Prognostic and predictive blood biomarkers in gastric cancer and the potential application of circulating tumor cells

Gastric cancer(GC), with its high incidence and mortality rates, is a highly fatal cancer that is common in East Asia particularly in China. Its recurrence and metastasis are the main causes of its poor prognosis. Circulating tumor cells(CTCs) or other blood biomarkers that are released into the circulating blood stream by tumors are thought to play a crucial role in the recurrence and metastasis of gastric cancer. Therefore, the detection of CTCs and other blood biomarkers has an important clinical significance; in fact, they can help predict the prognosis, assess the staging, monitor the therapeutic effects and determine the drug susceptibility. Recent research has identified many blood biomarkers in GC, such as various serum proteins, autoantibodies against tumor associated antigens, and cell-free DNAs. The analysis of CTCs and circulating cell-free tumor DNA(ctDNA) in the peripheral blood of patients with gastric cancer is called as liquid biopsy. These blood biomarkers provide the disease status for individuals and have clinical meaning. In this review, we focus on the recent scientific advances regarding CTCs and other blood biomarkers, and discuss their origins and clinical meaning.

Tumor heterogeneity of gastric cancer: From the perspective of tumor-initiating cell

Gastric cancer(GC) remains one of the most common and malignant types of cancer due to its rapid progression, distant metastasis, and resistance to conventional chemotherapy, although efforts have been made to understand the underlying mechanism of this resistance and to improve clinical outcome. It is well recognized that tumor heterogeneity, a fundamental feature of malignancy, plays an essential role in the cancer development and chemoresistance. The model of tumor-initiating cell(TIC) has been proposed to explain the genetic, histological, and phenotypical heterogeneity of GC. TIC accounts for a minor subpopulation of tumor cells with key characteristics including high tumorigenicity, maintenance of self-renewal potential, giving rise to both tumorigenic and non-tumorigenic cancer cells, and resistance to chemotherapy. Regarding tumor-initiating cell of GC(GATIC), substantial studies have been performed to(1) identify the putative specific cell markers for purification and functional validation of GATICs;(2) trace the origin of GATICs; and(3) decode the regulatory mechanism of GATICs. Furthermore, recent studies demonstrate the plasticity of GATIC and the interaction between GATIC and its surrounding factors(TIC niche or tumor microenvironment). All these investigations pave the way for the development of GATIC-targeted therapy, which is in the phase of preclinical studies and clinical trials. Here, we interpret the heterogeneity of GC from the perspectives of TIC by reviewing the above-mentioned fundamental and clinical studies of GATICs. Problems encountered during the GATIC investigations and the potential solutions are also discussed.

High circulating tumor cell concentrations in a specific subtype of gastric cancer with diffuse bone metastasis at diagnosis

AIM: To clarify the biological feature contributing to gastric cancer with diffuse bone metastases at diagnosis.METHODS: The participants visited the Department of Clinical Oncology, Akita University Hospital, from January 2014 to August 2015. The selection criterion for gastric cancer with diffuse bone metastases at diagnosis includes over 29 hot spots of bone scintigraphy. Circulating tumor cell were collected from 20 m L of peripheral venous blood drawn using a Cell Search kit and a Cell Tracks Auto Prep system by SRL, a clinical laboratory. The endpoints of this study were correlations between circulating tumor cells(CTC) count and therapeutic outcomes. RESULTS: Among 39 patients with gastric cancer, 5 patients met the criterion. The incidence of this subtype was 12.8%. CTC counts ranged from 235 to 6440 cells/7.5 m L of peripheral blood(median of 1724). These values were much higher than common gastric cancers(2 cells). In chemo-sensitive cases, CTC counts decreased within 14 d(median) from 275, 235 and 1724 to 2, 7 and 66, respectively. On the other hand, CTC counts increased after treatment failure or insensitive case from 2, 7 and 6440 to 787, 513 and 7885, respectively. The correlation between CTC count and survival time showed a trend, but did not reach significance(Y = 234.6- 0.03 X, P = 0.085).CONCLUSION: High CTC count is a biological hallmark of this subtype, and can be used as a direct and definitive indicator of therapeutic outcome.

Aneuploidy of chromosome 8 in circulating tumor cells correlates with prognosis in patients with advanced gastric cancer

Objective： Previous work indicated that aneuploidy of chromosome 8 in circulating tumor cells（CTCs）correlated with therapeutic efficacy for advanced gastric cancer（AGC） patients. In this follow-up study performed on the same population of AGC patients, we investigated whether and how aneuploidy of chromosome 8 in CTCs correlates with patients＇ clinical prognosis.Methods： The prospective study was performed on 31 patients with newly diagnosed AGC. Previously established integrated subtraction enrichment（SE） and immunostaining-fluorescence in situ hybridization（i FISH）platform was applied to identify, enumerate and characterize CTCs. Quantification of CTCs and analysis of their aneuploidy of chromosome 8 were performed on patients before and after therapy.Results： CTCs were measured in 93.5% of AGC patients, and two CTC subtypes with diverse threshold values were identified, multiploid CTCs with the threshold of ≥2 per 7.5 m L and multiploid plus triploid CTCs with the threshold of ≥4, which were found to significantly correlate with poor progression-free survival（PFS） and overall survival（OS）. In particular, patients with ≥10% increased multiploid CTCs after an initial 6 weeks of therapy had poor PFS and OS, whereas improved PFS and OS were observed on those who had ≥10% decreased multiploid CTCs. After adjusting for clinically significant factors, ≥10% increased post-therapy multiploid CTCs was the only independent predictor of PFS and OS.Conclusions： Aneuploidy of CTCs correlates with prognosis of AGC patients. Quantitative comparison monitoring multiploid CTCs before and after therapy may help predict improved or inferior prognosis and chemoresistance.

Update on a tumor-associated NADH oxidase in gastric cancer cell growth

Gastric cancer is one of the most common human malignancies, and its prevalence has been shown to be well-correlated with cancer-related deaths worldwide. Regrettably, the poor prognosis of this disease is mainly due to its late diagnosis at advanced stages after the cancer has already metastasized. Recent research has emphasized the identification of cancer biomarkers in the hope of diagnosing cancer early and designing targeted therapies to reverse cancer progression. One member of a family of growth-related nicotinamide adenine dinucleotide(NADH or hydroquinone) oxidases is tumor-associated NADH oxidase(t NOX; ENOX2). Unlike its counterpart CNOX(ENOX1), identified in normal rat liver plasma membranes and shown to be stimulated by growth factors and hormones, t NOX activity purified from rat hepatoma cells is constitutively active. Its activity is detectable in the sera of cancer patients but not in those of healthy volunteers, suggesting its clinical relevance. Interestingly, t NOX expression was shown to be present in an array of cancer cell lines. More importantly, inhibition of t NOX was well correlated with reduced cancer cell growth and induction of apoptosis. RNA interference targeting t NOX expression in cancer cells effectively restored non-cancerous phenotypes, further supporting the vital role of t NOX in cancer cells. Here, we review the regulatory role of t NOX in gastric cancer cell growth.

Evaluation of epithelial-mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer: Relevance to therapy response

