Melanoma pathology: new approaches and classification.

Cancer is caused by the accumulation of pathogenic alterations of the genome and epigenome that result in permanent changes that disrupt cellular homeostasis. The genes that become corrupted in this process vary among different tumour types, reflecting specific vulnerabilities and dependencies of the cell from which the cancer originated. This also applies to 'melanoma', a cancer that constitutes not one, but multiple diseases that can be separated based on their cell of origin, aetiology, clinical appearance and course, and response to treatment. In this article, we review the current classification of melanoma within distinct evolutionary pathways and the associated genetic alterations. In addition, we review the application of molecular diagnostics to the diagnosis of melanocytic tumours in the context of histopathological assessment.

Efficacy and safety of nilotinib in patients with KIT-mutated metastatic or inoperable melanoma: final results from the global, single-arm, phase II TEAM trial.

BACKGROUND: The single-arm, phase II Tasigna Efficacy in Advanced Melanoma (TEAM) trial evaluated the KIT-selective tyrosine kinase inhibitor nilotinib in patients with KIT-mutated advanced melanoma without prior KIT inhibitor treatment. PATIENTS AND METHODS: Forty-two patients with KIT-mutated advanced melanoma were enrolled and treated with nilotinib 400 mg twice daily. TEAM originally included a comparator arm of dacarbazine (DTIC)-treated patients; the design was amended to a single-arm trial due to an observed low number of KIT-mutated melanomas. Thirteen patients were randomized to DTIC before the protocol amendment removing this study arm. The primary endpoint was objective response rate (ORR), determined according to Response Evaluation Criteria In Solid Tumors. RESULTS: ORR was 26.2% (n = 11/42; 95% CI, 13.9%-42.0%), sufficient to reject the null hypothesis (ORR ≤10%). All observed responses were partial responses (PRs; median response duration, 7.1 months). Twenty patients (47.6%) had stable disease and 10 (23.8%) had progressive disease; 1 (2.4%) response was unknown. Ten of the 11 responding patients had exon 11 mutations, four with an L576P mutation. The median progression-free survival and overall survival were 4.2 and 18.0 months, respectively. Three of the 13 patients on DTIC achieved a PR, and another patient had a PR following switch to nilotinib. CONCLUSION: Nilotinib activity in patients with advanced KIT-mutated melanoma was similar to historical data from imatinib-treated patients. DTIC treatment showed potential activity, although the low patient number limits interpretation. Similar to previously reported results with imatinib, nilotinib showed greater activity among patients with an exon 11 mutation, including L576P, suggesting that nilotinib may be an effective treatment option for patients with specific KIT mutations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT01028222.

The combination of axitinib followed by paclitaxel/carboplatin yields extended survival in advanced BRAF wild-type melanoma: results of a clinical/correlative prospective phase II clinical trial.

BACKGROUND: Simultaneous chemotherapy with vascular endothelial growth factor (VEGF) inhibition has not shown additional benefit over chemotherapy alone in advanced melanoma. We tested administration of the potent VEGF inhibitor axitinib followed by paclitaxel/carboplatin to determine whether enhanced tumour proliferation during axitinib withdrawal leads to sustained chemosensitivity. METHODS: We conducted a prospective phase II trial in metastatic melanoma patients with ECOG performance status 0-1 and normal organ function. Axitinib 5 mg PO b.i.d. was taken on days 1-14 of each 21-day treatment cycle, and carboplatin (AUC=5) with paclitaxel (175 mg m(-2)) was administered on day 1 starting with cycle 2. 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT)-PET scans were performed in five patients to assess tumour proliferation on days 1, 14, 17, and 20 of cycle 1. Molecular profiling for BRAF was performed for all patients with cutaneous, acral, or mucosal melanoma. RESULTS: The treatment was well tolerated. The most common grade 3 AEs were hypertension, neutropenia, and anaemia. Grade 4 non-haematologic AEs were not observed. Four of five patients completing (18)F-FLT-PET scans showed increases (23-92%) in SUV values during the axitinib holiday. Of 36 evaluable patients, there were 8 confirmed PRs by Response Evaluation Criteria in Solid Tumors. Overall, 20 patients had SD and 8 had PD as the best response. The median PFS was 8.7 months and the median overall survival was 14.0 months. Five BRAF(V600E/K) patients had significantly worse PFS than patients without these mutations. CONCLUSIONS: Axitinib followed by carboplatin and paclitaxel was well tolerated and effective in BRAF wild-type metastatic melanoma. 3'-Deoxy-3'-(18)F-fluorothymidine-PET scans showed increased proliferation during axitinib withdrawal.

