
 basal cell carcinoma ( bcc ) and squamous cell carcinoma ( scc ) of the skin are the most common human solid malignant tumours .
one of the key biological processes that appear to be relevant to the establishment and prognosis of most forms of cancer , including skin cancer , is angiogenesis . using the mouse model eichten et al . demonstrated that during squamous skin carcinogenesis blood vessels undergo increase in endothelial cell proliferation , vessel diameter , vessel density and leakage .
although several studies evaluated the role of microvessel density in the progression of epithelial skin cancer , the proliferation of vascular endothelium or blood vessel area ( bva ) has not yet been investigated . for many years ,
the process of tumour vascularization was thought to proceed mainly through the sprouting of new blood vessels from the preexisting vasculature .
however , in recent years , additional endothelial - sprouting - independent mechanisms have been described , such as vascular cooption , intussusceptive angiogenesis , mosaic vessels , vasculogenesis , and/or vasculogenic mimicry .
two international consensus on quantification of angiogenesis in solid tumours were published [ 4 , 5 ] , considering the chalkley counting technique as a more reproducible and clinically applicable technique .
chalkley counting score reflects the relative vessel area , gibing an estimate of the total perfusion of the tumor .
it is important to note that two categories of endothelial cell specific antibodies are currently available for immunohistochemistry : the panendothelial cell markers , characterized by equal intensity of staining for small and large blood vessels , and antibodies that bind selectively to proliferating endothelium . in order to evaluate the degree of proliferating blood vessels we used the proliferating - endothelium marker cd105 and we compared the results with those obtained with cd31 immunostain .
a total of 70 formalin - fixed , paraffin - embedded surgical specimens of patients presenting with sccs and 70 with bccs at the university hospital  clnico lozano blesa 
the following clinicopathologic variables were recorded : sex , date of birth , date of diagnosis , anatomic site of the tumor , bcc histologic subtype ( superficial , nodular , and aggressive ) , scc histological grading ( well- , moderately , and poorly differentiated ) , lymphovascular invasion , and perineural invasion . in the  aggressive  bcc group ,
immunohistochemical stainings were performed using dako autostainer system and commercially available endothelial markers cd31 ( monoclonal mouse anti - human platelet endothelial cell adhesion molecule- ( pecam- ) 1 antibody , clone jc70a , dako , glostrup , denmark ; dilution : 1  :  50 for 1 hour ) and cd105 ( monoclonal mouse anti - human endoglin antibody , clone sn6h , dako , glostrup , denmark ; dilution : 1  :  100 for 1 hour ) . as positive control ,
the relative blood vessel area was determined both in intratumoral ( it ) and peritumoral ( pt ) areas using chalkley counting technique .
pt vessels were defined as cd34 positive vessels located at the interface of tumour and adjacent dermis ( chalkley method is based on the use of an eyepiece graticule containing 25 randomly positioned dots , which is rotated so that the maximum number of points is on or within the vessels ) .
the average of three highest values for each tumor section was used for the analysis .
the cd31 and cd105 chalkley counts did not follow the normal distribution ( kolmogorov - smirnov = 0.12 , p < 0.0001 , and kolmogorov - smirnov = 0.10 , p = 0.002 , resp . ) ; therefore , the mann - whitney test , wilcoxon signed - rank test , or spearman 's rank correlation was used as appropriate .
the intraobserver and interobserver agreement for classifying the tumours as chalkley count high or low ( using as a cut - off point the mean chalkley count value ) were evaluated by kappa statistic .
the median age of included patients was 77.6 years ( range 60.288.3 ) in scc group and 75.5 years ( range 53.588.9 ) in bcc group at the time of surgery ( p < 0.05 ) .
the main clinical and pathologic characteristics of patients included in the study are compiled in table 1 . in all histological slides cd31 or cd105 positive
the costaining of inflammatory cells has been observed in slides immunostained with cd31 , but these could be distinguished from endothelial cells on the basis of morphological differences .
median peritumoural- ( pt- ) cd31-chalkley count was 8.33 ( sd = 1.98 ) points for scc and 5.81 ( sd = 1.05 ) for bcc ( p < 0.001 ) ( figure 1(a ) ) , while median pt - cd105-chalkley count was 7.84 ( sd = 1.76 ) points for scc and 5.19 ( sd = 1.19 ) for bcc ( p < 0.001 ) ( figure 1(b ) ) .
median intratumoral- ( it- ) cd31-chalkley count was 4.67 ( sd = 1.23 ) for scc and 0.00 ( sd = 1.67 ) for bcc ( p < 0.001 ) , while median it - cd105-chalkley count was 2.33 ( sd = 1.71 ) and median it - cd105-chalkley count was 0.00 ( sd = 0.67 ) ( p > 0.001 ) .
moreover , both pt and it - rbva differed between superficial , nodular , and aggressive bcc ( pcd31 < 0.001 , pcd105 < 0.001 ) and between well- , moderately , and poorly differentiated scc ( pcd31 = 0.007 , pcd105 = 0.03 ) ( table 2 ) .   figure 2 shows samples of cd31 and cd105 immunostaining in bcc and scc . there was a strong correlation between cd31 and cd105 chalkley score ( pearson 's r = 0.8 ; p < 0.001 ) .