AIM: To evaluate the epithelial-to-mesenchymal transition(EMT) of circulating tumor cells(CTCs) in gastric cancer patients.METHODS: We detected tumor cells for expression of four epithelial(E^+) transcripts(keratins 8, 18, and 19 and epithelial cell adhesion molecule) and two mesenchymal(M^+) transcripts(Vimentin and Twist) by a quantifiable, dual-colorimetric RNA-in situ hybridization assay. Between July 2014 and October 2014, 44 patients with gastric cancer were recruited for CTC evaluation. Blood samples were obtained from selected patients during the treatment course [before surgery, after surgery and at the 6^(th) cycle of XELOX based chemotherapy(about 6 mo postoperatively)].RESULTS: We found the EMT phenomenon in which there were a few biphenotypic E^+/M^+ cells in primary human gastric cancer specimens. Of the 44 patients, the presence of CTCs was reported in 35(79.5%) patients at baseline. Five types of cells including from exclusively E^+ CTCs to intermediate CTCs and exclusively M^+ CTCs were identified(4 patients with M^+ CTCs and 10 patients with M^+ or M^+ > E^+ CTCs). Further, a chemotherapy patient having progressive disease showed a proportional increase of mesenchymal CTCs in the post-treatment blood specimens. We used NCI-N87 cells to analyze the linearity and sensitivity of Can Patrol^(TM) system and the correlation coefficient(R^2) was 0.999.CONCLUSION: The findings suggest that the EMT phenomenon was both in a few cells of primary tumors and abundantly in CTCs from the blood of gastric cancer patients, which might be used to monitor therapy response.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients,early detection and accurate monitoring of diseases are necessary.Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression,and analyses of these changes have been increasingly utilized for diagnostic,prognostic and therapeutic purposes in malignant diseases including gastric cancer(GC).Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations;however,those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities,which may change during the therapeutic process.Therefore,the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized.This concept,so called"liquid biopsy",would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of"tailor-made"cancer management programs.In the blood of cancer patients,the presence and potent utilities of circulating tumor cells(CTCs)and cell-free nucleic acids(cfNAs)such as DNA,mRNA and microRNA have been recognized,and their clinical relevance is attracting considerable attention.In this review,we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients,especially focusing on GC.

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie(XTSJ) decoction on gastric cancer stem-like cells(GCSCs). METHODS: The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor(VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells(GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction.RESULTS: CD44+ GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth(GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density(MVD)(GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium-(82.87% ± 6.53%) and high-dose XTSJ groups(77.43% ± 7.34%) was detected at 24 h in the CD44+ GCSCs group compared with the saline group(95.42% ± 5.76%) and the low-dose XTSJ group(90.74% ± 6.57%)(P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44- groups; significant differences were only detected in the high-dose XTSJ group at 48 h(78.57% ± 6.94%) and 72 h(72.12% ± 7.68%) when compared with the other CD44- groups(P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium(1.76 ± 0.15) and high-dose XTSJ(1.33 ± 0.081) groups compared with the GCSCs control group(2.72 ± 0.25) and the low-dose XTSJ group(2.51 ± 0.25)(P < 0.05). We also detected a remarkable decrease of MVD in the medium-(7.10 ± 0.60) and high-dose XTSJ(5.99 ± 0.47) groups compared with the GCSC control group(8.15 ± 0.42) and the low-dose XTSJ group(8.14 ± 0.46)(P < 0.05). Additionally, CD44 expression was decreased in these groups [medium-(4.43 ± 0.45) and high-dose XTSJ groups(3.56 ± 0.31) vs the GCSC control(5.96 ± 0.46) and low dose XTSJ groups(5.91 ± 0.38)](P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression.CONCLUSION: Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.

Aberrant expression of nuclear matrix proteins during HMBA-induced differentiation of gastric cancer cells

AIM: To investigate the aberrant expression of nuclear matrix proteins in human gastric cancer cells before and after hexamethylene bisacetamide (HMBA) treatment.METHODS: Proteomics analysis of differential nuclear matrix proteins was performed by two dimensional electrophoresis polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The expression levels of three nuclear matrix proteins were further confirmed by Western blotting and their locations in nuclear matrix filament were observed by quantum dots-based immunofluorescence.RESULTS: Proteomics analysis showed that 43 protein spots were significantly changed due to HMBA treatment.Fifteen proteins were identified in the HMBAinduced differentiation of gastric tumor cells.Eight proteins spots were down-regulated while seven were up-regulated.Among these proteins,prohibitin,nucleophosmin and hnRNP A2/B1 were significantly decreased in HMBA-treated human gastric cancer cells,and their locations in nuclear matrix were altered by HMBA.Our results proved the alteration of specific nuclear matrix proteins during the differentiation of human gastric cancer cells.And the aberrant expressions of nuclear matrix proteins were of significance in revealing the regulatory mechanism of tumor cell proliferation and differentiation.CONCLUSION: The aberrant expressions and intracellular redistributions of nuclear matrix proteins before and after HMBA treatment indicated that nuclear matrix proteins play a pivotal role in the differentiation of gastric cancer cells.

