barrett s oesophagus ( bo ) was endoscopically defined by the presence of at least 2 to 3 cm of columnar epithelium in the lower oesophagus .
recently , the global evidence - based consensus of montreal defined barrett oesophagus as columnar metaplasia lining the distal oesophagus , with specialized intestinal metaplasia ( i m ) with goblet cells , or gastric metaplasia with cardial type or fundi - coxyntic type mucosa . the main feature of intestinal metaplasia is the presence of alcian blue - positive mucin - producing goblet cells .
moreover , in clinical practice , the finding of cardial and/or fundic mucosa without intestinal metaplasia in the distal oesophagus is frequent and it has been reported as columnar - lined oesophagus ( clo ) .
however , it probably represents an early reflux - lesion preceding an evident barrett phenotype with goblet cells , in keeping with previous studies showing that ck7 is a marker related to early phase of gastroesophageal reflux , and it is present also in clo biopsies lacking of i m .
bo with i m represents an important risk factor for oesophageal adenocarcinoma , in keeping with the progression sequence metaplasia - dysplasia - adenocarcinoma .
hypothesized that patients with cardial metaplasia could potentially progress towards dysplasia and adenocarcinoma through i m .
instead , few and controversial data are reported in the literature about the progression risk of clo lacking of i m , giving rise to a question about the clinical management of this lesion . on this basis
, we studied a series of oesophageal biopsies from patients with endoscopic abnormalities related to columnar metaplasia , by performing some double and triple immunohistochemical / histo - chemical stains ( ck7/alcian / pas , p53/alcian/ pas , aurora a / alcian / pas ) in order to simultaneously assess the mucinous histochemical features of the cells with aberrant expression of such antigens .
the aim of the study was to evaluate if some immunophenotypic changes were present in clo cases without i m and if these changes could precede the appearance of the goblet cells or may be present independently of them .
aurora a maps in 20q13.2 , a chromosomal region frequently amplified and over - expressed in several human malignancies ( breast , bladder , prostate , ovary , colon and pancreas ) . recently its expression was evidenced in barrett adenocarcinoma and in clo . in experimental studies ,
the over - expression of aurora a was oncogenic and constantly associated with mutation of the gene tp53 that , becoming unable to block the kinase activity of the aurora a , leads to a strong accumulation of extra - copies of centrosomes .
cells without a functional p53 and with high levels of aurora a are characterized by repeated cell cycles , until they become aneuploid .
these observations identify the protein aurora a as a prognostic marker , being itself an accomplice of carcinogenesis .
to our knowledge , this is the first study in which a combined immunohistochemical/ histochemical method has been applied in the study of barrett pathology .
this is a retrospective study performed on 57 biopsies obtained from h. pylori - free patients showing columnar metaplasia of the lower oesophagus at the endoscopy .
the cases were retrieved from broader clinical records , consisting of 819 oesophageal biopsies collected between june 2006 and june 2012 and comprising 228 cases ( 27.8% ) of columnar metaplasia ( 135 with bo and 93 with clo ) and 26 cases of adenocarcinoma ( 3.1% ) .
formalin - fixed , paraffin - embedded specimens were retrieved from the archives of the department of pathology , university of palermo ( italy ) . for each patient , 4-quadrant biopsy specimens have been taken every 2 cm and stained with hematoxylin - eosin and alcian - pas .
the patients were divided into three groups : i ) group a , 20 bo cases consisting of i m , with alcian blue - positive goblet cells ; ii ) group b , 17 cases of clo consisting of pas+ columnar metaplasia without alcian blue positive goblet cells ; iii ) group c , 20 cases with intermediate features of transitional metaplasia ( tm ) , consisting of pas+ columnar mucosa with only few cells showing initial production of acid - alcian blue positive - mucins , but devoid of goblet cell morphology .
the combined staining method consists of two steps : in the first step an immunohistochemical staining was performed , according to the manufacturer s instructions ( universal lsaab kit , dako ) .
primary antibodies used in this study were : ck7 ( clone sp52 ; 1:100 ; ventana ) , p53 ( clone do7 ; 1:200 ; ventana ) and aurora a ( clone ep1008y ; 1:100 ; epitomics ) .
immunohisto -chemical positivity was considered significant when it was cytoplasmic for ck7 , nuclear for p53 and nuclear or nuclear / cytoplasmic for aurora a. as positive control for aurora a and p53 , we referred to a case of adenocarcinoma arising in the lower oesophagus ( barrett adenocarcinoma ) . for aurora
a and p53 , the positivity in at least 3% of the nuclei was considered significant . for ck7 , cytoplasmic staining in at least one gland
negative controls , lacking primary antibody incubations , were included for each immunohistochemical run . in the second step , a histochemical staining was performed on the same sections previously subjected to the immunohistochemical assay . at first ,
alcian blue staining was performed and the results of this double immunohistochemical / histochemical staining were observed .
