esophageal squamous cell carcinoma ( escc ) is the predominant histological subtype worldwide with high incidence rate in asia . to the best of our knowledge
, there is no data available regarding the incidence or epidemiology of escc in the study area .
however , in the neighboring state of assam in the north - east region of india , it is the leading cause of cancer in men and ranks second in women . 
 

although tremendous progress has been made in surgery and adjuvant chemoradiotherapy , prognosis of these patients remains poor .
a detailed search into these genetic alterations can provide crucial clues to discover novel biomarkers for improving diagnosis and guiding targeted therapy . 
 
 among the various genetic alterations studied as putative biomarkers in escc ,
gene amplification and over - expression of cyclin d1 have been frequently demonstrated in escc .
p16 is involved in the pathogenesis of esophageal cancer by influencing the cyclin kinase inhibitor cascade and dna mismatch repair processes . 
 
 in the present study ,
expressions of cyclin d1 and p16 in escc were evaluated and their association with various clinico - pathological parameters was evaluated to study their role as prognostic markers in escc . 


 the study was conducted in a group of 30 consecutive patients with primary escc without any prior history of chemotherapy or radiotherapy .
the diagnosis was based on endoscopic examination and histopathological examination of the esophageal endoscopic biopsies and/or resection biopsies of the patients admitted in neigrihms hospital from january 2011 to january 2012.union international cancer tnm classification guidelines were used for staging .
the study was approved by the research and ethics committee , neigrihms , shillong . 
 

tumors were histologically verified as escc and sub - typed based on the grade of differentiation as well differentiated ( g1 ) , moderately differentiated ( g2 ) or poorly differentiated ( g3 ) .
assessment of cyclin d1 and p16 were done immunohistochemically by horse radish peroxidase ( hrp ) method .
the paraffin embedded sections of the tumor were stained by a monoclonal antibody raised against cyclin d1 ( biogenex , ca , usa ) and p16 ( biogenex , ca , usa ) . 
 

moreover , 4 - 5 um sections were placed from paraffin blocks over the poly l - lysine coated slides .
the sections were baked by keeping the slides in a hot plate at 60 c for one hour and deparafinized by using xylene 1 and xylene 2 for 10 minutes each .
rehydration was done in graded alcohol ( absolute , 90% , 70% ) for 5 minutes each .
they were placed in 10 mmol / l citrate buffer ( ph=6.0 ) to unmask the epitopes .
after microwave antigen retrieval ( 5 min , 450 w ; 5 min , 600 w ) , the sections were allowed to cool down to room temperature ( approximately 20 min ) .
the slides were washed with phosphate buffer solution for three times with 1 minute interval . after blocking the non - specific protein binding sites with serum free protein block for 10 minutes at room temperature ,
the slides were again washed with phosphate buffer solution for three times with 1 minute interval .
primary antibody was then added and the slides were incubated in humidity chamber for 60 minutes at room temperature and washed with phosphate buffer solution thrice with 1 minute interval .
after addition of super enhancer for 30 minutes at room temperature and washing with phosphate buffer solution thrice with 1 minute interval , polymer hrp ( secondary antibody ) was added and incubated in humidity chamber for 30 minutes at room temperature .
the slides were washed with phosphate buffer solution thrice with 1 minute interval and 3,3 diaminobenzidine chromogen was added and incubated for 5 to 10 minutes at room temperature .
the slides were then washed in distilled water and counter stained with mayer s hematoxylin for 1 to 2 minutes .
the slides were put in running tap water for 5 minutes and dehydrated in graded concentration of alcohol ( 70% , 90% , absolute ) for 1 minute each .
each slide was examined under a light microscope without the knowledge of the patient s other data .
a minimum of 100 neoplastic cells were examined . according to the intensity of staining of neoplastic cells and the percentage of positive cells ,
immunoreactivity was classified into the following three categories based on the percentage of tumor cells showing nuclear reactivity : less than 10% , 10 - 50% and more than 50% .
immunointensity was classified as no immunostaining ( - ) , weakly immunostaining ( + ) , weak immunostaining ( + + ) and strongly positive immunostaining ( + + + ) .
for cyclin d1 staining , if strong nuclear staining was recognized in more than 10% of the tumor cells , the specimen was considered to be positive .
if strong nuclear staining was seen in less than 10% of the tumor cells , the specimen was judged to be negative .
positivity for p16 staining was considered if more than 80% of the tumor cells showed strong nuclear staining .
if less than 80% of the tumor cells showed nuclear staining , the specimen was considered to be negative . 
 