Multifocal G1-G2 gastric neuroendocrine tumors:Differentiating between Type Ⅰ, Ⅱ and Ⅲ, a clinicopathologic review

Gastric neuroendocrine tumors (gNETs) are a rare entity that is increasing in incidence.Different pathophysiological processes can lead to the development of these tumors,appropriate histological analysis is necessary to differentiate between grade 1 (G1) and grade 2 (G2) tumors as this will impact the management of these patients based on their increased risk of lymph node and distant metastases.To provide a comprehensive clinicopathologic review of multifocal gastric neuroendocrine tumors,with particular emphasis on G1 and G2 tumors and differentiating between types I,II and II and risk stratification based upon immunohistochemical profile.This review is based on peer-reviewed literature and the authors’ experience.gNETs are a heterogenous group of tumors that is rising in incidence.These lesions while arise from the same cell type,they have different etiologies.Identifying the type of gNETs is a collective effort of clinical and pathologic correlation.The correct grading and staging of these lesions are of paramount significance,due its impact on patient management and prognosis.

Coexistence of gastrointestinal stromal tumor, esophageal and gastric cardia carcinomas

Gastric gastrointestinal stromal tumor (GIST), esophageal squamous cell carcinoma and gastric cardia adenocarcinoma are distinct neoplasms originating from different cell layers; therefore, simultaneous development of such carcinomas is relatively rare. Auxiliary examinations revealed coexistence of esophageal and gastric cardia carcinoma with lymph node metastasis in a 77-year-old man. Intraoperatively, an extraluminal tumor (about 6.0 cm × 5.0 cm × 6.0 cm) at the posterior wall of the gastric body, a tumor (about 2.5 cm × 2.0 cm) in the lower esophagus, and an infiltrative and stenosing tumor (about 1.0 cm × 2.0 cm) in the gastric cardia were detected. Wedge resection for extraluminal gastric tumor, radical esophagectomy for lower esophageal tumor, and cardiac resection with gastroesophageal (supra-aortic arch anastomoses) were performed. Postoperative histological examination showed synchronous occurrence of gastric GIST, esophageal squamous cell carcinoma, and gastric cardia adenocarcinoma. Furthermore, immunohistochemistry indicated strong staining for c-Kit/CD117, Dog-1, Ki-67 and smooth muscle, while expression of S-100 and CD34 was negative.

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer

AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the Iso Flux platform. Paired specimens taken during the same blood draw(n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo(median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs(n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens(mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected(P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count(> 17) was associated with poorer overall survival(OS)(n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS(HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 cells.

STUDY ON EFFECTS OF ARSENIC TRIOXIDE ON GASTRIC CANCER CELL LINES

Objective To evaluate the effects of arsenic trioxide (As-2O-3) on apoptosis and differentiation of gastric cancer cell lines (GCCL). Methods MKN45 and SGC7901 cells were treated with As-2O-3 at different concentrations, then the apoptosis rates and cell cycle were determined by flow cytometry assays, the morphologic changes were observed under fluorescence microscopy and electronic microscopy, and the gene expressions were tested with immunohistologic staining. Results Higher apoptosis rates of GCCL were seen in the As-2O-3-treated group at concentrations of 5μmol and 10μmol, as compared with those in the 5-Fu-treated group. Cell-nuclear pyknosis and chromosomal condensation were observed. The As-2O-3 at a concentration of 0.5 μmol could induce the cell cycle changes of GCCL, revealing an increase in the proportion of G1/G0 phase cells and a decrease in the proportion of S phase cells. From the fifth day after treatment of SGC7901 with As-2O-3 at a low concentration, P53 and bcl-XL genes expression rates were reduced, Bax gene expression rate increased, and bcl-2 gene expression showed little change. Conclusion As-2O-3 could induce GCCL apoptosis at a high concentration and differentiation at a low concentration, but it could not completely reverse the malignant biological behaviours of cancer cells.