subsequently , pas staining was added ( triple staining ) and the results were observed again .
the histochemical stainings were performed with only minor modifications according to poletti et al . for alcian blue staining ,
the sections were stained with alcian blue solution ( ph 2.5 ) for 30 min at room temperature , washed in running tap water for 10 min , rinsed in di water , counterstained in mayer s hematoxylin for 5 min and washed in di water .
the acetic acid as mordant was not used because it has no effect on the final results . for pas staining ,
sections were oxidized in 0,5% periodic acid solution for 5 min , rinced in di water , placed in schiff reagent for 15 min , washed in tap water for 5 min , counterstained in mayer s hematoxylin for 1 min and washed in di water . finally , the slides were dehydrated , cleared and mounted .
differentiation with hydrochloric acid was omitted because it markedly reduces tissue antigenicity and causes fading of the chromogenic reaction when the immunohistochemical staining is performed before pas staining .
a case of adenocarcinoma of the lower oesophagus ( barrett adenocarcinoma ) is showed in figure 1a .
the tumor exhibited strong nuclear positivity for p53 and aurora a ( figure 1b ) and it was used as positive control for the immunohistochemical stains with these antibodies .
ck7 was positive in all the three groups ; it was more diffusely and strongly expressed in group a , while focally and more weakly in groups b and c. group a. both aurora a and p53 immunostainings were positive in the nuclei of alcian blue+ goblet cells in 6 of 20 cases ( figure 2a ) .
noteworthy , they were positive also in nuclei of pas+/alcian blue negative columnar cells , whereas adjacent alcian blue+ goblet cells were negative [ figure 2b ] .
group b. eight of 17 cases showed focal nuclear positivity for aurora a and p53 in pas+/alcian blue negative columnar cells ( figure 3 b , c ) .
noteworthy , ck7 was expressed just in the same aurora a / p53 positive areas ( figure 3d ) .
group c. four of 20 cases showed positive immunostaining both for aurora a and p53 .
aurora a was positive both in alcian blue - negative cells ( figure 4a ) and in cells with initial production of alcian blue - positive mucins , but lacking of goblet cell morphology ( cells with intermediate features between i m and clo ) ( figure 4b ) .
as in the previous group , p53 and ck7 were expressed in the same areas , despite the absence of i m .
noteworthy , the sequential histochemical steps of the double and triple staining did not affect the immunohistochemical results obtained in the previous step .
the combined use of immunohistochemical / histochemical staining methods has rarely been reported in the literature .
wong et al . used the double alcian blue / chromogranin a and alcian - blue / mib-1 staining in specimen of gastric mucosa with intestinal metaplasia and neuroendocrine hyperplasia , to distinguish the proliferating cellular component . to our knowledge
, a double histochemical / immunohistochemical method has not been applied to the study of oesophageal pathology . in our study , all the cases stained positively , at least focally , for ck7 , considered an early marker of gastroesophageal reflux because it is usually absent in normal fundic and cardial mucosa , but present in columnar metaplastic mucosa of the oesophagus , including cases lacking of i m .
furthermore , our study showed nuclear expression of aurora a kinase in columnar metaplasia lacking of goblet cells ( group b ) , often correlating with p53 nuclear expression .
the aurora a kinase belongs to serine / threonine group of kinases that operate as key regulators of mitosis and it has a role in the cell cycle progression .
several studies showed that a deregulation in the expression and activity of this protein leads to defects in the assembly of the centrosome and of the mitotic spindle , aneuploidy , genetic instability and tumourigenesis .
nuclear positivity has been reported in hormone - related ( breast , ovary and prostate ) human cancers associated with a poor outcome , but not in non - hormone responsive conditions , such as in the oesophageal columnar metaplasia and in the barrett adenocarcinoma .
previously , we evidenced the overexpression of aurora a with rt - pcr in cases of columnar mucosa with and without i m . in the present study , we evidenced aurora a immunohistochemical overexpression in the nuclei of pas+ cells . in the group
a we found nuclear aurora a - positive staining in some alcian blue+ goblet cells of barrett mucosa , but , noteworthy , other goblet cells did not stain for aurora a , whereas the adjacent pas+ columnar cells stained positively .
this is in keeping with the findings of group b cases , lacking of i m , in which aurora a was present in the nuclei of pas+ columnar cells .
the over - expression of aurora a in non - goblet cells may support the recently introduced conception of bo without intestinal metaplasia , in contrast with the definition in which the goblet cells represented an essential prerequisite for the diagnosis of bo and an obliged step in the cancer progression .
in fact , the definition of bo is still confusing and ambiguous . according to the american college of gastroenterology and to the american gastroeneterological association ,
intestinal metaplasia is the only type of oesophageal columnar epithelium that clearly predisposes to malignancy and it is still required for the diagnosis of bo .
furthermore , our findings are in keeping with kelty et al . , who observed that patients with clo lacking of i m on biopsy have a similar cancer risk to those with specialized i m , so they conclude that i m is not essential for cancer risk .
of course , since the carcinogenesis is a multistep process consisting of many overlapping mutations , the over - expression of aurora a in the columnar mucosa , with or without i m , should not be considered a condition necessarily leading to the tumour , but a potentially reversible condition after stopping or decreasing of the stimulus .
the appearance of aurora a and p53 nuclear positivity , together with the reflux - related ck7 cytoplasmic positivity , in areas lacking of i m , could suggest another way besides the traditional im - dysplasia - adenocarcinoma sequence . in keeping with our immunophenotypical findings
, we hypothesize a cancer sequence , in which ck7/ auroraa / p53/pas+ columnar mucosa could represent an intermediate step of the carcinogenesis , independently from the presence of intestinal alcian blue+ goblet cells ( figure 5 ) .
recently , in fact , adenocarcinoma of gastroesophageal junction arising from cardial type metaplasia have been described , suggesting again the hypothesis of a double pathway previously reported by us .
noteworthy , in our study , consisting of 819 oesophageal biopsies collected between june 2006 and june 2012 , we found 26 cases of adenocarcinoma ( 3.1% ) , 25 of which aroused in patients without a known clinical history of bo , lacking previous endoscopy and never treated with gerd therapy . on the contrary ,
none of 228 patients with endoscopic and histologic columnar - lined oesophagus ( 135 with bo and 93 with clo ) , treated with constant therapy for gerd symptoms , developed a high - grade dysplasia / adenocarcinoma , during a mean follow - up of 5 years ( range 1 - 12 ) with bi - annual endoscopy and biopsy .
only one case of clo , untreated , progressed to bo with dysplasia ( figure 6 ) .
given our results , we think that the immunophenotypical alterations ( such as aurora a and p53 expression ) in some cells of clo cases could be by themselves responsible of a risk progression , despite the absence of i m .
therefore , in our view , also clo patients require an appropriate management , because the outcome is not ab initio predictable .
of course , in consideration of the low risk of cancer progression , both in clo and bo patients treated with ppi therapy , and of the necessity to refine the surveillance program to obtain a good cost / benefit ratio , we think that a more long - term interval of endoscopies ( e.g. , a 3-year interval ) could be enough if bo and clo cases are properly treated .
ppi therapy , in fact , could be useful not only for the gerd symptoms but also to reduce the risk of cancer progression . in conclusion , the combined staining method allowed us to simultaneously evidence the type of mucins produced by the columnar cells and the immunophenotypical expression of aurora a , p53 and ck7 that may be indicative of an early phase of the carcinogenetic pathway independently from the i m step .
 intestinal metaplasia in barrett s oesophagus ( bo ) represents an important risk factor for oesophageal adenocarcinoma . 
 instead , few and controversial data are reported about the progression risk of columnar - lined oesophagus without intestinal metaplasia ( clo ) , posing an issue about its clinical management . 
 the aim was to evaluate if some immunophenotypic changes were present in clo independently of the presence of the goblet cells . 
 we studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metaplasia , by performing some immunohistochemical stainings ( ck7 , p53 , auroraa ) combined with histochemistry ( alcian - blue and alcian / pas ) , with the aim of simultaneously assess the histochemical features in cells that shows an aberrant expression of such antigens . 
 we evidenced a cytoplasmic expression of ck7 and a nuclear expression of aurora a and p53 , both in goblet cells of bo and in non - goblet cells of clo , some of which showing mild dysplasia . 
 these findings suggest that some immunophenotypic changes are present in clo and they can precede the appearance of the goblet cells or can be present independently of them , confirming the conception of bo as the condition characterized by any extention of columnar epithelium . 
 this is the first study in which a combined immunohistochemical / histochemical method has been applied to barrett pathology . 
Introduction
Materials and Methods
Results
Discussion