approximately , two - thirds of all gists occur in the stomach , followed by the small intestine , rectum , and esophagus . moreover , the body and fundus are the most frequent site of gists in stomach . endoscopy reveals a smooth mass with normal overlying mucosa or with mucosal ulceration in larger tumors . tissue sampling with standard endoscopic mucosal biopsy is mostly negative in gists for lying underneath normal appearing mucosa . endoscopic ultrasonography ( eus ) has the ability to obtain differential diagnosis by the wall layer of origin and echogenicity . on eus , gists are usually hypoechoic masses , which originate from the muscularis propria , also occasionally they can arise from the muscularis mucosa . in some larger tumors , the eus features include irregular extra - luminal border , echogenic foci , and cystic spaces . although only 10%-30% of gists are clinically malignant , all gists are known to have some degree of malignant potential . therefore , a simple classification of benign and malignant is not appropriate . at present , the modified fletcher classification system has been used most frequently to assess the malignant potential of gists [ table 1 ] . this risk classification system is composed of four factors : tumor size , tumor site , mitotic count , and presence of tumor rupture and classifies the malignant potential as four grade : very low , low , intermediate , and high grade . for patients with high risk of recurrence , preoperative neoadjuvant therapy with imatinib is recommended . among the four factors in fletcher system , tumor size , tumor site , and presence of tumor rupture are easy to determine by endoscopy and eus , whereas the mitotic count is generally obtained from resection specimens . although eus - guided fine needle aspiration ( fna ) and eus - guided trucut biopsy ( tcb ) can also obtain specimens before surgery , these techniques have risks of complications such as bleeding , localized abdominal pain , puncture site infection , and fever , and both have a limitation of inadequate specimen for immunohistochemical analysis . for these reasons , eus - fna or eus - tcb contrast - enhanced ultrasound ( ce - us ) has been applied in clinical practice for more than 10 years , especially in diseases of digestive and cardiovascular system . with the development of eus techniques and the advent of second generation contrast agent , ce harmonic ( ceh)-eus has been used for diagnosis of lesions of pancreas , liver , and gallbladder . in this prospective study , eus and ceh - eus the relationship between features of eus / ceh - eus and the malignant potential in gists was evaluated . between march 2015 and december 2015 , a total of 19 patients suspected of having a gist were enrolled in our study . the exclusion criteria include severe heart failure , severe chronic obstructive pulmonary disease , known allergic disposition to sonovue ( 59 mg , bracco societa per azioni , milan , italy ) , pregnancy or lactation , severe psychiatric disorders , and esophagogastric varices . this study was approved by the ethics committee of beijing friendship hospital , capital medical university . all patients provided informed consent . before undergoing ceh - ues , endoscopy features including tumor location , size , mucosa appearance , and other lesions then standard b - mode eus was performed in all patients to determine the tumor size , originating layer , echogenicity , and the growth patterns . color doppler mode was performed to detect intratumoral color doppler flow signals . for ceh - eus , the extended pure harmonic detection mode was used , with the mechanical index set at 0.25 and a transmitting frequency of 4.7 mhz . eus and ceh - eus were performed in the left lateral decubitus position under midazolam - induced conscious - sedation with heart rate and oxygen saturation monitoring . a bolus infusion of sonovue ( 59 mg/5 ml ) was administered via peripheral vein catheter , followed by a 10 ml saline flush . the agent arrival time ( aat ) was recorded , and the vascular structures were assessed in real time . us video sequences were continuously recorded and stored in the hard disk for off - line analyses . all eus and ceu - eus were performed by two experienced endoscopists using an olympus gf - ue260 ( olympus medical systems co. , ltd . , tokyo , japan ) connected to a us system ( prosound ssd -10 ; hitachi - aloka , tokyo , japan ) . routine blood count , liver and kidney function , and coagulation function were performed in all patients . ce - computed tomography ( ct ) and chest radiograph were obtained to exclude distant metastases . laparoscopic surgical procedures were performed in 3 cases with tumor size > 50 mm and/or with obviously extraluminal growth . endoscopic resections were performed in the other 16 patients under a complete general anesthesia . an olympus gif260j endoscope with an auxiliary water jet ( olympus medical systems co. , ltd . , tokyo , japan ) was used . the steps of endoscopic resection were as follows : ( 1 ) marking of the entire circumference using a dual knife at intervals of a few millimeters , approximately 25 mm laterally from the lesion ; ( 2 ) local injection of glycerin - fructose - methylene mixture ( consisted of 10% glycerin , 5% fructose , and methylene 4 mg in 250 ml normal saline solution ) ; ( 3 ) circumferential mucosal incision using dual knife ; ( 4 ) dissection of the tumor using it knife ; ( 5 ) en bloc resection of the tumor using snare ; ( 6 ) withdrawal of the resect specimen using the grasping forceps ; ( 7 ) wound closure with clips and hemostasis using hemostatic forceps or by argon plasma coagulation anytime when hemorrhage is observed . after surgery or endoscopic resection , fasting , water - deprivation , and intravenous fluid continued for at least 48 h. patients without complication started drinking water after 48 h and were discharged within 47 days . the histologic diagnosis of gist was defined as subepithelial tumors composed of spindle cells that stained positive for c - kit and cd34 . the immunohistochemistry examination included the following : cd34 , c - kit , dog-1 , smooth muscle actin ( sma ) , desmin , and ki67 . the independent sample t - test was used for the comparison of two groups regarding continuous variables . the chi - square test was used to compare the frequency data of two groups including clinical characteristics , eus , and ceh - eus features . statistical analyses were performed with spss software ( version 17.0 ; spss , chicago , il , usa ) . between march 2015 and december 2015 , a total of 19 patients suspected of having a gist were enrolled in our study . the exclusion criteria include severe heart failure , severe chronic obstructive pulmonary disease , known allergic disposition to sonovue ( 59 mg , bracco societa per azioni , milan , italy ) , pregnancy or lactation , severe psychiatric disorders , and esophagogastric varices . this study was approved by the ethics committee of beijing friendship hospital , capital medical university . all patients provided informed consent . before undergoing ceh - ues , endoscopy features including tumor location , size , mucosa appearance , and other lesions then standard b - mode eus was performed in all patients to determine the tumor size , originating layer , echogenicity , and the growth patterns . color doppler mode was performed to detect intratumoral color doppler flow signals . for ceh - eus , the extended pure harmonic detection mode was used , with the mechanical index set at 0.25 and a transmitting frequency of 4.7 mhz . eus and ceh - eus were performed in the left lateral decubitus position under midazolam - induced conscious - sedation with heart rate and oxygen saturation monitoring . a bolus infusion of sonovue ( 59 mg/5 ml ) was administered via peripheral vein catheter , followed by a 10 ml saline flush . the agent arrival time ( aat ) was recorded , and the vascular structures were assessed in real time . us video sequences were continuously recorded and stored in the hard disk for off - line analyses . all eus and ceu - eus were performed by two experienced endoscopists using an olympus gf - ue260 ( olympus medical systems co. , ltd . , tokyo , japan ) connected to a us system ( prosound ssd -10 ; hitachi - aloka , tokyo , japan ) . routine blood count , liver and kidney function , and coagulation function were performed in all patients . ce - computed tomography ( ct ) and chest radiograph were obtained to exclude distant metastases . laparoscopic surgical procedures were performed in 3 cases with tumor size > 50 mm and/or with obviously extraluminal growth . endoscopic resections were performed in the other 16 patients under a complete general anesthesia . an olympus gif260j endoscope with an auxiliary water jet ( olympus medical systems co. , ltd . , tokyo , japan ) was used . the steps of endoscopic resection were as follows : ( 1 ) marking of the entire circumference using a dual knife at intervals of a few millimeters , approximately 25 mm laterally from the lesion ; ( 2 ) local injection of glycerin - fructose - methylene mixture ( consisted of 10% glycerin , 5% fructose , and methylene 4 mg in 250 ml normal saline solution ) ; ( 3 ) circumferential mucosal incision using dual knife ; ( 4 ) dissection of the tumor using it knife ; ( 5 ) en bloc resection of the tumor using snare ; ( 6 ) withdrawal of the resect specimen using the grasping forceps ; ( 7 ) wound closure with clips and hemostasis using hemostatic forceps or by argon plasma coagulation anytime when hemorrhage is observed . after surgery or endoscopic resection , fasting , water - deprivation , and intravenous fluid continued for at least 48 h. patients without complication started drinking water after 48 h and were discharged within 47 days . the histologic diagnosis of gist was defined as subepithelial tumors composed of spindle cells that stained positive for c - kit and cd34 . the immunohistochemistry examination included the following : cd34 , c - kit , dog-1 , smooth muscle actin ( sma ) , desmin , and ki67 . the independent sample t - test was used for the comparison of two groups regarding continuous variables . the chi - square test was used to compare the frequency data of two groups including clinical characteristics , eus , and ceh - eus features . statistical analyses were performed with spss software ( version 17.0 ; spss , chicago , il , usa ) . between march 2015 and december 2015 , a total of 19 patients suspected of having a gist were enrolled in our study . the exclusion criteria include severe heart failure , severe chronic obstructive pulmonary disease , known allergic disposition to sonovue ( 59 mg , bracco societa per azioni , milan , italy ) , pregnancy or lactation , severe psychiatric disorders , and esophagogastric varices . this study was approved by the ethics committee of beijing friendship hospital , capital medical university . before undergoing ceh - ues , endoscopy features including tumor location , size , mucosa appearance , and other lesions were obtained . then standard b - mode eus was performed in all patients to determine the tumor size , originating layer , echogenicity , and the growth patterns . color doppler mode was performed to detect intratumoral color doppler flow signals . for ceh - eus , the extended pure harmonic detection mode was used , with the mechanical index set at 0.25 and a transmitting frequency of 4.7 mhz . eus and ceh - eus were performed in the left lateral decubitus position under midazolam - induced conscious - sedation with heart rate and oxygen saturation monitoring . a bolus infusion of sonovue ( 59 mg/5 ml ) was administered via peripheral vein catheter , followed by a 10 ml saline flush . the agent arrival time ( aat ) was recorded , and the vascular structures were assessed in real time . us video sequences were continuously recorded and stored in the hard disk for off - line analyses . all eus and ceu - eus were performed by two experienced endoscopists using an olympus gf - ue260 ( olympus medical systems co. , ltd . , tokyo , japan ) connected to a us system ( prosound ssd -10 ; hitachi - aloka , tokyo , japan ) . routine blood count , liver and kidney function , and coagulation function were performed in all patients . ce - computed tomography ( ct ) and chest radiograph were obtained to exclude distant metastases . laparoscopic surgical procedures were performed in 3 cases with tumor size > 50 mm and/or with obviously extraluminal growth . endoscopic resections were performed in the other 16 patients under a complete general anesthesia . an olympus gif260j endoscope with an auxiliary water jet ( olympus medical systems co. , ltd . , tokyo , japan ) was used . the steps of endoscopic resection were as follows : ( 1 ) marking of the entire circumference using a dual knife at intervals of a few millimeters , approximately 25 mm laterally from the lesion ; ( 2 ) local injection of glycerin - fructose - methylene mixture ( consisted of 10% glycerin , 5% fructose , and methylene 4 mg in 250 ml normal saline solution ) ; ( 3 ) circumferential mucosal incision using dual knife ; ( 4 ) dissection of the tumor using it knife ; ( 5 ) en bloc resection of the tumor using snare ; ( 6 ) withdrawal of the resect specimen using the grasping forceps ; ( 7 ) wound closure with clips and hemostasis using hemostatic forceps or by argon plasma coagulation anytime when hemorrhage is observed . after surgery or endoscopic resection , fasting , water - deprivation , and intravenous fluid continued for at least 48 h. patients without complication started drinking water after 48 h and were discharged within 47 days . the histologic diagnosis of gist was defined as subepithelial tumors composed of spindle cells that stained positive for c - kit and cd34 . the immunohistochemistry examination included the following : cd34 , c - kit , dog-1 , smooth muscle actin ( sma ) , desmin , and ki67 . the independent sample t - test was used for the comparison of two groups regarding continuous variables . the chi - square test was used to compare the frequency data of two groups including clinical characteristics , eus , and ceh - eus features . statistical analyses were performed with spss software ( version 17.0 ; spss , chicago , il , usa ) . one patient was pathologically diagnosed as middle differentiation adenocarcinoma and was excluded from this study . the other 18 patients were confirmed by pathology and immunohistochemistry examination . according to the modified fletcher classification system , 18 resected tumors consisted of 4 very low , 6 low , 6 intermediate , and 2 high malignant potential gists . we divided the 18 patients into 2 groups : group i ( lower malignant potential ) included low and very low malignant potential and group ii ( higher malignant potential ) included high and intermediate malignant potential . mean ( mean  standard deviation [ sd ] ) age was 51.5  10.0 years in group i and 58.9  14.1 years in group ii [ table 2 ] . one ( 1/10 ) patient of group i and 3 ( 3/8 ) patients of group ii were symptomatic . the other 14 patients were asymptomatic and were discovered incidentally or by other image examinations . the majority of tumors ( 7/10 vs. 5/8 ) located in fundus and cardia of stomach . no significant difference of these 3 features was found between group i and group ii ( p = 0.21 , 0.28 and 0.95 ) . clinical characteristics of 18 patients with gastrointestinal stromal tumors mean ( mean  sd ) diameter measured on eus was 14.6  5.8 mm and 32.1  8.4 mm [ table 3 ] and tumor size in group ii was significantly larger than that in group i ( p < 0.05 ) . furthermore , the number of patient with tumor size 20 mm was 3 of 10 in group i and 7 of 8 in group ii ( p < 0.05 ) . intraluminal growth was the predominant growth pattern in the two groups except that extraluminal growth was found in one patient in group ii , who underwent surgical resection . homogeneous hypoechogenicity was observed in all ten patients in group i while heterogeneous hypoechogenicity with focal hyperechoic area or anechoic area was found in four of eight patients in group ii ( p < 0.05 ) . after a bolus infusion of sonovue , the mean value of aat was 10.9  0.2 s in group i and 10.7  0.2 s in group ii ( p = 0.10 ) . no irregular intratumoral vessel was found in group i , regular fine intratumoral vessels were found in six patients , and no vessel was detected in other four patients . on contrast , in group ii , irregular intratumoral vessels were found in six of eight patients [ figure 1 ] and regular fine intratumoral vessels were found in other two patients . endoscopic ultrasonography and contrast enhanced harmonic - endoscopic ultrasonography features of 18 patients with gastrointestinal stromal tumors an intermediate malignant potential gastrointestinal stromal tumor of the stomach in a 76-year - old man . ( a ) white light endoscopy shows a ball - shaped mass with normal overlying mucosa ; ( b ) endoscopic ultrasonography shows a hypoechoic mass originated from the muscularis propria . ( c ) contrast enhanced harmonic -endoscopic ultrasonography shows irregular vessels except one patient with middle differentiation adenocarcinoma , the other 18 patients met gist diagnostic criteria . according to the modified fletcher classification system , tumor size and mitotic count were used for assessing the malignant potential , and no tumor rupture was found in all patients . in group i , four and six patients were very low and low malignant potential , respectively ; in group ii , six and two patients were intermediate and high malignant potential , respectively . sma was positive in one patient with very low malignant potential gist and one with low malignant potential gist . ki67 was positive in both two patients with high malignant potential gist and one with intermediate malignant potential gist [ figure 2 ] . ( a ) h and e ; ( b ) cd34 ; ( c ) c - kit ; ( d ) dog-1 ; ( e ) smooth muscle actin ; ( f ) desmin ; ( g ) ki67 ( all for 200 ) the modified fletcher classification system is the most commonly used standard for assessing the malignant potential of gists , which comprises four factors : tumor size , tumor site , mitotic count , presence of tumor rupture . the assessment of malignant potential is difficult to perform before tumor resection for the reason that the modified fletcher classification system depends on postoperative pathology . in the present study , we observed features of eus and ceh - eus preoperatively and evaluated the correlation between the features and malignant potential of gists . the modified fletcher classification system classifies the malignant potential as four grades : very low , low , intermediate , and high malignant potential . we collapsed patients with very low and low malignant potential gists into group i ( the lower group ) as well as intermediate and high malignant potential gists into group ii ( the higher group ) . no significant difference was found in the mean age , gender and tumor location between group i and group ii . the majority of patients in both groups were asymptomatic , of which subepithelial masses were detected incidentally during routine endoscopic screening or other image examinations . the tumor size is one of the four factors in the modified fletcher classification system , and the mean tumor size in group ii was significantly larger than that in group i ( 4.6  5.8 mm vs. 33.4  10.6 mm , p < 0.05 ) . the tumor size was < 20 mm in most patients in group i while only few in group ii ( 7/10 [ 70% ] vs. 1/8 [ 12.5% ] , p we interpret this result as that the percentage of larger size ( 20 mm ) in higher malignant potential gists is higher than that in lower malignant potential gists . furthermore , it does not mean that the malignant potential of a larger gist must be higher than that of a smaller gist . on the contrary , some smaller gists revealed higher malignant potential . on eus , gists are usually homogenous hypoechoic masses or heterogeneous hypoechoic masses with anechoic cystic spaces due to cystic degeneration and liquefaction necrosis . in our study , all of the ten gists in group i were homogenous hypoechoic and originated from the muscularis propria . on contrast , only four gists in group ii were heterogenous hypoechoic with focal hyperechoic area ( p < 0.05 ) . no severe complication such as bleeding or peritonitis was observed in all 18 patients . to date , no recurrence was found in all patients during endoscopy surveillance , and the follow - up is in progress . in some previous studies , researchers found that detection of intratumoral irregular vessels had important value in assessing the malignant potential of gists . in our study , we detected intratumoral irregular vessels by ceh - eus . in group ii , irregular vessels were detected in both two high malignant potential gists and four intermediate malignant potential gists . in group i , no irregular vessel was found in ten low and very low malignant potential gists . there was a significant difference for the detection of irregular vessels between the two groups . detection of irregular vessels had good sensitivity and specificity in discriminating higher and lower malignant potential gists , which are 75% and 100% , respectively . the positive predictive value of detection of irregular vessels to high malignant potential was 33% , and the negative predictive value was 100% . ce - ct , color , and pd - eus , ceh - eus are used for detection of intratumoral vessels clinically , and the efficacy of the former two methods is correlated to the diameter and flow of vessels . for ceh - eus , the harmonic signal of microbubbles was directly depicted , particularly in the vessels with small diameter and slow flow , which were hard to be identified by ce - ct or pd - eus . therefore , ceh - eus can be used to diagnose disease in various organs with abundant as well as poor blood supply . some researchers had reported the diagnostic value of ceh - eus in lesions of pancreas and gallbladder . found that aat of endometrial carcinoma was shorter than that of normal myometrium , and contributed this to by numerous and complex neovascularization in malignant lesions . in a study on evaluation of severity of chronic hepatitis c , ridolfi et al . also revealed that the mean hepatic vein arrival time decreased progressively with increasing severity of liver disease . however , no significant difference of aat was found between colorectal adenocarcinoma and adenoma . in this study , we also found no significant difference of aat between the two groups . the diagnosis of gists must be confirmed by pathology and immunohistochemistry examinations . at present , the diagnostic criterion of gists was defined as subepithelial tumors composed of spindle cells c - kit(+ ) and cd34(+ ) . eighteen patients , who met the criterion in this study except one patient , were diagnosed middle differentiated adenocarcinoma . dog-1 is a calcium dependent , receptor activated chloride channel protein expressed in gists , and it is positive in all 18 patients in our study . leiomyoma is another common subepithelial tumor in gastrointestinal tract , which is difficult to discriminate from gists by endoscopy and eus . however , cd34 and c - kit are rarely positive in leiomyoma , whereas sma and desmin are always positive . in this study , ki67 is a nuclear proliferation associated antigen expressed in the growth and synthesis phases of the cell cycle ( g1 , s , g2 , and m ) but not in the resting phase ( g0 ) . this antigen provides information about the proportion of active cells in the cell cycle and is correlated with tumor recurrence and prognosis . we observed that ki67 was positive in two high malignant potential gists and in one intermediate malignant potential gists in group i and was all negative in group ii . hence , strict follow - up and endoscopy surveillance are needed in these three patients with ki67(+ ) . . the limitations of this study are the small number of patients enrolled and the relatively short period of follow - up . the follow - up is still in progress , and further study with larger number of cases is needed .
objective : to evaluate the diagnostic value of endoscopic ultrasonography ( eus ) and contrast - enhanced harmonic ( ceh ) eus in patients with gastrointestinal stromal tumors ( gists).patients and methods : about 19 patients with suspected gists underwent eus and ceh - eus before tumor resection . 
 the malignant potential was assessed according to the modified fletcher classification system . 
 patients were divided into lower ( group i ) and higher ( group ii ) malignant potential group . 
 the clinical characteristics and eus / ceh - eus features were compared between two groups.results:the tumor size in group ii was significantly larger than that in group i ( 14.6  5.8 mm vs. 32.1  8.4 mm , p < 0.05 ) . 
 heterogeneous echogenicity was observed in 4 ( 4/8 ) cases in group ii and none in group i ( p < 0.05 ) . 
 irregular intratumoral vessels were detected in 6 cases in group ii and none in group i ( p < 0.05 ) . the sensitivity and specificity of irregular vessel detection for discriminating higher from lower malignant potential gists were 75% and 100% , respectively . 
 the positive predictive value and negative predictive value of detection of irregular vessels to high malignant potential gists were 33% and 100% , respectively.conclusion:detection of irregular intratumoral vessels can predict higher malignant potential before tumor resection . 
 the tumor size and echogenicity are assistant factors for malignant potential assessment . 
 endoscopic resection is an efficacious treatment with good security for appropriate patients .