circulating tumor cells ( ctcs ) are shed from primary or metastatic solid tumors into the circulation ( 1 ) . liquid biopsy  , enabling non - invasive and frequent monitoring of therapeutic responses in cancer patients in real time ( 1 - 6 ) . a study performed on 52 advanced gastric cancer ( agc ) patients in japan confirmed that patients with unfavorable ctc counts 2 or 4 weeks after initiation of chemotherapy had shorter median progression - free survival ( pfs ) and overall survival ( os ) compared with those with favorable ctc counts ( 7 ) . similar results were also obtained in our previous work with 136 agc patients , indicating that quantification of ctcs may be a promising approach for evaluating chemotherapeutic efficacy and predicting prognosis of agc patients . also , a persistently favorable ctc count or an early conversion to a favorable ctc count after therapy may indicate improved prognosis . in contrast , patients with persistently unfavorable ctcs or who converted to an unfavorable ctc count after therapy fared significantly worse , suggesting a limited treatment response . longitudinal changes of ctcs in response to therapy may help in rapidly indicating acquired resistance in individual patients before they are radiographically evaluated ( 8) . in addition to counting ctcs , pinpointing specific targeted - therapy - or chemotherapy - resistant ctc subtype classified by tumor biomarker expression and/or chromosome ploidy may be a better approach ( 3,9 - 11 ) . our previous study indicated that aneuploidy of chromosome 8 in ctcs of agc patients correlated with chemothera - peutic efficacy of paclitaxel ( ptx ) and cisplatin ( ddp ) ( 12 ) . the number of pre - treatment triploid ctcs in patients was inversely correlated with chemotherapeutic efficacy . after administration of ptx or ddp , triploid ctcs had intrinsic resistance to chemotherapeutic reagents , whereas multiploid ctcs ( 4 copies on chromosome 8) developing acquired resistance . similar observations were made in a ddp - treated gastric neuroendocrine cancer patient - derived xenograft ( pdx ) model ( 13 ) . identification of diverse ctc subtypes with distinct clinical significance could help guide more precise personal therapy and allow more robust analyses ( 14 ) . 
  in this study , we extended previous studies to investigate whether and how distinct ctc subtypes with diverse ploidy of chromosome 8 correlate with patients clinical prognosis in terms of pfs and os . subtraction enrichment ( se ) integrated with immunostaining - fluorescence in situ hybridization ( ifish ) was applied to enrich and characterize aneuploidy of chromosome 8 in ctcs ( 12 ) . correlations of quantified multiploid ctcs in patients under therapy with clinical prognosis were investigated to evaluate the utility of comparing multiploid ctcs for monitoring therapeutic response in agc patients . thirty - one newly diagnosed agc patients ( > 18 years ) were enrolled in the study at peking university cancer hospital from october 2013 to august 2014 . locally advanced , recurrent , metastatic gastric or gastroesophageal junction adenocarcinoma was histopathologically diagnosed and confirmed . patients who had not received treatment and those who had a karnofsky performance status > 60 , with adequate organ function and evaluable tumor lesions were eligible for this study . all enrolled patients were given first - line ptx or ddp - based chemotherapy ( 15 ) . those histopathologically diagnosed as human epidermal growth factor receptor 2 ( her2)-positive cancer received anti - her2 targeted therapy and ddp chemotherapy ( 16 ) . six weeks ( 2 cycles ) after therapy , evaluation of clinical response was performed using computed tomography ( ct ) according to response evaluation criteria in solid tumors ( recist ) version 1.1 criteria ( 17 ) . responses were categorized as partial response ( pr , at least a 30% decrease in the sum of diameters of target lesions ) , progressive disease ( pd , at least a 20% increase in the sum of diameters of target lesions ) , or stable disease ( sd , neither sufficient shrinkage to qualify as pr nor a sufficient increase to qualify as pd ) . written informed consent was obtained from all subjects . the study was approved by the ethics committee of peking university cancer hospital and was performed according to the declaration of helsinki principles . peripheral blood samples were collected from patients before and 6 weeks ( the first evaluation of efficacy ) after therapy initiation . ctcs were enriched using se - ifish ( cytelligen , san diego , ca , usa ) according to a previously published protocol ( 12 ) . briefly , 7.5 ml of patient blood was collected into a tube containing acid - citrate - dextrose ( acd ) anticoagulant ( becton dickinson , franklin lakes , nj , usa ) , followed by thoroughly mixing and overlaying on 3 ml of hctc separation matrix . supernatants above red blood cells were collected and incubated with immunomagnetic particles conjugated to anti - leukocytes monoclonal antibodies at room temperature for 10 min with gentle shaking . the magnetic particle - free solution was spun down at 500 g for 2 min at room temperature . sedimented cells were thoroughly mixed with cell fixative and applied onto cytelligen - coated ctc slides . samples were subsequently subjected to immunostaining with a cocktail of alexa fluor 594-conjugated monoclonal anti - cd45 and alexa fluor 488-conjugated anti - pan - cytokeratin ( panck ) ( ck4 , 5 , 6 , 8 , 10 , 13 and 18 ) for 1 h in the dark , followed by fish with centromere probe ( cep ) 8 spectrum - orange ( vysis , abbott laboratories , abbott park , il , usa ) using a s500 statspin thermobrite slide hybridization / denaturation system ( abbott molecular , des plaines , il , usa ) . correlation of ctc positivity with clinicopathologic characteristics and clinical responses was examined using fisher s exact test . the threshold of ctcs was established with nonparametric receiver operating characteristic ( roc ) analysis , and optimal threshold values were determined to maximize the youden index ( sensitivity+specificity-1 ) , which was determined by selecting a point that maximizes the number of subjects correctly classified , and giving equal weight to sensitivity and specificity ( 18 ) . pfs was defined as the duration from initial blood collection to the time that clinical progression was confirmed or the participants were censored . os was defined as the time from initial blood collection to the date that death occurred or the participants were censored . kaplan - meier survival plots for pfs or os were generated based on numbers of total or aneuploid ctcs before and after therapy , and survival curves were compared using log - rank tests . cox proportional hazards regression was applied to determine the univariate and multivariate hazard ratios ( hr ) for pfs and os . pre- and post - therapy total ctc counts , subtypes of multiploid chromosome 8 ctcs , and standard clinical factors including gender , age , liver and peritoneal metastases , lauren classification , her2 status and the type of treatment were subjected to univariate analyses for pfs and os . p<0.05 was considered statistically significant , and all p values were two - sided . thirty - one newly diagnosed agc patients ( > 18 years ) were enrolled in the study at peking university cancer hospital from october 2013 to august 2014 . locally advanced , recurrent , metastatic gastric or gastroesophageal junction adenocarcinoma was histopathologically diagnosed and confirmed . patients who had not received treatment and those who had a karnofsky performance status > 60 , with adequate organ function and evaluable tumor lesions were eligible for this study . all enrolled patients were given first - line ptx or ddp - based chemotherapy ( 15 ) . those histopathologically diagnosed as human epidermal growth factor receptor 2 ( her2)-positive cancer received anti - her2 targeted therapy and ddp chemotherapy ( 16 ) . six weeks ( 2 cycles ) after therapy , evaluation of clinical response was performed using computed tomography ( ct ) according to response evaluation criteria in solid tumors ( recist ) version 1.1 criteria ( 17 ) . responses were categorized as partial response ( pr , at least a 30% decrease in the sum of diameters of target lesions ) , progressive disease ( pd , at least a 20% increase in the sum of diameters of target lesions ) , or stable disease ( sd , neither sufficient shrinkage to qualify as pr nor a sufficient increase to qualify as pd ) . written informed consent was obtained from all subjects . the study was approved by the ethics committee of peking university cancer hospital and was performed according to the declaration of helsinki principles . peripheral blood samples were collected from patients before and 6 weeks ( the first evaluation of efficacy ) after therapy initiation . ctcs were enriched using se - ifish ( cytelligen , san diego , ca , usa ) according to a previously published protocol ( 12 ) . briefly , 7.5 ml of patient blood was collected into a tube containing acid - citrate - dextrose ( acd ) anticoagulant ( becton dickinson , franklin lakes , nj , usa ) , followed by thoroughly mixing and overlaying on 3 ml of hctc separation matrix . supernatants above red blood cells were collected and incubated with immunomagnetic particles conjugated to anti - leukocytes monoclonal antibodies at room temperature for 10 min with gentle shaking . the magnetic particle - free solution was spun down at 500 g for 2 min at room temperature . sedimented cells were thoroughly mixed with cell fixative and applied onto cytelligen - coated ctc slides . samples were subsequently subjected to immunostaining with a cocktail of alexa fluor 594-conjugated monoclonal anti - cd45 and alexa fluor 488-conjugated anti - pan - cytokeratin ( panck ) ( ck4 , 5 , 6 , 8 , 10 , 13 and 18 ) for 1 h in the dark , followed by fish with centromere probe ( cep ) 8 spectrum - orange ( vysis , abbott laboratories , abbott park , il , usa ) using a s500 statspin thermobrite slide hybridization / denaturation system ( abbott molecular , des plaines , il , usa ) . statistical analyses were performed with ibm spss statistics ( version 21.0 ; ibm corp . , new york , usa ) . correlation of ctc positivity with clinicopathologic characteristics and clinical responses was examined using fisher s exact test . the threshold of ctcs was established with nonparametric receiver operating characteristic ( roc ) analysis , and optimal threshold values were determined to maximize the youden index ( sensitivity+specificity-1 ) , which was determined by selecting a point that maximizes the number of subjects correctly classified , and giving equal weight to sensitivity and specificity ( 18 ) . pfs was defined as the duration from initial blood collection to the time that clinical progression was confirmed or the participants were censored . os was defined as the time from initial blood collection to the date that death occurred or the participants were censored . kaplan - meier survival plots for pfs or os were generated based on numbers of total or aneuploid ctcs before and after therapy , and survival curves were compared using log - rank tests . cox proportional hazards regression was applied to determine the univariate and multivariate hazard ratios ( hr ) for pfs and os . pre- and post - therapy total ctc counts , subtypes of multiploid chromosome 8 ctcs , and standard clinical factors including gender , age , liver and peritoneal metastases , lauren classification , her2 status and the type of treatment were subjected to univariate analyses for pfs and os . p<0.05 was considered statistically significant , and all p values were two - sided . quantification of total and aneuploid ctcs ( 4 copies on chromosome 8) in pre - treatment patients by se - ifish was performed on all of the 31 enrolled agc patients . the median number of ctcs detected in whole population of patients was 3 ( range , 0720 ) and the mean number ( \begin{document}$\bar { x}\!\!\;$\end{document}s ) was 32128 . and examination of correlation between ctc numbers and various clinico - pathological characteristics is summarized in table 1 . all patients with mixed type carcinoma were positive for ctcs ( 2 per 7.5 ml ) , whereas patients with intestinal and diffuse type cancer had a ctc positivity of 83.3% and 33.3% , respectively . further pairwise comparison revealed that positivity of ctc in intestinal and mixed type patients was significantly higher than that of diffuse type ( p=0.038 , p=0.021 , respectively ) . no obvious correlation was found in other clinicopathological characteristics , including gender , age , primary tumor sites , metastatic sites , and her2 status . correlation of ctcs in pre - treatment patients with clinical characteristics ( n=31 ) of the 31 enrolled agc patients , 22 had completed two cycles of therapy and a post - therapy ctc evaluation . at the time of analysis , 18 of 22 patients had pd , and 14 died . the median pfs and os were 8.1 [ 95% confidence interval ( 95% ci ) , 6.79.4 ] months and 18.7 ( 95% ci , 12.724.6 ) months , respectively . to establish an optimal threshold of ctcs for predicting pfs and os , roc curves and youden index were estimated for both pre- and post - treatment ctcs ( supplementary figure s1 ) ( 18 ) . we observed that < 4 ctcs per 7.5 ml was a favourable threshold , whereas 4 was an unfavorable value in this study . 
