| OBJECTIVES: To review the experience of diagnosis and treatment on gastrointestinalstromal tumors (GIST) in our hospital. To explore the effect of clinical features,pathological features and treatment manners on GIST prognosis. To analyze the effect ofmutation characteristics of c-kit and PDGFRA on GIST prognosis. By using the methodof “small samples for high throughput screening+large samples for validation”, to findthe risk-related microRNAs and encoding-genes in GISTs. To improve the level ofclinical diagnosis and treatment of GIST by studying the biological function and itsmolecular mechanism of these molecules.METHODS:(1) The clinicopathological and follow-up data of479GIST patientsbetween1997and2012admitted in Renji Hospital Shanghai Jiaotong University Schoolof Medicine were collected and retrospectively analyzed. The clinical features,pathological characteristics, operative techniques and drug therapy modality were studied.The impact factors on prognosis were analyzed.(2) The mutation statuses of c-kit andPDGFRA in204GISTs were detected by PCR amplification and direct sequencing. Theclinicopathological features, prognostic factor mutation status and their relationship wereanalyzed.(3) The differential expression profiling of microRNA in12GIST patients wasstudied by utilizing miRCURY LNA microRNA expression array. The screeningresults of array were validated in191formalin-fixed paraffin-embedded GIST tissues byTaqman qPCR. The relative expressions of differential microRNAs in different risk GISTwere compared with One-way ANOVA. The relationship between candidate microRNA expression and patient’s prognosis was analyzed by using Kaplan-Meier model. In vitrocell experiments were used to study the effect of candidate microRNA on cell biologicalfunction including proliferation, migration, invasion and apoptosis. The target gene andrelated signal pathway were predicted by bioinformatics analysis. Finally, the target geneof candidate microRNA was validated by using qPCR, western-blot and luciferase reportgene. Therefore the clinical significance, biological function and its mechanism of themicroRNA were clarified.(4) The differential expression profiling of encoding gene in12GIST patients was studied by utilizing NimbleGen microarray. The screening resultswere validated by SYBR qPCR in fresh frozen GIST tissue. The GIST tissue array wasestablished to validate the differential gene with immunohistochemical staining. Theprotein product of differential gene in GIST and non-GIST population serum wasexamined by ELISA technique.RESULT:(1) Out of479GISTs,276(57.6%) were located in stomach,111(23.2%) insmall intestine,28(5.8%) in duodenum,7(1.7%) in colon,21(4.4%) in rectum,3(0.6%)in esophagus and32(6.7%) in other site (including omentum, peritoneum andmesenterium). According to NIH risk classification,479cases were classified as very lowrisk (33cases,6.9%), low risk (183cases,38.2%), intermediate risk (81cases,16.9%)and high risk (182cases,38.0%) respectively. In immunohistochemical examination, thepositive rate of DOG1, CD117, CD34, S-100and SMA is90.7%,88.1%,79.3%,14.4%and35.3%respectively.92.5%(443cases) operations were radical while7.5%(36cases)operations were palliative.106patient received drug treatment with imatinib mesylate.Among IM treatment cases, the drug was administrated as neoadjuvant therapy in8cases,as adjuvant therapy in81cases and as advanced-stage therapy in25cases respectively. Ofthe435GIST patients who were successfully followed-up, the1-,3-,5-year overallsurvival rate was96.9%,89.2%and82.2%respectively. And the1-,3-,5-yearrelapse-free survival rate was93.7%,80.1%and75.7%respectively. The1-,3-,5-yearoverall survival rate according to risk classification (very low risk, low risk, intermediaterisk and high risk) was100%,100%,89.2%and61.6%respectively. And the1-,3-, 5-year relapse-free survival rate was100%,97.8%,86.8%and47.6%respectively.Univariate analysis revealed sex, tumor site, tumor size, mitotic rate, NIH classification,CD34expression and the radical extent of operation were associated with overall andrelapse-free survival. COX hazard proportional model (Forward LR) revealed that tumorsize, risk classification and radical extent of operation were independent risk factors ofoverall survival in GIST patients. The model also revealed that mitotic rate, riskclassification and radical extent of operation were independent risk factor of relapse-freesurvival in GIST patients. The patients received IM adjuvant therapy had better overallsurvival and relapse-free survival than those who didn’t received IM adjuvant treatment(94.4%v.s.70.6%on5-year OS and81.6%v.s.51.5%on5-year RFS, respectively). Thelate stage GIST patients received IM therapy had better overall survival andprogression-free survival than those who didn’t received IM treatment (23.1%v.s.0.0%on5-year OS and30.8%v.s.10.0%on2-year PFS, respectively).(2) The overallmutation rate of c-kit and PDGFRA in204GISTs was93.6%(191/204). c-kit exon11mutation was the majority of mutation type (159/182,87.3%). More than half (84/159,52.8%) of c-kit exon11mutations were deletion mutations. The area from NO.557to NO.560codon of c-kit exon11was a hot zone of all types of mutations. The recurrence rate ofwild-type GISTs (5/13,38.5%) was high than mutant GISTs. The rate of tumor arisingfrom stomach was higher in GISTs without c-kit exon11mutation (19/24,79.2%) thanGISTs with c-kit exon11mutation (66/159,41.5%). GISTs with c-kit exon11deletionmutation (37/89,41.6%) had a high proportion of with high mitotic rate than GISTs withother types mutation in exon11. GISTs with c-kit exon11deletion mutation had lower3-year survival rate than those GISTs with other types mutation in exon11. The3-yearoverall and relapse-free survival rate of GISTs were decreased when the mutationinvolved more than3codons.(3)27risk-related microRNAs were screened out in12GIST patients by microarray. Bioinformatics analysis revealed that these microRNAswere related to many tumor signal pathway and biological function of transcriptionregulation, protein binding and cell nucleus. miR-29c-5p and miR-148b-3p were validated to be related to GIST risk in FFPE tissue. Furthermore miR-148b-3p was related toprognosis of GIST patients, which was extra down-regulated in high-risk GIST with badprognosis (P<0.0001). The in vitro cell experiment revealed that the migration andinvasion capacities of GIST cells were promoted by applying microRNA inhibitor, whichalso inhibited apoptosis of GIST cells. miR-148b-3p may play a important role in GISTtumorigenesis and development by directly targeting c-kit, which was proven by us withbioinformatics analysis, qPCR, western-blot and luciferase report gene.(4)59encodinggenes were screened out in12GIST patients. RGS5(P=0.012) and DKK4(P=0.0072)was validated to be related to GIST risk. By immunohistochemical staining of DKK4in aset of tissue array involving326GIST cases, we found the expression rate of DKK4is farmore higher in GIST (92.5%) than adjacent normal tissue (26.3%)(P<0.001). Theexpression intensity of DKK4is related to tumor size (P=0.002), mitotic rate (P=0.004)and risk classification (P<0.001). The overall survival (P=0.004) and relapse-free survival(P<0.001) of GIST patients with high-level DKK4expression was lower than GISTpatients with low-level DKK4expression. The concentration of DKK4protein in serumwas significantly higher in GIST population (359.5±156.4pg/ml) than in non-GISTpopulation (54.92±4.705pg/ml)(P=0.0347) when using ELISA as detection method.CONCLUSION:(1) Tumor site, tumor size, mitotic rate, NIH classification, CD34expression and the radical extent of operation were associated with overall andrelapse-free survival of GIST patients. Tumor size, risk classification and radical extent ofoperation were independent risk factors of overall survival in GIST patients. Mitotic rate,risk classification and radical extent of operation were independent risk factor ofrelapse-free survival in GIST patients. IM therapy could significantly improve prognosisof intermediate-high risk GIST patients and late stage GIST patients.(2) The area fromNO.557to NO.560codon of c-kit exon11was a hot zone of all types of mutation. Therecurrence rate of wild-type GISTs was high than mutant GISTs. c-kit exon11deletionmutation and mutation involved more than3codons are poor prognostic factors in GISTpatients.(3) miR-148b-3p was related to clinicopathological characteristics and prognosis of GIST patients, down-regulation of miR-148b-3p in high-risk GIST was a poorprognostic factor. miR-148b-3p could affect biological function (including migration,invasion and apoptosis) of GIST cells by directly targeting KIT.(4) The expressionintensity of DKK4is related to tumor size, mitotic rate, and risk classification. The overallsurvival and relapse-free survival of GIST patients with high-level DKK4expression waslower than GIST patients with low-level DKK4expression. The concentration of DKK4protein in serum was significantly higher in GIST population than in non-GISTpopulation. |
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