Imatinib is the first-line therapy for gastrointestinal stromal tumor (GIST), significantly enhancing patient prognosis. However, its clinical efficacy and safety are highly dependent on plasma concentration, which exhibits considerable interindividual variability. The purpose of this study is to determine the optimal monitoring time for imatinib concentration in postoperative patients with GIST, analyze single nucleotide polymorphisms affecting plasma concentration, and investigate their correlation with efficacy and adverse drug reactions (ADRs), offering valuable insights for personalized therapy. This study was conducted on 116 postoperative patients with GIST treated with imatinib in China between 2014 and 2018. Imatinib peak and trough concentrations (Cmax and Cmin) were measured in 608 samples using liquid chromatography-tandem mass spectrometry to determine the optimal monitoring time for steady-state concentration. DNA was extracted from 60 patients’ peripheral blood to Genotype 22 single nucleotide polymorphisms in genes such as CYP3A4, CYP3A5, ABCB1, SLC22A1, and SLC22A5 (OCTN2). Therapeutic efficacy was evaluated based on recurrence-free survival (RFS), and ADRs were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 5.0. Statistical analyses were performed using SPSS 26.0, with p < 0.05 considered statistically significant. The Cmax and Cmin of imatinib and its metabolite N-desmethyl imatinib reached their highest levels within the first month of treatment, and were stabilized by the third month. Genetic polymorphisms in ABCG2 (rs2231137), SLC22A1 (rs628031, rs3605065), and SLC22A5 (OCTN2) (rs2631372) were significantly associated with variations in imatinib Cmin. Genetic polymorphisms in CYP3A5 (rs776746), ABCG2 (rs2231137), and SLC22A1 (rs755828176) influenced imatinib Cmax. Patients carrying the CG and CC genotypes of SLC22A5 (OCTN2) (rs2631372) exhibited longer RFS and lower disease progression risk compared to those with the GG genotype (p < 0.05). Additionally, there was no significant correlation between the occurrence of ADRs and the studied genetic polymorphisms. Imatinib plasma concentration was stabilized by the third month in postoperative patients with GIST. Genetic polymorphisms in ABCG2, SLC22A1, and SLC22A5 (OCTN2) were associated with imatinib plasma concentration, while SLC22A5 (OCTN2) also influenced recurrence rates. These results provide a reference for personalized therapy and concentration monitoring.

To compare the efficacy and feasibility of laparoscopic resection with those of open surgery for gastric gastrointestinal stromal tumors (GISTs) in unfavorable locations using the propensity score weighting (PSW) method. Clinicopathological and follow-up data from 170 patients found to have gastric GISTs in unfavorable locations from January 2017 to December 2021 were analyzed retrospectively. Among the 170 patients, 121 underwent laparoscopic surgery, and 49 underwent open surgery. The PSW was used to create balanced cohorts and adjusted for six clinically validated covariates: sex, age, BMI, tumor size, mitotic rate, and recurrence risk. After PSW, 325.2 patients, consisting of 167 laparoscopic (Lap group) and 158.2 open surgery (Open group) patients, were matched. The Lap group had shorter operation times (84.7 ± 37.7 min versus 123.4 ± 46.9 min, P < 0.001) and shorter hospital stays (7.0 [5.0, 8.0] days versus 9.0 [8.0, 11.0] days, P < 0.001) than the Open group. Although the Open group exhibited a numerically higher complication rate (10.24

Netrin-1 is a laminin-related protein found to promote proliferation and invasion in multiple types of cancers. Recent studies have identified the function role of netrin-1 in several cancers; however, the influence of netrin-1 in human gastric cancer(GC) remains largely unknown. In this study, we found netrin-1 was upregulated in human GC tissues, where its expression correlated inversely with cancer stage and lymph node metastasis. We detected netrin-1 and its receptor knockdown significantly suppressed GC cells proliferation and invasion, while overexpression netrin-1 reversed these effects. Xenografted analyses using GC cells displayed significantly inhibition of tumor growth and metastasis by netrin-1 depletion. Furthermore, we identified that netrin-1 as a regulator of PI3K/AKT pathway to modulate GC cells proliferation and invasion abilities via its receptor neogenin. Taken together, our findings argued that netrin-1 and its receptor neogenin might act synergistically in promoting GC cells proliferation and invasion through the PI3K/AKT signaling pathway. It is conceivable that netrin-1 could be new therapeutic target to GC therapy.

Autophagy plays a multifaceted role in tumorigenesis. However, the association between genetic variants in autophagy and gastric cancer susceptibility remains unclear. We evaluated the association between single-nucleotide polymorphisms (SNPs) of autophagy-related genes and gastric cancer risk using a cohort of 1,625 cases and 2,100 controls. Next, transcriptomic data were used to analyze differential gene expression and characterize the tumor immune microenvironment (TME). Single-cell RNA sequencing analysis was performed to investigate cell-type-specific expression profiles. In vitro gain/loss-of-function experiments were conducted to explore the biological roles of TRAF6 in cancer cells. We found that TRAF6 rs5030437 G > A conferred an increased risk of gastric cancer (OR = 1.23, 95% CI: 1.06-1.43, p = 1.28 × 10-3), particularly in males (OR = 1.24, 95% CI: 1.07-1.44, p = 3.69 × 10-3) and older individuals (OR = 1.41, 95% CI: 1.18-1.68, p = 1.53 × 10-4). TRAF6 expression was significantly upregulated in tumor tissues, correlating with poor prognosis in gastric cancer patients. Enrichment analyses implicated TRAF6 in immune response and inflammation signaling, manifested as its characteristic cellular expression distribution and influence on the abundance of specific immune cells. In vitro experiments demonstrated that TRAF6 positively regulated autophagy activity and promoted cell viability, migration, and proliferation of gastric cancer cells, which were reversed by autophagy inhibition. This study elucidated the genetic association of rs5030437 G > A and gastric cancer as well as the impacts of TRAF6 on TME dynamics and cancer biology, which provided novel insights into gastric carcinogenesis and the tumor ecosystem.

Intraoperative hemorrhage is an important factor affecting intraoperative safety and postoperative patient recovery in percutaneous nephrolithotomy (PCNL). This study aimed to identify the factors that influence intraoperative hemorrhage during PCNL and develop a predictive nomogram model based on these factors.A total of 118 patients who underwent PCNL at the Department of Urology, The Affiliated Huai’an No.1 People’s Hospital of Nanjing Medical University from January 2021 to September 2023 was included in this study. The patients were divided into a hemorrhage group (58 cases) and a control group (60 cases) based on the decrease in hemoglobin levels after surgery. The clinical data of all patients were collected, and both univariate analysis and multivariate logistic regression analysis were conducted to identify the independent risk factors for intraoperative hemorrhage during PCNL. The independent risk factors were used to construct a nomogram model using R software. Additionally, receiver operating characteristic (ROC) curves, calibration curves and decision curve analysis (DCA) were utilized to evaluate the model.Multivariate logistic regression analysis revealed that diabetes, long operation time and low psoas muscle mass index (PMI) were independent risk factors for intraoperative hemorrhage during PCNL (P < 0.05). A nomogram model was developed incorporating these factors, and the areas under the ROC curve (AUCs) in the training set and validation set were 0.740 (95

BACKGROUND:Autophagy plays a crucial role in sustaining the homeostasis in various malignant diseases. It has also been reported to promote tumor development in multiple cancers. Glutaminolysis instead of Warburg Effect produce adequate ATP and provide nitrogen and carbon to replenish the TCA cycle which has been discovered to be a new energy source for tumor cells recently. By means of degrading intracellular particles including amino acids, nucleotides, fatty acids, sugars and aged organisms, autophagy can recycle the aforementioned particles into bioenergetics and biosynthesis pathways, finally favoring tumor cells. MicroRNA is a kind of noncoding RNA that regulates the targeting gene expression mostly at post-transcription level. Among these miRNAs, microRNA-133a-3p is reported to be a tumor suppressor in numerous cancers. METHODS:We characterized the down-regulated expression level of microRNA-133a-3p in gastric cancer via TCGA database. Subsequently, we verified the tumor suppressor role of microRNA-133a-3p in gastric cancer cells through a series biological function assay. We used immunofluorescence and transmission electron microscope to observe the negative effect of microRNA-133a-3p on autophagy and used dual-luciferase report assay to identify the candidate gene GABARAPL1 of microRNA-133A-3p.Then we used high performance liquid phase mass spectrometry and seahorse analysis to detect whether miR-133a-3p could block the glutaminolysis metabolism through autophagy. At last, we confirmed the tumor suppressor role of microRNA-133a-3p in vivo on PDX mice model. RESULTS:We demonstrated that microRNA-133a-3p overexpression could block the activation of autophagy to ruin the abnormal glutaminolysis and further inhibit the growth and metastasis of gastric cancer cells. We successfully proved gastric cancer cells can replenish glutaminolysis via autophagy and microRNA-133a-3p could block aforementioned pathway by targeting core autophagy participants GABARAPL1 and ATG13.We then verified the negative function of microRNA-133a-3p on autophagy-mediated glutaminolysis both in PDX model and human gastric cancer organoid model. CONCLUSIONS:MicroRNA-133a-3p targets GABARAPL1 to block autophagy-mediated glutaminolysis, further repressing gastric cancer growth and metastasis.

Background: Recently, the use of immunochemotherapy in the treatment of advanced gastric cancer (GC) has been increasing and programmed cell death protein 1 (PD-1) inhibitors combined with chemotherapy has become the first-line treatment for advanced GC. However, few studies with small sample sizes have examined this treatment regimen to assess its effectiveness and safety in the neoadjuvant treatment phase of resectable local advanced GC. Materials and methods: Herein, we systematically searched PubMed, Cochrane CENTRAL, and Web of Science for clinical trials on neoadjuvant immunochemotherapy (nICT) in advanced GC. The primary outcomes were effectiveness [evaluated by major pathological response (MPR) and pathological complete response (pCR)] and safety [assessed by grade 3-4 treatment-related adverse events (TRAEs) and postoperative complications]. A meta-analysis of non-comparative binary results was performed to aggregate the primary outcomes. Direct comparative analysis was used to compare pooled results of neoadjuvant chemotherapy (nCT) with nICT. The outcomes emerged as risk ratios (RR). Results: Five articles with 206 patients were included, and all of them were from the Chinese population. The pooled pCR and MPR rates were 26.5% (95% CI: 21.3%-33.3%) and 49.0% (95% CI: 42.3%-55.9%), while grade 3-4 TRAEs and post-operative complication rates were 20.0% (95% CI: 9.1%-39.8%) and 30.1% (95% CI: 23.1%-37.9%), respectively. Direct comparison showed that with the exception of grade 3-4 TRAEs and postoperative complications, all outcomes including pCR, MPR, and R0 resection rate favoured nICT to nCT. Conclusion: nICT is a promising strategy for use as an advisable neoadjuvant treatment for patients with advanced GC in Chinese population. However, more phase III randomized controlled trials (RCTs) will be required to further consolidate the efficacy and safety of this regimen.

S1.Detailed SNP information, S2.Relationship of additional SNP and the incidence of rash

Correlation between tumor size, Ki-67 and positive rate of ctDNA mutation.

Background:Colorectal cancer (CRC) is the third most common cancer worldwide and one of the leading causes of cancer-related death. Peptidomics, considered a novel branch of proteomics, has an increasing range of applications in the screening, diagnosis, prognosis, and even monitoring of cancer. However, there is little information for peptidomics analysis in CRC.Methods:In this study, a comparative peptidomic profiling was analyzed in 3 CRC tissue samples and 3 adjacent intestinal epithelial tissue samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Results:Among the total 133 nonredundant peptides identified, 59 were significantly differentially expressed in the CRC samples and benign colonic epithelium conditions [fold change (FC) >2, P<0.05]. Totals of 25 up-regulated and 34 down-regulated peptides were detected, respectively. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to predict the possible functions of these relevant precursor proteins. To clearly identify the potential interaction network of peptide precursors, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to determine protein interactions and a possible central role in CRC.Conclusions:Our results for the first time revealed the differentially expressed peptides between the serous CRC tissue and the adjacent intestinal epithelial tissue samples, and these prominently variable peptides might have an important potential role in occurrence and development of CRC.

Supplementary table 1 Univariate analysis of influence factors of concordance between ctDNA and tissue DNA detections; Supplementary table 2 Concordance analysis between ctDNA and tissue DNA detections in genetic analysis (Tumor size>10cm, n=23); Supplementary table 3 Concordance analysis between ctDNA and tissue DNA detections in genetic analysis (Ki-67>5%, n=19); Supplementary table 4 Univariate analysis of influence factors of prognosis (Log-rank Test)

膀胱癌是泌尿系统最常见的恶性肿瘤之一,具有较高的发病率和病死率.虽然近十几年来针对膀胱癌的治疗手段已经得到了很大的提升,但其治疗效果仍不理想.因此,探索膀胱癌发生发展的具体分子机制以开发新的、更为有效的诊断和治疗方法具有极为重要的临床意义.转移相关的肺腺癌转录物1(MALAT1)是长链非编码RNA家族的重要成员之一,已被证明在膀胱癌的发生发展和转移中起着关键的作用.本文综述了 MALAT1在膀胱癌中的作用机制及其作为膀胱癌诊断、治疗和预后的生物标志物的潜力.

Imatinib dosages and operation treatment.

[This retracts the article DOI: 10.1016/j.omtn.2017.10.008.].

Abstract Objective Laparoscopic-assisted total gastrectomy (LATG) is the most common methods of gastrectomy for gastric cancer (GC). However, totally laparoscopic total gastrectomy (TLTG) is uncommon because reconstruction is difficult, especially for the intracorporeal esophagojejunostomy. In this study, we compared short-term oncologic outcomes in TLTG group with linear anastomosis and LATG group. Methods The retrospective cross-sectional study was conducted. The clinic-pathological data of 108 patients underwent TLTG and 153 patients underwent LATG who were admitted to the First Affiliated Hospital of Nanjing Medical University between May 2016 and October 2019 were collected. The clinicopathological characteristics and surgical outcomes before and after propensity score matching (PSM) were compared between the two groups. Furthermore, a systematic review and meta-analysis were conducted. Results Besides the estimated blood loss(P < 0.001) and the length of incision(P < 0.001) in TLTG group was less than those in LATG group, no other differences were found between the two groups in operative time (P = 0.993), the number of harvested lymph nodes (P = 0.181), the time of first flatus(P = 0.076), start of liquid diet (P = 0.240), start of soft diet (P = 0.147), the time of removing drainage (P = 0.725), postoperative hospital stay (P = 0.688)and postoperative morbidity (P = 1.000) after PSM. The meta-analysis also demonstrated no significant differences in above surgical outcomes among the groups, although the number of harvested lymph nodes was higher and estimated blood loss was lower in the TLTG group than that in the LATG group. Conclusions TLTG for GC is feasible and safe. However, the further validation of high-quality randomized controlled trial is still needed.

BACKGROUND:Previous studies have revealed the critical role of transglutaminase 2 (TGM2) as a potential therapeutic target in cancers, but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer (GC) are not fully understood. In this study, we examined the role and potential mechanism of TGM2 in GC.METHODS:Western blotting, immunohistochemistry, CCK8, colony formation and transwell assays were used to measure TGM2 expression in the GC cells and tissues and to examine the in vitro role of TGM2 in GC. Xenograft and in vivo metastasis experiments were performed to examine the in vivo role of TGM2 in GC. Gene set enrichment analysis, quantitative PCR and western blotting were conducted to screen for potential TGM2 targets involved in GC. Gain/loss-of-function and rescue experiments were conducted to detect the biological roles of STAT1 in GC cells in the context of TGM2. Co-immunoprecipitation, mass spectrometry, quantitative PCR and western blotting were conducted to identify STAT1-interacting proteins and elucidate their regulatory mechanisms. Mutations in TGM2 and two molecules (ZM39923 and A23187) were used to identify the enzymatic activity of TGM2 involved in the malignant progression of GC and elucidate the underlying mechanism.RESULTS:In this study, we demonstrated elevated TGM2 expression in the GC tissues, which closely related to pathological grade, and predicted poor survival in patients with GC. TGM2 overexpression or knockdown promoted (and inhibited) cell proliferation, migration, and invasion, which were reversed by STAT1 knockdown or overexpression. Further studies showed that TGM2 promoted GC progression by inhibiting STAT1 ubiquitination/degradation. Then, tripartite motif-containing protein 21 (TRIM21) was identified as a ubiquitin E3 ligase of STAT1 in GC. TGM2 maintained STAT1 stability by facilitating the dissociation of TRIM21 and STAT1 with GTP-binding enzymatic activity. A23187 abolished the role of TGM2 in STAT1 and reversed the pro-tumor role of TGM2 in vitro and in vivo.CONCLUSIONS:This study revealed a critical role and regulatory mechanism of TGM2 on STAT1 in GC and highlighted the potential of TGM2 as a therapeutic target, which elucidates the development of medicine or strategies by regulating the GTP-binding activity of TGM2 in GC.

目的 建立测定伊马替尼及其代谢物(N-去甲基伊马替尼)血浆蛋白结合率的方法,并应用于胃肠道间质瘤(GIST)患者.方法 以伊马替尼-d8为内标,采用甲醇沉淀蛋白处理样品,以平衡透析法结合液相色谱-串联质谱法测定,并同法检测GIST患者血浆中伊马替尼及其代谢物的游离浓度.结果 伊马替尼在120、4000 ng/mL浓度下,与白蛋白、α1-酸性糖蛋白、球蛋白的血浆蛋白结合率分别为(92.5±1.0)％和(91.7±0.4)％、(56.6±2.0)％和(62.6±2.6)％、(56.3±3.1)％和(68.0±8.6)％;N-去甲基伊马替尼在60、2000 ng/mL浓度下,上述结合率分别为(90.6±3.5)％和(91.3±1.5)％、(54.1±5.1)％和(63.7±1.3)％、(56.2±7.6)％和(67.5±7.3)％.与低浓度伊马替尼(120 ng/mL)及其代谢物(60 ng/mL)比较,高浓度伊马替尼(4000 ng/mL)及其代谢物(2000 ng/mL)与α1-酸性糖蛋白、球蛋白的血浆蛋白结合率显著提高(P<0.05),但与白蛋白的血浆蛋白结合率比较,差异无统计学意义(P>0.05).空白血浆中,高浓度伊马替尼(4000 ng/mL)及其代谢物(2000 ng/mL)的血浆蛋白结合率显著低于低(120、60 ng/mL)、中(750、375 ng/mL)浓度(P<0.01).GIST患者血浆中,伊马替尼及其代谢物的平均血浆蛋白结合率分别为(99.0±0.3)％、(99.2±0.3)％,伊马替尼及其代谢物的浓度与血浆蛋白结合率的相关系数分别为-0.2985、-3.3323(P均小于0.05).结论 成功建立了测定伊马替尼及其代谢物血浆蛋白结合率的方法;GIST患者伊马替尼及其代谢物的血浆蛋白结合率与药物浓度呈负相关.

662 Background: Gastrointestinal stromal tumors (GISTs) are prone to multi-drug resistance after drug treatment, but tissue samples from advanced patients with drug resistance are hard to obtain, impeding the study of the mechanism of drug resistance. In this study, plasma samples from TKI-resistant GIST patients were collected for next generation sequencing (NGS) to detect circulating tumor DNA (ctDNA), which were then used to generate mutation profiles leading to TKI resistance. Methods: A total of 82 GIST patients who acquired TKI resistance were enrolled from Apr. 2016 to Jun. 2021. Plasma ctDNA samples after drug resistance were collected for NGS analysis of 425 genes. Results: Among 82 patients, 54 received monotherapy with imatinib, 24 with 2 TKIs (21 with imatinib and subsequent sunitinib, 2 with imatinib subsequent regorafenib, and 1 with imatinib subsequent anlotinib), and 4 with 3 TKIs (imatinib subsequent sunitinib and regorafenib). Fifty patients (61%) were ctDNA-positive and their mutation profiles were further analyzed. Detection rate of ctDNA in imatinib-resistant patients was 57% (31/54). Among these 31 patients, 12 (39%) showed KIT gene mutation (8 with the known drug-resistant KIT mutations, 6 of these patients exhibited single-drug resistance while 2 showed multi-drug resistance due to KIT mutations mainly in Exon 13 V654A, Exons 17 Y823D, D820Y, N822Y, and N822K). The remaining 19/31 cases (61%) could be attributed to other gene mutations, with 47% due to cancer-associated mutations or other downstream signaling pathways (such as, cell cycle, TP53, RAS and PI3K). Among patients resistant to imatinib followed by other TKIs, ctDNA detection rate was 68% (19/28). Among these 19 patients, 10 (53%) showed KIT gene mutation (including 5 with the known drug-resistant KIT mutations, 4 of these patients exhibited single-drug resistance, while 1 showed multi-drug resistance due to mutations mainly in Exon 13 V654A, Exons 17 D820A, and D820G). The remaining 9/19 cases (47%) could be attributed to other gene mutations, with 44% due to cancer-associated mutations or other in downstream signaling pathways (such as Wnt, cell cycle, TP53, RAS and PI3K). Results indicated a growing trend of mutations leading to resistance among GIST patients treated with multiple TKIs (baseline vs. imatinib resistance vs. multiple TKIs resistance, 3.95% vs. 25.81% vs. 26.32%). Conclusions: Advanced GIST patients who received one or more TKI(s) may be resistant to single or multiple drugs due to complex mutations. NGS can be used to detect ctDNA without the need to obtain tumor tissue samples. This approach facilitates the monitoring of drug resistance in patients with advanced tumors, especially those with multiple metastases. NGS allows detection of mutations at multiple secondary sites and helps to overcome tumor heterogeneity, ctDNA-based NGS analysis will play an increasingly important role in assessment of drug resistance in GIST patients.

AbstractBackgroundChemotherapy can significantly improve the disease‐free survival and overall survival of patients with advanced gastric cancer (GC). 5‐fluorouracil (5‐FU) is frequently applied in the clinic, acting as a first‐line chemotherapy drug of advanced GC, which could be used alone or combining platinum drugs. However, its efficacy is significantly attenuated by chemoresistance, which is associated with patients’ poor survival. Recently, there is evidence suggesting that dysregulation of autophagy may contribute to drug resistance in cancer, and circular RNAs (circRNAs) also take part in chemoresistance. However, whether circRNAs participate in 5‐FU chemoresistance through autophagy remains largely unknown.MethodsRNA sequencing technologies and bioinformatics analysis were performed in GC. Sanger sequencing, Actinomycin D assay and RNase R assay confirmed the circular structure of circular CPM (circCPM). Various cell line models and animal models were used to explore related functions in vitro and in vivo. Quantitative Real‐time PCR (qRT‐PCR), fluorescence in situ hybridization, ribonucleic acid; (RNA) pulldown assays, RNA binding protein immunoprecipitation assays and Luciferase reporter assays were applied to explore involved pathways.ResultscircCPM was up‐regulated in 5‐FU resistant GC cell lines and tissue. Moreover, high circCPM expression is positively associated with poor survival. Silencing circCPM greatly improved chemosensitivity in vitro and in vivo. Mechanistically, it directly binds to miR‐21‐3p in the cytoplasm and therefore increases the expression of PRKAA2, contributing to the activation of autophagy and chemoresistance.ConclusionOur results reveal that circCPM has a crucial role in regulating GC autophagy and 5‐FU resistance by targeting PRKAA2. It may function as a new theory basis for assessing the curative effect of GC and reversing 5‐FU chemoresistance.

Background: This study aimed to compare patient outcomes after laparoscopic distal gastrectomy with uncut Roux-en-Y reconstruction for gastric cancer between a group who underwent a duodenal stump reinforcement procedure and those who did not.Methods: Data from 233 patients with gastric cancer (GC) undergoing distal gastrectomy under laparoscope combined with uncut Roux-en-Y reconstruction were retrospectively investigated. Patients were divided into two groups. The non-reinforcement group (NR) underwent surgery from June 2014 to March 2015 with no reinforcement of the duodenal stump (n=54) and the reinforcement group (R) underwent surgery from April 2015 to June 2018 with reinforcement of the duodenal stump (n=179). In group R, the duodenum was divided using an endoscope-assisted linear stapler, which was reinforced by a purse-string suture along the duodenal staple line. Results: Duodenal stump leakage was observed in 2 patients from group NR (3.7%), while no duodenal stump leakage or fistula was detected in group R. In addition, no significant difference was observed in the patient characteristics between group NR and R.Conclusions: The incidence of duodenal stump leakage can be reduced by reinforcement with a purse-string suture.