  in pre - treatment patients , those with 4 ctcs had the median pfs and os as shown in figure 1a , b and these were not significantly different from patients with < 4 ctcs ( p=0.487 , p=0.533 , respectively ) . however , 6 weeks after therapy initiation , the median pfs of patients with 4 post - therapy ctcs was 4.5 ( 95% ci , 3.85.2 ) months , which was significantly shorter than that of patients with < 4 post - therapy ctcs [ 8.4 ( 95% ci , 8.08.8 ) months , p=0.001 ] ( figure 1c ) . for median os , the patients with 4 post - therapy ctcs had inferior median os [ 14.3 ( 95% ci , 6.721.9 ) months ] compared with those with < 4 post - therapy ctcs [ 20.0 ( 95% ci , 14.925.0 ) months , p=0.044 ) ( figure 1d ) . data suggest that a threshold of unfavorable post - therapy ctcs ( 4 ) may help predict inferior pfs and os . selection of the optimal cut - off value of ctcs for predicting overall survival ( os ) . ( a ) receiver operating characteristic ( roc ) curves for pre- and post - therapy ctcs predicting os ; ( b ) youden index for different cut - off values of ctcs based on the roc curves in ( a ) [ youden index = sensitivity+specificity-1 ] ; ( c , d ) roc areas under the curve ( aucs ) for ctc thresholds of 1 to 5 in pre- ( c ) and post - treatment ( d ) patients . the cut - off points of 1 and 2 for pre - therapy ctcs and 1 to 5 for post - therapy ctcs all exhibit larger youden index . when the aucs for ctc thresholds of 1 to 5 are compared , a cut - off value of 4 for post - therapy ctcs maximizes the auc area ( 0.668 for post - therapy ctcs ) kaplan - meier curves for progression - free survival ( pfs ) ( a , c ) and overall survival ( os ) ( b , d ) in patients with 4 and < 4 ctcs either pre- ( a , b ) or post - therapy ( c , d ) . our previous work indicated that aneuploidy of chromosome 8 in ctcs correlated with therapeutic response in agc patients . triploid chromosome 8 ctcs were constantly chemoresistant , whereas multiploid ctcs may develop acquired resistance after chemotherapy ( 12 ) . in the current study , correlation of enumeration of triploid and multiploid chromosome 8 ctcs ( hereafter referred to triploid and multiploid ctcs respectively ) with patient prognosis was investigated . 
  based on estimated roc curves and youden index , 1 triploid ( data not shown ) and 2 multiploid ( supplementary figure s2 ) ctcs were selected as an unfavorable threshold in this study . although neither pre- nor post - treatment triploid ctcs were correlated with pfs and os ( data not shown ) , patients with 2 post - therapy multiploid ctcs had significantly shorter median pfs and os compared with those with < 2 post - therapy multiploid ctcs [ 4.5 ( 95% ci , 3.85.2 ) months vs. 8.4 ( 95% ci , 7.89.0 ) months , p=0.011 ; 11.4 ( 95% ci , 10.212.6 ) months vs. 22.6 ( 95% ci , 16.828.2 ) months , p=0.001 ] . no significant correlation of pre - treatment multiploid ctcs with pfs and os was found ( figure 2 ) . six weeks after therapy , inferior pfs and os were observed in patients with 2 multiploid ctcs compared with patients with < 2 multiploid . thus , unfavorable ( 2 ) post - therapy multiploid ctcs may predict inferior pfs and os for agc patients . selection of the optimal cut - off value of multiploid ctcs for overall survival ( os ) . ( a ) receiver operating characteristic ( roc ) curves for pre- and post - therapy multiploid ctcs predicting os ; ( b ) youden index for different cut - off values of multiploid ctcs based on the roc curves in ( a ) ; ( c , d ) roc areas under the curve ( aucs ) for multiploid ctc thresholds of 1 to 3 in pre- ( c ) and post - treatment patients ( d ) . the threshold of 2 multiploid ctcs is selected as unfavorable because of the maximal auc area ( 0.769 for post - therapy ctcs ) . correlation of multiploid ctcs in pre - treatment ( a , b ) and post - treatment ( c , d ) patients with progression - free survival ( pfs ) ( a , c ) and overall survival ( os ) ( b , d ) . to investigate the significance of multiploid ctcs , variations of quantified multiploid ctcs before and after treatment in response to therapy were compared . patients after 6 weeks of therapy were classified into two groups : group 1 with the number of multiploid ctcs decreased 10% following therapy ; and group 2 with the number of multiploid ctcs increased 10% . as shown in figure 3a , the median pfs of group 2 was significantly shorter than that of group 1 [ 4.5 ( 95% ci , 1.27.8 ) months vs. 8.3 ( 95% ci , 7.79.1 ) months , p=0.003 ] . group 2 had inferior median os compared to group 1 [ 11.4 ( 95% ci , 3.823.0 ) months vs. 20.0 ( 95% ci , 18.022.0 ) months , p=0.004 ] ( figure 3b ) . statistical analysis showed that quantitative comparison of multiploid ctcs after therapy may help evaluate therapeutic response and patient prognosis . patients with 10% decreased multiploid ctcs after the first 2 cycles of therapy may have improved pfs and os and those with 10% increased multiploid ctcs after the first 2 cycles of therapy may have inferior pfs and os . correlation of variations of multiploid chromosome 8 ctcs after therapy with progression - free survival ( pfs ) ( a ) and overall survival ( os ) ( b ) . patients are categorized into two groups according to the trends of changes of multiploid ctcs from pre - treatment to post - treatment . group 1 , multiploid ctcs are decreased 10% ; group 2 , multiploid ctcs are increased 10% . to compare the predictive value of variable multiploid ctcs with the one - site unfavorable ctcs , cox proportional hazards regression model was used and the results showed that several unfavorable parameters were associated with inferior pfs and os ( figure 4 ) . a 10% increase of multiploid ctcs after therapy indicated the highest risk of progression and mortality . moreover , multivariate analysis demonstrated that after adjusting for clinically significant factors ( including post - therapy ctcs , post - therapy multiploid ctcs and increase of multiploid ctcs which were significant predictors in cox univariate analysis ) , only 10% increase in multiploid ctcs was an independent predictor of pfs ( hr , 9.697 ; 95% ci , 1.58259.444 ; p=0.014 ) and os ( hr , 6.833 ; 95% ci , 1.35034.583 ; p=0.020 ) . thus , comparing multiploid chromosome 8 ctcs before and after treatment can be used to evaluate therapeutic efficacy . forest plots displaying cox univariate analysis of variables predicting to progression - free survival ( pfs ) and overall survival ( os ) . variables include clinicopathological characteristics , numbers of total ctcs , subtypes of triploid and multiploid chromosome 8 ctcs in pre- and post - treatment patients , as well as variation of multiploid ctcs after therapy . quantification of total and aneuploid ctcs ( 4 copies on chromosome 8) in pre - treatment patients by se - ifish was performed on all of the 31 enrolled agc patients . the median number of ctcs detected in whole population of patients was 3 ( range , 0720 ) and the mean number ( \begin{document}$\bar { x}\!