We present a case of a 75-year-old woman who initially sought medical attention in 1993 for a 1.2-cm pigmented skin lesion of the upper back, just right of the midline, which was diagnosed on biopsy as melanoma, 1.9 mm in thickness, with an in situ component that extended to the lateral specimen margins. The patient underwent surgical re-excision of the biopsy site, without lymph node dissection, approximately one month later; this re-excision specimen was free of residual melanoma. The patient then presented 15 months later with left axillary lymphadenopathy and a slightly enlarged right axillary lymph node, prompting bilateral axillary lymph node dissections. One of thirteen left axillary lymph nodes contained metastatic melanoma comprised of sheets of dyshesive polygonal cells with abundant amphophilic cytoplasm and enlarged, irregular nuclei; these cells stained positively for melanoma markers by immunohistochemistry (S100 +, melanoma cocktail (HMB-45/tyrosinase) +; ), and were negative for cytokeratin (). Nodal metastases were not identified on the right side. The patient then underwent an autologous vaccine protocol for one year.
Immunohistochemical Results for Left Axillary Lymph Node Metastasis and Right Axillary Lymph Node Biphasic Metastatic Deposit with Features of Melanoma and Adenocarcinoma
The patient was followed closely and had an uneventful medical history until 12 years later, when she presented with another axillary mass, now on the right side, that was enlarging over a two-week interval. Upon right axillary lymph node dissection, a single 4.9-cm lymph node contained metastatic tumor, and the remaining 14 lymph nodes were cancer-free. Histologic examination of the positive lymph node revealed a biphasic metastasis with histologic and immunohistochemical characteristics of both melanoma and adenocarcinoma (). Interestingly, the adenocarcinomatous component was nested within the melanomatous component, with no clear intervening stroma or fibrous capsule. The outer rim of tumor contained sheets and nests of dyshesive polygonal cells with abundant amphophilic cytoplasm, enlarged pleomorphic nuclei with densely hyperchromatic chromatin and prominent nucleoli, and without significant pigment deposition (). This aspect of the tumor exhibited patchy, but strong cytoplasmic and nuclear immunohistochemical staining for S100 (), as well as staining for Melan-A (), NSE, and weak, patchy staining with a cytokeratin cocktail (AE1/AE3 and Cam 5.2, ), but was negative for the melanoma markers HMB-45andMiTF-1, as well asCK7, CK19, CK20, HMWK-903, CDX-2, Ber-EP4, mCEA, glypican-3, HepPar-1, and TTF-1 (). By contrast, the central portion of the tumor consisted of somewhat smaller cells arranged in gland-like and microcystic structures with apparent mucin, colloid, or proteinaceous debris within the lumina (). The nuclei of these cells were smaller and had a more vesicular chromatin pattern than the nuclei in the adjacent melanomatous component. Quite intriguingly, the cells in the adenocarcinomatous area stained strongly with the keratin cocktail (), Ber-EP4 (), CK19, CK20, CDX-2, and glypican-3 (), but were negative for S100 (), Melan-A (), HMB-45, MiTF-1, NSE, mCEA, HepPar-1, HMWK-903, CK7, and TTF-1 (). The differential diagnosis of this area thus included metastasis of an adenocarcinoma, possibly of gastrointestinal, hepatic, or pancreatobiliary origin, verses true transdifferentiation of the adjacent melanoma into adenocarcinoma. Importantly, neither the patient’s history nor a careful work-up revealed any evidence of a second malignancy beside the prior melanoma, which arose in a drainage area of this right axillary lymph node metastasis. No evidence of adenocarcinomatous differentiation was noted in the patient’s primary melanoma or the left axillary metastasis from 12 years earlier, and immunohistochemical analysis of this material showed no cytokeratin expression (). The patient was next seen six months later, at which time a follow-up PET-CT study revealed widespread metastases in the pelvis, vertebrae, and lungs. No colonic, hepatic, gastric, or pancreatobiliary lesions were seen. At this point, the patient was lost to follow-up.
Biphasic metastatic tumor deposit in a right axillary lymph node
To further evaluate these two tumors at a molecular level, DNA was extracted from both tumor areas and from the original melanoma metastasis from the left axilla, resected 12 years earlier, and subjected to comparative genomic hybridization (CGH)13
and DNA sequence analysis for mutations in BRAF
oncogenes commonly mutated in the majority of melanomas originating from the trunk.13
Multiple chromosomal aberrations in both tumor areas were identified by CGH, the majority of which were shared between the two areas indicating a clonal relationship: loss of chromosomes 1p, 4, 9, 10q, 11p, 15, 17p, 21, and 22, as well as gains of chromosomes1q, 2, 5p, 13, 17q, 19, and 20 (); this pattern of chromosomal aberrations is typical for melanoma.7
A common clonal origin of the two tumor areas was further suggested by the presence of a narrow deletion of the distal part of chromosome 6q, which was present in both histologically distinct tumors (see arrows in ); analysis of the original metastasis by CGH was unsuccessful due to the small size of the tumor and normal tissue contamination. Finally, the notion of a clonal relationship between the different tumor populations was confirmed by detecting an NRAS
Q61K mutation caused by an identical nucleotide substitution in both tumor areas (, third and fourth panels), as well as in the metastasis resected 12 years earlier (, second panel). This mutation was somatically acquired as the patient’s normal tissue showed no abnormalities (, top panel). Both tumors were wild type for BRAF
. We note that cross-contamination of our microdissected sample can be ruled out as an explanation for finding the mutation in both areas from the biphasic right axillary lymph node metastasis. A careful manual microdissection was performed on keratin-stained slides to help distinguish the two regions. Also, the peak heights of the sequencing traces indicate that the mutant allele is the most abundant allele in both samples (, third and fourth panels). The opposite would be expected if a minority of mutant cells from one area had been accidently included in the microdissected tissue of the other area. NRAS
maps to chromosome 1p, which showed loss of one copy in both tumor areas by CGH. The increased abundance of the mutant allele in both samples thus indicates that the chromosomal arm with the wild type copy was deleted. Interestingly, the chromosomal alterations in both tumor areas are not entirely identical by CGH. Some aberrations, such as a loss of chromosome 5q, gain of chromosomes 7 and 18, and loss of distal chromosome 14, were only found in the area of overt melanoma, whereas the adenocarcinomatous portion demonstrated gains of 6p and distal 8q, which are absent in the melanoma (). Together, the genetic findings demonstrate that the two morphologically distinct tumor cell populations arose from a common ancestor cell, but represent genetically distinct subclones. Dual-color immunofluorescence using S100 (red) and keratin cocktail (green), performed as previously described,15
showed strong keratin positivity in the adenocarcinomatous area and S100 positivity in the melanoma region as expected (). Interestingly, a few cells along the transition zone between the two areas were positive for both S100and keratin (arrows in ).
Molecular genetic studies of metastatic melanoma with striking adenocarcinomatous differentiation
These molecular results also verify that this metastasis is from the patient’s primary melanoma on the back, as the metastatic melanoma resected from the contralateral axilla 12 years prior harbors the same NRAS
mutation (). Approximately 20% of melanomas on the trunk harbor NRAS
mutations, most of which occur at codon 61, as in our case.21
By contrast, NRAS
mutations are infrequent in adenocarcinoma, with the exception of follicular thyroid carcinoma, in which codons 12 and 13 are frequently involved.27