“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 mRNA when sensitive molecular approaches such as 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 because the nodal tissue is destroyed during preparation for RT-PCR. Nonetheless 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 the 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 the cells that express that mRNA. Cells containing MART-1 mRNA were detected in 6/21 SN (29%) and 2/16 NSN (13%) that were tumor-negative on hematoxylin and eosin and 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 2/7 NSN (29%) that were histologically tumor-free. MART-1 mRNA positive cells were also detected in tumor negative SN sections from 6/7 (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 while 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 co-localized 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.

Circulating Tumor Cells in Melanoma Patients

“…Cells similar to the captured CTCs have recently been described in histologically negative sentinel lymph nodes [28]. Itakura et al described 2 subpopulations of melanoma-related cells, which seem analogous to the 2 subpopulations of cells we report here from blood.…”

“…The putative CTCs consisted of large cells, about half of which stained for both pancytokeratins (pan-KRT) and for CD-45. These captured/cultured melanoma CTCs appear to be analogous to the melanoma-related cells recently been reported in “histologically negative” sentinel lymph nodes [28]. These authors described MLANA-RNA-expressing cells which appeared consistent with melanoma cells, as well as putative macrophage-melanoma hybrids.…”

Analysis of Melanoma Recurrence Following a Negative Sentinel Lymph Node Biopsy

“…Another source of possible FN SLNBx is surgical failure, wherein the operative surgeon fails to identify the SLN or samples non-sentinel lymph nodes in the targeted basin [7,[23][24]. Furthermore, pathologic misclassification has been described as a potential source of FN SLNBx, with re-examination of sentinel lymph nodes demonstrated that 27 to 80% of patients who developed clinical progression of disease secondary to a FN SLNBx had histologic evidence of metastatic disease at the time of initial SLNBx [7,16,25].…”

Indocyanine Green SPY Elite–Assisted Sentinel Lymph Node Biopsy in Cutaneous Melanoma

“…13,14 Indocyanine green is a small substance molecule (774.96 Da) tricarbocyanine dye and is the only near-infrared fluorophore that is approved by the U.S. Food and Drug Administration for human use. 15 Indocyanine green offers several inherent advantages over other modalities of sentinel lymph node biopsy.…”