Roland Böni

Serum S100 – A Marker for Disease Monitoring in Metastatic Melanoma

BACKGROUND S100 proteins are low-molecular-weight calcium-binding proteins and appear to play an important role in various cellular processes such as cell division and differentiation. In histopathology, S100 is widely accepted as the marker of choice for immunohistochemical identification of malignant melanoma. When S100 was detected in the serum of patients with malignant melanoma, it was suggested that serum S100 may be a useful marker for the stage of disease. OBJECTIVE The aim of this study was to examine serum S100 concentrations of patients with different stages of malignant melanoma and to determine the value of serum S100 in the follow-up of melanoma patients during treatment. METHODS Sera were obtained from 73 melanoma patients in different stages of the disease. The control group consisted of 130 healthy subjects. In 4 patients with metastatic melanoma, serum S100 was measured serially. Serum levels were measured by a commercially available immunoradiometric assay. RESULTS While only 1 out of 25 stage I/II patients and 3 of 14 patients with lymph node metastases (stage III, 21.4%) showed detectable serum S100 levels, 27 of 34 patients with disseminated disease (stage IV, 79.4%) had elevated serum S100. Interestingly, rising levels of serum S100 in the serial measurement indicated progression of the disease, and a complete decline reflected 2 patient remissions. CONCLUSION The data support the value of serum S100 as a clinical marker for progression of metastatic melanoma and serological monitoring during systemic therapies.

Staging of metastatic melanoma by whole-body positron emission tomography using 2-fluorine-18-fluoro-2-deoxy-D-glucose

Summary Metastatic melanoma was staged in l5 patients using whole‐body positron emission tomography (PET) and the radiopharmaceutical 2‐fluorine‐18‐fluoro‐2‐deoxy‐D‐glucose (FDG). PET correctly demonstrated 30 metastases in lung, brain, pancreas, nasal cavity, skin and subcutaneous tissue, and lymph nodes. It detected 97% of all metastases exceeding its spatial resolution (>5mm). Two cutaneous metastases (approximately 3 mm) did not show increased FDG uptake; the overall detection sensitivity was 91%. Two false‐positive lesions in one patient were due to severe wound infection. PET correctly excluded malignancy in four cases where suspicious lesions were found with conventional cross‐sectional imaging modalities but later ruled out by fine‐needle biopsy.

Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution

Monoclonal antibody T311 specifically detects tyrosinase protein expression. Tyrosinase‐derived peptides are recognized by CD8+ T‐celis and applied in immunotherapy. We examined formalin‐fixed paraffin‐embedded tissue of 50 melanoma (primary n=31, metastatic n=19) and 41 control cases (junctional, dermal, compound, Spitz, Reed, balloon‐cell nevi) by immunochemistry using the alkaline phosphatase‐anti‐alkaline phosphatase method after antigen retrieval. Staining with mAb T311 showed a sensitivity of 94% for melanoma with a very high specificity for melanocytic cells. Immunopositivity (94% of melanomas overall) correlated inversely with clinical stage: clinical stage I and stage II showed 100%, stage III and stage IV 86% immunoreactivity each. Staining changed from an exclusively homogeneous pattern in early stages to a more heterogeneous pattern in later stages. Melanocytic control tissue like nevi of different subtypes all showed weak to moderate, homogeneous immunoreactivity with polarity towards the epidermis. RT‐PCR ELISA analysis of short‐term melanoma cell cultures displayed mRNA expression in only half of the originally immunopositive tumors only, suggesting rapid mRNA expression loss in culture. mAb T311 allows detection of melanoma‐associated tyrosinase protein expression and thus profiling of melanomas using routine archival tissue suited for immunotherapy approaches involving tyrosinase derived epitopes.

Melan A/MART-1 immunoreactivity in formalin-fixed paraffin-embedded primary and metastatic melanoma: frequency and distribution

Monoclonal antibody (MAb) A103 specifically detects Melan A/MART-1 protein expression. Melan A/MART-1 -derived peptides are recognized by CD8+ T-cells and are used in immunotherapy. We examined formalin-fixed paraffin-embedded tissue from 57 melanomas (34 primary, 23 metastatic) and 39 control cases (junctional, dermal, compound, Spitz, Reed and balloon-cell naevi) using the alkaline phosphatase and anti-alkaline phosphatase immunochemical method after antigen retrieval. Immunoreactivity was rated as low, medium or high, and staining pattern as homogeneous or heterogeneous. Staining with MAb A103 showed a sensitivity of 88% for melanoma, with a very high specificity for melanocytic cells. Immunopositivity decreased along with clinical stage, with stage I showing 100%, stage II 88%, stage III 90% and stage IV 75% immunoreactivity. Staining changed from an exclusively homogeneous pattern in the early clinical stages to a more heterogeneous pattern in the later stages. Melanocytic control tissues consisting of naevi of different subtypes all showed weak to moderate homogeneous immunoreactivity, with polarity towards the epidermis. Analysis of short-term melanoma cell cultures using reverse transcription-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) demonstrated mRNA expression in only one third of the originally immunopositive tumours, suggesting rapid mRNA expression loss in culture. MAb A103 allows the detection of melanoma-associated Melan A/MART-1 protein expression in routine archival tissue and thus enables the profiling of melanomas suited for immunotherapy approaches involving Melan A/MART-1 derived epitopes.

MIB-1 immunoreactivity correlates with metastatic dissemination in primary thick cutaneous melanoma

BACKGROUND The proliferation rate of transformed cells is a putative prognostic indicator. MIB-1 is a murine monoclonal antibody to a Ki-67 epitope that detects a nuclear antigen found only in proliferating cells. OBJECTIVE The aim of this study was to test for a correlation between MIB-1 immunoreactivity and the metastatic potential of malignant melanoma. METHODS MIB-1 reactivity (% total tumor nuclei) was assessed in 34 formalin-fixed, paraffin-embedded primary cutaneous melanomas and correlated with metastatic potential and overall survival (follow-up, 10.5 +/- 1.8 years). RESULTS Whereas no correlation was found between MIB-1 reactivity and metastases in primary thin cutaneous melanoma (Breslow thickness, < 0.75 mm; mean thickness, 0.39 +/- 0.16 mm), good correlation was found (p = 0.0001) in primary thick cutaneous melanoma (Breslow thickness, > 1.5 mm; mean thickness, 3.0 +/- 1.3 mm). MIB-1 reactivity was 12.3% +/- 7.7% and 0.7% +/- 1.3% with and without metastases, respectively, and was highest in the primary melanomas that later metastasized. However, overall survival in patients with thick cutaneous melanoma and metastases did not correlate with MIB-1 reactivity. CONCLUSION MIB-1 proliferative activity is a useful prognostic indicator in primary cutaneous melanomas thicker than 1.5 mm and may predict the development of metastases.

Ca2+-binding proteins S100A6 and S100B in primary cutaneous melanoma*

Purpose: Commercially available polyclonal antibodies against a mixture of bovine brain S100 proteins have become an established marker for immunohistochemical characterization of malignant melanoma. However, the commercially available antibodies used are undefined and to date, 13 different human S100 proteins are known. The purpose of this study was to examine the expression of 4 newly available polyclonal antibodies against the human recombinant Ca2+‐binding S100 proteins, S100A1, S100A2, S100A4 and S100A6, in cutaneous melanoma and to correlate these findings with the standard S100 staining as well as with the metastatic potential of the primary.

The PTEN tumour suppressor gene and malignant melanoma.

A candidate tumour suppressor gene, PTEN, has recently been identified within chromosome 10q23, the locus of the Cowden syndrome/Lhermitte Duclos disease susceptibility gene. Cowden disease is an autosomal dominant cancer predisposition syndrome associated with tumours of the breast, thyroid and, less frequently, malignant melanoma. Based on the identification of mutations in sporadic breast, brain and prostate tumours, we decided to examine the potential role of PTEN in sporadic malignant melanoma. Frozen tissue from primary cutaneous melanomas (n - 23) and metastases (n=17) were microdissected, and microsatellite markers D10S541 and D10S547, flanking the gene on both sides, were used to search for loss of heterozygosity (LOH) in the PTEN gene locus. To identify mutations within the putative tumour suppressor gene, we performed single strand conformation polymorphism (SSCP) analysis using intronic primers to amplify exons 5, 6, 7 and 8 of the PTEN gene. No LOH was detected using the polymorphic markers D10S541 and D10S547. SSCP analysis revealed no aberrant bands in the tumour specimen. Our results suggest that the PTEN gene does not play a major role in the initiation and progression of melanoma.

No detection of HTLV-I proviral DNA in lesional skin biopsies from Swiss and German patients with cutaneous T-cell lymphoma

Summary The search for an infective agent linked to cutaneous T‐cell lymphoma (CTCL) has also included the human T‐cell lymphotropic virus type I (HTLV‐I). Using sensitive techniques such as Southern blotting under low stringency conditions of hybridization and polymerase chain reaction (PCR) with primer sets designed to match pol, env and pX sequences of HTLV‐I, we have screened lesional skin biopsies of 50 Swiss and German patients suffering from CTCL. No evidence of proviral integration of HTLV‐I could be demonstrated in any of our patients. Our results, as well as a review of the literature, indicate that at least for European patients, HTLV‐I does not seem to play a role in the aetiology of CTCL.

Expression of the proliferation and apoptosis-associated CAS protein in benign and malignant cutaneous melanocytic lesions.

We have examined the expression of the cellular apoptosis susceptibility protein, a nuclear transport factor that plays a role in apoptosis and cell proliferation, in benign and malignant melanocytic lesions. Tissue samples of 55 formalin-fixed, paraffin-embedded melanoma (primary n=32, metastatic n=23) and of 27 control cases (junctional dermal, compound, Spitz, Reed, blue nevi, balloon-cell nevus, lentigo maligna) were analyzed by immunohistochemistry with anti-cellular apoptosis susceptibility antibodies. The percentage of cellular apoptosis susceptibility-positive cells as well as the intensity on a four-point scale was evaluated. In normal skin, expression of cellular apoptosis susceptibility was primarily found in the basal cell layer of the epidermis. Benign melanocytic lesions that stained positive for cellular apoptosis susceptibility (13 of 27) showed a homogeneously distributed staining pattern with a mean of 5+/-12% cellular apoptosis susceptibility positive cells. Five out of 7 lentigo maligna melanoma, 11 out of 12 superficial spreading melanoma and all acrolentiginous (n=7) and nodular (n=6) melanoma showed immunoreactivity of medium (++) to high ( ) intensity. Vertical growth phases of primary cutaneous melanoma stained stronger than horizontally growing cell clusters. All metastases (n= 23) stained strongly positive, the staining pattern being inhomogeneous. Cellular apoptosis susceptibility detection in clinical stages according to UICC showed an increase from 43+/-34% cellular apoptosis susceptibility positive cells in stage I, to 53+/-26% in stage II, 68+/-24% in stage III and 72+/-24% in stage IV, respectively. Because the expression of cellular apoptosis susceptibility correlates predominantly with advanced stages of melanoma, staining with anti-cellular apoptosis susceptibility antibodies may be useful for diagnosis of melanoma and possibly as an immunohistochemical prognostic factor in cutaneous melanocytic lesions.

Syringocystadenoma papilliferum: a study of potential tumor suppressor genes.

Syringocystadenoma papilliferum (SP) is a benign tumor most commonly located on the scalp or face, which frequently arises from a nevus sebaceus (NS). Transition of SP to basal cell carcinoma (BCC) and, albeit rarely, to metastatic adenocarcinoma may occur. Allelic deletions of the human homologue of the drosophila patched gene (PTCH) occur in both NS and BCC. To search for genetic changes in SP, a microdissection-based genetic analysis using polymorphic markers at 9q22 (PTCH; D9S15, D9S303, D9S287, D9S252) as well as markers at 9p21 flanking the tumor suppressor gene p16 (IFNA, D9S171) was performed. Glandular epithelium consisting of two rows of cells as well as adjacent normal tissue or inflammatory infiltrates in the stroma, when present, was dissected and subjected to single-step DNA extraction and loss of heterozygosity (LOH) analysis. Two of 10 informative SP cases showed LOH at 9q22 (PTCH). Three of 7 informative SP cases showed allelic deletions at 9p21 (p16). Allelic loss at 9q22 is consistant with the clinical observation of transition of SP to BCC. The finding of frequent allelic loss at 9p21 is unlikely to be related to the rare transition of SP to metastatic adenocarcinoma. Our study supports the hypothesis of a gatekeeper role of the tumor suppressor gene p16 in a variety of benign and malignant tumors, including SP.