Use Of Immunohistochemistry

It is absolutely essential that all lymph nodes be examined by immunohistology using antibodies to S-100 protein and HMB-45, unless the node contains overt tumor on gross inspection or review of H&E-stained slides. The antibody MART-1 (melanoma-associated antigen recognized by T lymphocytes) (Melan-A) can be substituted for HMB-45. Immunohistology will always increase the frequency of sentinel lymph nodes found to contain tumor. The proportion of sentinel nodes that require immunohistology to identify occult tumor decreases as pathologists gain experience in evaluating sentinel nodes. This is the pathologist's equivalent of the surgeon's learning curve [24].

S-100 protein is a highly robust marker for melanoma cells, staining virtually 100% of melanomas [14,16]. We look for epithelioid, oval, or spindle-shaped cells (usually located in the subcapsular sinus) that show S-100 protein positivity in both the cytoplasm and the nucleus (Figs. 2a-2c). There are other cells within the lymph nodes that contain S-100 protein. The dendritic leukocytes of the para-cortex are the most prominent of these confounding cells. Identification of these cells is not difficult in reactive paracortices where they are polydendritic (Fig. 3a). Difficulty may be encountered in inactive lymph nodes where the dendritic leukocytes show either no or minimal dendrite formation (Fig. 3b). S-100 protein positivity may also be found in capsular nevi (Fig. 4b) [26] and in the Schwann cells of node-associated nerves (Fig. 5) [27].

HMB-45 is a more specific marker for melanoma cells but does not stain the cells of between 10% and 15% of melanomas. In contrast to S-100 protein, HMB-45-positive epitopes are confined to the cytoplasm. Nonetheless, antibodies to HMB-45 have the advantage that they do not stain dendritic leukocytes and either do not stain or stain weakly capsular nevocytes (Fig. 4c). The antibody MART-1 (Melan-A) may be used in a role similar to HMB-45, but it suffers from the same defect that a proportion of melanomas do not stain positively with this reagent.

(a)
Lymph Nodes What TheyPositive Lymph Nodes
(c)
Reactive Lymph Does The Lymph Node Have Artery

Figure 3 (a) Polydendritic S-100-protein-positive paracortical dendritic leukocytes in a reactive lymph node. Polyclonal antibody to S-100 protein. Original magnification x240. (b) Nondendritic S-100-protein-positive paracortical dendritic leukocytes in a nonreactive sentinel lymph node. Polyclonal antibody to S-100 protein. Original magnification x240.

Figure 3 (a) Polydendritic S-100-protein-positive paracortical dendritic leukocytes in a reactive lymph node. Polyclonal antibody to S-100 protein. Original magnification x240. (b) Nondendritic S-100-protein-positive paracortical dendritic leukocytes in a nonreactive sentinel lymph node. Polyclonal antibody to S-100 protein. Original magnification x240.

Figure 2 (a) Single S-100-protein-positive melanoma cells in the subcapsular sinus of a sentinel node. Note that there is both cytoplasmic and nuclear staining. Polyclonal antibody to S-100 protein. Original magnification x240. (b) Microfocus of S-100-protein-positive melanoma cells, predominantly in the subcapsular sinus. Polyclonal antibody to S-100 protein. Original magnification x180. (c) Macrofocus of S-100-protein-positive melanoma cells in the subcapsular sinus and extending into the adjacent lymphoid tissue. Polyclonal antibody to S-100 protein. Original magnification x180.

Melanoma Lymph Node MappingNerves Groin Area
Figure 5 S-100-protein-positive perinodal nerve. Nerves that traverse the node and are cut transversely may present interpretative difficulty. Polyclonal antibody to S-100 protein. Original magnification x240.

One potential source of error with HMB-45 is that in lymph nodes with trabecular calcification (mainly in nodes in the groin or iliac area), extracellular HMB-45 reactivity may be identified.

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