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“Stealth” Melanoma Cells in Histology-negative Sentinel Lymph Nodes

Itakura, Eijun MD, PhD*; Huang, Rong-Rong MD*; Wen, Duan-Ren MD*; Cochran, Alistair J. MD, FRCP(Glasg), FRCPath*,†,‡,§

The American Journal of Surgical Pathology: November 2011 - Volume 35 - Issue 11 - p 1657–1665
doi: 10.1097/PAS.0b013e3182322cf7
Original Articles

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.

*Departments of Pathology and Laboratory Medicine

Surgery, David Geffen School of Medicine at UCLA

Jonsson Comprehensive Cancer Center, UCLA, Los Angeles

§John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica, CA

Conflicts of Interest and Source of Funding: Supported by NIH/NCI Grant P01 CA 29605 (administered by John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica, CA). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

Correspondence: Eijun Itakura, MD, PhD, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave., CHS 1P-162, Los Angeles, CA 90095-1732 (e-mail:

© 2011 Lippincott Williams & Wilkins, Inc.