Randomized clinical trial on D2 lymphadenectomy versus D2 lymphadenectomy plus complete mesogastric excision in patients undergoing gastrectomy for cancer (DCGC01 study).

BACKGROUND: It is unknown whether D2 lymphadenectomy + complete mesogastric excision for gastric cancer improves survival compared with just D2 lymphadenectomy. METHODS: Between September 2014 and June 2018, patients with advanced gastric cancer were randomly assigned (1 : 1) to laparoscopic D2 lymphadenectomy or D2 lymphadenectomy + complete mesogastric excision gastrectomy. The modified intention-to-treat population was defined as patients who had pathologically confirmed gastric adenocarcinoma (pT1 N1-3 M0 and pT2-4 N0-3 M0). The primary endpoint was 3-year disease-free survival. Secondary endpoints were the recurrence pattern and overall survival. RESULTS: The median follow-up of patients in the D2 lymphadenectomy group (169 patients) and patients in the D2 lymphadenectomy +complete mesogastric excision group (169 patients) was 55 (interquartile range 37-60) months and 51 (interquartile range 40-60) months respectively. Recurrence occurred in 50 patients in the D2 lymphadenectomy group (29.6%) versus 33 patients in the D2 lymphadenectomy + complete mesogastric excision group (19.5%) (P = 0.032). The 3-year disease-free survival was 75.5% (95% c.i. 68.3% to 81.3%) in the D2 lymphadenectomy group versus 85.0% (95% c.i. 78.7% to 89.6%) in the D2 lymphadenectomy + complete mesogastric excision group (log rank P = 0.042). The HR for recurrence in the D2 lymphadenectomy + complete mesogastric excision group versus the D2 lymphadenectomy group was 0.64 (95% c.i. 0.41 to 0.99) by Cox regression (P = 0.045). The 3-year overall survival rate was 77.5% (95% c.i. 70.4% to 83.1%) in the D2 lymphadenectomy group versus 85.8% (95% c.i. 79.6% to 90.2%) in the D2 lymphadenectomy + complete mesogastric excision group (log rank P = 0.058). The HR for death in the D2 lymphadenectomy + complete mesogastric excision group versus the D2 lymphadenectomy group was 0.64 (95% c.i. 0.41 to 1.02) (P = 0.058). CONCLUSION: Compared with conventional D2 dissection, D2 lymphadenectomy + complete mesogastric excision is associated with better disease-free survival, but there is no statistically significant difference in overall survival. REGISTRATION NUMBER: NCT01978444 (http://www.clinicaltrials.gov).

Surgical and oncological outcomes of laparoscopic right hemicolectomy (D3 + CME) for colon cancer: A prospective single-center cohort study.

BACKGROUND: Complete mesocolic excision (CME) or D3 lymphadenectomy led to survival benefits for locally advanced right colon cancer, but with vague definitions in anatomy and debated surgical hazard in clinic. Aiming to achieve a precise definition of it in anatomy, we proposed laparoscopic right hemicolectomy (D3 + CME) as a novel procedure for colon cancer. However, the surgical and oncological results of this procedure in clinic were uncertain. METHODS: We performed a cohort study involving prospective data collected from a single-center in China. Data from all patients who underwent right hemicolectomy between January 2014 and December 2018 were included. We compared the surgical and oncological outcomes between D3 + CME and conventional CME. RESULTS: After implementation of exclusion criteria, a total of 442 patients were included. D3 + CME group performed better in lymph nodes harvested (25.0 [17.0, 33.8] vs. 18.0 [14.0, 25.0], P < 0.001) and the proportion of intraoperative blood loss ≥ 50 mL (31.7% vs. 51.8%, P < 0.001); no significant difference was observed in the complication rates between two groups. Kaplan-Meier analysis demonstrated that a better cumulative 5-year disease-free survival (91.3% vs. 82.2%, P = 0.026) and a better cumulative 5-year overall survival (95.2% vs. 86.1%, P = 0.012) were obtained in the D3 + CME group. Multivariate COX regression revealed that D3 + CME was an independent protective factor for disease-free survival (P = 0.026). CONCLUSION: D3 + CME could improve surgical and oncological outcomes simultaneously for right colon cancer compared to conventional CME. Large-scale randomized controlled trials were further required to confirm this conclusion, if possible.

Short-term outcomes of D2 lymphadenectomy plus complete mesogastric excision for gastric cancer: a propensity score matching analysis.