Genetic polymorphism in Methylenetetrahydrofolate Reductase chloride transport protein 6 (MTHFR CLCN6) gene is associated with keratinocyte skin cancer in a cohort of renal transplant recipients.

Background: Renal transplant recipients (RTRs) are at increased risk of keratinocyte cancer (KC), especially cutaneous squamous cell carcinoma (cSCC). Previous studies identified a genetic variant of the Methylenetetrahydrofolate Reductase (MTHFR) gene, C677T, which conferred a risk for diagnosis of cSCC in Irish RTRs. Objective: We sought to find further genetic variation in MTHFR and overlap genes that may be associated with a diagnosis of KC in RTRs. Methods: Genotyping of a combined RTR population (n = 821) from two centres, Ireland (n = 546) and the USA (n = 275), was performed. This included 290 RTRs with KC and 444 without. Eleven single nucleotide polymorphisms (SNPs) in the MTHFR gene and seven in the overlap gene MTHFR Chloride transport protein 6 (CLCN6) were evaluated and association explored by time to event analysis (from transplant to first KC) using Cox proportional hazards model. Results: Polymorphism at MTHFR CLCN6 (rs9651118) was significantly associated with KC in RTRs (HR 1.50, 95% CI 1.17-1.91, p < 0.00061) and cSCC (HR 1.63, 95% CI 1.14-2.34, p = 0.007). A separate SNP, MTHFR C677T, was also significantly associated with KC in the Irish population (HR 1.31, 95% CI 1.05-1.63, p = 0.016), but not American RTRs. Conclusions: We report the association of a SNP in the MTHFR overlap gene, CLCN6 and KC in a combined RTR population. While the exact function of CLCN6 is not known, it is proposed to be involved in folate availability. Future applications could include incorporation in a polygenic risk score for KC in RTRs to help identify those at increased risk beyond traditional risk factor assessment.

Nodular lesions arising in a large congenital melanocytic naevus in a newborn with eruptive disseminated Spitz naevi.

Congenital malignant melanoma within a pre-existing large congenital melanocytic naevus (CMN) is exceedingly rare. Its incidence is difficult to determine due to the small number of reported cases and because of problems associated with diagnosis. Some benign nodular proliferations (called proliferative nodules) arising in CMN, while rare, are significantly more common and can mimic malignant melanoma clinically or histologically. There are no reported cases of congenital melanoma or benign proliferative nodules in CMN in patients who also had eruptive disseminated Spitz naevi. We describe a girl who was noted to have a dark-brown plaque with several large erythematous nodules affecting the scalp at delivery, in addition to multiple erythematous dome-shaped papules that developed in a disseminated manner over several months, beginning at 10 days of age. It was difficult, not only clinically but also histologically, to determine the benign or malignant nature of all of these lesions. As primary cutaneous melanoma, atypical proliferative nodules in CMN, bland CMN or CMN with foci of increased cellularity and Spitz naevi show clear differences in the genetic aberration patterns, comparative genomic hybridization (CGH) could be a diagnostic help in ambiguous cases such as this. CGH performed on this patient showed multiple DNA copy number changes in the most atypical nodule, but such alterations could not be found in the remainder of the lesions. CGH showed differences between the nodular lesions that occurred in the CMN and helped us in supporting the diagnosis of this unique case of benign proliferative nodules and a possible congenital melanoma arising in a large CMN, associated with multiple widespread eruptive Spitz naevi.

Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma.

BACKGROUND: Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin. METHODS: GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH. RESULTS: Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS. CONCLUSION: The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.