we next proceeded to evaluate the degree of proliferating endothelium by dividing the cd105-chalkley score by the cd31-chalkley score .
the mean cd105/cd31 quotient was 0.96 for bcc ( sd = 0.16 ) and 0.79 for scc ( sd = 0.16 ) ( p = 0.024 ) . in a subset of cases
( n = 30 ; 15 sccs and 15 bccs ) , the intraobserver and the interobserver agreement were evaluated by kappa statistic ( both for cd31 and cd105 ) .
the intraobserver agreement between the two examinations by ip for classifying the tumors as high or low chalkley counting values ( taking the mean value for each antibody as a cut - point ) was k = 0.65 for cd31 and k = 0.80 for cd105 , whereas the interobserver agreement between ip and tgc was k = 0.54 for cd34 and k = 0.67 for cd105 ( k - values between 0.41 and 0.60 indicate moderate strength of agreement and k - values between 0.61 and 0.80-substantial strength of agreement ) .
in our present study , we found statistically greater relative pt and it blood vessel areas in scc compared to bcc , both with cd31 and cd105 antibodies .
these findings seem to be logical , since high degree of vascularization provides oxygen and nutrients for tumour growth , explaining , at least in part , the differences in clinical behavior between bcc and scc . while most studies evaluating the role of angiogenesis in cancer , including epithelial skin tumours , used the technique described by weidner et al .
, based on the number of microvessels quantification , we evaluated the blood vessel area , reflecting the global tumour perfusion .
investigated the angiogenesis pattern in bcc and scc of the skin , demonstrating that the majority of samples with high vascularization belonged to the squamous histotype , while the majority of bcc showed a significantly lower number of microvessels .
chin et al . evaluated vascular patterns in sccs , bccs , and trichoepitheliomas ( tes ) using cd31 mab .
differences between the neovascularization in bcc with benign course and bcc with aggressive behavior have been found [ 9 , 10 ] . from the clinical perspective
, the phase ii clinical trial based on 31 patients with advanced cutaneous scc treated with cetuximab showed a disease control rate in 69% and response rate in 28% of included patients .
however and surprisingly , bcc exhibited significantly higher proportion of proliferating endothelium than scc , apparently contradicting the abovementioned results .
there is at least one explanation for these findings , based on angiogenesis - independent mechanisms of tumour vascularization .
intussusceptive angiogenesis occurs by the insertion of the interstitial tissue column into the lumen of preexisting vessels inducing partition of the vessel lumen .
this type of angiogenesis is faster and more economical than sprouting , occurs within hours of even minutes , and does not primarily depend on ec proliferation .
the ability for growing by coopting the massive vascular plexus present in the peritumoural connective tissue was described in aggressive tumours localized in highly vascularized tissues .
thus , our results suggest that scc , characterized by more rapid growth and aggressive clinical behaviour , explores other mechanisms of tumour vascularization which allow faster oxygen and nutrient transport , while bcc growth mainly depends on proliferation of vascular endothelium .
importantly , angiogenesis - independent mechanisms of tumour vascularization were described to be more resistant to antiangiogenic therapy .
angiogenesis is a dynamic process , making its quantification inherently difficult . since no decisions have to be made on whether adjacent stained structures are the reflection of one single microvessel or two separate microvessels , chalkley counting is considered a more objective approach than weidner technique .
we evaluated the reproducibility of this technique , showing both inter- and intraobserver moderate to substantial strength of agreement .
slightly lower agreement in cd31-immunostained slides could be explained by the presence of costaining of inflammatory cells , which could result in the erroneous incorporation of inflammatory cell count into the final score . in conclusion
, our results suggest that the existence of angiogenesis - independent mechanisms of tumour vascularization may be considered as a hallmark of epithelial tumors with more aggressive clinical behaviour .
 
 aims . to evaluate the vascularization in basal cell carcinomas ( bccs ) and squamous cell carcinomas ( sccs ) of the skin 
 . methods . 
 we performed cd31 ( i.e. , panendothelial marker ) and cd105 ( i.e. , proliferating endothelium marker ) immunostaining on samples of 70 sccs and 70 bccs of the skin . 
 we evaluated the relative blood vessel area using the chalkley counting method in each histologic subtype of these tumours . 
 we calculated the degree of proliferation of blood vessel endothelium dividing cd105-chalkley score by cd31-chalkley score . 
 results . 
 we found significantly higher peritumoral and intratumoral blood vessel area in scc when compared to bcc ( both with cd31 and cd105 ) . 
 chalkley counts differed significantly between groups with different bcc histologic subtypes and scc with different grade of differentiation . 
 surprisingly , the degree of proliferation of blood vessel endothelium was higher in bcc when compared to scc . conclusions . 
 while scc exhibited significantly higher intratumoral and peritumoral blood vessel areas compared to bcc , the relatively low rate of proliferating endothelium in this tumour type suggests the existence of endothelial - sprouting - independent mechanisms of vascularization in scc . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion