Capsule Summary
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D2-40 antibody specifically marks lymphatic channels in tissue sections.
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D2-40 immunohistochemistry enhances detection of lymphatic channels, improving identification of lymphatic invasion by melanoma tumor.
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Sixty-four melanoma biopsies/excisions, with Breslow depth of 2.0 mm or less, were retrospectively evaluated for multiple clinical and pathologic characteristics and their correlation with sentinel node status.
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D2-40–enhanced detection of lymphatic invasion was the most significant predictor of sentinel node status.
The incidence of melanoma continues to rise in the United States and other countries with a considerable mortality rate.1, 2 The American Cancer Society estimates there were 62,480 cases of melanoma in the United States in 2008 with 8420 melanoma related deaths.3 In the United States, with increased awareness and heightened evaluation, melanomas with a thickness less than 1 mm are most commonly diagnosed, comprising approximately two thirds of all new diagnoses, whereas those larger than or equal to 2.0 mm in thickness comprise only approximately 15% of all new diagnoses.1, 2, 4 Therefore approximately 85% of all melanomas are less than 2.0 mm in thickness. Tumor thickness and ulceration are known to be the greatest pathologic prognostic indicators from the primary lesion.4, 5 Other factors, including site, age, Clark level, mitotic rate, tumor infiltrating lymphocytes, regression, lymphangiogenesis, and angiolymphatic invasion, have been considered to affect prognosis.4, 6, 7, 8, 9, 10 After an established diagnosis, treatment with wide local excision with or without a sentinel lymph node biopsy has become the standard of care.10 Involvement of regional lymph nodes (clinically or microscopically by sentinel lymph node biopsy) is known to be one of the most important predictors of melanoma recurrence and survival, with current risk stratification and adjuvant therapy based on sentinel lymph node biopsy status.5, 11
Currently, different institutions use different criteria identified on the primary biopsy, including varying Breslow depths and other factors to determine who should be offered sentinel node biopsy.5, 12, 13, 14 Multiple studies have examined the importance of various factors identified on the primary biopsy and their impact on sentinel node status and therefore melanoma prognosis.6, 7, 8, 9, 12, 13, 15, 16 While tumor thickness and presence or absence of ulceration have been considered major indicators of tumor prognosis and are incorporated into melanoma staging, more recently the presence or absence of angiolymphatic invasion has begun to emerge as a possible important prognosticator for melanoma outcome. In one study, the presence of angiolymphatic invasion tripled the odds of detecting a positive sentinel node biopsy.6 In a separate study of 526 patients with primary melanoma, the presence of angiolymphatic involvement was the second most important prognostic factor after tumor thickness in predicting survival.17 However, until recently, distinguishing invasion of blood vessels from invasion of lymphatic vessels has been difficult. Previously, angiolymphatic invasion was detected with the use of routine hematoxylin-eosin (H&E) staining alone or with the aid of endothelial cell immunohistochemical markers, such as CD31, CD34, or UEA-1 (Ulex europaeus). H&E alone can lead to false-positive findings, in which areas of retraction artifact are misidentified as lymphatic invasion, and to false-negative findings because of difficulty in identifying tumor emboli that obliterate the lumens of lymphatic vessels. The immunostains mentioned above help with these problems, but are limited in their ability to distinguish lymphatic from blood vessel invasion.18
More recently, lymphatic-specific endothelium markers, LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), D2-40, and podoplanin have been utilized to enhance the detection of lymphatic invasion. LYVE-1 binds to hyaluronan on the luminal surface of lymphatic vessels.19 D2-40 binds to M2A antigen (a monomeric molecular mass 40,000 surface sialoglycoprotein that is extensively glycosylated with O-linked carbohydrate structures)20 and specifically reacts with lymphatic vessels,21 allowing identification of lymphatic vessel invasion by tumor.18 D2-40 has been found to be roughly equivalent to the antibody to podoplanin in its sensitivity and specificity in highlighting lymphatic channels, rather than vascular channels,22 and it has been proposed that anti-podoplanin and D2-40 react against the same molecule.23
The importance of lymphatic invasion and its increased detection by the use of D2-40 immunostaining has been studied in numerous tumors, such as breast, lung, esophagus, colon, gastric, and uterine tumors.24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 In many of these studies, lymphatic invasion correlated with worsening prognosis including increased risk of nodal metastasis, decreased survival, and long-term or distant recurrence. The ability of LYVE-1 and D2-40 immunostaining to detect lymphatic invasion in melanoma and how this may impact sentinel node status has recently begun to be studied.36, 37, 38 Niakosari et al37 reported immunostaining with D2-40 on primary melanomas that resulted in an enhanced detection rate of lymphatic invasion from 0 to 16% (7 of 44). Utilizing LYVE-1, Sahni et al38 showed an enhanced detection rate of lymphatic invasion from no cases of angiolymphatic invasion reported using routine H&E to 16.7% (6 of 36 cases) when LYVE-1 was utilized. Dadras et al7 utilized LYVE-1 immunostaining to show that increased lymphangiogenesis within melanomas correlated with poorer prognosis. In work published in abstract form, Dadras and Doeden39 showed that D2-40 and LYVE-1 immunohistochemistry enhances lymphatic vascular invasion detection by 14% and 11%, respectively, and patients with lymphatic vascular invasion were more likely to have nodal metastasis than those without lymphatic vascular invasion. More recently, in melanomas of 1 mm or more in depth, lymphatic invasion in the primary melanoma biopsy as detected by D2-40 immunostaining was found to be the most significant predictor of sentinel node positivity.36
In the present study, we sought to determine if melanoma lymphatic invasion, as detected by immunostaining with D2-40, correlated with sentinel lymph node status in thin to intermediate depth melanomas (Breslow thickness, ≤2.00 mm). Previous studies have included stage T3a (Breslow thickness 2.01-4 mm, non-ulcerated) and above36, 37, 38; however, the greatest number of melanomas diagnosed are 2.0 mm or less in thickness and cases with a thickness of less than 1.0 mm may present the greatest difficulty in determining whether sentinel node biopsy should be performed. Therefore we were particularly interested in determining whether immunostaining with D2-40 would enhance detection of lymphatic invasion in melanomas of thin to intermediate thickness (pathologic T stage 2B or less) and whether D2-40-positive lymphatic invasion would correlate with sentinel lymph node biopsy status in these cases. Hence, we restricted our study to melanomas with a Breslow thickness of 2.00 mm or less. We also compared lymphatic invasion as detected by immunostaining with D2-40 to other clinical (age and sex) and histologic prognostic features (Breslow thickness, ulceration, and mitotic rate).