\!\;$\end{document}s ) was 32128 . and examination of correlation between ctc numbers and various clinico - pathological characteristics is summarized in table 1 . all patients with mixed type carcinoma were positive for ctcs ( 2 per 7.5 ml ) , whereas patients with intestinal and diffuse type cancer had a ctc positivity of 83.3% and 33.3% , respectively . further pairwise comparison revealed that positivity of ctc in intestinal and mixed type patients was significantly higher than that of diffuse type ( p=0.038 , p=0.021 , respectively ) . no obvious correlation was found in other clinicopathological characteristics , including gender , age , primary tumor sites , metastatic sites , and her2 status . of the 31 enrolled agc patients , 22 had completed two cycles of therapy and a post - therapy ctc evaluation . at the time of analysis , 18 of 22 patients had pd , and 14 died . the median pfs and os were 8.1 [ 95% confidence interval ( 95% ci ) , 6.79.4 ] months and 18.7 ( 95% ci , 12.724.6 ) months , respectively . to establish an optimal threshold of ctcs for predicting pfs and os , roc curves and youden index were estimated for both pre- and post - treatment ctcs ( supplementary figure s1 ) ( 18 ) . we observed that < 4 ctcs per 7.5 ml was a favourable threshold , whereas 4 was an unfavorable value in this study . 
  in pre - treatment patients , those with 4 ctcs had the median pfs and os as shown in figure 1a , b and these were not significantly different from patients with < 4 ctcs ( p=0.487 , p=0.533 , respectively ) . however , 6 weeks after therapy initiation , the median pfs of patients with 4 post - therapy ctcs was 4.5 ( 95% ci , 3.85.2 ) months , which was significantly shorter than that of patients with < 4 post - therapy ctcs [ 8.4 ( 95% ci , 8.08.8 ) months , p=0.001 ] ( figure 1c ) . for median os , the patients with 4 post - therapy ctcs had inferior median os [ 14.3 ( 95% ci , 6.721.9 ) months ] compared with those with < 4 post - therapy ctcs [ 20.0 ( 95% ci , 14.925.0 ) months , p=0.044 ) ( figure 1d ) . data suggest that a threshold of unfavorable post - therapy ctcs ( 4 ) may help predict inferior pfs and os . selection of the optimal cut - off value of ctcs for predicting overall survival ( os ) . ( a ) receiver operating characteristic ( roc ) curves for pre- and post - therapy ctcs predicting os ; ( b ) youden index for different cut - off values of ctcs based on the roc curves in ( a ) [ youden index = sensitivity+specificity-1 ] ; ( c , d ) roc areas under the curve ( aucs ) for ctc thresholds of 1 to 5 in pre- ( c ) and post - treatment ( d ) patients . the cut - off points of 1 and 2 for pre - therapy ctcs and 1 to 5 for post - therapy ctcs all exhibit larger youden index . when the aucs for ctc thresholds of 1 to 5 are compared , a cut - off value of 4 for post - therapy ctcs maximizes the auc area ( 0.668 for post - therapy ctcs ) . kaplan - meier curves for progression - free survival ( pfs ) ( a , c ) and overall survival ( os ) ( b , d ) in patients with 4 and < 4 ctcs either pre- ( a , b ) or post - therapy ( c , d ) . our previous work indicated that aneuploidy of chromosome 8 in ctcs correlated with therapeutic response in agc patients . triploid chromosome 8 ctcs were constantly chemoresistant , whereas multiploid ctcs may develop acquired resistance after chemotherapy ( 12 ) . in the current study , correlation of enumeration of triploid and multiploid chromosome 8 ctcs ( hereafter referred to triploid and multiploid ctcs respectively ) with patient prognosis was investigated . 
