urinary bladder cancer is the 13 leading cause of cancer  related death among men and women worldwide .
an estimated 386,300 new cases of bladder cancer occurred in 2008 , making it the 9 most common cause of cancer worldwide .
the highest incidence rates are found in western countries , but incidence is increasing in india . according to recent statistics from the indian cancer registry ,
bladder cancer is 9 most common cancer recorded in india , contributing 3.9% of all cancer cases , irrespective of gender .
much controversy , however , surrounds the grading of bladder tumors . arising from the need to develop a commonly accepted classification system that pathologists , urologists , and oncologists could use , various new classification systems
have been proposed , including the world health organization ( who ) 1973 and the international society of urological pathology ( isup ) 1998 and 2004 classifications . according to 1998 who / isup , 1999 who blue books and the 2004 who classifications , the noninvasive urothelial neoplasms have been divided into three major subgroups  flat , endophytic and papillary .
papillary urothelial neoplasms have been further categorized as urothelial papilloma ( up ) , inverted papilloma , papillary urothelial neoplasm of low malignant potential ( punlmp ) , low  grade papillary urothelial carcinoma , and high  grade papillary urothelial carcinoma . despite the introduction of the new 2004 who / isup classification
the literature indicates that there is still much debate as to whether the 2004 classification should be used exclusively and whether the 1973 who form should be abandoned . reporting both grades
the best grading system should not only be easy to apply , but also allow for division of tumors into groups with different biologic characteristics that correlate with different clinical outcomes .
therefore , the need for objective grading is high , which would increase the reproducibility of a classification system .
proliferation markers , including molecular markers , mitotic activity index and the quantification of nuclear features using morphometry may have a valuable role in further refinements of the classification system , and could be correlated very well with histologic grade in bladder cancer , in addition to being highly reproducible .
such a multiparameter study is very useful as these features are reliable , relatively cheap and easy to assess , and discrimination between different grades may increase when these quantitative and proliferative parameters are used as an adjunct to routine histopathological grading , all with an increase in reproducibility .
the mean profile area of the 10 largest nuclei ( mna10 ) found in a histological tumour section has proved to be valuable as an independent marker for grade and prognosis .
additional studies demonstrate a significant correlation between the ki67 index and the tumor grade and stage of urinary bladder cancer .
it has been noted that ki67 offers good discriminatory power between low malignant potentialcases and carcinoma .
mitotic activity index ( mai ) could be applied in the routine examination of urothelial tumor diseases with increased risk of malignancy , primarily because it is quick and easy to apply , and secondly because of its high degree of fidelity and objectivity .
thus quantitative nuclear parameters ( mna10 nuclei ) and proliferative parameters ( ki67 and mai ) are accurate , well reproducible , easy to assess , progression predictors and can aid in grading all patients with urothelial cell carcinomas .
the purpose of our study on transitional cell carcinomas ( tcc ) of the urinary bladder is to develop a reproducible method for grading that is simple and robust , and may thus be used in daily patient care .
the study group was confined to fifty cases of urothelial neoplasm of different grades obtained either by transurethral resection or by biopsy performed at the departments of urology .
the tissue was fixed in 4% buffered formaldehyde , dehydrated , and embedded in paraffin wax .
all fifty cases of tccs were evaluated according to the world health organisation / isup 2004 classification and were classified as reactive hyperplasia , papilloma , punlmp , lmp(low malignant potential ) and hmp ( high malignant potential ) .
the worst differentiated areas were taken into consideration for mitoticcounts and mna10 measurements , avoiding the necrotic , damaged , inflamed , and benign or lower grade parts of the section .
mna10 measurement was performed on h&e stained sections for the subjectively 10 largest nuclei using an olympus bx 51 microscope at a total magnification of 400x , and at a magnification of 400x on the host computer , processing was done by image  analyzing software ( microsoft image pro  plus version 6.3 ) .
the percentage of tumor cells with nuclear staining was determined by counting at least 1000 tumor cells in the ten selected fields displaying the highest immunoreactivity .
ihc for ki67 was performed by mounting 45 m sections on slides coated with suitable tissue adhesive , deparaffinization of sections in xylene and rehydration through graded alcohols ; antigen retrieval was done in a pressure cooker .
the wash was repeated and diluted primary antibody was applied for 60 minutes , washed , and incubated with post  primary block for 30 minutes .
the slides were rinsed again with the buffer before being incubated with polymer for 30 minutes , further washed and incubated in diaminobenzidine ( dab ) solution for 10 minutes .
counterstaining was done with mayer 's hematoxylin . finally , the sections were dehydrated in graded alcohols and xylene , and mounted with distrene 80 dibutyl phthalate xylene ( dpx ) .
the stained sections were examined and each lesion was graded according to the 2004 who / isup classification .
these grades were correlated and compared with quantitative morphometric ( mna10 nuclei ) and proliferative ( mitotic activity index and ki67 labelling index ) parameters with routine histopathological grade .
new grading was done by using these three parameters as an adjunct to the routine histopathological grade .
the values of mna10 nuclei ( m ) , mitotic activity index ( /10 hpf ) and ki67 labelling index ( % ) were entered into a master spreadsheet ( microsoft excel ) and analyzed using spssv20 software .
descriptive statistics ( mean , standard deviation , range , frequency distribution , percentages ) were applied wherever appropriate .
all the values showed significant correlation with each other and with an increase in the grade of the urothelial neoplasm .
value was significant ( < 0.05 ) for the grading according to these parameters . 

