
 almost 40%60% of patients with colorectal cancer ( crc ) develop metastasis , predominantly in the liver .
tumor angiogenesis has been implicated as a major factor in the development and spread of these metastases .
the recent discovery of vascular endothelial growth factor - c ( vegf - c ) and vegf receptor-3 ( vegfr-3 ) involvement in lymphatic vessel development   and specific lymphatic markers has provided new insights into the field of lymphangiogenesis . using these markers ,
studies have suggested that lymphangiogenesis plays an active role in the formation and spread of colorectal liver metastases ( clm ) [ 4 , 5 ] .
the patterns of intratumoral lymphatics may have potential clinical significance as a predictive marker of disease recurrence and patient survival . in this study , we investigated the patterns of tumor lymphangiogenesis using monoclonal d2 - 40 and lyve-1 antibody , as a predictive marker for disease - free survival in patients with clm .
blood vessels were examined using cd34 antibody to differentiate the blood vessels from the lymphatic vessels .
tissue was obtained from the victorian cancer biobank for 49 patients who underwent hepatic resection for clm .
informed consent had been obtained from these patients at the time of surgery for long - term storage of specimen samples and subsequent research according to well - established protocols .
formal ethics approval was obtained from the human research ethics committee ( hrec ) of the austin hospital for the study ( hrec submission number : h2012/04618 ) .
the tumor mass was divided into four regions for each parameter measured ; tumor periphery , tumor centre , liver immediately adjacent to tumor , and host liver distal to tumor .
previously , we demonstrated that , following the treatment with a novel vascular destructive agent ( vda ) , oxi4503 , the bulk of the tumor died leaving a viable rim of tumor cells at the periphery extending one hundred microns from the tumor - host interface towards the tumor centre .
based on findings from our previous study , the tumor periphery is defined as the area covering the tumor - host interface and extending one hundred microns towards the tumor centre . the tumor centre is the remaining bulk of the tumor without considering the periphery .
the liver immediately adjacent to the tumor is defined as the area at the tumor - host interface and extending one hundred microns away from the tumor .
the host liver distal to the tumor is host liver which is farther than one hundred microns from the tumor .
tissue samples from the specimen were fixed in 10% buffered formalin , embedded in paraffin , and cut into four m thick sections .
d2 - 40 is a new selective monoclonal antibody for lymphatic endothelium which does not cross react with blood vessel endothelium . for d2 - 40 and lyve-1 staining , heat antigen retrieval was performed using tris buffer ( 50  mm ) ph 9.5 for 15 minutes at 99c .
the sections were immunostained with mouse monoclonal antibody d2 - 40 used at 0.03475  mg / ml ( d2 - 40 , dako , victoria , australia ) and lyve-1 used at 0.0067  mg / ml ( abcam , cambridge , usa ) .
cd34 is an established endothelial vessel marker normally expressed on tumor vessels and host vessels undergoing neovasculature . for cd34 , no antigen retrieval was required . normal goat serum ( 20% )
cd34 was used at 0.005  mg / ml ( abcam , cambridge , usa ) . a polymer - based detection kit containing goat anti - mouse immunoglobulins ( igg ) coupled with horseradish peroxidase ( hrp ) ( envision plus , dakocytomation pty , ltd , botany , nsw , australia ) was used .
images of positively stained vessels were captured using a digital light microscope ( nikon coolscope , nikon corporation , japan ) between 10x and 400x magnification .
the images of tumor fields were captured to be representative of the entire tumor , using a raster pattern which allowed for captured fields to be random and avoid overlap .
the images were analyzed using image - pro plus ( version 5 , media cybernetics , perth australia ) .
vessels were assessed as the number of positively stained vessels per tumor area to provide a microvascular density index .
the lymphatic vessel density ( lvd ) assessments were performed by researchers blinded to the patient outcome data .
pairwise comparisons of group means for parametric data were performed using analysis of variance ( anova ) with post hoc analysis as appropriate .
nonparametric data were performed using mann - whitney u or kruskal - wallis tests , as appropriate .
disease - free survival was calculated from the date of surgery to the date of progression or the date of last follow up .
overall survival was not used in this study as a result of a relatively small number of deaths ( 7/49 ) within the follow up period .
receiver operating characteristic ( roc ) analysis was conducted to assess the discriminative performance and the predictive capability of lvd in each region ( tumor periphery , tumor centre , and adjacent liver ) with tumor recurrence as the end point .
an area under the curve ( auc ) of 1 represents a perfect test ; an area of 0.5 represents a worthless test . cut - off points that maximized sensitivity and specificity were established by analyzing roc curve coordinate points . using optimal cut - off thresholds determined from roc analysis ,
kaplan - meier survival curves were generated to compare survival between groups of high and low lvd in the three regions .
all statistical analyses were performed using spss ( statistics package for social sciences , spss , chicago , il ) .
median follow - up time for all patients was 27 months ( range 495 months ) .
all patients had been assessed by a multidisciplinary team consisting of radiologists , hpb surgeons , oncologists , and nuclear physicians prior to commencement of treatment .
standard indications for liver resection of clm were followed after excluding extrahepatic metastases by multidetector computed tomography ( mdct ) of chest and triple phase mdct of abdomen and pelvis in addition to whole body positron emission tomography with fluorodeoxyglucose integrated with computed tomography ( fdg pet / ct ) scans .
patients received a combination chemotherapy regimen including oxaliplatin ( folfox or capecitabine / oxaliplatin ) or irinotecan ( folfiri ) based regimen .
cd34 appears to only stain blood vessel endothelial cells , leaving lymphatic endothelial cells negative .
serial immunohistochemistry staining using cd34 and d2 - 40 antibodies demonstrates the specificity of the markers .
no overlapping of vessels was observed between cd34 and d2 - 40 ( figure 1 ) .
quantification of d2 - 40 staining ( figure 2 ) revealed greater density at the tumor periphery compared to the tumor centre ( 49.22  24.3 positive lvd / mm versus 22.1  11.5 positive lvd / mm , p < 0.001 ) , adjacent liver ( 49.22  24.3 positive lvd / mm versus 4.3  4.9 positive lvd / mm , p < 0.001 ) , normal liver ( distal to the tumor ) ( 49.22  24.3 positive lvd / mm versus 6.7  3.1 positive lvd / mm , p < 0.001 ) , and benign liver ( 49.22  24.3 positive lvd / mm versus 8.7  6.2 positive lvd / mm , p < 0.001 ) .
lyve-1 was found to be expressed in the liver sinusoids but absent from the tumor ( figure 3 ) .
the roc curve in figures 4(a ) , 5(a ) and 6(a ) shows the ability of lvd in the tumor periphery , centre , and adjacent liver to be used as a prognostic marker to predict the likelihood of disease recurrence following hepatic resection .
the roc graph shows a statistically significant ability of peripheral ( p < 0.01 ) , central ( p < 0.05 ) , and adjacent liver ( p = 0.01 ) lvd to predict disease recurrence . peripheral lvd was the most discriminative , with an area under the curve ( auc ) of 0.713 , followed by lvd in the adjacent liver with an auc equal to 0.708 and central lvd with an auc equal to 0.692 .
according to the optimal cut - off values provided by roc analysis , patients were categorized into two groups : low lvd and high lvd in different regions .
low d2 - 40 stained lvd in the tumor periphery ( p < 0.01 ) , centre ( p = 0.01 ) , and liver adjacent to the tumor ( p = 0.018 ) ( figures 4(b ) , 5(b ) and 6(b ) , resp . ) correlated significantly with disease - free survival . 

