Identification of melanoma cells in H&E stained sections of sentinel nodes is usually straightforward when they form masses or clusters of tumor cells. However, early metastases may be seen as single melanoma cells, making their detection in H&E sections more challenging as such cells can show a considerable range of morphological variation, and are often amelanotic. Lightly pigmented or unpigmented melanoma cells may closely simulate sinus and other histiocytes that are often abundant in lymph nodes. Extended histopathological search for small metastatic deposits by serial sectioning and immunohistochemistry reportedly detects occult melanoma cells in up to 32% of SN (39
). Micrometastases may be missed if the wrong part of a node is examined or if tumor cells do not express melanoma-associated markers detectable by immunohistochemistry.
There are reports of patients with an intermediate prognosis who have an enhanced signal for mRNA for melanoma-associated genes detected by RT-PCR, in nodes that showed no evidence of melanoma cells on microscopic evaluation (44
). The RT in situ
PCR technique is valuable in basic research and clinical science and permits detection of mRNA in specifically identifiable cells and tissues. In this study, we identified MART-1 mRNA-positive cells in SN and NSN in which prior search for cells expressing MART-1 protein by immunohistochemistry was negative. Some of these cells are truly occult melanoma cells that despite containing MART-1 mRNA do not have detectable cytoplasmic MART-1 protein. Such “stealth” melanoma cells were also detected in microscopically tumor-free NSN from patients with a tumor-positive SN When a completion lymph node dissection (CLND) is performed for patients with a positive SN, tumor is detected in the NSN of 10 to 20% of patients (21
). We showed that MART-1 mRNA-positive cells were also present in microscopically negative NSN. The frequency of tumor-positive NSN may increase when nodal tissues are evaluated by sensitive molecular analyses (4
). The biological significance of nodes found to contain tumor by special and extended evaluation remains unknown. Equally the biological potential and significance of “stealth” melanoma cells remains to be determined.
Human tumors express antigens recognized by autologous T lymphocytes. Tumour progression is often accompanied by alterations in the expression of such “differentiation” antigens, in some instances leading to tumor escape from immunosurveillance. Although MART-1 is detectable in most melanomas and is considered a suitable target for immunomodulation therapy, metastatic melanomas often shows heterogeneous expression of MART-1 (15
). Melanocyte-specific microphthalmia-associated transcription factor (Mitf-M) regulates the expression of melanocyte-associated genes, and downregulation of Mitf-M results in the loss or reduced expression of melanoma-associated antigens in melanoma cells (16
). Loss of melanoma-associated antigens may also be induced by soluble factors produced by melanoma cells (37
), including oncostatin M (19
). Thus, when attempting to detect micrometastases in SNs by molecular methods, attention should be paid to the possibility of downregulation of genes for tumor-associated antigen. The use of multiple melanoma markers can significantly improve the specificity of molecular testing (65
), but conversely may affect sensitivity, because alteration of melanoma-associated antigens has implications for tumor growth and metastasis and these melanoma-associated antigens are downregulated in metastatic melanoma cells.
Alternative cellular sources for mRNA for melanoma-associated genes in lymph nodes include macrophages that have phagocytosed materials from melanoma cells. MART-1 mRNA was detected in macrophages (melanophages) containing intracellular granules of melanin. Co-localization of CD68 and the RT in situ
PCR enhanced signal for MART-1 mRNA was found, further supporting the view that these cells are macrophages. Melanophage accumulation is typically observed at the primary site in the early stage of melanoma evolution. Single isolated melanophages and groups of melanophages forming small granulomas often mimic micrometastasis in regional lymph nodes. Large irregularly shaped intracellular granules of pigment, comprised of aggregated melanosomes are morphological characteristics that help distinguish melanophages from melanoma cells where, in most instances, the melanosomes are small, regular in size and shape and diffusely dispersed. Electron microscopic examination has revealed that melanophages contain partially or fully melanized melanosomes that may be located within phagosomes. Melanosomes can also be found in dermal melanophages in postinflammatory hyperpigmentation (42
) or in tumor cells in cutaneous neoplasms other than melanoma such as pigmented squamous cell carcinoma and pigmented basal cell carcinoma (63
). Melanosomes often exhibit immunoreactivity for HMB-45 (56
), and HMB-45-positive melanophages have been demonstrated by immunoelectron microscopy. On the other hand, MART-1 is not localized to melanosomes but it is located in electron-lucent cytoplasmic vacuoles and vesicles in melanocytes (64
). Our study revealed that the MART-1 epitope is also be detectable in melanophages, possibly indicating phagocytosed materials retained in their cytoplasm. However, we can not entirely exclude the possibility that the highly sensitive molecular biology technique that we employed may augment acquired nucleotides in the cytoplasm of macrophages that have phagocytosed melanoma cell components. MART-1- or HMB-45-positive melanophages may be difficult to differentiate from melanoma cells on light microscopy.
Melanoma-macrophage hybridization is another possible explanation for cells that express characteristics of both melanoma cells and macrophages. Until recently, the hypothesis that fusion of cancer cells and normal cells may facilitate metastasis has received limited attention. It has been suggested that cancer cells may spontaneously fuse with host cells to produce hybrids (17
). Evidence in support of cancer cell fusion with macrophages or other bone marrow-derived cells has been reported from studies of animal models of human cancer (6
). Hybrid cells formed from macrophages and melanoma cells may possess the malignant potential of the melanoma cells and the migratory phenotype of the macrophages. Such hybrid cells contain coarse intracellular melanin granules of the type typically seen in melanophages (53
). In the tumor microenvironment, macrophages often constitute a major component of the tumor stroma. Macrophages are conventionally regarded as promoters of tumor progression (68
) and are recruited to melanoma by expression of chemokines including CCL2 (MCP-1). However, it is difficult to further track the underlying macrophages and demonstrate hybridization with tumor cells in human tissues. A recent study reported that epithelial cancers arise by transdifferentiation of bone marrow stem cells, a process that did not involve hybridization since these cells were diploid without evidence of fusion of donor and recipient cells (23
). Transdifferentiation of bone marrow stem cells to cancer cells may be another possible explanation of tumor dormancy and cancer cell metastasis.
In contrast, melanoma cells, particularly metastatic melanoma cells may exhibit an aberrant immunophenotype and may express many of the known lysosomal and phagocytic markers, such as CD68, α1 antitrypsin and α1 chymotrypsin that are typical of macrophages. They also frequently show immunoreactivity with macrophage markers including, MAC387 and HAM56 (1
). Some melanoma cells express MHC class II molecules that are normally present on antigen presenting cells and activated T cells. Metastatic melanoma cells may show macrophage-like phagocytic activities that involve cytoplasmic vesicles (41
). Macrophage-like properties have also been observed in gliomas (29
), which can preferentially express melanoma-associated antigens such as gp100, MART-1, tyrosinase, TRP-2, MAGE-A1 and MAGE-A3 (12
). It remains unclear how melanoma cells develop aberrant macrophage-like immunophenotypes. The possibility that they are the result of melanoma-macrophage hybridization is clearly worthy of further consideration.
Another pitfall of the use of molecular biology techniques is that the “tumor-associated” genes used for detection of melanoma cells by RT-PCR are not absolutely specific to melanoma cells. These genes are mostly genes for melanocyte-lineage antigens that are also expressed by normal melanocytes and benign nevus cells. Nevus cells in lymph nodes represent a well known potential diagnostic pitfall in the RT-PCR analysis (61
). Histologically, nodal nevi appear as aggregates of nevus cells in the lymph node capsule and trabeculae, and are a potential source of confusion with metastatic melanoma. Discrimination of benign nevocytes from metastatic melanoma requires close consideration of cytologic features of the cells which are negative or at most faintly positive for HMB-45 (8
Though infrequent, benign inclusions in lymph nodes represent the presence of heterologous elements not normally found in lymph nodes. These include salivary inclusions in peri-parotid lymph nodes, thyroid inclusions in cervical lymph nodes, mammary gland inclusions in axillary lymph nodes, or endometrial glandular inclusions in abdominal and pelvic lymph nodes (30
). The occurrence of such inclusions provides an additional potential pitfall in the use of molecular biology to detect cancer metastases.
The prognostic significance of histology-negative RT-PCR-positive sentinel nodes remains debatable. There are no objective data to support or contradict complete lymph node dissection for histology-negative, molecular-positive sentinel nodes. A definitive answer must await completion of MSLT-2, a multicenter randomized trial in the United States, Europe and Australia (44
). Although the clinical importance of the lymphatic system is well recognized, the actual molecular mechanisms involved remain poorly understood. As part of the elucidation of these mechanisms improvements in the techniques and criteria involved in the detection of (early) lymph node metastasis of melanoma will be of great importance. These investigations will determine the clinical significance of “stealth” melanoma cells and the other MART-1 mRNA positive cells.