"Stealth" melanoma cells in histology-negative sentinel lymph nodes

Abstract

A proportion of patients who develop regional and distant recurrences of melanoma after a pathologically negative sentinel lymph node (SN) biopsy are reported to have enhanced signals for melanoma-associated messenger ribonucleic acid (mRNA) when sensitive molecular approaches such as reverse transcriptase polymerase chain reaction (RT-PCR) are used to evaluate their SN tissue. The significance of these findings remains controversial, because the cellular source of the augmented signals cannot be known as the nodal tissue is destroyed during preparation for RT-PCR. Nevertheless, it is claimed that the source of the augmented signal is covert metastatic melanoma cells. To determine whether there are histologically occult metastases in SN and whether there are sources of augmentable melanoma-associated mRNA other than melanoma cells, we applied reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) to formalin-fixed paraffin-embedded nodal tissue. This approach amplifies small amounts of melanoma-associated mRNA and permits identification of cells that express that mRNA. Cells containing MART-1 mRNA were detected in 6 of 21 SNs (29%) and 2 of 16 nonsentinel lymph node (NSNs) (13%) that were tumor negative on hematoxylin and eosin and on immunohistochemical assessment for S-100, MART-1, and HMB-45. In patients with microscopic evidence of melanoma in their SN, MART-1 mRNA-positive cells were identified in 2 of 7 NSNs (29%) that were histologically tumor free. MART-1 mRNA-positive cells were also detected in tumor-negative SN sections from 6 of 7 (86%) nodes that had tumor present in areas of the node not represented in the studied sections. Some cells that expressed MART-1 mRNA that was diffusely distributed in the cytoplasm appeared to be melanoma cells, whereas others resembled macrophages. The latter cells expressed augmented mRNA on granules that were intermixed with melanin granules. In other cases, MART-1 mRNA-positive macrophage-like cells contained nuclei and nucleoli more typical of melanoma cells and may represent the macrophage-melanoma hybrids that have been previously reported. Combination of RT in situ PCR for MART-1 mRNA and immunohistochemistry for CD68 revealed that CD68 was colocalized in some cells that expressed MART-1 mRNA. Some lymph nodes that are tumor negative by histology and immunohistochemistry contain cells that express mRNA for MART-1. Some of these cells may be interpreted as "stealth" melanoma cells in which, despite the presence of MART-1 mRNA, there is an absence of immunohistochemically detectable MART-1 protein. Other cells that contain MART-1 mRNA are clearly not melanoma cells or may represent melanoma hybrids. These findings should be taken into account when interpreting and applying the results of RT-PCR analysis of nodal (and other) tissues.

Figure 1

“Stealth” melanoma cells identified by enhanced signal for MART-1 mRNA detected by RT in situ PCR (blue/purple color) (Panels A-C). In some original H&E slides, melanoma cells were present in areas separate from the RT in situ PCR-detected “stealth” melanoma cells (Panels D&E). Some MART-1 mRNA positive cells contained microdispersed melanin granules, a pattern that is typical of melanogenic cells, but lacked a nucleus which would have permitted determination of their malignant or benign status by H&E (Panel F). Small clusters of macrophage-like cells containing nuclei and nucleoli more typical of melanoma cells and microdispersed melanin granules (Panel G). These cells show positivity for MART-1 mRNA by the RT in situ PCR (blue/purple color) (Panel H). MART-1 mRNA is not seen in adjacent melanophages with coarse brown pigmented granules.

Figure 2

MART-1 mRNA (blue/purple color) is seen by RT in situ PCR in melanophages with coarse pigment granules (Panels A&B). Immunohistochemistry for MART-1 shows a weakly positive signal in scattered large cells with “normal” nuclei and coarse melanin granules, consistent with melanophages (Panel C). Immunohistochemistry for HMB-45 shows a positive signal in scattered large cells, consistent with melanophages (Panel D). Double staining of MART-1 mRNA (blue/purple color) and CD68 antigen (red color) shows that some cells co-express both MART-1 mRNA and CD68 (arrow), while MART-1 mRNA and CD68 are separately expressed by other cells. (Panels E&F). Sentinel node (Panel G) and NSN (Panel H) from a breast cancer patient are negative for MART-1 mRNA.