table 2 shows that patients , with high lvd in the periphery , centre , and liver adjacent to the tumor , appear to demonstrate close correlation to disease recurrence within the first and second year ( p < 0.05 ) after resection .
however , only patients with high lvd in the tumor periphery significantly correlated with disease - free survival within the third year ( p = 0.009 ) after resection . in cox multivariate analysis , taking into consideration the other variables including sex , age , total tumor volume ( ttv ) , largest tumor volume ( ltv ) , and number of lesions , only high lvd in the normal liver adjacent to the tumor showed significant correlation ( p = 0.046 ) to disease recurrence following resection ( table 3 ) .
the potential role of lymphangiogenesis in the process of tumor metastasis has been largely overshadowed by the role of angiogenesis . for decades ,
angiogenesis , the formation of new blood vessels , has been the main focus of research in the pathogenesis of tumor metastases . over the decades
, much knowledge has accumulated linking angiogenesis as an essential step in tumor growth and development .
it has been reported that , in the absence of active angiogenesis , the growing tumor may undergo necrosis and apoptosis beyond 1 - 2  mm in diameter due to limitation imposed by diffusion .
several studies have focused on the microvessel density ( mvd ) as a prognostic tool ; hasan et al . observed elevated levels of mvd in crc to be associated with poor prognosis .
our study is in concordance with those findings and demonstrates significant differences in mvd between different regions of the tumor and adjacent liver .
this highlights the spatial differences within the tumor microenvironment and the heterogeneity of the tumor .
in contrast to the extensive characterization of molecular mechanisms involved in angiogenesis , research into the role and mechanisms of lymphangiogenesis in cancer has been hampered by singular lack of specific markers .
however , recent discovery of lymphangiogenic factor , vascular endothelial growth factor - c ( vegf - c ) , and specific lymphatic markers ( lyve-1 and d2 - 40 ) now allow researchers to identify and focus on the role of lymphangiogenesis in tumor metastases and to determine whether they have prognostic implications
. the recent introduction of lyve-1 and d2 - 40 has paved the way for exciting research into the field of lymphangiogenesis : its mechanisms and the possible role these vessels play in the spread and dissemination of tumor cells [ 7 , 13 ] . despite the recent focus on lymphangiogenesis research , literature on the role of lymphatic vessels in tumor metastasis
early studies reported the absence of lymphangiogenesis within the tumor , initially believed to be due to the high interstitial pressure created by rapidly proliferating tumor cells . due to the absence of intratumoral lymphatic vessels ,
it was suggested that lymphatic vessels at the periphery were responsible for the spread of tumors .
however , more recent studies have reported the presence of intratumoral lymphatics in several different cancers such as breast cancer   and colon cancer . dadras et al . investigated the possibility of using lvd as a marker for prognosis ; the study reported high intratumoral lvd in metastatic lesions compared to primary lesions significantly correlated with poor disease - free survival in cutaneous melanoma . in agreement ,
saad et al . also reported the presence of intratumoral lymphatics in 46% of cases with stage 1 endocervical adenocarcinoma ; however , they observed small and flattened vessels within the tumor , in contrast to the wide open lymphatic vessels found in the tumor periphery , casting a doubt of functionality of intratumoral lymphatic vessels .
in contrast to dadras ' findings , saad et al . showed a significant correlation with peritumoral d2 - 40 lvd and depth of invasion .
due to many conflicting results and the absence of a general consensus on the prognostic value of lvd , different types of cancers need to be investigated separately to identify the prognostic value of lymphangiogenesis .
few studies have investigated lvd as a prognostic marker in clm and the results have been contradictory [ 1921 ] . despite recent progress in this field , the potential use of lvd as a prognostic marker in clm remains unclear .
investigating the expression of vegf - c , a marker associated with lymphangiogenesis , matsumoto et al . reported that vegf - c overexpression significantly correlated with tumor invasion , lymphatic invasion , and lymph node metastases .
in contrast , using lyve-1 as a marker for lymphatic vessels , brundler et al . observed no significant correlation between lvd and clinical outcome and concluded that lvd had no prognostic value in esophageal adenocarcinoma .
one possible explanation for the results reported by brundler et al . maybe due to the antibody used to identify lymphatic vessels .