normal esophageal epithelia , which represented a positive control , were included in each run and the negative control section was carried out by omitting the primary antibody .
we also considered the adjacent non - neoplastic squamous epithelia to compare the positive staining in tumors . in cyclin
d1 immunostaining , adequate nuclear staining was observed in the benign controls in the normal epithelia in all cases , which represented a positive control .
the benign controls showed p16 expression in less than 20% of cells with weak intensity mainly confined to the basal epithelial cells . 
 

association between cyclin d1 and p16 expression with clinicopathological parameters were evaluated by chi - square ( x ) test and fischer s exact test . probability ( p ) less than 0.05 was considered statistically significant . 


 a total of 30 patients with histologically confirmed squamous cell carcinoma of the esophagus were included in the study .
the age of the patients ranged from 36 years to 79 years with a mean of 53.2 years .
histological grading of the 30 patients was as follows : 2 patients were well differentiated escc ( g1 ) , 14 patients were moderately differentiated escc ( g2 ) and 14 patients were poorly differentiated escc ( g3 ) . there were 7 cases in stage i , 8 in stage ii , 11 in stage iii , and 4 in stage iv .
in all the 30 patients of escc , the association of p16 and cyclin d1 expression and the clinicopathological parameters like tumor 
 
 site , tumor size , tumor invasion to adventitia , lymph node metastasis and histological grade of tumors were analyzed . 
 

out of the 30 patients , positive expression of cyclin d1was detected in 26 ( 86.7% ) patients and the remaining 4 patients were cyclin d1 negative ( 13.3% ) . the percentage of tumors with invasion to the adventitia ( 88.2% ) , lymph node metastasis ( 87.5% ) , and tumors which were poorly differentiated ( 92.9% ) were higher in the cyclin d1 positive tumors than in the cyclin d1 negative tumors .
however , no significant association was found between cyclin d1 expression and the different clinicopathological parameters ( table 1 ) . 
 

we found that 22 ( 73.3 % ) patients had escc which showed negativity for p16 . the remaining 8 ( 26.7% ) patients were positive for p16 .
the percentage of tumors with invasion to the adventitia ( 82.4% ) and poorly differentiated tumors ( 92.9% ) were higher in the p16 negative tumors than in the p16 positive tumors .
however , there were no significant association with regard to site , size and lymph node status of the tumors and p16 expression ( table 2 ) . 


 cyclin d1 gene encodes a protein that complexes with a cyclin dependent protein kinase ( cdk ) to phosphorylate prb protein and promote cell s advancement from the g1 phase to the s phase .
overexpression of cyclin d1 is thought to override the g1 checkpoint , driving tumor cell proliferation .
amplification of cyclin d1 results in growth advantage for tumor cells and enhances tumorigenesis . in escc
studies on escc have shown that cyclin dl overexpression , evaluated by immunohistochemical staining , results from cyclin di amplification . 
 
 in the present study , positive expression of cyclin d1
the percentage was higher than in other high incidence areas like japan , south africa , china and other regions of india . in a study done in japan , the researchers found 25% cyclin d1 overexpression .
similarly in south africa , chetty and co - workers demonstrated 29% of escc immunopositivity for cyclin d1 and in china , lin and colleagues demonstrated 56.5% cyclin d1 immunopositivity . in a study done in india , 67% immunopositivity for cyclin d1
these differences in results reflect geographical and epidemiological variations . 
 
 local dietary habits consist of rice along with fish or meat preparations .
moreover hot chilli , smoked meat and hot tea are quite popular . in a study done by phukan and colleagues in the north - east region of india , it was found that consumption of very spicy foods , hot foods and beverages , a diet containing high amounts of chilli and leftover food was positively associated with the risk of esophageal cancer .
moreover tobacco smoking , betel quid chewing and alcohol consumption are the major known risk factors for esophageal cancer .
betel quid chewing , a common habit in south - east asia has been found to increase the risk of developing escc by 4.7 - 13.3 fold , although other exogenous risk factors may also be involved . the north - east indian variety of betel nut , locally known as 
kwai , is raw , wet and consumed unprocessed with betel - leaf and slaked lime and contains higher alkaloids , polyphenol and tannins , which has been found to be genotoxic .
this assumes importance since using fermented areca nuts with any form of tobacco is a common habit in the area of the study and is a potential risk factor of escc in this region . 
 

