mast cells have a wide variety of functions , which not only encompass regulation of immune responses , but also autoimmunity , tolerance to graft rejection , promotion of or protection from cancer , wound healing , angiogenesis , cardiovascular diseases , diabetes mellitus , obesity , and other significant diseases . in the previous days
, these cells were considered as important due to their role in ig - e driven allergic reactions . however , today the wide spectrum of properties of mast cells is well - recognized .
they play an important role in the induction of angiogenesis in the early - stages of development of tumor .
the association of angiogenesis and cancer has been credited to the visionary pioneer folkman ( 1933 - 2008 ) , who first stated that tumor growing was directly dependent on the blood vessel network development .
experimentally induced tumors have shown mast cell accumulation close to the tumor cells before the onset of angiogenesis . on the other hand , tumors induced in mast cell - deficient mice
acikalin et al . found that microvessel density ( mvd ) was a significant prognostic factor in colorectal carcinoma ( ca ) .
they concluded that microvessel counts and mast cell density ( mcd ) have a significant correlation with one another , which prompted them to suggest that mast cells may play a role in promoting angiogenesis thereby assisting in tumor progression . in their study
they found in their study that mvd is an important prognostic factor in case of invasive transitional cell ca of bladder .
concluded that tumor neovascularization might play a vital role in the prognosis of patients with squamous cell ca of the head and neck .
invasive squamous cell carcinoma ( scc ) of the cervix is the most common malignant tumor of the female genital tract in most countries , especially in developing countries like india and the most frequent neoplasm among women in many of them .
this study was done to evaluate the relationship between mcd and mvd in carcinoma in situ ( cis ) , microinvasive carcinoma ( microinvasive ca ) and invasive squamous cell cervical ca .
the term cis was employed when there was no differentiation at any level of the cervical squamous epithelium ( despite some occasional flattening of the surface cells ) and the basal cell was disorganized .
the term cis has now been incorporated within the domain of high grade squamous intraepithelial lesion , under the bethesda classification . even though this classification was originally designed for the cytologic specimens ,
microscopically , three major categories of cervical scc exist : large cell nonkeratinizing , keratinizing , and small cell .
this study was carried out over a period of 2 years from august , 2011 to june , 2013 , in our department .
we studied 6 cases of cis , 4 cases of microinvasive scc ( microinvasive scc ) and 38 cases of invasive cervical ca ( invasive ca ) .
all these tissue sections were obtained from patients who had undergone total abdominal hysterectomy with or without bilateral oopherectomy .
cervical punch biopsies and cases of cervical intraepithelial neoplasia ( cin ) grades 1 and 2 were excluded from our study .
the samples were fixed in 10% formalin and embedded in paraffin , according to standard procedures .
5 m thick sections were cut and mounted on glass slides . for each case , three sections were obtained : one for routine hematoxylin and eosin staining , one for toluidine blue staining and the third for immunohistochemical analysis with cd-34 . for immunohistochemistry , the sections were mounted on poly - l - lysine coated slides .
the toluidine blue stain gives a light blue background to the section with mast cells appearing red - purple in color .
ten  hot spots  ( areas with the highest mcd ) from each slide were identified .
the total of the counts of the ten fields was recorded as the mast cell count per 10 hpf .
however , we found that the toluidine blue staining method was simpler and it produced better results .
single endothelial cell or clusters of endothelial cells positive for cd-34 were considered as a microvessel .
the presence of blood cells or fibrin without any detectable endothelial cells is not sufficient to define a microvessel .
three  hot spots  ( areas with the highest microvessel concentration ) from each slide were identified .
mvd of the sample was estimated as a mean of mvd in three histological fields and it was recorded as the mvd per hpf .
we studied 48 cases , of which 38 were of invasive ca , 4 of microinvasive scc and 6 of cis .
the mean diameter of these tumors was 4.6 cm . among the 38 cases of invasive ca ,
12 were found to be in the stage pt1b1 and 26 in the stage pt1b2 .
all the 4 cases of microinvasive ca were in the stage pt1a2 . among the 38 cases of invasive cervical ca , 34 cases were of large cell nonkeratinizing type .
four cases were of large cell keratinizing type [ figure 1 ] . ( a ) gross appearance of a case of invasive cervical carcinoma ; ( b ) microphotograph showing nonkeratinizing invasive squamous cell carcinoma ( scc ) of cervix , 100 ; ( c ) microphotograph showing nonkeratinizing invasive scc of cervix , 400 ; ( d ) microphotograph showing nonkeratinizing invasive scc of cervix with clear cell differentiation , 400 we found that both mcd and mvd increased from normal samples through cis to invasive cervical ca [ figures 2 and 3 ] . in the four cases of microinvasive ca , the mcd and mvd were more than that of the control samples but less than that of the six cases of cis [ table 1 ] .
microphotograph showing mast cells in invasive cervical squamous cell carcinoma ; ( a ) giemsa stain , 400 ; ( b ) toluidine blue stain , 400 microphotograph showing microvessels by cd-34 staining in ( a ) control cervical tissue samples , 400 ; ( b ) microinvasive carcinoma of cervix , 400 ; ( c ) invasive cervical squamous cell carcinoma of cervix , 400 mcd and mvd in control samples , cis , microinvasive ca and invasive ca the mast cells were mostly found located at the interface between the tumor tissue and underlying stroma .
statistical significance of mcd and mvd as determined by student 's t - test is shown in tables 2 and 3 .
statistical significance of mcd when compared by student 's t - test statistical significance of mvd when compared by student 's t - test
the presence of mast cells in tumor tissue was first reported by ehrlich in 1878 .