  based on estimated roc curves and youden index , 1 triploid ( data not shown ) and 2 multiploid ( supplementary figure s2 ) ctcs were selected as an unfavorable threshold in this study . although neither pre- nor post - treatment triploid ctcs were correlated with pfs and os ( data not shown ) , patients with 2 post - therapy multiploid ctcs had significantly shorter median pfs and os compared with those with < 2 post - therapy multiploid ctcs [ 4.5 ( 95% ci , 3.85.2 ) months vs. 8.4 ( 95% ci , 7.89.0 ) months , p=0.011 ; 11.4 ( 95% ci , 10.212.6 ) months vs. 22.6 ( 95% ci , 16.828.2 ) months , p=0.001 ] . no significant correlation of pre - treatment multiploid ctcs with pfs and os was found ( figure 2 ) . six weeks after therapy , inferior pfs and os were observed in patients with 2 multiploid ctcs compared with patients with < 2 multiploid . thus , unfavorable ( 2 ) post - therapy multiploid ctcs may predict inferior pfs and os for agc patients . selection of the optimal cut - off value of multiploid ctcs for overall survival ( os ) . ( a ) receiver operating characteristic ( roc ) curves for pre- and post - therapy multiploid ctcs predicting os ; ( b ) youden index for different cut - off values of multiploid ctcs based on the roc curves in ( a ) ; ( c , d ) roc areas under the curve ( aucs ) for multiploid ctc thresholds of 1 to 3 in pre- ( c ) and post - treatment patients ( d ) . the threshold of 2 multiploid ctcs is selected as unfavorable because of the maximal auc area ( 0.769 for post - therapy ctcs ) . correlation of multiploid ctcs in pre - treatment ( a , b ) and post - treatment ( c , d ) patients with progression - free survival ( pfs ) ( a , c ) and overall survival ( os ) ( b , d ) . to investigate the significance of multiploid ctcs , variations of quantified multiploid ctcs before and after treatment in response to therapy were compared . patients after 6 weeks of therapy were classified into two groups : group 1 with the number of multiploid ctcs decreased 10% following therapy ; and group 2 with the number of multiploid ctcs increased 10% . as shown in figure 3a , the median pfs of group 2 was significantly shorter than that of group 1 [ 4.5 ( 95% ci , 1.27.8 ) months vs. 8.3 ( 95% ci , 7.79.1 ) months , p=0.003 ] . group 2 had inferior median os compared to group 1 [ 11.4 ( 95% ci , 3.823.0 ) months vs. 20.0 ( 95% ci , 18.022.0 ) months , p=0.004 ] ( figure 3b ) . statistical analysis showed that quantitative comparison of multiploid ctcs after therapy may help evaluate therapeutic response and patient prognosis . patients with 10% decreased multiploid ctcs after the first 2 cycles of therapy may have improved pfs and os and those with 10% increased multiploid ctcs after the first 2 cycles of therapy may have inferior pfs and os . correlation of variations of multiploid chromosome 8 ctcs after therapy with progression - free survival ( pfs ) ( a ) and overall survival ( os ) ( b ) . patients are categorized into two groups according to the trends of changes of multiploid ctcs from pre - treatment to post - treatment . group 1 , multiploid ctcs are decreased 10% ; group 2 , multiploid ctcs are increased 10% . to compare the predictive value of variable multiploid ctcs with the one - site unfavorable ctcs , cox proportional hazards regression model was used and the results showed that several unfavorable parameters were associated with inferior pfs and os ( figure 4 ) . a 10% increase of multiploid ctcs after therapy indicated the highest risk of progression and mortality . moreover , multivariate analysis demonstrated that after adjusting for clinically significant factors ( including post - therapy ctcs , post - therapy multiploid ctcs and increase of multiploid ctcs which were significant predictors in cox univariate analysis ) , only 10% increase in multiploid ctcs was an independent predictor of pfs ( hr , 9.697 ; 95% ci , 1.58259.444 ; p=0.014 ) and os ( hr , 6.833 ; 95% ci , 1.35034.583 ; p=0.020 ) . thus , comparing multiploid chromosome 8 ctcs before and after treatment can be used to evaluate therapeutic efficacy . forest plots displaying cox univariate analysis of variables predicting to progression - free survival ( pfs ) and overall survival ( os ) . variables include clinicopathological characteristics , numbers of total ctcs , subtypes of triploid and multiploid chromosome 8 ctcs in pre- and post - treatment patients , as well as variation of multiploid ctcs after therapy . clinical validity of the ctcs shed from various solid tumors including agc has been published elsewhere ( 1,3,4,11 ) . nevertheless , clinical application of a conventional epithelial cellular adhesion molecule ( epcam)-dependent strategy for ctc measurement is limited due to heterogeneous expression of epcam or the absence of both epcam and cytokeratin ( ck ) induced by epithelial - mesenchymal transition ( emt ) ( 4,19 - 21 ) . our previous work confirmed that epcam - independent se - ifish was better than an epcam - dependent strategy for measuring ctcs in agc patients ( 12 ) . here , we report that most patients had 1 ctc per 7.5 ml as measured by se - ifish . post - therapy patients with 4 ctcs had worse pfs and os compared with those with < 4 ctcs . triploid ctcs measured before or after treatment did not correlate with pfs and os , perhaps due to the intrinsic chemoresistance of trisomy ctc , causing an unchanged number of triploid ctcs after therapy ( 12 ) . however , unfavorable ( 2 per 7.5 ml ) multiploid ctcs in post - therapy patients was significantly associated with inferior pfs and os . moreover , patients with 10% decrease in multiploid ctcs after the first 6 weeks of therapy may have improved pfs and os , whereas those with 10% increase in multiploid ctcs had inferior pfs and os . compared with one - site unfavorable total ctcs and the subtype of multiploid chromosome 8 ctcs , a 10% increase in post - treatment multiploid ctcs had the greatest highest hr for pfs and os , and it was the only independent prognostic value according to cox multivariate analysis . although the optimal cut - off value for variation of multiploid ctcs needs to be further analyzed in expanded studies , data in this study show a correlation of multiploid ctcs with acquired drug resistance ( 11 ) . also , multiploid ctc counts in post - therapy patients might predict the course of agc as they offer a more rapid method of evaluation compared with radiographic imaging . karyotypic characterization of enriched ctcs during therapy might replace imaging to assess therapeutic resistance , and guide the clinical treatment of cancer patients . molecular mechanisms of aneuploidy of ctcs and its correlation to diverse therapeutic sensitivities warrant the study . on - going next generation sequencing ( ngs ) analysis of a single ctc may reveal genomic landscapes of ctc subtypes possessing distinct clinical significance ( 22 - 25 ) , which should facilitate the elucidation of evolutionary mechanisms involved in therapy resistance . unfavorable ( 4 ) post - therapy ctcs may predict inferior prognosis in agc patients . previous work showed that triploid ctcs in agc patients were constantly resistant to chemotherapy , and multiploid ctcs developed an acquired chemoresistance ( 11 ) . the data from this study suggested that unfavorable ( 2 ) multiploid ctcs in post - therapy patients were significantly associated with inferior pfs and os . thus , ctcs may be used to assess therapeutic efficacy and predict inferior prognosis . the se - ifish platform for ctcs detection was free provided and optimized by daisy dandan wang and peter ping lin in cytelligen , san diego , ca . 81301323 , 81472789 ) ; beijing natural science foundation ( no . 7161002 ) ; and the capital health research and development of special ( no .
objectiveprevious 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.methodsthe prospective study was performed on 31 patients with newly diagnosed agc . 
 previously established integrated subtraction enrichment ( se ) and immunostaining - fluorescence in situ hybridization ( ifish ) 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 . 
 resultsctcs 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 ml 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.conclusionsaneuploidy 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 .