BACKGROUND: Our previous study has demonstrated the surgical advantages of D2 lymphadenectomy plus complete mesogastric excision (D2 + CME) in gastric cancer surgery. To further verify the safety of D2 + CME procedure, we conducted this large-scale, observational cohort study and applied propensity score matching (PSM) approach to compare D2 + CME with conventional D2 in terms of short-term outcomes in gastric cancer patients. METHODS: Data on 855 patients from Tongji Hospital who underwent laparoscopic-assisted distal gastrectomy (LADG) with R0 resection (496 in the conventional D2 cohort and 359 in the D2 + CME cohort) between Dec 12, 2013 and Dec 28, 2017 were retrieved from prospectively maintained clinical database. After PSM analysis at a 1:1 ratio, each cohort included 219-matched patients. Short-term outcomes, including surgical results, morbidity, and mortality within 30 days after the operation, were collected and analyzed. RESULTS: In this large-scale, observational cohort study based on PSM analysis, the D2 + CME procedure showed less intra-laparoscopic blood loss, more lymph node harvest, and faster postoperative flatus than the conventional D2 procedure. However, both the overall and severe postoperative adverse events (Clavien-Dindo classification grade ≥ III a) seemed comparable between two cohorts. CONCLUSION: The present study showed that D2 + CME was associated with better short-term outcomes than conventional D2 dissection for patients with resectable gastric cancer.

An Optimal Approach for Laparoscopic D3 Lymphadenectomy Plus Complete Mesocolic Excision (D3+CME) for Right-Sided Colon Cancer.

BACKGROUND: It is common knowledge that high ligation of blood vessels at the D3 level and complete mesocolic excision (CME) are two critical points of right hemicolectomy for right colon cancer (RCC). 1-5 To date, a safe strategy for completing these two procedures under laparoscopic surgery has not been extensively described. The authors provide a video to demonstrate laparoscopic right hemicolectomy (D3 + CME) with an optimal mesentery-defined approach. By identifying three "tri-junctions," this approach facilitates dissection of the entire mesocolon along the embryologic planes as far centrally as possible and enables the high tie of feeding vessels at bifurcation. The authors propose that this approach is safe, decreases blood loss, and is a secure method for right colon cancer intervention. METHODS: Between June 2014 and June 2015, the study recruited 36 patients with informed consent, and these patients underwent laparoscopic D3+CME for right colon cancer by a single surgeon. All the participants provided informed written consent to participate in the study. This study was approved by the Tongji Hospital Ethics Committee. The patients' demographics, oncologic charac- teristics, postoperative outcomes within 30 days, and follow-up data were collected. The perioperative outcomes included blood lost, number of retrieved lymph nodes, postoperative hospital length of stay, and morbidity. The postoperative 30-day morbidity included cardiovascular, pulmonary, and urinary complications, as well as wound infection, anastomotic leakage, and postoperative ileus. The complications were diagnosed and categorized based on relevant clinical manifestations. For this procedure, all patients are placed in the Trendelenburg position, with five trocars inserted. Carbon dioxide (CO2) is inflated through the intraumbilical trocar, maintaining steady intraabdominal pressure. The operating surgeon stands between the patient's legs, with the camera holder on the left and the assistant on the right. The operation table will be rotated left side up to redistribute the small bowels. The standard surgical procedures shown in the video are as follows. First, the surgeon identifies the first "tri-junction" (TJ1) in the ileocolic area (TJ1 is the fusion point of the mesocolon, the visceral peritoneum, and the intestinal mesentery). The surgeon then incises along the fusion fascia and separates the loose connective tissues with an ultrasonically activated device. Mobilization is continued to the origins of the ileocolic vessels, which are clipped and cut. The posterior mesocolic fascia is bluntly separated from the inferior mesentery bed, which is formed by duodenum, Gerota's fascia, and nearby structures. The second part of duodenum and the head of pancreas are exposed. Next, the surgeon mobilizes along the superior mesentery vein (SMV) and superior mesentery artery (SMA), with blunt dissection of the covering fascia and loose connective tissue to preserve the entire mesocolon completely and as far centrally as possible. Careful dissection is continued until the middle colic vessels (middle colic vein and middle colic artery) are reached. Afterward, the superior right colic vein of Henle's trunk is exposed and divided at the root. One pack of gauze is inserted beneath the mobilized mesocolon. Second, the surgeon divides the greater omentum. Entrance to the omental bursa is established after the second "tri-junction" (TJ2) is identified (TJ2 is the fusion point of the transverse mesocolon, the mesogastrium and the greater omentum). The fusion plane is bluntly separated between the transverse mesocolon (TM) and the right gastroepiploic mesentery (RGEM) until the previously placed gauze is exposed. Finally, the third "tri-junction" (TJ3) is identified (TJ3 is the fusion point of the retroperitoneum, the mesocolon, and the lateral peritoneum) at the inferior attachments of cecum. The ascending colon is freed up with mobilization of the lateral retroperitoneal attachments from the cecum to the hepatic flexture. Special attention should be paid to avoid breaking the fascia renalis. The tumor carrying the colon is exteriorized through an abdominal incision with a wound protector. Continuity of the digestive tract is performed extracorporeally with side-to-side ileotransverse colon anastomosis using a linear stapler. All the treatments follow standardized recovery protocols. RESULTS: This study recruited 20 males and 16 females. The median age was 56.5 years, and the median body mass index (BMI) was 22.1 kg/m2. Twelve patients had experienced previous abdominal surgery. No intraoperative complications occurred. The tumor was located in the ileocecus of 14 patients and in the hepatic flexture of 22 patients (Supplemental Table 1). The median number of retrieved lymph nodes was 20 (interquartile range [IQR], 14.8-27 (Supplemental Table 2). The median volume of blood lost was 5 ml (IQR 5-10 ml). The median postoperative hospital stay was 10 days (IQR 9-12.3 days). One patient received treatments from the intensive care unit (ICU). One patient underwent reoperation for incision dehiscence. Seven patients had a postoperative complication diagnosed within 30 days (Supplemental Table 3). The median follow-up period was 12 months (IQR 3-20) months. All the patients received adjuvant chemotherapy, with no case of recurrence (Supplemental Table 4). CONCLUSION: An optimal mesentery-defined approach for laparoscopic D3 + CME allows for ligation of feeding vessels at their bifurcation and for CME to be performed simultaneously with technical efficiency. This procedure is safe and strongly practical for advanced right colon cancer intervention.

Short-term outcomes of laparoscopic D2 lymphadenectomy with complete mesogastrium excision for advanced gastric cancer.

BACKGROUND: D2 lymphadenectomy has been widely accepted as a standard procedure of surgical treatment for local advanced gastric cancer [1, 2]. However, neither the dissection boundary nor the extent of the excision for perigastric soft tissues has been described [3-7]. Our previous researches demonstrate the existence of disseminated cancer cells in the mesogastrium [8, 9] and present an understandable mesogastrium model for gastrectomy [10]. Hence, the D2 lymphadenectomy plus complete mesogastrium excision (D2 + CME) is firstly proposed in this study, aiming to assess the safety, feasibility and corresponding short-term surgical outcomes. METHODS: All of these patients underwent laparoscopy assisted D2 + CME radical gastrectomy with a curative R0 resection, and all the operations were performed by Prof. Jianping Gong, chief of GI surgery of Tongji Hospital, Huazhong University of Science and Technology. All participants provided informed written consent to participate in the study. This study was approved by the Tongji Hospital Ethics Committee. The standard surgical procedures in the video are described as follows. Dissect along the gastrocolic ligament and then toward the left colic flexture with special made gauze. Bluntly separate the adipose tissues to find fascia plane. Expose along the plane toward the splenic inferior polar area. Precede to the origins of left gastroepiploic vessels (LGEVs), clip and cut. All the mobilized adipose tissues in this area are defined as left gastroepiploic mesentery (LGEM) [10]. Next, turn to infra-pyloric area. Dissect the fascia plane between right gastroepiploic mesentery (RGEM) and transverse mesocolon. Turn to the pancreas head, remove the covering adipose tissues, identify the superior mesentery vein and expose the origins of right gastroepiploic vessels (RGEVs). Clip and cut. All the surrounding mobilized adipose tissues are defined as RGEM [10]. Move to the superior boarder of pancreas with the stomach reflected cephalad, incise the serosa and bluntly mobilize through the plane with gauze. Turn to the common hepatic artery (CHA), remove the adherent adipose tissue. Expose the root of left gastric vein, clip and cut. Dissect the thick sheath of left gastric artery, expose at the root, trip clip and cut. All mobilized lateral adipose tissues and dorsal parts are defined as left gastric mesentery (LGM) [10]. Toward right, dissect follow the CHA and hepatic portal vein (HPV). Next, move toward the left side of LGM and dissect along the splenic artery until reaching the posterior gastric wall. Move to the anterior area of stomach and divide the lesser omentum. Clean up the adipose tissue and nerves along the lesser curvature up to the gastroesophageal junction. Expose and cut the right gastric vessels (RGVs) where the mobilized adipose tissues are defined as right gastric mesentery (RGM) [10]. Reconstruction of the alimentary tract was done by extracorporeal anastomosis. Standard recovery protocols were followed in postoperative treatments. RESULTS: Fifty-four patients between September 2014 and March 2015 have been recruited with informed consent and underwent laparoscopic D2 + CME by a single surgeon. The mean number of retrieved regional lymph nodes was 35.04 ± 10.70 (range 14-55). The mean volume of blood loss was 12.44 ± 22.89 ml (range 5-100). The mean laparoscopic surgery time was 127.82 ± 17.63 min (range 110-165). The mean hospitalization time was 11.09 ± 4.28 days (range 8-28). No operative complication was observed during the hospitalization. CONCLUSION: The anatomical boundary of mesogastrium is well described and dissected within D2 + CME surgical process. It proves to be safely feasible and repeatable with less blood lost, qualified lymph nodes retrieval results and other improved short-term surgical outcomes in advanced gastric cancer. Meanwhile, potential disseminated cancer cells fall into the mesogastrium can be eradicated by D2 + CME.

Protocol for analyzing invadopodia formation and gelatin degradation.

Invadopodia are actin-rich protrusions on the tumor cell membrane that degrade the extracellular matrix and play a crucial role in tumor cell invasion and metastasis. Here, we present a protocol to examine invadopodia's ability to form and degrade the extracellular matrix during tumor invasion and metastasis. We detail the procedure for using immunofluorescence staining to indirectly detect invadopodia formation and assess their extracellular matrix degradation capability via the gelatin degradation assay. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis.

A single nucleotide polymorphism in the DAB2IP gene is associated with risk of aggressive prostate cancer (PCa), and loss of DAB2IP expression is frequently detected in metastatic PCa. However, the functional role of DAB2IP in PCa remains unknown. Here, we show that the loss of DAB2IP expression initiates epithelial-to-mesenchymal transition (EMT), which is visualized by repression of E-cadherin and up-regulation of vimentin in both human normal prostate epithelial and prostate carcinoma cells as well as in clinical prostate-cancer specimens. Conversely, restoring DAB2IP in metastatic PCa cells reversed EMT. In DAB2IP knockout mice, prostate epithelial cells exhibited elevated mesenchymal markers, which is characteristic of EMT. Using a human prostate xenograft-mouse model, we observed that knocking down endogenous DAB2IP in human carcinoma cells led to the development of multiple lymph node and distant organ metastases. Moreover, we showed that DAB2IP functions as a scaffold protein in regulating EMT by modulating nuclear beta-catenin/T-cell factor activity. These results show the mechanism of DAB2IP in EMT and suggest that assessment of DAB2IP may provide a prognostic biomarker and potential therapeutic target for PCa metastasis.

DAB2IP suppresses invadopodia formation through destabilizing ALK by interacting with USP10 in breast cancer.

Invadopodia, being actin-rich membrane protrusions, play a vital role in tumor cell invasion and metastasis. Our previous studies have revealed some functions of the DOC-2/DAB2 interacting protein (DAB2IP) as a tumor suppressor. Nevertheless, the specific role and mechanism of DAB2IP in invadopodia formation remain unclear. Here, we find that DAB2IP effectively suppresses invadopodia formation and metastasis in breast cancer, both in vitro and in vivo. Additionally, DAB2IP could downregulate anaplastic lymphoma kinase (ALK), resulting in the inhibition of tyrosine phosphorylation of Cortactin and the prevention of invadopodia formation. DAB2IP competitively antagonizes the interaction between the deubiquitinating enzyme Ubiquitin-specific peptidase 10 (USP10) and ALK, leading to a decrease in the abundance of ALK protein. In summary, DAB2IP impairs the stability of ALK through USP10-dependent deubiquitination, suppressing Cortactin phosphorylation, thereby inhibiting invadopodia formation and metastasis of breast cancer cells. Furthermore, this study suggests a potential therapeutic strategy for breast cancer treatment.

Expression of cyclins in high-density cultured cells and in vivo tumor cells.

It is widely assumed that during the mammalian cell cycle, four major cyclins, including G1 cyclins (D, E) and mitotic cyclins (A, B1), are expressed in an orderly scheduled pattern in exponentially cultured cells. In high-density cultured cells and in vivo growing cells, whether these cyclins are expressed in the same pattern remains unknown. In this study, we investigated the expression of cyclins by flow cytometry and western-blotting in cultured MOLT-4 and HepG2 cells at high-density. We found that, in high-density cultures, the expression levels of cyclin D, E, and A within each cell cycle phase was less than their levels in low-density cultures. Cyclin B1 was slightly reduced in G2/M phase and unexpectedly expressed in G1 phase. The cyclin B1 expressed in G1 phase was localized mainly in the cytoplasm, and its level was positively correlated with apoptosis. Moreover, we obtained similar cyclins expression pattern in inoculated HepG2 tumor cells from immune-deprived mice and leukemic cells from leukemia patients. These findings describe an altered pattern of cyclin expression in high-density cultured cells as well as in vivo tumor cells. We suggest that high-density cultures might mimic the in vivo growth conditions and such experimental situation may be useful for cell cycle research.

DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis.

In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G(0)/G(1) cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP(-/-) animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.

YAP1 induces invadopodia formation by transcriptionally activating TIAM1 through enhancer in breast cancer.

Yes-associated protein 1 (YAP1), a central component of the Hippo pathway, plays an important role in tumor metastasis; however, the underlying mechanism remains to be elucidated. Invadopodia are actin-rich protrusions containing multiple proteases and have been widely reported to promote cell invasiveness by degrading the extracellular matrix. In the present study, we report that YAP1 induces invadopodia formation and promotes tumor metastasis in breast cancer cells. We also identify TIAM1, a guanine nucleotide exchange factor, as a target of the YAP1-TEAD4 complex. Our results demonstrate that YAP1 could promote TEAD4 binding to the enhancer region of TIAM1, which activates TIAM1 expression, subsequently increasing RAC1 activity and inducing invadopodia formation. These findings reveal the functional role of Hippo signaling in the regulation of invadopodia and provide potential molecular targets for preventing tumor metastasis in breast cancer.

DAB2IP suppresses tumor malignancy by inhibiting GRP75-driven p53 ubiquitination in colon cancer.

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibitory role in many signals associated with tumorigenesis. However, the underlying mechanism of this function remains unclear. Our study shows that DAB2IP was positively associated with a good prognosis in patients with colorectal cancer and wild-type p53 expression. An in vitro assay showed that DAB2IP elicited potent tumor-suppressive effects by inhibiting cell invasiveness and colony formation and promoting cell apoptosis in wild-type p53 colon cancer cells. In addition, DAB2IP improved the stability of wild-type p53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we revealed that DAB2IP and p53 interacted with the ubiquitin ligase-related protein GRP75. Mechanistically, DAB2IP is competitively bound to GRP75, thus reducing GRP75-driven p53 ubiquitination and degradation. Moreover, the Ras-GAP domain was required for the DAB2IP-GRP75 interaction and DAB2IP-mediated p53 ubiquitination. Finally, animal experiments revealed that DAB2IP inhibited tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, providing new insight into DAB2IP-induced tumor suppression and a novel molecular interpretation of the p53 pathway.

Complete mesogastric excision for locally advanced gastric cancer: short-term outcomes of a randomized clinical trial.

Implementation of complete mesogastric excision in gastric cancer surgery, named D2 lymphadenectomy plus complete mesogastric excision (D2+CME), has recently been proposed as an optimal procedure. However, the safety and efficacy of D2+CME remain uncertain. In this randomized controlled trial, patients receiving D2+CME exhibit less intraoperative blood loss, more lymph node harvesting, and earlier postoperative flatus than patients receiving conventional D2 radical surgery. Univariate Cox regression analysis reveals that the risk ratio for postoperative flatus in D2+CME group is 1.247 (p = 0.044). Overall postoperative complications are comparable between the two groups, but complications are significantly less severe in the D2+CME group than the D2 group (Clavien-Dindo classification grade ≥ IIIa: 4 D2+CME patients [11.8%] versus 9 D2 patients [33.3%]; p = 0.041). In conclusion, our work shows that D2+CME is associated with better short-term outcomes and surgical safety than conventional D2 dissection for patients with advanced gastric cancer.

Genome-wide identification of CpG island methylator phenotype related gene signature as a novel prognostic biomarker of gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most fatal cancers in the world. Results of previous studies on the association of the CpG island methylator phenotype (CIMP) with GC prognosis are conflicting and mainly based on selected CIMP markers. The current study attempted to comprehensively assess the association between CIMP status and GC survival and to develop a CIMP-related prognostic gene signature of GC. METHODS: We used a hierarchical clustering method based on 2,082 GC-related methylation sites to stratify GC patients from the cancer genome atlas into three different CIMP subgroups according to the CIMP status. Gene set enrichment analysis, tumor-infiltrating immune cells, and DNA somatic mutations analysis were conducted to reveal the genomic characteristics in different CIMP-related patients. Cox regression analysis and the least absolute shrinkage and selection operator were performed to develop a CIMP-related prognostic signature. Analyses involving a time-dependent receiver operating characteristic (ROC) curve and calibration plot were adopted to assess the performance of the prognostic signature. RESULTS: We found a positive relationship between CIMP and prognosis in GC. Gene set enrichment analysis indicated that cancer-progression-related pathways were enriched in the CIMP-L group. High abundances of CD8+ T cells and M1 macrophages were found in the CIMP-H group, meanwhile more plasma cells, regulatory T cells and CD4+ memory resting T cells were detected in the CIMP-L group. The CIMP-H group showed higher tumor mutation burden, more microsatellite instability-H, less lymph node metastasis, and more somatic mutations favoring survival. We then established a CIMP-related prognostic gene signature comprising six genes (CST6, SLC7A2, RAB3B, IGFBP1, VSTM2L and EVX2). The signature was capable of classifying patients into high-and low-risk groups with significant difference in overall survival (OS; p < 0.0001). To assess performance of the prognostic signature, the area under the ROC curve (AUC) for OS was calculated as 0.664 at 1 year, 0.704 at 3 years and 0.667 at 5 years. When compared with previously published gene-based signatures, our CIMP-related signature was comparable or better at predicting prognosis. A multivariate Cox regression analysis indicated the CIMP-related prognostic gene signature was an independent prognostic indicator of GC. In addition, Gene ontology analysis indicated that keratinocyte differentiation and epidermis development were enriched in the high-risk group. CONCLUSION: Collectively, we described a positive association between CIMP status and prognosis in GC and proposed a CIMP-related gene signature as a promising prognostic biomarker for GC.

An Optimal Surgical Approach for Suprapancreatic Area Dissection in Laparoscopic D2 Gastrectomy with Complete Mesogastric Excision.

BACKGROUND: During the radical operation, the suprapancreatic area is featured by anatomical complexity, and the lymph node dissection for this area is technically difficult and demanding.1-4 Previously, we have demonstrated the presence of disseminated cancer cells in the mesogastrium5,6 and presented a mesogastrium model for gastrectomy.7 As a consequence, laparoscopic D2 lymphadenectomy plus complete mesogastric excision (D2+CME) was proposed as a new concept in the surgical treatment of advanced gastric cancer.8 D2+CME procedure has been shown to be associated to lower number of free intraperitoneal cancer cells and with a better disease-free survival than conventional D2 gastrectomy.9 Under the concept of mesogastrium model, the proposed D2+CME procedure could help surgeons better define the anatomical boundaries of suprapancreatic mesogastrium, thus using it to achieve a complete and standard excision of the suprapancreatic area dissection. Here, we briefly present perioperative results of our case series with the laparoscopic curative subtotal gastrectomy and D2+CME with a R0 resection and present a video to detail the technical aspects of a laparoscopic D2+CME approach for suprapancreatic area dissection. METHODS: All patients in this study underwent laparoscopic subtotal gastrectomy (D2+CME) with a curative R0 resection. This study was approved by the Tongji Hospital Ethics Committee (Unique Reference Number: TJ-IRB20180811). The procedures in the video are described as follows. Based on our previous mesogastrium model (also named "Table model", Supplemental Figure 1), the suprapancreatic mesogastrium is attached to the lesser curvature or the posterior gastric wall and extended to the suprapancreatic area, respectively.7 Surgeon stands on patient left side, and the assistant lifts the stomach upward and cephalic to expose the suprapancreatic mesogastrium including left gastric mesentery (LGM), right gastric mesentery (RGM) and posterior gastric mesentery (PGM). First of all, towards to the left side of the suprapancreatic area, the "tri-junction" point of LGM is exposed. Using an energy devise, surgeon opens serosa layer and identifies the retrogastric space. The LGM and PGM are mobilized bluntly, between which a fusion retrogastric space is revealed. Both the LGM and PGM are covered by smooth and shiny surfaces of fascial propria and regarded as the "meso-bed" mutually. Secondly, at the inner side of duodenum, surgeon bluntly separates the adjuvant tissues along gastro-duodenal artery (GDA) upward and exposes right gastric mesentery (RGM). Next, after mobilizing the left gastric mesentery, surgeon removes the adipose tissue adherent to the common hepatic artery (CHA) and exposes the root of the left gastric artery after dissecting the perivascular sheath with triple-clips. Then, surgeon dissects RGM along the CHA and portal vein (HPV) towards the right side of the suprapancreatic area; afterwards, the right gastric vessels and RGM are identified and ligated. Lastly, the superior border of splenic vessels is dissected. The anterior lobe of the PGM is raised up with ligated posterior gastric vessels. Remarkably, posterior gastric vessels may be absent in some cases. Reconstruction of the alimentary tract is Roux-en-Y method. Standard recovery protocols are followed in postoperative treatments. RESULTS: Between August 28th 2017 and December 27th 2018, 107 patients receiving laparoscopic curative subtotal gastrectomy (D2+CME) with a R0 resection were retrospective collected in this study. After exposing the suprapancreatic mesogastrium including RGM, PGM and LGM with D2+CME procedure, the LNs and fat tissues around 7, 9, 8a, 12a and 11p were removed en bloc in all patients. This study recruited 67 males and 40 females. The median age was 55 years, with body mass index (BMI) 23.0 kg/m2 (Supplemental Table 1). The median number of retrieved regional lymph nodes was 31 (range 25-41), including 22 (range 17-27.5) suprapancreatic lymph nodes. The median volume of blood loss was 14 ml (range 6-34). The median total operation time was 287 min (range 265.5-313.5) and laparoscopic surgery time was 132 min (range 116-142) (Supplemental Table 2). Postoperative morbidity occurred at a rate of 9.3 %, and the mortality rate was 0% (Supplemental Table 3). The median follow-up was 10 months (range 8-13). No patient was lost during follow-up (Supplemental Table 4). CONCLUSION: A laparoscopic subtotal gastrectomy with D2+CME procedure provides for a complete and standardized en bloc excision of the suprapancreatic area dissection.

[Silencing of cell cycle checkpoint kinase gene enhances cisplatin-induced apoptosis of lung cancer cells].

OBJECTIVE: To investigate the changes in cell cycle induced by cisplatin (DDP) and the effect of antisense oligonucleotide (AsODN) targeting Chk1/2 on DDP-induced apoptosis in lung cancer cell line A549 cells. METHODS: The characteristics of cell cycle and apoptosis induced by DDP were detected by flow cytometry using SubG1 method. Chk1/2 mRNA and protein expression were assayed by RT-PCR and Western blot under best condition of transfection of AsODN targeting Chk1/2 by lipofection. Apoptosis of A549 cells induced by DDP was determined by flow cytometry using AnnexinV-FITC staining after transfection of Chk1/2 AsODN. RESULTS: Asynchronized A549 cells were treated with 10 micromol/L DDP, and significant S-phase arrest was observed at 12 h later. Transfection with antisense oligonucleotide targeting Chk1/2 inhibited the Chk1/2 expression at both mRNA and protein levels. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 100% - 200%, compared with that in the sODN control (P < 0.05), but combined use of Chk1- and Chk2-specific AsODN did not show synergistic effects as compared with that induced by treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). CONCLUSION: Chk1 and Chk2 may be regarded as effective targets of chemotherapy for lung cancer. Silencing the key effector Chk1 and Chk2 genes may significantly increase the chemosensitivity of lung cancer cells.

[Down-regulation of Chk1/Chk2 gene expression increases apoptosis in irradiated HeLa cells and its mechanism].

OBJECTIVE: To explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action. METHODS: Asynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry. RESULTS: Apoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P < 0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN. CONCLUSION: The radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.