table 1 shows mna10 in different grades of tumor and a comparison with the initial and final grade , using quantitative and proliferative parameters . 
 
comparison of mna10 ( m ) in various grades of tumor 
 table 2 gives the ranges and means with standard deviation of mai/10 hpf , in addition to the comparison with initial and final grade after using quantitative and proliferative parameters . 
 
comparison of mai ( /10 hpf ) in various grades of tumor 
 table 3 , presents the ki67 labelling index in the various grades of tumor .
an increasing trend with an increase in the grade of the tumor is observed . 
 
comparison of ki67 in various grades of tumor in table 4 , a comparison is made between the routine histological grading done using the 2004 who / isup classification and the grading performed in our study according to the quantitative and proliferative parameters .
discrepancy arose in seven cases giving an overall correct classification of 86% with the 2004 who / isup classification . 
 
comparison of grading on the basis of histopathology alone and according to mna10 , mai and ki 67 finally , table 5 shows the distribution and variation in five out of 15 cases graded as lmp in routine reporting , and their subsequent change into punlmp ( 2 ) , lmp ( 10 ) and hmp ( 03 ) after using the quantitative and proliferative parameters 
  distribution of cases of lmp with maximum variation ( 5 cases out of 15 )
mna10 measurement was performed on h&e stained sections for the subjectively 10 largest nuclei using an olympus bx 51 microscope at a total magnification of 400x , and at a magnification of 400x on the host computer , processing was done by image  analyzing software ( microsoft image pro  plus version 6.3 ) .
mai was assessed by counting mitotic figures using well  established criteria . where histological interpretation was in doubt , a mitosis was not registered .
the percentage of tumor cells with nuclear staining was determined by counting at least 1000 tumor cells in the ten selected fields displaying the highest immunoreactivity .
ihc for ki67 was performed by mounting 45 m sections on slides coated with suitable tissue adhesive , deparaffinization of sections in xylene and rehydration through graded alcohols ; antigen retrieval was done in a pressure cooker .
the wash was repeated and diluted primary antibody was applied for 60 minutes , washed , and incubated with post  primary block for 30 minutes .
the slides were rinsed again with the buffer before being incubated with polymer for 30 minutes , further washed and incubated in diaminobenzidine ( dab ) solution for 10 minutes .
counterstaining was done with mayer 's hematoxylin . finally , the sections were dehydrated in graded alcohols and xylene , and mounted with distrene 80 dibutyl phthalate xylene ( dpx ) .
the stained sections were examined and each lesion was graded according to the 2004 who / isup classification . these grades were correlated and compared with quantitative morphometric ( mna10 nuclei ) and proliferative ( mitotic activity index and ki67 labelling index ) parameters with routine histopathological grade .
new grading was done by using these three parameters as an adjunct to the routine histopathological grade .
the values of mna10 nuclei ( m ) , mitotic activity index ( /10 hpf ) and ki67 labelling index ( % ) were entered into a master spreadsheet ( microsoft excel ) and analyzed using spssv20 software .
descriptive statistics ( mean , standard deviation , range , frequency distribution , percentages ) were applied wherever appropriate . spearman 's correlation test was used to determine the correlation between various variables .
all the values showed significant correlation with each other and with an increase in the grade of the urothelial neoplasm .

 table 1 shows mna10 in different grades of tumor and a comparison with the initial and final grade , using quantitative and proliferative parameters . 
 
comparison of mna10 ( m ) in various grades of tumor 
 table 2 gives the ranges and means with standard deviation of mai/10 hpf , in addition to the comparison with initial and final grade after using quantitative and proliferative parameters . 
 
comparison of mai ( /10 hpf ) in various grades of tumor 
 table 3 , presents the ki67 labelling index in the various grades of tumor .
an increasing trend with an increase in the grade of the tumor is observed . 
 
comparison of ki67 in various grades of tumor in table 4 , a comparison is made between the routine histological grading done using the 2004 who / isup classification and the grading performed in our study according to the quantitative and proliferative parameters .
discrepancy arose in seven cases giving an overall correct classification of 86% with the 2004 who / isup classification . 
 
comparison of grading on the basis of histopathology alone and according to mna10 , mai and ki 67 finally , table 5 shows the distribution and variation in five out of 15 cases graded as lmp in routine reporting , and their subsequent change into punlmp ( 2 ) , lmp ( 10 ) and hmp ( 03 ) after using the quantitative and proliferative parameters 
  distribution of cases of lmp with maximum variation ( 5 cases out of 15 )
bladder cancer is morphologically heterogeneous . over ninety percent of bladder cancer cases are urothelial ( transitional cell )
carcinomas , whereas primary squamous cell carcinoma , adenocarcinoma and other tumors are less common .
the pathologic grade is usually critical in determining the clinical management and prognosis of patients .
the previously widely  used 1973 who three  tiered system ( grade 1 to 3 ) was converted into the two  tiered ( low grade [ lg ] vs. high grade [ hg ] ) 2004 who classification or 1998 who / international society of urological pathology classification , which delineate a set of specific and standardized architectural and cytologic criteria for each grade .
the 2004 system introduced a new category , papillary urothelial neoplasm of low malignant potential ( punlmp ) , to encompass cases with even lower potential for progression than typical lg carcinomas .
multiple studies assessing the 2004 who classification have been performed , but universal acceptance of this grading system has not been achieved because of persistent questions regarding its validity and usefulness .
high interobserver variability has been seen , especially in distinguishing punlmps from lg carcinomas [ 14 , 15 ] ( figure 1 & 2 ) .
considering the heterogeneity and broad range of morphologic features in both lg and hg papillary carcinomas , one key issue still awaiting investigation is whether the two  tiered morphologic grading scheme reliably and sufficiently reflects the biology and clinical behavior of these tumors and , more practically , whether there are adjunct immunohistochemical or molecular markers that could be helpful in the classification ( figure 2 , 3 ) .
punlmp ( 1a ) h&e ; 200x ( 1b ) ki67 ; 100x . low grade tumor ( 2a ) h&e ; 200x,(2b ) h&e ; 400x , ( 2c ) ki67 ; 200x , ( 2d ) ki67 ; 400x .
high grade tumor ( 3a ) h&e ; 200x ( 3b ) h&e ; 400x ( 3c ) ki67 ; 200x ( 3d ) ki67 ; 400x .
it is rare in people younger than 50 ; the median age at time of diagnosis is 70 years for each gender . the age ( 6069 years ) of diagnosis
was found to be similar in various studies from asian , middle east and western countries [ 1520 ] .
the incidence of tcc increases with age and is higher among males than females , with a ratio of 3.5:1 .
this is similar to the ratio ( 4:1 ) reported in the united states   and globally [ 1 , 18 ] .
however , higher male : female ratios , varying from 6:1 to 8.6:1 , have also been reported in literature [ 2 , 15 , 17 , 21 ] . according to the 2004 who classification ,
50 bladder biopsies were classified histopathologically as reactive hyperplasia , papilloma , punlmp , lmp and hmp .
the majority were high grade ( 50% ) , 30% were low grade and 12% were punlmp .
the tumors varied in size from 1 to 2.5 cm in lmp and punlmp , and to more than 2.5 cm in hmp .
we identified lesions of , the lateral wall ( 42% ) , posterolateral wall ( 28% ) , posterior wall ( 16% ) neck ( 10% ) and dome ( 4% ) .
the posterior wall showed a relatively high incidence for hmp ( 50% ) and the lateral or posterior wall were involved relatively in all grades [ 2224 ] .
however due to subjectivity , the reproducibility of the grading system is low [ 25 , 26 ] . according to epstein et al .
, morphometric grading into two groups support who / isup grading of tcc as low grade and high grade urothelial neoplasm . in our study , the values of the mean nuclear area of the 10 largest nuclei of hmp are significantly higher than in punlmp and lmp , but there is considerable overlap in the range between reactive and punlmp and lmp ( table 1 ) .
mai ( /10 hpf ) in hmp ranged from 15 to 30/10 hpf ( mean 21.36 5.3/10 hpf ) and in lmp , from 5 to 14/10 hpf ( mean 6.42 1.68/10 hpf ) ( table 2 ) .
we found mai to be significantly lower , 2 to 4/10 hpf , in both punlmp ( mean 3.29 1.49/10 hpf ) and reactive lesions(mean 3 1.16 ) .
various authors have also documented similar findings , and it has been suggested that grading of superficial tcc could be based on m / v index ( volume corrected mitotic index ) alone ; it is the best histological variable for grading and prognosis ) [ 2830 ] .
ki67 is a nuclear protein and is used to predict proliferative activity and thus biological aggressiveness of the tumor . in our study
ki67 labelling index showed an increasing trend with an increase in grade ( figure 1 , 2 , 3 ) .
although an overlap was observed in punlmp and reactive lesions , punlmp , lmp and hmp showed significant correlation between histological grade and ki67 positivity ( table 3 ) .
high tumor proliferation or ki67 labelling index may increase the value of the who grading system as well as predictor of stage progression [ 8 , 34 , 35 ] .