mast cells were thought to play a pivotal role in angiogenesis due to their close association with blood vessels and lymphatic channels .
these cells were also found to accumulate in substantial numbers in richly vascularized tissues such as hemangiomas , polyps , and tumors .
the data on relationship between mcd and mvd in cervical cancers and pretumoral conditions are scanty . in our study
, we found that both mcd and mvd increased from normal cervical samples through cis to invasive ca .
but , in the four cases of microinvasive scc in our study , both mcd and mvd were higher than that of the control samples but less than that of cis .
we found highest mcd ( 102/10 hpf ) in a case of large cell nonkeratinizing scc with clear cell differentiation ( stage pt1b2 ) .
lowest mcd ( 20/10 hpf ) was found in two cases of large cell nonkeratinizing scc ( stage pt1b1 ) . in our study ,
lowest mvd ( 2/hpf ) was found in a case of large cell nonkeratinizing scc ( stage pt1b1 ) .
we found highest mvd ( 35/hpf ) in another case of large cell nonkeratinizing scc ( stage pt1b2 ) .
reported that the mcd remained constant through the grades 1 - 3 of cin , but it significantly increased in invasive cervical ca .
they concluded that the mast cells provide an effective mechanism to create the vascularized microenvironment necessary for tumor cells to proliferate and disseminate .
utrera - barillas et al . proved by their results that mast cells and macrophages are of vital importance in the development of tumor associated blood and lymphatic capillaries in cervical ca .
bentez - bribiesca et al . found that the mvd in normal epithelium and in dysplasias was similar but they observed a significant increase in cis and invasive cervical ca .
found an increase in mvd from the control samples through cis to microinvasive cervical ca .
wilk et al . found similar increase both in mcd and mvd in their study . on the other hand ,
naik et al . reported that mast cells increase in inflammatory conditions of cervix , but decrease in malignant conditions .
vieira et al . compared 3 endothelial markers , anticd-31 , anticd-34 and bnh9 , for evaluation of angiogenesis in cervical cancers .
they concluded that anticd-34 and bnh9 have higher sensitivity than anticd-31 . in this study , anticd-34 has been used for assessment of mvd . in our study , all the 38 cases of invasive cervical ca belonged to stage pt1 .
this is probably because surgical treatment or hysterectomy is considered only for early - stage cervical ca .
we also noted that during our study period of 2 years , we obtained a large number of cases of invasive cervical ca ( 38 ) , compared to the number of cases of cis ( 6 ) and microinvasive ca ( 4 ) .
we attributed this discrepancy with the poor socioeconomic conditions and lack of awareness among the patients of developing countries like ours .
the patients fail to seek medical help at an early - stage of the disease , thus contributing to the large number of cases of invasive ca that we encountered .
several studies have found a correlation between mvd and prognosis in cases of cervical cancers .
our study is limited by the fact that a similar assessment could not be done , since we lost most of our patients to follow - up .
randall et al . concluded in their study that mvd is an important prognostic factor in high - risk early - stage cervical cancer .
independently reported in their studies that higher mvd is associated with poor overall survival rates .
found similar significance of mvd and they suggested that in future , this criterion may be used for selection of patients for antiangiogenesis therapy . a correlation between mcd and tumor progression has been reported in malignancies of other organs as well .
mukherjee et al . found that mcd was higher in well - differentiated gastric cancers than in control subjects .
they also found that poorly - differentiated gastric cancers had lower mcd than well - differentiated ones .
reported in their study that mcd is correlated with angiogenesis and progression of tumor in patients with gastric ca .
elezoglu and tolunay et al . found that there is an association between mcd and mvd in colorectal ca .
they reported that the grade of colorectal ca increased with the number of mast cells , while survival decreased with an increase in mcd . in our study
, we found that both mcd and mvd are higher in invasive cervical ca than in cis or microinvasive scc .
mast cell accumulation is related with angiogenesis in cis , microinvasive ca and invasive cervical ca .
the values of mcd and mvd are higher in invasive cervical cas when compared to those in cis , microinvasive ca and control samples .
therapeutic strategies against mast cell mediators or angiogenesis may be of help in control of progression of early - stage cervical ca .
however , further studies with larger sample size are required before drawing any definite conclusion .
 background : mast cells are involved in induction of angiogenesis in the early - stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.aims and objectives : this study was carried out to evaluate the association between mast cell density ( mcd ) and microvessel density ( mvd ) in carcinoma in situ ( cis ) , microinvasive carcinoma ( ca ) and invasive squamous cell ca of cervix.materials and methods : six cases of cis , four cases of microinvasive ca and 38 cases of invasive ca were studied over a period of 2 years from august , 2011 to june , 2013 . 
 ten control samples were included in the study . 
 routine histologic examination was done . 
 toluidine blue stain was used for mcd determination . 
 immunohistochemical analysis with cd-34 was done for assessing mvd . 
 student 's t - test was used to calculate the statistical significance of mcd and mvd.results:both mcd and mvd increased from normal samples through cis to invasive cervical ca . 
 in the four cases of microinvasive ca , the mcd and mvd were more than that of the control samples , but less than that of the six cases of cis.conclusion:there is a correlation between mast cell accumulation and angiogenesis in cis , microinvasive ca and invasive cervical squamous cell ca . 
 mcd and mvd in invasive ca exceed those in cis and microinvasive ca . 
 it gives us an opportunity to postulate that therapeutic strategies against mast cell mediators and angiogenesis may be of benefit in patients of early - stage cervical ca . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION