ARTICLE |
Correspondence to: Noriyuki Nagai, Dept. of Oral Pathology & Medicine, Graduate School of Medicine & Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8525, Japan. E-mail: nori@md.okayama-u.ac.jp
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Summary |
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Angiogenesis is an essential process in the progression of malignant tumors. However, little is known of the angioarchitecture in primary oral malignant melanoma. We sought to determine this by the use of periodic acidSchiff (PAS) stain, endothelial markers (CD34, CD105) and laminin, and by transmission electron microscopy in two cases. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and these stained positively for PAS, laminin, and endothelial markers. Mosaic and tumor-lined vessels were infrequently encountered. Most PAS-positive patterned networks and loops ultrastructurally represented intratumor microhemorrhages that probably arose secondary to tumor vessel leakiness. Vascular channels of the vasculogenic mimicry type were rare. They stained for laminin but not for endothelial markers.
(J Histochem Cytochem 50:15551562, 2002)
Key Words: tumor microvasculature, oral malignant melanoma, mosaic vessels, co-opted vessels, vascular channels, intracellular channels, periodic acidSchiff stain, immunohistochemistry, transmission electron, microscopy
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Introduction |
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ANGIOGENESIS is a complex process involving a series of sequential events that include extracellular matrix (ECM) remodeling as well as proliferation and migration of endothelial cells, leading to blood vessel neoformation from pre-existing microvessels (
Recently,
Although cutaneous and uveal melanomas represent the two most common forms of malignant melanomas (
Although there have been many reported studies on the microcirculation patterns and their functional significance in uveal and cutaneous malignant melanoma (
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Materials and Methods |
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Tissue Specimens
From the series of eight cases of primary oral malignant melanoma diagnosed at the Department of Oral Pathology and Medicine, Okayama University, two cases representing the most recently diagnosed (1998 and 1999) were selected for this preliminary study. This is to minimize the inherent problems of preservation of formalin-fixed, paraffin-embedded tissue blocks that may compromise optimal immunohistochemical staining. These slides were reviewed and confirmed as primary oral malignant melanomas. New 45 µm sections were cut and stained routinely with hematoxylineosin and periodic acidSchiff (PAS) without hematoxylin counterstaining so as to reduce background cellular details that may mask the detection of PAS-positive patterned networks.
Immunohistochemistry
For immunohistochemistry, three primary antibodies were selected, two known to react with the cell membrane of endothelial cells and one with the basal lamina. CD34, a pan-endothelial marker, is a transmembrane glycoprotein of 115 kD. It is present on immature hematopoietic precursor cells and endothelial cells, but its function awaits clarification (
For immunohistochemical staining, deparaffinized sections were pretreated for antigen retrieval by autoclave heating (132C, 3 min) in 10 nM citrate buffer (pH 3.3, 5 min). These sections were then immersed in 0.3% methanol containing 1% H2O2 for 30 min to block endogenous peroxidase and rinsed in 0.05 M TBS (5 min, three times) before immersion in blocking solution (Funakoshi; Tokyo, Japan) for 10 min at room temperature. Then the sections were covered with the primary antibody and incubated overnight at 4C. The immunoreaction was performed using an EnVision/AP kit (Dako; Kyoto, Japan). The antigenic sites were demonstrated by reacting sections with New Fuchsin kit according to manufacturer's instructions (Dako). The nuclei were counterstained with hematoxylin. For negative control, sections were reacted with normal rat serum or with the secondary antibody alone. All the control sections were negative. Positive staining controls were included for each antibody and, where present in the specimens, internal staining controls were also checked for appropriate reactions with each antibody.
Transmission Electron Microscopy
Tissue samples containing small regions of tumor with vascular loops localized from light microscopic sections were microdissected out, postfixed in 2.5% glutaraldehyde, and postfixed further in a solution of 1% OsO4. These were dehydrated and embedded according to standard procedure, and thin sections were cut and stained with uranyl acetatelead citrate and examined with a EF LEO912 transmission electron microscope.
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Results |
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Light Microscopic and Immunohistochemical Observations
Microscopically, both tumors consisted of solid sheets of melanoma cells disposed in a predominant lobular growth pattern (Fig 1A). Melanin pigmentation was patchy and of a moderate amount. Except for one case in which the surface oral mucosa was ulcerated and covered with a fibrinopurulent membrane, necrosis was not observed in the multiple tumor samples examined.
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Hematoxylineosin-stained sections showed tumor microvasculature consisting of scattered irregularly shaped blood vessels and many slit-like channels also containing blood or fibrinous material (Fig 1A and Fig 1B). PAS staining demonstrated the formation of complex networks and closed loops within the vertical growth phase of these tumors, each loop surrounding smaller lobules or nests of melanoma tumor cells (Fig 1C and Fig 1D). Blood vessels were embedded in the PAS-positive pattern (Fig 1C and Fig 1D).
Immunohistochemical examination showed that some intratumor vessels were positively stained with pan-endothelial marker anti-CD34 antibodies (Fig 2A) but were not positively stained with anti-CD105 antibodies, indicating that these represented normal vessels entrapped within tumor tissues. Other intratumor blood vessels stained positively for both anti-CD34 and anti-CD105 antibodies but with strong expression of the latter marker (Fig 2B), thus indicating that these represented areas of activated endothelial cells and tumor-associated neoangiogenesis (Fig 2B). Moreover, CD105 stained melanoma cells, suggesting that activated melanoma cells may potentially show endothelial cell-like characteristics, which results in vasculogenic mimicry (Fig 2B). However, in areas of PAS-positive vascular loops and patterned networks, a weak to negative reaction for these endothelial markers was observed. Laminin regularly stained the basal lamina of the normal vessels, intratumor blood vessels, and areas containing PAS-positive networks and loops (Fig 2C). In the latter, laminin positivity produced a reticular meshwork pattern (Fig 2C).
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Transmission Electron Microscopic Observations
Tumor-associated vessels, consisting of capillary-like structures lined by normal endothelial cells (Fig 3), were identified within the tumor mass. These probably represented entrapped normal vessels and/or new tumor vessels co-opted from the existing host vasculature. In the PAS-positive networks and loops, many of these loops were hollow irregular channels, containing red blood cells and fibrinous material and lined with melanoma tumor cells (Fig 4). Most were intratumor microhemorrhages that probably occurred secondary to tumor vessel leakiness. Residual collagen fibrils were identified within them, confirming that these were stromal compartments and not vascular lumens (data not shown). True tumor-lined vessels and mosaic vessels in which endothelial and melanoma tumor cells formed the luminal surfaces were infrequently encountered. Vascular channels composed of solid or hollow ECM columns were also rare. In these channels, red blood cells and an internal basal lamina lining were identified (Fig 5A and Fig 5B). Melanoma cells lined these channels externally. Endothelium was absent.
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Discussion |
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Angiogenesis is an essential process in the progression of malignant tumors during growth and metastasis. This study investigated for the first time the light microscopic, transmission electron microscopic, and immunohistochemical characteristics of the microvasculature in two primary oral malignant melanomas. In these cases, the tumors presented clinically as large pigmented nodular masses arising from the mucoperiosteum of the hard palate and maxillary gingival tissues. Extensive bilateral cervical nodal metastases were present in one case but these tissues were not included for evaluation here.
The present study confirmed earlier reports that the tumor vasculature in malignant melanoma is structurally heterogeneous (
In this study, we also observed that endothelium-lined tumor vessels in primary oral malignant melanomas differ from those found in the normal palatal mucosa and gingival tissues in these aforementioned aspects. The normal palatal mucosa derives its blood supply from the greater and lesser palatine vessels, and the gingival tissues in the maxilla are supplied by superior alveolar blood vessels. These feeder and exit major vessels were not observed in the melanoma tumor tissue samples examined here. The tumor vessels in this study were distinguished from normal vessels entrapped within tumor tissue by their differential expression of CD105, which detects activated endothelial cells and neoangiogenesis (
By the criteria of
Intratumor hemorrhages are believed to be a manifestation of tumor vessel leakiness caused by a defective endothelial monolayer lining these vessels (
Mosaic blood vessels are characterized by the presence of endothelial and tumor cells that jointly participate to form the internal lining of these vessels (
The significant contribution of the tumor microvasculature in oral malignant melanomas as metastatic pathways remains unclear. This is because oral malignant melanoma, like its cutaneous counterpart, has a propensity to spread via the lymphatic system. Although enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, the occurrence of intratumor lymphangiogenesis is questionable (
In conclusion, the results of this preliminary study suggest that primary oral malignant melanoma has a heterogeneous tumor microvasculature consisting predominantly of endothelium-lined blood vessels and, to a lesser extent, tumor cell-lined blood vessels and rare matrix-rich vascular channels.
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Acknowledgments |
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Supported by a grant-in-aid for scientific research B from the Ministry of Education, Science, Sports and Culture (no. 12470385 to NN).
Received for publication December 17, 2001; accepted May 29, 2002.
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