when all three parameters were combined with the routine histological grade , there were discrepancies in the grading of seven cases .
only 86% cases were graded correctly using only the 2004 isup / who classification ( table 4 ) .
the grades of five from a total of 15 lmp cases were revised after using quantitative and proliferative parameters ( table 5 ) .
out of these five cases , two were revised to high grade on the basis of all three parameters that were found to be within the range of high grade .
murphy et al . concluded that discrepancies were higher in discriminating punlmp and lmp and that quantitative and proliferative parameters had high reproducibility and could be used as an adjunct to routine histopathology in differentiating various grades .
one case of lmp was also upgraded to hmp on the basis of high proliferation , with an increase in mai and ki67 , but where mna10 nuclei remained within the range .
zainab et al . concluded that morphometric analysis should be used cautiously in dealing with tcc , especially when trying to discriminate between grade ii and iii .
two cases of lmp were downgraded to punlmp , as mai and ki67 were within the range for punlmp .
shim et al . proposed a scoring system suggesting that mitosis may be the best diagnostic parameter for reproducibility of punlmp .
mai and ki67 again proved to be more reproducible in differentiating between punlmp and lmp .
mna10 was within the range of hmp , but mitotic activity and ki67 were very low . again in this case mai and ki67 alone were much more reproducible than using only mna10 nuclei .
however , its reproducibility in grading is lower than mai and ki67 , which are highly reproducible as an adjunct to histopathological grading .
concluded that the discriminating power of mna10 , when combined with ki67 and mai , was found to be stronger than that of mna10 alone .
pich et al . also observed many overlaps of values between g1 and g2 tumors , but no overlapping was found between g3 and g1/g2 tumors .
mna10 nuclei alone could be used significantly in distinguishing low and high grades and its use is reproducible .
an advantage of mai is high reproducibility and simplicity of assessment as it can be determined from paraffin embedded sections and also allows for visualization of proliferating cells and adequate histopathological assessment .
mna10 in combination with ki67 and mai was found to be stronger than mna10 alone .
mai and ki67 are reproducible and can be routinely used as an adjunct to histopathological grading in classifying tumors .
this multivariate grading model should be applied in routine grading to overcome interobserver variability and increase the reproducibility of grading .
these biomarkers are non  invasive , rapidly and easily assessed , more objective and accurate with high sensitivity and specificity , correlating well with the grade .
 introductionmean nuclear area of 10 nuclei ( mna10 ) , mitotic activity index ( mai ) and ki67 are highly reproducible and can be routinely used as adjuncts to histopathological grading in classifying tumors . 
 assays of these biomarkers are non  invasive , rapid , easy to perform , more objective and accurate , with high sensitivity and specificity , and correlate well with tumor grade.material and methodsthis study was conducted at the department of pathology pgims , rohtak on 50 cases , of which 25 cases were high  grade , 15 low  grade , 6 papillary urothelial neoplasm of low malignant potentialand 4 reactive lesions as per the 2004 isup / who classification . 
 mna10 , mai and ki67 immunoquantitation were performed on stained sections.resultsthe age of the patients varied from 35 to 87 years . 
 male : female ratio was 3.5:1 . 
 the mean mna10 ( m2 ) for high grade malignant potential was 104.52 25.64 m2 , which was significantly higher than in punlmp ( 47.64 10.23 ) and lmp ( 51.57 15.66 ) . 
 mai ( /10 hpf ) showed an increasing trend from reactive lesions to hmp , with a mean of ( 3 1.16)/10 hpf to ( 21.36 5.31)/10 hpf respectively . 
 ki67 labelling index , a proliferative marker , revealed increasing trend lowest with reactive lesions ( 10 2.83% ) and highest in high grade tumors ( 65.96 14.44 ) . 
 spearman 's correlation showed maximum correlation between mai and ki67 and the increasing grade of tumor.conclusionsmna10 in combination with ki67 and mai was found to be stronger than mna10 alone . 
 mai has high reproducibility in differentiating low and high grade , with simple assessment in paraffin embedded sections allowing adequate histopathological analysis and visualization of proliferating cells simultaneously . 
 this multivariate grading model should be applied in routine grading to overcome interobserver variability and to increase reproducibility of grading . 
INTRODUCTION
MATERIAL AND METHODS
Quantitative analysis
Proliferative parameters
Immunoquantitation
Procedure for Immunohistochemical (IHC) staining [
Interpretation and statistical analysis
Data analysis
Observations
DISCUSSION
CONCLUSIONS