the percentage of tumors with invasion to the adventitia ( 88.2% ) , lymph node metastasis ( 87.5% ) and tumors which were poorly differentiated ( 92.9% ) were higher in the cyclin d1 positive tumors than in the cyclin d1 negative tumors .
however , there were no significant association with cyclin d1 expression with regard to site , size , tumor invasion , lymph node status and histological grade of the tumors .
takeuchi and colleagues observed that the percentage of cyclin d1 positive tumors was higher in tumors with invasion to the adventitia and lymph node metastasis .
however in both no significant association of cyclin d1 positivity with tumor invasion , lymph node metastasis or differentiation was observed . 
 

alterations of p16 occur by multiple mechanisms , including mutation , loss of heterozygosity ( loh ) and promoter hypermethylation .
immunohistochemical staining is useful for the detection and localization of aberrant p16 expressionin tumor samples .
takeuchi and co - workers detected loss of p16 in expression 50% , mathew and colleagues in 45% of escc , and taghavi and colleagues in 56% . in the present study the percentage of p16 negativity was higher than the other studies .
increased loss of p16 expression in the present study may be a consequence of various environmental and lifestyle factors specific to this region associated with an increased susceptibility to escc as mentioned above . 
 

the study on association of p16 expression and the clinicopathological parameters showed that the percentage of tumors which were more than 3 cm ( 73.3% ) , tumors with invasion to the adventitia ( 82.4% ) , lymph node metastasis ( 68.8% ) and tumors which were poorly differentiated ( 92.9% ) were p16 negative .
however , there was no significant statistical association in p16 expression with any of these parameters .
there was a tendency for a decreased percentage of p16 negativity in poorly differentiated escc compared with moderately differentiated escc .
the association of p16 alterations with advanced - stage escc suggests that p16 alterations confer tumor cells with invasiveness and aggressiveness . from the therapeutic point of view
this is important as cells lacking p16 are resistant to dna damage - induced growth arrest compared with cells that retain p16 . 
 
 in the present study ,
in another study , 71% of the patients were positive for cyclin d1 and negative for p16 .
the mechanism underlying the correlation between cyclin dl overexpression and loss of pl6 expression has not been identified , but these results suggest that accumulation of many kinds of gene alterations occurs during oncogenesis and tumor progression .
furthermore , the p16 and cyclin d1 alterations may be linked because these cell cycle regulators are associated with cdk4-mediated phosphorylation of prb . 
 

a small sample size was the major limitation of the study and a larger cohort over a longer duration is required to arrive at a conclusive result .
however the present study attempts to analyze the role of p16 and cyclin d1 in escc in a region , where limited data is available regarding escc . 
 
 in conclusion , we report that alterations of cyclin d1 and p16 play an important role in escc .
loss of p16 expression was associated with poor differentiation however cyclin dl overexpression was independent of clinicopathological factors in this study . 

 background 
 esophageal squamous cell carcinoma ( escc ) is one of the lethal cancers 
 with a high incidence rate in asia . 
 many genes including cyclin d1 and p16 
 play important role in its carcinogenesis . 
 we aimed to analyze the expressions 
 of cyclin d1 and p16 with the various clinicopathological characteristics of 
 escc . 
 
 methods 
 we examined 30 biopsy samples of escc for cyclin d1 and p16 protein expressions 
 using immunohistochemistry . 
 immunointensity was classified as no 
 immunostaining ( - ) , weakly immunostaining ( + ) , weak immunostaining ( + + ) 
 and strongly positive immunostaining ( + + + ) . 
 
 results 
 out of the 30 cases , positive expression of cyclin d1 was detected in 26 cases 
 ( 86.7% ) . 
 the percentage of tumors with invasion to the adventitia ( 88.2% ) , 
 lymph node metastasis ( 87.5% ) , and tumors which were poorly differentiated 
 ( 92.9% ) were higher in cyclin d1 positive tumors than in the cyclin d1 negative 
 tumors . 
 however no significant association was found between cyclin d1 
 expression and the different clinicopathological parameters.there were 22 
 cases of escc ( 73.3 % ) which showed negativity for p16 . 
 the percentage of 
 tumors with invasion to the adventitia ( 82.4% ) and poorly differentiated tumors 
 ( 92.9% ) were higher in the p16 negative tumors than in the p16 positive 
 tumors . 
 there was significant association between the histological grade and 
 p16 expression ( p=0.012 ) . 
 however , there were no significant association with 
 regard to site , size and lymph node status of the tumors and p16 expression . 

 
 conclusion 

 the study shows that alterations of cyclin d1 and p16 play an important 
 role in escc . 
 loss of p16 expression was associated with poor differentiation . 
 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONFLICT OF INTEREST