Oxyntic gland adenoma endoscopically mimicking a gastric neuroendocrine tumor: A case report

Gastric adenocarcinoma is one of the most common malignancies worldwide.Histochemical and immunohistologic analyses classify the phenotypes of gastric adenocarcinoma into several groups based on the variable clinical and pathologic features.A new and rare variant of gastric adenocarcinoma with chief cell differentiation(GA-CCD)has recently been recognized.Studies reporting the distinct clinicopathologic characteristics proposed the term oxyntic gland polyp/adenoma because of the benign nature of the GACCD.Typically,GA-CCD is a solitary mucosal lesion that develops either in the gastric cardia or fundus.Histologically,this lesion is characterized by tightly clustered glands and anastomosing cords of chief cells.Immunohistochemically,GA-CCD is diffusely positive for mucin(MUC)6 and negative for MUC2and MUC5AC.However,other gastric tumors such as a gastric neuroendocrine tumor or fundic gland polyp have been difficult to exclude.Because GA-CCD tends to be endoscopically misdiagnosed as a neuroendocrine tumor or fundic gland polyp,comprehensive assessment and observation by an endoscopist are strongly recommended.Herein,we report a rare case of oxyntic gland adenoma endoscopically mimicking a gastric neuroendocrine tumor that was successfully removed by endoscopic mucosal resection.

Tumor progression-dependent angiogenesis in gastric cancer and its potential application

Despite improvements in the early diagnosis,prognosis and therapeutic strategies for gastric cancer(GC),human GC remains one of the most frequently diagnosed malignant tumors in the world,and the survival rate of GC patients remains very poor.Thus,a suitable therapeutic strategy for GC is important for prolonging survival.Both tumor cells themselves and the tumor microenvironment play an important role in tumorigenesis,including angiogenesis,inflammation,immunosuppression and metastasis.Importantly,these cells contribute to gastric carcinogenesis by altering the angiogenic phenotype switch.The development,relapse and spreading of tumors depend on new vessels that provide the nutrition,growth factors and oxygen required for continuous tumor growth.Therefore,a state of tumor dormancy could be induced by blocking tumor-associated angiogenesis.Recently,several antiangiogenic agents have been identified,and their potential for the clinical management of GC has been tested.Here,we provide an up-to-date summary of angiogenesis and the angiogenic factors associated with tumor progression in GC.We also review antiangiogenic agents with a focus on the anti-vascular endothelial growth factor receptor(VEGFR)-mediated pathway for endothelial cell growth and their angiogenesis ability in GC.However,most antiangiogenic agents have reported no benefit to overall survival(OS)compared to chemotherapy alone in local or advanced GC.In phase III clinical trials,only ramucirumab(anti-VEGFR blocker)and apatinib(VEGFR-TKI blocker)have reported an improved median overall response rate and prolonged OS and progression-free survival outcomes as a 2 nd-line agent combined with chemotherapy treatment in advanced GC.By providing insights into the molecular mechanisms of angiogenesis associated with tumor progression in GC,this review will hopefully aid the optimization of antiangiogenesis strategies for GC therapy in combination with chemotherapy and adjuvant treatment.

Cell-type specificity of β-actin expression and its clinicopathological correlation in gastric adenocarcinoma

