Laboratory Investigation (2017) 97, 118–129 © 2017 USCAP, Inc All rights reserved 0023-6837/17 $32.00 PATHOBIOLOGY IN FOCUS Lymphatic invasion and angiotropism in primary cutaneous melanoma Andrea P Moy, Lyn M Duncan and Stefan Kraft Access of melanoma cells to the cutaneous vasculature either via lymphatic invasion or angiotropism is a proposed mechanism for metastasis. Lymphatic invasion is believed to be a mechanism by which melanoma cells can disseminate to regional lymph nodes and to distant sites and may be predictive of adverse outcomes. Although it can be detected on hematoxylin- and eosin-stained sections, sensitivity is markedly improved by immunohistochemistry for lymphatic endothelial cells. Multiple studies have reported a significant association between the presence of lymphatic invasion and sentinel lymph node metastasis and survival. More recently, extravascular migratory metastasis has been suggested as another means by which melanoma cells can spread. Angiotropism, the histopathologic correlate of extravascular migratory metastasis, has also been associated with melanoma metastasis and disease recurrence. Although lymphatic invasion and angiotropism are not currently part of routine melanoma reporting, the detection of these attributes using ancillary immunohistochemical stains may be useful in therapeutic planning for patients with melanoma. Laboratory Investigation (2017) 97, 118–129; doi:10.1038/labinvest.2016.131; published online 19 December 2016 Primary cutaneous melanoma has a propensity for metastasis, However, despite multiple studies, controversy remains both to lymph nodes and to distant sites, which is also the regarding the clinical relevance of lymphatic invasion in main cause of mortality in patients with melanoma. Sentinel melanoma. As yet, it is not a criterion for pathologic staging, lymph node (SLN) metastasis is one of the most powerful although the presence of lymphatic invasion may prompt SLN predictors of survival for patients with melanoma and biopsy in patients with thin melanoma.7 intravascular spread of tumor cells within lymphatics is Extravascular migratory metastasis (EVMM), in which widely accepted as a key mechanism of metastasis.1,2 Multiple tumor cells migrate along the external surfaces of vessels, is prognostic factors, including tumor thickness, mitotic rate, another potential mechanism of spread of melanoma to and ulceration, are incorporated into the current American nearby or distant sites.8 Angiotropism, believed to be the Joint Committee on Cancer melanoma staging system and histopathologic correlate of EVMM, has been defined by used to guide recommendations regarding which patients melanoma cells cuffing the endothelium of vessels in a should undergo SLN biopsy. However, these factors are pericytic location, located at the advancing front of the tumor limited in their ability to reliably predict which patients will mass. Although the presence of angiotropism within a develop metastasis in the SLN basin, disease progression, or primary melanoma has been associated with melanoma – recurrence, distinguishing them from patients cured by metastasis,9 11 the frequency of angiotropism in melanomas, excision of the primary cutaneous tumor. Thus, many its relationship to intravascular and lymphatic invasion, as investigators have studied additional histopathologic para- well as its clinical significance remain unclear. meters with the aim of identifying melanoma biomarkers associated with aggressive disease. DETECTION OF LYMPHATIC INVASION The prognostic value of lymphatic invasion, defined as Lymphatic invasion in primary cutaneous melanomas can be tumor cells located within lymphatic vessels, in melanoma has difficult to detect on hematoxylin- and eosin (H&E)-stained been the subject of much investigation recently, as immuno- sections alone. Foci of intralymphatic tumor cells may be histochemical markers of lymphatic endothelium have misinterpreted for multiple reasons. For example, tumor become available. In addition, lymphatic invasion has been emboli completely filling or distending lymphatic vessels may shown to be an unfavorable factor in other cancer types.3–6 compress and obliterate the lymphatic endothelial cells, Dermatopathology Unit, Pathology Service, Massachusetts General Hospital, Boston, MA, USA Correspondence: Dr S Kraft, MD, Dermatopathology Unit, Pathology Service, Massachusetts General Hospital, 55 Fruit Street, Warren 829A, Boston, MA 02114, USA. E-mail: [email protected] Received 3 August 2016; revised 24 October 2016; accepted 8 November 2016 118 Laboratory Investigation | Volume 97 February 2017 | www.laboratoryinvestigation.org PATHOBIOLOGY IN FOCUS Lymphatic invasion and angiotropism in melanoma AP Moy et al leading to a false-negative