Our study of LVI in a large prospectively accrued cohort of melanoma patients demonstrated that the use of endothelial markers D2-40 and CD34 significantly increased the rate of LVI detection over routine histology alone. We also showed that the additional LVI-positive cases identified using IHC markers were significantly associated with adverse clinical variables, DFS, and OS. These results suggest that routine histology fails to detect a substantial number of LVI-positive cases in primary melanoma, which may have clinically relevant prognostic implications for the patient.
Although previous studies have reported increased rates of LVI positivity with IHC markers, it was unclear whether these additional cases had additional prognostic relevance. In many studies evaluating IHC-detected LVI, there were no cases of LVI detectable by routine histology, which thus precluded a comparison between the 2 methodologies.15,19,21
Most IHC-detected cases of LVI remained undetectable even upon review of the H&E-stained sections; thus, it is not known whether these additional cases are true or false positives. Some studies have used SLN positivity as a proxy for the gold standard in evaluating the sensitivity and specificity of IHC-detected LVI.5
However, there are many molecular events that occur between vascular invasion and the ultimate seeding of the SLN, and many patients with LVI-positive tumors do not undergo SLN biopsy, which limits the applicability of SLN status as an appropriate benchmark. Our data demonstrate that the use IHC markers enhances the detection rate of LVI in primary melanoma. Furthermore, LVI-positive cases that would have been “missed” by routine histology are associated with poor DFS and OS on univariate analysis (). These “missed” LVI-positive cases also have unfavorable clinicopathologic features such as increased thickness and ulceration; thus, the association loses significance in the multivariate analysis that includes all of these variables. However, the enhanced rate of LVI detection using markers may help to identify patients at higher risk, tailor treatment accordingly, and possibly improve outcomes.
All LVI-positive channels in our melanoma cohort were either intratumoral or localized to the tumor edge. The clinical and biological relevance of intratumoral LVI has been contested. Previous studies have shown a relatively high incidence of intratumoral LVI that correlates with adverse clinical outcome19
and with SLN positivity,16
whereas others have reported that LVI is more common11
or only detected21
in the peritumoral area. The predominance of intratumoral LVI noted in our cohort and in others may explain, at least in part, the difficulty in detecting LVI by routine histology and the subsequent increase in detection noted with the use of endothelial markers. This observation is similar to previously reported data in breast carcinoma in which LVI is more reliably detected in peritumoral tissue by routine histology but more frequently detected in intratumoral tissue by IHC methods.24
However, only peritumoral LVI is considered in the St Gallen criteria for adjuvant treatment of breast cancer.6
Similarly, functional studies of xenograft mice have suggested that intratumoral lymphatics are not functional and that lymphatic vessels at the tumor margin are solely responsible for metastatic dissemination.18
As the rate of peritumoral LVI noted in our cohort was so low, we were not able to compare outcomes between patients with peritumoral versus intratumoral LVI. However, our results showing that intratumoral LVI is associated with worse DFS and OS independent of stage suggest that intratumoral lymphatics in primary melanoma are functional and that the detection of LVI in an intratumoral location has important prognostic implications.
Our results demonstrated a strong association between LVI and ulceration, which was significant for both the IHC-detected cases and the cases detected by routine histology. A previous study of primary melanomas evaluated for LVI by routine histology alone found that ulceration and LVI had virtually the same effect on risk of relapse and death.10
Another study of IHC-detected LVI in melanoma reported the same association between ulceration and LVI as noted in our study,21
and previous investigations have also noted an association between ulceration and the lymphatic vessel area.11
Although current evidence strongly suggests a link between the 2 variables, it is currently unknown whether one is the cause and the other the effect or whether there is an underlying molecular mechanism simultaneously driving both processes. It has been demonstrated in vitro that melanoma cells overexpress proangiogenic factors such as vascular endothelial growth factor in response to hypoxia but not to other external stimuli such as ultraviolet radiation.22
Thus, it is possible that ulceration is indicative of a hypoxic state in primary melanoma that promotes lymphangiogenesis, which in turn results in a larger vessel area that provides more conduits by which melanoma cells can spread to regional and distant sites. Also unknown is the relative contribution of the melanoma cell versus the lymphatic endothelial cell in promoting invasion. Previous studies have concluded that intratumoral vessels play a passive role in the process.18
Other investigations that emphasize the role of the tumor microenvironment, however, suggest that the endothelium may also secrete cytokines that attract tumor cells and facilitate vascular invasion.7,26
An important finding from our study and from others5
is that the lymphatic endothelial cells in LVI-positive cases are usually intact, suggesting that the process of invasion is more intricate and not merely an effect of mass or volume.
We did not observe a significant correlation between LVI and SLN status. Previous studies using endothelial markers have shown that the extent of lymphangiogenesis in the primary melanoma, irrespective of the identification of tumor cells inside the vasculature, is predictive of SLN positivity and worse OS.3,4,23
However, previous studies investigating the association between vascular invasion and SLN status have yielded conflicting results, with some studies showing a correlation between the 2 variables5,10,16,19
and others showing no correlation20
(). Our data show that neither IHC-detected LVI nor LVI detected by routine histology was correlated with SLN status, suggesting that it should not figure prominently in the decision to perform SLN biopsy in a patient who would otherwise not be a candidate based on currently accepted criteria. Nonetheless, we did note that LVI was a negative prognostic variable in terms of its association with worse DFS and OS. Thus, it is possible that, although LVI is an early event in the metastatic cascade, it is a better predictor of later events and the ultimate outcome rather than of more immediate events such as SLN positivity. Further study is warranted to evaluate why unequivocal evidence of LVI in the primary tumor does not necessarily translate into SLN positivity.
Summary of Published Reports Using Lymphatic Endothelial Markers to Detect LVI in Primary Melanoma
Another consideration to be taken into account is that previous investigations demonstrating an association between LVI and SLN positivity were retrospective and only included cases in which an SLN was performed. Thus, the melanoma cases included in these studies are often thicker (>1.00 mm) and the rate of SLN positivity higher than what would be expected in a consecutively accrued patient cohort from an academic medical center. The cases in our study were prospectively followed up and selected for inclusion without regard to SLN status; thus, many of the cases were thin, and only 11% of patients had a positive SLN biopsy. The selection criteria reflect the aims of the study, which were not to assess the ability of IHC-detected LVI to predict SLN status but to evaluate IHC-detected LVI in terms of the association with adverse clinicopathologic variables and survival.
In a multivariate model inclusive of stage, IHC-detected LVI was significantly associated with worse DFS and OS. One previous survival analysis reported a significant association between IHC-detected LVI and OS in a multivariate model in which ulceration also remained significant.19
Another study, however, consisting of a larger cohort of cases noted no association between IHC-detected LVI and DFS or OS on univariate analysis.5
It is possible that case selection and the length of follow-up contribute to the disparate results noted among these 3 studies. Our results suggest that LVI detected using IHC adds prognostic information beyond that obtainable by conventional staging alone.
In conclusion, the use of endothelial markers significantly increased the rate of detection of LVI in primary melanoma. Thus, the use of endothelial markers should be considered in primary melanoma cases.