to date , many studies have reported on the prognostic significance of circulating tumour cells ( ctcs ) detection in metastatic breast cancer ( mbc ) . the most currently used approach to detect ctcs , particularly in clinical trials , is the cellsearch system ( veridex , warren , nj , usa ) , which is the only fda cleared device for the enumeration of ctc in whole blood . it performs automated immunomagnetic epithelial cell adhesion molecule ( epcam ) based enrichment followed by cytokeratins ( ck ) staining of cells in blood samples . according to cellsearch , the standard definition of ctc is an epcam , ck and cd45 nucleated object . circulating cells with these characteristics are detected in about 60% of mbc patients , where their presence was found an independent predictor of overall survival ( os ) and progression free survival ( pfs ) . in the remaining 40% of patients with metastasis disease , , it is theoretically possible that ctc that express epcam will not express cytokeratins , thus resulting undetected by the cellsearch system . in breast cancer , specifically , loss of ck has been described as an indicator of aggressive disease , and found associated to reduced os and unfavourable prognostic factors as hr negativity . in this context , it has been suggested that epithelial - to - mesenchymal transition ( emt ) , which is characterized by loss of ck and gain of mesenchymal markers as vimentin and fibronectin , may be implicated in ctc formation in breast cancer . recently , it has been observed   that a higher proportion of mbc patients with unfavourable prognostic factors ( high grade , triple negative disease , brain metastases ) had undetectable ctc status , suggesting that an underestimation of ctc by cellsearch may be partly because of ctc undergoing emt . aim of this study was to evaluate the prognostic significance of a cell population characterized by lack of ck expression ( thus undetected by cellsearch ) and gain of mesenchymal markers in blood of mbc patients . this study analysed a population of 55 patients treated between october 2007 and december 2008 for mbc at sapienza university of rome . median follow - up period was 24 months . before starting any systemic therapy , all patients characteristics of breast cancer patients blood samples were maintained at room temperature and processed within a maximum of 12 hrs after blood drawing . ctcs were isolated from 10 cc of peripheral blood by cellection dynabeads coated with the monoclonal antibody towards the human epcam . cellection epithelial enrich is designed to optimally enrich bead - free , viable epithelial tumour cells . for each 10 ml blood sample we added 250 l cellection magnetic beads coated with berep4 . we then lysed the enriched cells with the lysis buffer supplied and added 20 l dynabeads oligo(dt ) 25 to capture poly a+ mrna . from the captured mrna the method has been extensively validated , and is able to detect 1 tumour cell / ml of blood avoiding illegitimate transcription from leukocytes . to verify the integrity of extracted rnas , 5 l of each cdna were amplified in pcr buffer containing 25 pmol each of upstream and downstream gapdh primers as housekeeping gene and 1.25 units of platinum taq polymerase ( life technologies ) . the absence of leukocytes in the pellet of epcam positive cells was routinely confirmed by pcr amplifications for cd45 and cytokeratin 8/18/19 ( ck8 , ck18 , ck19 ) , used as markers of epithelial cells . according to standard definition , ctcs were defined as all epcam positive cells negative for cd45 expression but expressing ck8/18/19 . samples negative for cd45 and negative for ck8/18/19 expression were marked as samples negative for presence of ctcs ( as classically defined ) . all samples ( ck and ck ) were then investigated for the presence of vimentin and fibronectin used as markers of emt . sixteen blood samples from healthy volunteers have been analysed for the presence of vimentin and fibronectin . statistical analysis was performed with bmdp statistical software , version 7 ( statistical solutions , saugus , ma , usa ) and spss ( chicago , version 15.00 for windows ) . pfs was defined as the time elapsed between the date of blood sampling and the date of clinical disease progression or death for any cause . meier product - limit method was used to correlate pfs with ck and emt markers on ctc . this study analysed a population of 55 patients treated between october 2007 and december 2008 for mbc at sapienza university of rome . median follow - up period was 24 months . before starting any systemic therapy , all patients blood samples were maintained at room temperature and processed within a maximum of 12 hrs after blood drawing . ctcs were isolated from 10 cc of peripheral blood by cellection dynabeads coated with the monoclonal antibody towards the human epcam . cellection epithelial enrich is designed to optimally enrich bead - free , viable epithelial tumour cells . for each 10 ml blood sample we added 250 l cellection magnetic beads coated with berep4 . we then lysed the enriched cells with the lysis buffer supplied and added 20 l dynabeads oligo(dt ) 25 to capture poly a+ mrna . from the captured mrna the method has been extensively validated , and is able to detect 1 tumour cell / ml of blood avoiding illegitimate transcription from leukocytes . to verify the integrity of extracted rnas , 5 l of each cdna were amplified in pcr buffer containing 25 pmol each of upstream and downstream gapdh primers as housekeeping gene and 1.25 units of platinum taq polymerase ( life technologies ) . the absence of leukocytes in the pellet of epcam positive cells was routinely confirmed by pcr amplifications for cd45 and cytokeratin 8/18/19 ( ck8 , ck18 , ck19 ) , used as markers of epithelial cells . according to standard definition , ctcs were defined as all epcam positive cells negative for cd45 expression but expressing ck8/18/19 . samples negative for cd45 and negative for ck8/18/19 expression were marked as samples negative for presence of ctcs ( as classically defined ) . all samples ( ck and ck ) were then investigated for the presence of vimentin and fibronectin used as markers of emt . sixteen blood samples from healthy volunteers have been analysed for the presence of vimentin and fibronectin . statistical analysis was performed with bmdp statistical software , version 7 ( statistical solutions , saugus , ma , usa ) and spss ( chicago , version 15.00 for windows ) . pfs was defined as the time elapsed between the date of blood sampling and the date of clinical disease progression or death for any cause . meier product - limit method was used to correlate pfs with ck and emt markers on ctc . after ctc isolation , 5/55 ( 9% ) samples were found positive for cd45 and discarded . among the remaining 50 samples , 28 ( 56% ) were found ck / cd45 , thus ctc positive , according to standard definition . on the contrary , 22/50 ( 46% ) these samples , because of the lack of ck expression , would have been considered negative for ctc presence . the median pfs of the ck - group was 10.2 months versus 11.8 of the ck . difference in progression free survival between patients with ck positive versus negative ctcs ( a ) . difference in progression free survival between patients with mesenchymal markers positive versus negative ctcs ( b ) . difference in progression free survival of patients according to concomitant expression of ck and mesenchymal markers on ctc ( c ) . fibronectin and/or vimentin were found expressed in 24/50 ( 48% ) samples . independently of the expression of ck , the median pfs of patients with emt markers on ctcs was 6.6 months versus 15.3 months of those with emt . according to the expression of cytokeratins and emt markers in ctc we had four subgroups of patients : ( 1 ) 10/50 ( 20% ) were ck / emt , ( 2 ) 12/50 ( 24% ) were ck / emt , ( 3 ) 16/50 ( 32% ) were ck / emt ans ( 4 ) 12/50 ( 24% ) were ck / emt . the median pfs of the subgroups were : 16.4 , 5.1 , 14.6 and 8.1 months , respectively . the difference in pfs between patients with ck / emt and those with ck / emt ctc was found statistically significant . a similar significant difference in pfs was found between patients with ck / emt and those with ck / emt ctc ( p = 0.000 ) ( fig . the presence of mesenchymal markers in ctc lacking ck expression is shown in figure 2 . expression of cd45 , ck8/18/19 , vimentin and fibronectin in 5 mbc patients ( lanes 15 ) . m : molecular size marker . in all 16 blood samples from healthy donors we failed to find expression of vimentin and fibronectin ( fig . 3 ) . expression of vimentin and fibronectin in blood from healthy volunteers ( lanes 116 ) . in the era of the  microscopic revolution cancer has been rethought at a single - cell level . data are indeed increasing which support the prognostic value of ctc , in terms of pfs and os , in metastatic breast cancer patients . liquid biopsy offered an alternative strategy to monitor cancer evolution and response to therapies . despite this , a grey area is emerging regarding currently available methods for the detection of ctc . based on current definition of these cells ( epcam / ck / cd45 ) approximately one third of metastatic breast cancer patients have no ctc in peripheral blood . in a very recent work , questions have been raised as to whether negative ctcs and  undetectable ctcs should be differently interpreted . indeed , in a higher proportion of patients with poor prognosis disease , ctcs were not detected using the cellsearch system . this could be partially because of an underestimation of ctc displaying mesenchymal traits , after activation of emt program . in this study we explored the hypothesis that circulating cells lacking ck expression and gaining mesenchymal markers , thus undetected through standard ctc isolation methods , may have prognostic significance in metastatic breast cancer patients . our analysis demonstrates that , independently of the expression of ck on ctc , the groups of patients with worse prognosis are those with ctc expressing mesenchymal markers . indeed , among the four subgroups of patients examined , a substantial difference in pfs has been observed consistently with the gain of mensenchymal markers . thus , we may speculate that the expression of ck alone on ctc may not adequately predict prognosis in mbc patients . in fact cells lacking cytokeratins and expressing mesenchymal markers would not have been counted through cellsearch analysis , because classical definition criteria for ctc were not met . although the potential loss of ctc during the enrichment steps has been widely suggested , whether this cell loss has a negative impact in the detection of prognostic ctc was still unanswered . to our knowledge , this is the first study assessing the prognostic relevance of a different ctc population , selected for the expression of mesenchymal markers ( vimentin , fibronectin ) even in absence of ck expression . our results suggest that , because of the loss of epithelial antigens by ctc during emt , assays targeting epithelial antigens may miss the most invasive pool of ctc . thus , there is an urgent need to improve detection methods to identify , count and characterize all ctc after emt .
abstractcirculating tumour cells ( ctcs ) are independent predictor of prognosis in metastatic breast cancer . nevertheless , in one third of patients , circulating tumour cells are undetected by conventional methods . 
 aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients . 
 we isolated ctc from blood of 55 metastatic breast cancer patients . 
 ctc were characterized for cytokeratins and markers of epithelial mesenchymal transition . 
 the gain of mesenchymal markers in ctc was correlated to prognosis of patients in a follow - up of 24 months . 
 the presence of mesenchymal markers on ctc more accurately predicted worse prognosis than the expression of cytokeratins alone . because of the frequent loss of epithelial antigens by ctc , assays targeting epithelial antigens may miss the most invasive cell population . 
 thus , there is an urgent need to improve detection methods to identify ctc which undergone epithelial mesenchymal transition program .