Dual inactivation of RB and p53 pathways in RAS-induced melanomas.

The frequent loss of both INK4a and ARF in melanoma raises the question of which INK4a-ARF gene product functions to suppress melanoma genesis in vivo. Moreover, the high incidence of INK4a-ARF inactivation in transformed melanocytes, along with the lack of p53 mutation, implies a cell type-specific role for INK4a-ARF that may not be complemented by other lesions of the RB and p53 pathways. A mouse model of cutaneous melanoma has been generated previously through the combined effects of INK4a(Delta2/3) deficiency (null for INK4a and ARF) and melanocyte-specific expression of activated RAS (tyrosinase-driven H-RAS(V12G), Tyr-RAS). In this study, we made use of this Tyr-RAS allele to determine whether activated RAS can cooperate with p53 loss in melanoma genesis, whether such melanomas are biologically comparable to those arising in INK4a(Delta2/3-/-) mice, and whether tumor-associated mutations emerge in the p16(INK4a)-RB pathway in such melanomas. Here, we report that p53 inactivation can cooperate with activated RAS to promote the development of cutaneous melanomas that are clinically indistinguishable from those arisen on the INK4a(Delta2/3) null background. Genomewide analysis of RAS-induced p53 mutant melanomas by comparative genomic hybridization and candidate gene surveys revealed alterations of key components governing RB-regulated G(1)/S transition, including c-Myc, cyclin D1, cdc25a, and p21(CIP1). Consistent with the profile of c-Myc dysregulation, the reintroduction of p16(INK4a) profoundly reduced the growth of Tyr-RAS INK4a(Delta2/3-/-) tumor cells but had no effect on tumor cells derived from Tyr-RAS p53(-/-) melanomas. Together, these data validate a role for p53 inactivation in melanomagenesis and suggest that both the RB and p53 pathways function to suppress melanocyte transformation in vivo in the mouse.

Chromosomal gains and losses in primary cutaneous melanomas detected by comparative genomic hybridization.

The analysis of genetic changes in primary cutaneous melanoma has been limited by the need for fixation for diagnostic purposes. However, comparative genomic hybridization is able to analyze such specimens. We have applied comparative genomic hybridization to 32 primary melanomas to discover and map genomic regions with aberrant DNA copy numbers. The analysis was performed on native, nonamplified DNA extracted from manually dissected tumor sections. Loss of chromosome 9 was detected in 81% of the tumors, most commonly affecting the p arm. Additional common losses occurred on chromosomes 10 (63%), 6q (28%), and 8p (22%). Gains in copy number involved chromosomes 7 (50%), 8 (34%), 6p (28%), 1q (25%), 20 (13%), 17 (13%), and 2 (13%). Amplifications indicating areas harboring potential oncogenes were seen at 4q12, 5p14.3-pter, 7q33-qter, 8q12-13, 11q13.3-14.2, and 17q25. Correlations among the regions with copy number changes indicate that losses of chromosomes 9 and 10 occur early on in melanoma progression, whereas gains of chromosome 7 occur later. This sequence of events was further substantiated by an intratumoral comparison of copy number changes in areas with radial and vertical growth phase patterns. The overall pattern of regions affected by copy number changes is consistent with cytogenetic data from metastatic melanoma; however, the frequencies of involvement differ, providing further insight into the course of genetic events.

Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin.

Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each (P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11-13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary, thereby revealing one mechanism of local recurrence.

HDM2 protein overexpression, but not gene amplification, is related to tumorigenesis of cutaneous melanoma.

