Intratumoral lymphatic vessels have been noted in certain human cancers, including some characterized by frequent metastases to regional nodes such as melanomas, head and neck carcinomas, papillary thyroid and breast cancers [5
]. It has been established that dissemination of metastases may occur via the lymph nodes [13
]. In melanoma and head and neck cancer, it has been postulated that cure might follow from the removal of regional metastases in a minority of those without evidence of disseminated disease [14
]. Our use of specific IHC staining for lymphatic vessels highlighted the near ubiquity of lymph vessels within VGP melanomas.
Metastatic spread of tumor cells is responsible for the majority of cancer deaths. In this study, we tested the hypothesis that LI and other evidence of intraprimary metastasis (“local metastasis”) are associated with regional nodal metastatic disease, as well as disease disseminated beyond the region and melanoma death. Several previous studies using antibodies recognizing LYVE-1 or podoplanin to highlight the lymphatic vessels in melanomas have showed that the extent of tumor lymphatic density was significantly associated with regional lymph node and disseminated metastasis [2
]. It has been proposed that the increase in lymphatic surface area increases the chances of intravasation and subsequent dissemination of neoplastic cells [19
]. A recent study by Shields et al described both lymphatic endothelial cell (LEC) chemotaxis and proliferation in response to metasatic melanoma cells and secretion by endothelial cells of chemotactic agents that attract melanoma cells, suggesting that bidirectional interactions between these partners may promote lymphatic invasion [20
]. Consistent with this hypothesis, the present study unequivocally showed that lymphatic invasion is common in primary VGP melanomas. Interestingly, Shields reported that peritumoral lymphatic density for melanomas is no greater than that found in normal skin. Although we did not address whether intra- and peritumoral lymphatic invasion are associated with lymph node metastasis, we showed that intra-tumoral and peri-tumoral lymphatic vessels are prevalent and that they and tumoral lymphatic invasion are associated with both metastasis and melanoma-related death.
These considerations raise the important biological and clinical questions of whether some primary melanomas initially metastasize solely via a regional lymphatic route and whether this is translatable into a therapeutic advantage, that is, whether removal of “early” metastatic disease by sentinel lymphdenectomy is associated with improved overall survival (as suggested by Morton et al’s secondary analysis of their randomized trial of the impact the procedure on survival [14
]or only with informative prognostic information as suggested by Gervasoni et al [21
]. We preliminarily addressed this issue in an analysis that examined whether patients with regional metastatic failure would more frequently have lymphatic invasion than those who failed with disseminated disease. In the present study, patients with evidence of regional skin/nodal metastatic disease (n=30) were more likely to have lymphatic invasion compared to those with disseminated disease and no evidence of skin/nodal disease (n=10). The lymphatic invasion rates for these two groups were 50% and 30%, respectively. As expected in this small dataset, these percentages were not significantly different (Fisher’s exact test, two-sided, p=0.4645). Patients with evidence of regional skin/nodal disease also more frequently had “local metastasis” compared to those with disseminated disease and no evidence of skin/nodal disease. The local metastasis rates for these two groups were 63% and 40%, respectively. These percentages were not significantly different (Fisher’s exact test, two-sided, p=0.2743). LI in the form of tumor emboli has been reported in up to 5–8% of primary lesions, similar to our study’s rate of 5% using H&E stained slides. Most studies only stain lymphatic vessels without simultaneously staining for tumor cells. This would be expected both to miss the single or non-aggregated and infrequent intralymphatic tumor cells [1
] and to misinterpret other intralymphatic cells as tumor cells. With double IHC staining and MSI, we found a surprising 35% of the cases had LI. We believe it is potentially of clinical importance to identify LI. Its presence likely reflects a step in tumor progression beyond lymphangiogenesis explaining the association of lymphangiogenesis with worse prognosis. It, perhaps as part of a composite biomarker “local metastasis”, may also be an independent predictive factor for regional metastasis. The test of the latter hypothesis and of the hypothesis of a therapeutically exploitable lymphatic pathway awaits larger studies that also include makers of blood vascular invasion, that specifically address the status of SLN biopsies, and that have long term follow-up for outcomes that include patterns of metastatic failure and disease-specific survival.
This study demonstrates the potential utility of MSI in quantifying biomarkers (e.g., with image analysis) and interpreting the spatial and anatomical distribution of biomarkers of high order processes (e.g., lymphatic vascular invasion). We have demonstrated that MSI can be used effectively to separate multiple dyes on archival formalin-fixed paraffin-embedded histological sections of melanomas. With the development of new visualization methods, such as quantum dot labeled antibodies [23
], it will be possible to extensively phenotype tumor cells and their relationship with their microenvironment on a single histological section.
We conclude that LI is an under-observed phenomenon in primary melanomas. Detection of LI in primary melanomas can be significantly improved by IHC and MSI. LI is a strong candidate as a clinically useful predictor of the state of regional nodes that may influence clinical decision-making (e.g., with respect to whether or not to offer patients with primary melanomas SLN biopsies). The use of MSI to detect biomarkers and to reveal them in an anatomical context can be widely generalized to address other issues in tumor biology and prognostication.