AIM:To investigate cell type specific distribution ofβ-actin expression in gastric adenocarcinoma and its correlation with clinicopathological parameters.METHODS:β-actin is a housekeeping gene,frequently used as loading control,but,differentially expresses in cancer.In gastric cancer,an overall increased expression ofβ-actin has been reported using tissue disruptive techniques.At present,no histological data is available to indicate its cell type-specific expression and distribution pattern.In the present study,we analyzedβ-actin expression and distribution in paired normal and tumor tissue samples of gastric adenocarcinoma patients using immunohistochemistry(IHC),a tissue non-disruptive technique as well as tissue disruptive techniques like reverse transcriptase-polymerase chain reaction(RT-PCR)and western blotting.Correlation ofβ-actin level with clinicopathological parameters was done using univariate analysis.RESULTS:The results of this study showed significant overexpression,at both mRNA and protein level in tumor tissues as confirmed by RT-PCR(1.47±0.13 vs2.36±0.16;P<0.001)and western blotting(1.92±0.26 vs 2.88±0.32;P<0.01).IHC revealed thatβ-actin expression is majorly distributed between epithelial and inflammatory cells of the tissues.Inflammatory cells showed a significantly higher expression compared to epithelial cells in normal(2.46±0.13 vs 5.92±0.23,P<0.001),as well as,in tumor tissues(2.79±0.24 vs6.71±0.14,P<0.001).Further,comparison of immunostaining between normal and tumor tissues revealed that both epithelial and inflammatory cells overexpressβ-actin in tumor tissues,however,significant difference was observed only in inflammatory cells(5.92±0.23vs 6.71±0.14,P<0.01).Moreover,combined expression in epithelial and inflammatory cells also showed significant increase(4.19±0.15 vs 4.75±0.14,P<0.05)in tumor tissues.In addition,univariate analysis showed a positive correlation ofβ-actin level of inflammatory cells with tumor grade(P<0.05)while epithelial cells exhibited negative correlation(P>0.05).CONCLUSION:In gastric cancer,β-actin showed an overall higher expression predominantly contributed by inflammatory or tumor infiltrating immune cells of the tissue microenvironment and correlates with tumor grade.

叶酸受体阳性循环肿瘤细胞检测在胃癌患者中的临床意义及相关性分析

目的 研究叶酸受体阳性循环肿瘤细胞(folate receptor positive circulating tumor cells, FR+CTC)检测在胃癌患者中的临床意义。方法 回顾性分析术前检测过FR+CTC表达量的胃癌手术患者的临床资料。绘制FR+CTC、白蛋白、总蛋白、血红蛋白、CA125、CA199及CEA预测胃癌患者合并腹膜转移、淋巴结转移、脉管侵犯、神经侵犯、肿瘤突破浆膜层及进展期胃癌的ROC曲线。研究FR+CTC值与胃肠道肿瘤标志物和临床常用营养指标相关的相关性。探讨FR+CTC值及临床相关指标在不同类型胃癌中表达量的统计学差异情况。结果 FR+CTC诊断胃癌患者合并腹膜转移、淋巴结转移、脉管侵犯、神经侵犯、肿瘤突破浆膜层及Ⅲ~Ⅳ期胃癌的AUC值分别为0.933、0.653、0.614、0.628、0.714、0.703,明显优于传统肿瘤标志物(CEA、CA199、CA125)。通过Spearman相关性分析显示,FR+CTC值与白蛋白含量呈负相关(R=-0.26,P=0.0043)。FR+CTC表达量在腹膜转移、淋巴结转移、脉管侵犯、神经侵犯、肿瘤突破浆膜层及Ⅲ~Ⅳ期胃癌患者中明显增加,差异均有统计学意义(P<0.05)。结论 FR+CTC值较传统肿瘤标志物能更准确地预测胃癌患者合并腹膜转移、淋巴结转移、脉管侵犯、神经侵犯、肿瘤突破浆膜层及肿瘤处于Ⅲ~Ⅳ期的概率,并且FR+CTC在这些类型胃癌患者中表达量明显增加,可常规作为新型的临床肿瘤标志物。

胃癌循环肿瘤细胞与临床病理特征及预后的相关性研究

目的研究胃癌循环肿瘤细胞(CTCs)及其亚型与临床病理特征及预后的相关性。方法采取前瞻性研究方法,随机选择2015年5月至2019年1月期间在广西医科大学附属肿瘤医院胃肠外科就诊,经病理诊断为胃腺癌并符合入组条件的初始治疗患者作为研究对象,利用Canpatrol TM技术平台富集分离入组病例术前外周血CTCs,采取多重原位mRNA杂交技术对CTCs进行鉴定、分型并计数;分析CTCs及其亚型与患者年龄、性别、分化程度、神经侵犯、脉管侵犯、Lauren分型、肿瘤部位、浸润深度(T分期)、淋巴结转移(N分期)、AJCC病理分期之间的相关性;对所有入组病例进行随访,分析CTCs与预后相关性。结果共入组病例58例,其中男性37例,女性21例,平均年龄54.4岁;基于Canpatrol TM技术平台,将CTCs分为上皮型(eCTCs)、混合型(mixCTCs)及间质型(mCTCs)3个亚型;CTCs总检出率为90.7%,其中eCTCs检出率68.5%,mixCTCs检出率74.1%;mCTCs检出率61.1%;CTCs及其亚型检出数量与年龄及Lauren分型相关,年龄<60岁的胃癌病例术前外周血CTCs总数及mixCTCs显著低于年龄≥60岁的胃癌病例,P=0.016;Lauren肠型胃癌中CTCs总数及eCTCs、mCTCs数量显著高于弥漫型胃癌(P<0.05);CTCs及其亚型检出数量与病理分期无显著相关性(P>0.05),但当按Lauren分型进行分层后,肠型胃癌组eCTCs数量与病理分期显著正相关(P<0.05);CTCs及其亚型检出数量与性别、神经脉管侵犯、肿瘤部位、浸润深度(T分期)、淋巴结转移(N分期)等指标均无明显相关(P>0.05);本组病例平均随访时间50个月尚未到达中位生存时间,进一步COX回归分析提示肿瘤TNM分期、Lauren分型、神经侵犯是胃癌独立预后因子,CTCs检出数量与生存预后无相关性。结论胃癌外周血CTCs检测到不同上皮-间质标记亚群,提示胃癌外周血CTCs存在上皮-间质转化(EMT)现象;胃癌外周血CTCs及其亚型数量与Lauren分型显著相关,肠型胃癌CTCs检出数量与病理分期关系密切,提示胃癌CTCs的检测在肠型胃癌的预后监测方面可能更有应用价值。

骨形态发生蛋白拮抗剂GREM1作为胃癌肿瘤微环境免疫活性预测指标的临床价值

目的:筛选出可预测胃癌(GC)肿瘤微环境(TME)免疫活性的预后基因。方法:收集55例GC患者术后石蜡组织标本及其对应的癌旁组织;从TCGA数据库和GEO数据库中共下载了976例GC患者的转录组数据及临床数据;采用估计算法(ESTIMATE)和反卷积算法(CIBERSORT)分别评估各样本中免疫/基质得分及免疫细胞浸润程度;R语言中的“limma”包筛选差异表达基因(DEGs);采用单变量Cox回归分析筛选具有预后价值的DEGs;qRT-PCR检测枢纽基因mRNA的表达情况;GSEA探究GREM1的潜在生物学功能。采用TISIDB和CellMiner数据库分析GREM1与免疫特征分子及药物敏感性的相关性。结果:免疫评分与GC患者的预后呈正相关;在免疫得分和基质得分高、低分组中共筛选出40个共享的TME相关DEGs;通过对DEGs进行单变量Cox回归分析,得到GREM1、SFRP2、CYP1B1和MGP共4个具有预后意义的共享DEGs。通过比较基因在肿瘤与癌旁组织的表达差异及与免疫微环境的密切程度,发现GREM1最可能对TME中的免疫重塑发挥作用;GREM1的表达与GC患者的临床病理特征(TNM)呈正相关,而与生存率呈负相关;GSEA结果显示GREM1高表达组主要富集于免疫相关通路,GREM1的表达与M2型巨噬细胞呈正相关,而与CD8+T细胞呈负相关;GREM1与免疫抑制剂TGF-β1、免疫增强剂ENTPD1、趋化因子CCL14及受体CCR2呈正相关;GREM1高表达的肿瘤细胞对抗肿瘤药物维莫德吉的治疗敏感性最强。结论:GREM1可作为反映GC TME免疫抑制状态的临床指标。