interpretation. Alternatively, some using IHC for both S100 and D2-40 has been most studied artifacts may lead to a false-positive interpretation. For and rates of detection in melanoma have ranged from 33 to instance, stromal retraction artifact adjacent to tumor cells 47% of cases.26–29 Xu et al28 showed that the fraction of can mimic a lymphatic space. Infolding of the wall of melanomas with lymphatic invasion detected using dual IHC lymphatic vessels with abluminal tumor cells may simulate increased with increasing tumor thickness and mitotic rate; intralymphatic tumor depending on the plane of section interestingly, lymphatic invasion was identified in 32% examined. In addition, tumor cells may be artificially (40/125) of thin vertical growth phase (VGP) melanomas introduced into lymphatic spaces during the surgical measuring o1.0 mm in thickness. The utility of other procedure or tissue processing. Thus, careful histopathologic endothelial cell or melanoma markers has also been evaluation is essential. In a multicenter study, Egger et al12 investigated. Sahni et al30 used dual S100 and LYVE IHC to evaluated melanomas 41.0 mm in thickness from 2183 detect definite lymphatic invasion in 17% (6/36) of patients and reported lymphovascular invasion in 7.8% of melanomas, none of which showed lymphatic invasion on cases, as detected on H&E-stained sections. Nagore et al13 initially stained H&E sections. Feldmeyer et al31 used double identified lymphatic invasion in 5.7% (47/823) of melanomas IHC with MiTF, a nuclear melanocytic marker, and D2-40 to (see Table 1 for an overview of studies). identify lymphatic invasion in 38% (21/56) of cases, as The detection of lymphatic invasion is enhanced with compared with 22% (13/64) with D2-40 IHC alone and immunohistochemistry (IHC) using antibodies that are overall 4% (5/120) with H&E staining. specific to the lymphatic endothelium, such as lymphatic Although there is currently no gold standard for the vessel endothelial hyaluronan receptor 1 (LYVE-1) and detection of lymphatic invasion, in our experience, dual IHC podoplanin. LYVE-1 binds to hyaluronan on the luminal with S100 and D2-40 is most helpful in the identification of surface of lymphatic vessels and is absent on blood vessels.14 lymphatic invasion in primary cutaneous melanomas The monoclonal antibody D2-40 detects an epitope on (Figure 1). We recently studied 105 melanomas with dual podoplanin, a transmembrane sialoglycoprotein, and is highly S100/D2-40 IHC and detected lymphatic invasion in 23%.32 sensitive and specific for the endothelium of lymphatic Unequivocal lymphatic invasion can be recognized when vessels.15–18 With the use of LYVE-1 or D2-40 IHC, Doeden S100-positive cytologically atypical cells, stained with a red et al19 detected lymphatic invasion in 16% (15/94) of chromogen, are seen circumferentially surrounded by a thin melanomas studied. Fohn et al20 evaluated 64 melanomas D2-40-positive lymphatic endothelium, stained with a brown measuring 2.0 mm or less in thickness and reported a chromogen. Either solid tumor emboli or single tumor cells sevenfold increase in the detection of lymphatic invasion may be seen within the lymphatic spaces. In contrast, it can be with the use of D2-40 IHC, from 3.1% detected on H&E- difficult to assess the precise identity of cells within stained sections at the time of biopsy to 21.9% detected in lymphatic vessels on D2-40 staining only, as histiocytes and retrospective review with the use of IHC. Additional studies other inflammatory cells may be mistaken for melanocytic using D2-40 IHC have detected lymphatic invasion in cells. Although not specific for melanoma, S100 is a useful 15–33% of cases (Table 1).21–25 Of note, Petersson et al23 stain to aid in this task, as it is most sensitive melanocytic did not detect lymphatic invasion in any of the four marker. In addition, dual staining with S100 and D240 may desmoplastic melanomas nor any of the 29 melanomas also increase specificity, as some of the artifacts seen on o1.0 mm in thickness that were studied. H&E-stained sections can be detected. For example, infolding In addition to highlighting foci of lymphatic invasion, IHC of abluminal tumor cells may be detected with D24-0 staining can confirm the identity of a vessel as lymphatic, as blood even when no endothelial cell nuclei can be seen. However, as vessel invasion may be mistaken for lymphatic invasion on occasional histiocytic and dendritic cells may express S100, H&E-stained sections. Although few studies investigating the IHC evaluation may lead to a false-positive interpretation. presence and clinical significance of blood vessel invasion Therefore, intralymphatic cells must display nuclear have been done, it has been shown that blood vessel