approximately 1 million cases are diagnosed each year of which 42% are from the developing world .
pakistan shows the highest reported incidence of breast cancer from asia , as per the available literature [ 4 , 5 ] .
it is the most common malignancy found among women in pakistan [ 6 , 7 ] .
there has been a recent paradigm shift in the surgical management of breast cancer from radical surgery to breast conserving therapy ( bct ) with better cosmetic results . for early invasive cancer , survival rates , after bct ,
, however , there is a risk of local recurrence . among the predictors of local recurrence
, surgical margin analysis has proven to be the strongest [ 811 ] . despite a few recent guidelines ,
no clear consensus has been reached globally regarding the surgical margin analysis [ 10 , 11 , 13 ] .
this prompted us to conduct a cross - sectional study on breast cancer specimen , to find out the effect of formalin on the closest tumor - free surgical margin .
the study was carried out at a tertiary care hospital over a period of ten months .
our inclusion criteria involved all patients with histopathologically proven breast cancer undergoing surgery as the primary treatment modality .
these patients were undergoing different surgical procedures , namely , wide local excisions , quadrantectomies , and all types of mastectomy ( including simple mastectomy and modified radical mastectomy ) .
the types of cancer included were invasive cancers comprising ductal , lobular , metaplastic , medullary , papillary , and ductal cancers with neuroendocrine differentiation .
in addition , noninvasive cancers , including ductal carcinoma in situ and noninvasive papillary carcinoma , which could be macroscopically analyzed , were also included .
patients who had previously undergone chemotherapy or radiation before the operation were excluded as well .
the specimen was collected from the operation theatre and was handed over to the pathologist . a questionnaire , prepared after thorough literature search ,
was used to record the surgical margins and additional information such as age , stage of the disease , type of surgery , and type and grade of tumor .
the breast specimens were delivered fresh without immersion in formalin to the pathologist , who then prepared them .
continuous demographic variable , that is , age , was described as mean  standard deviation and categorical variables , namely , tumor grade , stage of the disease , type of surgery , final tumor type , and tumor grades , were described as percentages .
the measurements of the closest tumor - free surgical margins in relation to specimen fixation were compared by paired t - test .
using tumor - free surgical margin as the outcome of interest , stratification was used to assess the impact of variables described in the questionnaire , namely , age , tumor stage , tumor type , and tumor grade .
the mean age of the sample size came out to be 53 years with a standard deviation of 13 years .
the patient population in the sample size predominantly underwent mastectomy , including modified radical mastectomy and simple mastectomy .
breast conservation surgery , that is , wide local excision and quadrantectomy , was done in 10% ( table 1 ) .
ninety - two patients in the study population were found to have invasive cancer , whereas carcinoma in situ which was exclusively ductal carcinoma in situ ( dcis ) was found in the remaining eight patients ( table 1 ) .
the outcome variable was the closest tumor - free margin reduction after fixation of the specimen with formalin .
as shown in table 2 , after applying paired sample t - test , the mean reduction in margin was found to be 2.14  mm which was statistically significant ( p value 0.0001 ) .
using the reduction in tumor - free margin after formalin fixation as our outcome of interest , stratification was used to assess the impact of variables described in the questionnaire , namely , age , tumor stage , tumor type , and tumor grade ; mean margin reduction was also evaluated for each of these variable groups and was found to be statistically insignificant ; that is , p value > 0.05 for all ( table 3 ) .
pakistan is considered one of the high risk regions as far as breast cancer is concerned .
current treatment strategies take into account properties of the individual patient 's tumor biology , as well as the size and location of tumor , to guide treatment [ 19 , 20 ] .
the treatment options include both surgical resection for local disease and medical therapy for systemic disease .
surgical resection , being the mainstay of treatment , has come a long way from the time of radical mastectomy to the breast conservation therapy being extensively practiced nowadays . due to the lack of screening program and population awareness in pakistan , most of the patients would present at a younger age with a more advanced disease such as stage 3 or stage 4 and are lymph node positive [ 5 , 21 ] .
this is one of the reasons behind most of the patients in our study having mastectomy rather than bct .
also , at the time period of this study , we were in process of shifting to bct with a cautious approach towards neoadjuvant chemotherapy to make breast conservation possible . with the increasing number of breast conservation surgeries
being performed worldwide for breast cancers , the analysis of surgical margins has gained an important value in determining the outcome of treatment .
many a time , it has been categorized as the most important factor for predicting local recurrence after surgery [ 811 ] .
several studies have reported the effect of positive margins for tumor cells on the rate of local recurrence of cancer for in situ and invasive disease . in 2002 , singletary reviewed 10 studies , with more than 4000 patients of early invasive cancer with stages i and ii diseases .
the recurrence rates ranged from 8 to 25% ( mean 16% ) for positive margins , whereas , for negative margins , the recurrence rates varied between 2 and 7% ( mean 4% ) .
they also defined the positive margin as tumor cells touching the cut margin of the specimen .
there exists significant variation in perception of surgical margins not only amongst various centers but also between the multidisciplinary teams , including surgeons , pathologists , and radiologists involved in the management of the disease .
the debate is over the negative margin definition and the exact margin width to describe negative margins .
houssami et al . found an impact of surgical margins therapy for invasive breast meta - analysis , based on 21 studies , on the local recurrence in breast conservation cancer .
they concluded that the adoption of wider margins ( e.g. , 5  mm ) was unlikely to have a substantial additional benefit for long - term local control over using a narrow margin width ( i.e. , 1 - 2  mm ) for declaring negative margins in invasive breast cancer .
only recently , some regional guidelines have come to support the concept of  no tumor at inked margin  to be taken as negative margin . the practice varies worldwide including pakistan [ 24 , 25 ] .
furthermore , for in situ cancer , the least acceptable margin described is 2  mm   and as high as a 5  mm margin is taken as acceptable negative margin in some regions . hence , shrinkage of margin after formalin fixation may still hold strong implications on further treatment strategy of breast cancer .
although margin shrinkage is much more relevant for bcs specimen , as far as implications are concerned , our study population at that particular cross - section of time underwent mastectomy mostly due to the reasons already described above .
it was not possible for us to complete the needed sample size if we took bct specimen only .
hence , we aimed to describe shrinkage of tumor - free margin after formalin fixation affecting both types of specimen , that is , mastectomy and bct . this can be taken as one of the limiting factors of our study .
formalin fixation of surgical specimen has been described in various types of tumors as being responsible for shrinkage of tumor specimen and its tumor - free margins .
the various tumors that have been studied include colorectal , esophageal , skin , and , more recently , breast tumors [ 15 , 16 , 27 , 28 ] .
the comparison of preformalin fixation macroscopic margin and postformalin fixation microscopic margin in principle may not seem compatible ; however , it is the most convenient and practical way .
this method has already been described in assessing esophageal and colorectal as well as breast tumors in the past [ 27 , 28 ] .
the study confirms a striking shrinkage of tumor - free margin in their specimen with a mean reduction of 3.5  mm which was statistically significant .
, however , contradicts these results and does not show any shrinkage after formalin fixation in sixty - eight breast specimens . in order to see the impact of variables like age , tumor type , tumor grade , tumor stage , and type of surgery on the margin shrinkage ,
what seemed prominent was the fact that the shrinkage was slightly less for carcinoma in situ and breast conservation surgery specimens .
this may have been a result of a small sample size of these two subsets of our study population .
firstly , it was a cross - sectional study due to which long - term outcome in the form of recurrence could not be analyzed .
secondly , the mastectomy specimens in our study had only one margin to be described , which was the posterior tumor - free margin , even though all margins are equally important in predicting local recurrence .
we used the closest tumor - free margin as an accurate representative of the shrinkage of the rest of the margins as well .
firstly , it can facilitate the journey towards establishing a universal definition of margin analysis .
there is also a need for more research on the techniques of transport and fixation of specimen to avoid handling errors .
we would also like to suggest that more research be conducted on the effect of formalin fixation , preferably on a larger sample size and on bct specimen only for better inference .
prospective randomized control trials can also be considered to compare the recurrence of tissue analyzed with and without formalin fixation .
our breast cancer specimens show considerable shrinkage of their tumor - free surgical margins due to tissue fixation with formalin .
this inference may have effects on the postoperative management plan of these patients and can end up in decisions for further surgery or adjuvant radiotherapy .
 margin analysis in breast surgery is an important predictor of local recurrence and can have vital impact on the postoperative treatment planning . 
 objective . 
 the aim was to assess the mean reduction in the closest tumor - free surgical margin in millimeters of breast cancer specimens following formalin fixation . 
 materials and methods . 
 we conducted a cross - sectional study at the aga khan university hospital from march 30 , 2010 to january 20 , 2011 . 
 one hundred consecutive breast tumour surgical specimens which had macroscopically visible tumour were included . 
 the cancer type included both in situ and invasive cancers . 
 excluded were the patients who had previous surgery or systemic / radiation therapy . 
 the closest tumor - free margin was recorded and compared with the margin after formalin fixation . 
 p value of < 0.05 was considered significant . results . 
 the mean age of our 100 patients was 53 years with the majority of the patients having undergone mastectomy for predominantly invasive ductal carcinoma . 
 following formalin fixation , the mean reduction of the closest tumor - free margin was noted as 2.14  mm which was found statistically significant . conclusion . 
 considerable shrinkage of tumor - free surgical margins of breast cancer specimen was noted after formalin fixation . 
 this inference can have implications on the postoperative management plan . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion