peripheral vascular disease occurred.CONCLUSION Our results suggest that the appearance of peripheral vascular disease may be related to a prolonged response to gemcitabine.

A novel synthesized prodrug of gemcitabine based on oxygen-free radical sensitivity inhibited the growth of lung cancer cells

In the present study,we introduced the H2O2-sensitive thiazolidinone moiety at the 4th amino group of gemcitabine(GEM)to synthesize a new target compound named GEM-ZZQ,and then we confirmed its chemical structure by nuclear magnetic resonance spectroscopy.We further confirmed that GEM-ZZQ had a good chemical stability in different pH solutions in vitro and that it could be activated by H2O2 to release GEM.Pharmacodynamic studies revealed that the growth inhibition of human normal epithelial cells was weaker by GEM-ZZQ than by GEM treatment and that the inhibition of various lung cancer cell lines by GEM-ZZQ was similar to that of GEM.For the lung cancer cell lines that are resistant to the epidermal growth factor receptor(EGFR)-targeting inhibitor osimertinib,GEM-ZZQ showed less growth inhibition than GEM;however,GEM-ZZQ in combination with cisplatin showed better synergistic effects than GEM in the low-dose groups.In summary,we provided a new anti-cancer compound GEM-ZZQ for treating lung cancer by modifying the GEM structure.

Gemcitabine治疗晚期非小细胞肺癌的临床研究

目的：评价Ｇｅｍｃｉｔａｂｉｎｅ（中文译名：健择）单药及其与顺氯铵铂联合化疗方案治疗ＩＩ、ＩＶ期非小细胞肺癌（ＮＳＣＬＣ）的临床疗效及其不良反应。材料与方法：１健择单药研究：从１９９７年９月至１９９８年５月入选ＩＩ、ＩＶ期ＮＳＣＬＣ病人２１例，以前未接受过任何化疗、放疗。ＩＩ期病人１１例，ＩＶ期病人１０例。健择１０００ｍｇ／ｍ２，第１，８，１５天各注一次，每２８天为一疗程。２健择与顺氯铵铂联合化疗研究：从１９９７年１０月至１９９８年７月入选ＩＩ、ＩＶ期ＮＳＣＬＣ病人４８例，以前未接受过任何化疗、放疗。ＩＩ期病人１９例，ＩＶ期病人２９例。健择１０００ｍｇ／ｍ２，第１，８，１５天各注一次；顺氯铵铂１００ｍｇ／ｍ２，第一天用，每２８天为一疗程。结果：１健择单药研究：可评价疗效的有１９例，６例获部分缓解（ＰＲ），其中２例（１０５％）经４周以上复查证实。总有效率３１５％［９５％ＣＩ，９４％～４５１５％］。全组均可评价不良反应，少数病人发生轻微胃肠道反应、白细胞下降、血红蛋白下降和血小板下降，只有４８％（各１例）患者发生ＩＩ度的恶心呕吐及白细胞下降，有１例患者发生ＩＩ度感染。全组中位生存期６１３月。２?

Navelbine治疗晚期肺癌的Ⅱ期临床研究报告

1992年7月至1993年2月,用NAVELBINE治疗35例Ⅲ—Ⅳ期的非小细胞肺癌患者,所有病例均经病理组织学证实,包括鳞癌12例,腺癌18例、肺泡癌3例、腺样囊腺瘤和大细胞癌各1例。实际可评价的病人34例,其中Navelbine单药治疗16例(25mg/m^2/周×4),5例PR,有效率为31.3％。联合治疗(Navelbine+CDDP)18例,CR1例(5.6％)PR8例(44.4％),总有效率为50％。主要毒性为骨髓抑制,白细胞减少Ⅲ—Ⅳ级发生率为32.4％,其中一例高龄者白细胞严重减少,合并败血症而死亡。该研究提示,Navelbine对非小细胞肺癌的治疗价值是肯定的,主要剂量限制毒性为骨髓抑制,应在肿瘤学专家的指导下使用。

过表达Plk-1对gemcitabine诱导的胰腺癌细胞化疗敏感性的影响

目的观察Plk-1基因过表达对gemcitabine诱导胰腺癌细胞AsPC-1化疗敏感性的影响。方法①将携带有人外源性Plk-1基因的真核表达质粒转染AsPC-1细胞系设为处理组,转染空载体设为对照组,无处理组设为空白组,转染24 h后,提取细胞总RNA和总蛋白,利用RT-PCR法及Western blot鉴定AsPC-1细胞内Plk-1基因和蛋白表达水平。②采用流式细胞术检测转染组细胞凋亡的改变。③不同浓度gemcitabine作用于转染细胞,MTT法测定转染48 h后细胞增殖能力。结果①测序结果表明成功的从AsPC-1细胞总RNA中扩增Plk-1基因。RT-PCR结果表明:未转染组AsPC-1细胞组、转染空载体pcDNA3.1组及pcDNA3.1/Plk-1组24 h各组细胞内Plk-1 mRNA相对量分别为(2.14±0.16)、(2.18±0.15)、(2.58±0.18),转染pcDNA3.1/Plk-1组与其他各组相比,差异有高度统计学意义(P<0.01)。Western blot结果显示:未转染组AsPC-1细胞组、转染空载体pcDNA3.1及pcDNA3.1/Plk-1组24 h各组细胞内Plk-1基因的蛋白表达水平为(0.989±0.018)、(1.022±0.021)、(1.243±0.143),转染pcDNA3.1/Plk-1组与其他各组相比,差异有高度统计学意义(P<0.01)。②在gemcitabine作用下,pcDNA3.1/Plk-1转染组细胞凋亡率明显低于未转染组及空载体转染组,差异有高度统计学意义(P<0.01)。③AsPC-1/Plk-1转染组细胞的生长抑制率亦明显高于未转染组及空载体转染组,差异有高度统计学意义(P<0.01)。结论胞浆过表达Plk-1活性分子可明显降低gemcitabine诱导的AsPC-1细胞凋亡,增强其对化疗药物的耐受性。

吉西他滨(Gemcitabine)联合铂类药物治疗老年中晚期非小细胞肺癌(NSCLC)的临床观察

目的 :探讨吉西他滨 (Gemcitabine)联合铂类药物治疗老年中晚期非小细胞肺癌 (NSCLC)的临床疗效和毒性。方法 :予吉西他滨 (Gemcitabine) 10 0 0mg/m2 第 1、 8天静滴并联合铂类药物治疗老年非小细胞肺癌(NSCLC) 2 0例 ,三周为一治疗周期 ,两周期后评价疗效和毒副作用 ,随访缓解期和生存期。结果 :本组 2 0例病例中 ,治疗总有效 4 0 % ,中位缓解期 7个月 ,中位生存期 9个月 ,主要毒副作用为骨髓抑制和胃肠道反应。其中白细胞减少发生率为 80 % ,血小板减少发生率为 6 0 %。结论 :吉西他滨 (Gemcitabine)对老年非小细胞肺癌(NSCLC)有较好疗效 ,主要毒性反应为较严重的血液学毒性 ,老年患者最好辅以G -CSF或GM -CSF以及免疫与营养支持治疗 ,则能顺利完成化疗 ,吉西他滨 (Gemcitabine)联合铂类药物可做为老年非小细胞肺癌 (NSCLC)的一线治疗方案。

Treatment of Primary Liver Cancer with Postoperative After-Loading Radiotherapy and Gemcitabine

Objective： To evaluate the curative effectiveness of postoperative after-loading radiotherapy with the use of gemcitabine in 22 patients with primary liver cancer. Methods： From Oct. 1999 to Dec. 2001, 22 patients with primary liver cancer underwent postoperative after-loading radiotherapy 3-10 days after hepatectomy and chemotherapy using gemcitabine （1400 mg every week for 3 weeks, repeated after one week interval, total cycles were 6） and compared with 22 cases of sole hepatectomy. Three-six catheters were placed for irradiation after hepatectomy. The single-dose of after-loading radiotherapy was 10 Gy, 24 sessions per person. Results＇. The rate of AFP negative-reversion was 100% （17/17） in the treated group, higher than in control group （62.5%, 10/16, P〈0.05）. In the treated group, the 1-year relapse rate, metastasis rate and survival rate were 18.2% （4/22）, 0 and 100% （22/22） respectively, while in the control group they were 45.5% （10/22）, 13.6% （3/22） and 77.3% （17/22） respectively. There were significant differences between the two groups in relapse rate, metastasis rate and survival rate within a year （P〈0.05）. Conclusion： Postoperative after-loading radiotherapy with gemcitabine is an effective way for the treatment of primary liver cancer.

Treatment of Unresectable Carcinoma of Pancreas with ^(125)I Implantation and ^(125)I Plus Gemcitabine

Objective: To study the role of 125 I and 125 I plus gemcitabine (GEM) in treatment of unresectable carcinoma of pancreas. Methods: From April 2000 to April 2003, 38 untreated patients with locally advanced pancreatic cancer (LAPC) were collected and randomized into two groups: Arm A 125 I (18 patients) and Arm B 125 I+GEM (20 patients). Eligibility criteria were: cytologically and pathologically proven pancreatic carcinoma, Karnofsky performance status (kps) 60 80, age 18 75 years, adequate hematological, renal and liver function, and controllable pain. Arm A patients were treated with 125 I implants. Arm B patients started chemotherapy within 10 14 d post operatively following the implant procedure. Chemotherapy doses were as follows: GEM 1 000 mg/m 2 weekly × 3 followed by 1 week of rest for 3 cycles. In addition, all patients underwent laparotomy and surgical staging. The surgical procedures performed were biopsy, gastric bypass and biliary bypass. The total activity and number of seeds used were as recommended by Anderson. The mean activity, minimal peripheral dose (MPD), and volume of implants were 20 mCi, 14 000 cGy, and 53 cm 3, respectively. Results: Overall response rate (CR+PR) in Arm A was 37.6% and in Arm B it was 44.5% ( P >0.05). PR median duration in Arm A was 6.7 months and in Arm B it was 4.8 months ( P <0.05). Clinical benefit response was experienced by 11.7 % of Arm A compared with 42.1% of Arm B ( P <0.05). The incidences of hematological toxicity (such as neutropenia) between Arm A and Arm B were 5.8% and 21.1%, respectively ( P >0.05). The survival rates of 12 and 24 month were 32.5%, 16.3% for Arm A and 61%, 38.7% for Arm B ( P =0.04). The rate of complication of Arm A was lower than that of Arm B without statistical significance. Conclusion: To some extent, 125 I or 125 I plus GEM is able to lead to a moderate objective response for LAPC with obstructive jaundice on the base of biliary bypass or/and gastric bypass, but 125 I plus GEM is more effective than 125 I in improvement of the quality of life and survival rate in patients with LAPC.

基因芯片技术筛选Gemcitabine诱导胰腺癌Panc-1细胞凋亡相关基因的研究

目的研究抗癌药吉斯他滨（Gemcitabine）诱导胰腺癌Panc-1细胞凋亡的相关基因。方法应用基因芯片技术检测50．33μg／mL Gemcitabine作用于胰腺癌Panc-1细胞48h后凋亡基因表达的变化。结果Gemcitabine抑制细胞表达14种凋亡相关基因，包括IRF-4、Scotin、14．3．3gamma、PDE4、GRPS、TID1、AFP、STAT5、Sox-4、c-Rel、MYCN、Htt、CatD、Gd。相反，gemcitabine促进了3种凋亡相关基因的表达，包括CyclinB1、ASK1、AKT2。结论Gemcitabine诱导胰腺癌Panc-1细胞凋亡通过非依赖caspase ASK1途径。

hENT1 expression is predictive of gemcitabine outcome in pancreatic cancer: A systematic review

High human equilibrative nucleoside transporter 1(hENT1)-expression has shown a survival benefit in pancreatic cancer patients treated with gemcitabine in several studies.The aim of this systematic review was to summarize the results and try to assess the predictive value of hENT1 for determining gemcitabine outcome in pancreatic cancer.Relevant articles were obtained from PubMed,Embase and Cochrane databases.Studies evaluating hENT1-expression in pancreatic tumor cells from patients treated with gemcitabine were selected.Outcome measures were overall survival,disease-free survival(DFS),toxicity and response rate.The database searches identified 10 studies that met the eligibility criteria,and a total of 855 patients were included.Nine of 10 studies showed a statistically significant longer overall survival in univariate analyses in patients with high hENT1-expression compared to those with low expression.In the 7 studies that reported DFS as an outcome measure,6 had statistically longer DFS in the high hENT1 groups.Both toxicity and response rate were reported in only 2 articles and it was therefore hard to draw any major conclusions.This review provides evidence that hENT1 is a predictive marker for pancreatic cancer patients treated with gemcitabine.Some limitations of the review have to be taken into consideration,the majority of the included studies had a retrospective design,and there was no standardized scoring protocol for hENT1-expression.

Propofol induces apoptosis and increases gemcitabine sensitivity in pancreatic cancer cells in vitro by inhibition of nuclear factor-κ B activity

AIM:To investigate the effect of propofol on human pancreatic cells and the molecular mechanism of propofol action.METHODS:We used the human pancreatic cancer cell line MIAPaCa-2 for in vitro studies measuring growth inhibition and degree of apoptotic cell death induced by propofol alone,gemcitabine alone,or propofol followed by gemcitabine.All experiments were conducted in triplicate and carried out on three or more separate occasions.Data were means of the three or more independent experiments±SE.Statistically significant differences were determined by two-tailed unpaired Student’s t test and defined as P<0.05.RESULTS:Pretreatment of cells with propofol for 24 h followed by gemcitabine resulted in 24%-75% growth inhibition compared with 6%-18%when gemcitabine was used alone.Overall growth inhibition was directly correlated with apoptotic cell death.We also showed that propofol potentiated gemcitabine-induced killing by downregulation of nuclear factor-κB(NF-κB).In contrast,NF-κB was upregulated when pancreatic cancer cells were exposed to gemcitabine alone,suggesting a potential mechanism of acquired chemoresistance.CONCLUSION:Inactivation of the NF-κB signaling pathway by propofol might abrogate gemcitabineinduced activation of NF-κB,resulting in chemosensitization of pancreatic tumors to gemcitabine.

Nedaplatin/Gemcitabine Versus Carboplatin/Gemcitabine in Treatment of Advanced Non-small Cell Lung Cancer: A Randomized Clinical Trial

Objective： To evaluate the efficacy and safety of nedaplatin/gemcitabine （NG） and carboplatin/gemcitabine （CG） in the management of untreated advanced non-small cell lung cancer （NSCLC）. Methods： Sixty-two patients with previously untreated advanced NSCLC were recruited between June 2006 and November 2007. Subjects were randomly assigned to the NG arm （n=30） and the CG arm （n=32）. Only patients （24 and 25 in the NG and CG arms, respectively） who completed 〉2 chemotherapy cycles were included in the data analysis. The primary outcome measure was the objective response rate （ORR）. The secondary outcome measures included progression-free survival （PFS）, overall survival （OS） and adverse events. Results： There were no statistically significant differences in the efficacy measures （ORR, P=0.305; median PFS, P=0.298, median OS, P=0.961） or in the major adverse events （grade 3/4 neutropenia, P=0.666; grade 3/4 anemia, P=0.263; grade 3/4 thrombocytopenia, P=0.222） between the two treatment arms. However, there was a trend towards higher ORR （37.5% vs. 24.0%）, longer PFS （6.0 vs. 5.0 months）, and less adverse events in the NG arm. Conclusion： NG regimen seems to be superior over CG regimen for advance NSCLS, but further investigation is needed to validate this superiority.