lyve-1 does not appear to be a sensitive and reliable marker for lymphatic vessels in all solid tumors . in agreement with previous studies
lyve-1 stained liver sinusoidal endothelial cells and was not able to identify lymphatic vessels in the tumor .
lyve-1 is a known hyaluronan ( ha ) receptor found on lymphatic endothelial cells ; however , the liver sinusoidal endothelial cells play a major role in ha catabolism and as a result also display ha receptors . in addition , ichida et al . reported elevated ha levels associated with liver injury including cirrhosis and hepatocellular carcinoma ( hcc ) .
it is believed that reduced expression of the scavenging lyve-1 ha receptors during liver injury and hcc leads to increased ha serum levels .
indeed , in contrast to brundler et al . , saad et al . reported a significant correlation between lvd and lymph node metastasis and lymphovascular invasion and tumor stage in esophageal adenocarcinoma using d2 - 40 as a marker for lymphatic vessels .
due to these few and conflicting results , clear consensus on the potential use of lvd as a prognostic marker remains to be elucidated .
using d2 - 40 as a specific marker for lymphatic vessels , our data demonstrated that lvd in the tumor periphery and centre and adjacent liver significantly correlated with disease - free survival .
lvd in the tumor periphery and centre and adjacent liver correlated with recurrence within the first two years following resection , while lvd in the tumor periphery continued to correlate with disease recurrence three years after resection .
however , in the multivariate analysis , only the lvd in the adjacent liver was significantly correlated with disease - free survival .
the contradictory results regarding the prognostic significance of lvd in tumor metastases may be due to different patient cohorts , specific tumors included in the analysis , or the method / markers used to detect lymphatic vessels .
one of the limitations of this study is that samples were obtained only from a single tumor from each patient .
this , therefore , may not necessarily reflect the lvd in other tumors in the same patient and , hence , be a confounding factor .
this study has demonstrated that d2 - 40 is effective in identifying lymphatic vessels in human clm .
the monoclonal antibody strongly labeled lymphatic vessels without staining blood vessels as observed in the serial staining of d2 - 40 and cd34 .
d2 - 40 was , therefore , found to be an appropriate and selective marker for lymphatic vessels in clm .
our results further demonstrate the potential predictive value of lvd detected by d2 - 40 , as a prognostic marker clm . despite the limitations imposed by the retrospective nature of the study , relatively short follow - up period , and sampling of single lesions from each patient ,
the results have established a foundation for investigating what appears to be a potentially significant predictive factor in the long - term survival of patients with clm .
determining the lymphatic development within tumors may further play a significant role in the selective use of biological agents   with the ability to target lymphatics being currently under development .
 
 background . colorectal cancer ( crc ) is the most common form of cancer diagnosed in australia across both genders . 
 approximately , 40%60% of patients with crc develop metastasis , the liver being the most common site . 
 almost 70% of crc mortality can be attributed to the development of liver metastasis . 
 this study examines the pattern and density of lymphatics in colorectal liver metastases ( clm ) as predictors of survival following hepatic resection for clm . 
 methods . 
 patient tissue samples were obtained from the victorian cancer biobank . 
 immunohistochemistry was used to examine the spatial differences in blood and lymphatic vessel densities between different regions within the tumor ( clm ) and surrounding host tissue . 
 lymphatic vessel density ( lvd ) was assessed as a potential prognostic marker . 
 results . 
 patients with low lymphatic vessel density in the tumor centre , tumor periphery , and adjacent normal liver demonstrated a significant disease - free survival advantage compared to patients with high lymphatic vessel density ( p = 0.01 , p > 0.01 , and p = 0.05 , resp . ) . 
 lymphatic vessel density in the tumor centre and periphery and adjacent normal liver was an accurate predictive marker of disease - free survival ( p = 0.05 ) . 
 conclusion . 
 lymphatic vessel density in clm appears to be an accurate predictor of recurrence and disease - free survival . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion
5. Conclusion