A novel N-ras mutation in malignant melanoma is associated with excellent prognosis.

Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.

Expression of the endothelin-B receptor in pigment cell lesions of the skin. Evidence for its role as tumor progression marker in malignant melanoma.

Endothelins (ETs) exert several functions in human melanocytes, including proliferation, dendrite formation, and melanin synthesis. Among the ET receptors, the non-selective endothelin-B (ETB) receptor is the major receptor in melanocytes and malignant melanoma (MM) cells. In spite of the important role of ETs and their receptors in the growth and differentiation of melanocytes, the distribution and expression levels of ETB receptors in tissue sections of benign and malignant pigment cell lesions is still unknown. We combined immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to study ETB receptor expression in benign and malignant pigment cell lesions and in normal skin. Immunohistochemistry on paraffin-embedded tissue sections of 159 cases revealed a significant increase in intensity of ETB receptor expression from common nevi over dysplastic nevi and primary MM to metastatic MM. Quantitative PCR using realtime detection on 75 samples confirmed the immunohistochemical results. These data add the ETB receptor to the growing list of tumor progression markers in MM and suggest that ETs play a role in the progression of MM in the skin.

Subcutaneous lymphoid hyperplasia arising at site of ethnic scarifications and mimicking subcutaneous panniculitis-like T-cell lymphoma.

The case of a 40-year-old black man, who developed a very unusual tumour-like lymphoid hyperplasia involving primarily the subcutaneous tissue, is reported. The lesion, which arose at a site of tribal scarifications, displayed a deceptive morphology that closely resembled subcutaneous panniculitis-like T-cell lymphoma (SPTCL). An accurate diagnosis could only be made following detailed immunohistochemical and molecular studies. Although SPTCL has been thought to represent a very specific clinicopathologic entity, the present case illustrates that its histological appearance can, however, be closely mimicked by reactive and benign conditions.

Analysis of N- and K-ras mutations in the distinctive tumor progression phases of melanoma.

Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma. To investigate whether these mutations occur in early or late tumor progression phases, we searched for point mutations in the N- and K-ras genes in 69 primary cutaneous melanoma, 35 metastases, and seven nevocellular nevi in association with cutaneous melanoma. Lesions were microdissected in order to procure pure tumor samples from the distinctive growth phases of the cutaneous melanoma; the very sensitive denaturing gradient gel electrophoresis technique was used to visualize the mutations, and was followed by sequencing. Point mutations in the N-ras gene but not in the K-ras gene were detected on denaturing gradient gel electrophoresis. Twenty-three primary (33%) and nine metastatic (26%) melanomas showed bandshifts for N-ras. In the majority of cases, mutations occurring in early growth phases (i.e., the "intraepidermal" radial growth phase), were preserved in later growth phases (i.e., the invasive radial growth phase, vertical growth phase, and metastatic phase), which proves the clonal relationship between the successive growth phases. In three cases, however, the mutations differed between the distinctive growth phases within the same cutaneous melanoma, due to the occurrence of an additional mutation (especially in codon 61) in a later tumor progression phase. Our approach also permitted us to analyze the mutational status of nevi, associated with cutaneous melanoma. Six out of seven associated nevi carried the same sequence (mutated or wild-type) as the primary cutaneous melanoma, whereas in one case the sequence for N-ras differed between the primary melanoma and the associated nevus. In conclusion, this approach allowed us to demonstrate the clonal relationship between subsequent growth phases of melanoma and associated nevi; our results suggest that N-ras exon 1 mutations preferentially occur during early stages of tumor progression and hence may be involved in melanoma initiation, whereas those in N-ras exon 2 are found preferentially during later stages and hence are more probably involved in metastatic spread of cutaneous melanoma.

Evaluation of B cell lymphoid infiltrates in bone marrow biopsies by morphology, immunohistochemistry, and molecular analysis.

AIMS: Morphological criteria to distinguish between reactive and neoplastic B cell lymphocytoid infiltrates in trephines have been defined but are not always reliable. Polymerase chain reaction (PCR) analysis of the CDR3 region of the immunoglobulin heavy chain (IgH) gene which, by demonstrating monoclonality, can provide additional arguments in favour of lymphoid malignancy is now frequently used for the detection and follow up of B cell lymphoma (NHL). The aim of this study was to investigate the usefulness of morphological findings in bone marrow biopsies in comparison with data obtained by PCR analysis. METHODS: Eighty nine bone marrow biopsies displaying lymphoid infiltrates were evaluated by morphology and immunohistochemistry as well as by CDR3-PCR using consensus framework 3 (FRW3) and JH primers. RESULTS: The presence of a clonal B cell proliferation was demonstrated by PCR analysis in 45 biopsies, including 21 samples considered to be positive, 17 to be suspicious, and seven to be negative by morphology. In the remaining 44 trephines we found no evidence of clonality, although 12 of these trephines were thought to be positive by morphology. CONCLUSIONS: These results, revealing an incomplete correlation between CDR3-PCR data and immunomorphological findings, indicate that molecular analysis may be more sensitive and specific in general. However, false negative PCR results do occur, which emphasises the necessity to combine both diagnostic tools in the evaluation of lymphoid infiltrates.