We investigated the role of alterations of HDM2, the human homologue of murine mdm2, in the tumorigenesis and progression of cutaneous melanoma. A well-characterized cohort of 172 cases representing different points in the spectrum of melanocyte transformation (16 dysplastic nevi, 11 melanomas in situ, 107 invasive primaries, and 38 metastatic lesions), as well as 11 human melanoma cell lines were examined by immunohistochemistry and Western blotting for HDM2 protein expression, and by either Southern blotting (SB) or fluorescence in situ hybridization for HDM2 gene amplification. HDM2 overexpression, defined as >20% tumor cells showing nuclear immunoreactivity, was observed in 1 of 16 (6%) dysplastic nevi, 3 of 11 (27%) melanomas in situ, and 81 of 145 (56%) invasive primary and metastatic melanomas. Comparable frequencies of HDM2 overexpression were observed among invasive primary cases with differing tumor thicknesses as well as among the metastatic cases: 21 of 40 (53%) at < or =1.5 mm; 31 of 50 (62%) at 1.6-3.9 mm; 10 of 17 (58%) at >4 mm; and 19 of 38 (50%) metastases. HDM2 amplification was observed in 1 of 88 (1%) primary cases using fluorescence in situ hybridization, and in 0 of 12 (0%) metastatic cases that overexpressed HDM2 using SB. Melanoma cell lines expressed HDM2 protein, but there was no evidence of amplification by SB. Our data suggest that HDM2 protein overexpression is common in invasive and metastatic melanoma. Observing HDM2 overexpression in noninvasive melanoma suggests that expression of this oncogene may play an early role in melanocyte transformation. HDM2 amplification occurs infrequently, and other mechanisms that up-regulate HDM2 expression are under investigation.

Molecular cytogenetic analysis of Spitz nevi shows clear differences to melanoma.

Spitz nevus is a benign neoplasm of melanocytes that can be difficult or impossible to distinguish from melanoma by clinical and histopathologic examination. We studied genomic DNA from 17 Spitz nevi by comparative genomic hybridization (CGH). Thirteen lesions showed no chromosomal aberrations, three cases had a gain involving the entire p-arm of chromosome 11, and one case showed a gain of chromosome 7q21-qter. Fluorescence in situ hybridization (FISH) on lesional tissue with a probe for the p-arm of chromosome 11 showed 6-10 p-arm signals per nucleus in those cases with a CGH-detected gain of chromosome 11p. One case with a normal CGH profile also showed increased copy number of 11p by FISH. Thus, the majority of Spitz nevi have a normal chromosomal complement at the level of CGH resolution; however some may contain gains, with 11p apparently being the most frequently involved location. These findings differ significantly from the previously reported changes in primary cutaneous melanoma, which show frequent deletions of chromosomes 9p (82%), 10q (63%), 6q (28%), and 8p (22%), as well as gains of chromosomes 7 (50%), 8 (34%), 6p (28%), 1q (25%) by CGH analysis. These clear differences in the location and frequencies of chromosomal aberrations in Spitz nevi and primary cutaneous melanomas could represent a basis for developing adjunctive techniques for refining accuracy in the difficult differential diagnosis of spitzoid melanocytic neoplasms.

Inhibition of human skin phospholipase A2 by "lipocortins" is an indirect effect of substrate/lipocortin interaction.

Proteins of the annexin/lipocortin family have been claimed to mediate the anti-inflammatory action of glucocorticosteroids by the inhibition of phospholipases A2. This hypothesis has been challenged by the finding that annexins do not directly interact with the enzyme in a classical enzyme/inhibitor behavior, but more likely block the access of the phospholipase A2 to its substrate by binding to phospholipids. Because former studies with skin phospholipase A2 suggested a specific regulation by annexin-1, we investigated the substrate dependence of this effect. For this purpose phospholipase A2 activities in human epidermis and dermis homogenates were measured in the presence of various amounts of annexins-1, -2, or -5. The respective annexin was preincubated in separate series either with the substrate or with the enzyme. We found a partial inhibition of both epidermal and dermal phospholipase A2 activities with all annexins tested (annexin-5 >> annexin-2 > annexin-1). The inhibitory effect was absolutely dependent on the annexin/phospholipid ratio and occurred only at very high annexin concentrations relative to the amount of substrate. Our data demonstrate that the inhibition of human skin phospholipase A2 by annexins depends on the substrate concentrations, as has been shown for phospholipases A2 of other origins as well. All observations can be explained by the current "substrate depletion model" characterizing the indirect effects of annexins on phospholipase A2 activities. It is therefore rather unlikely that annexins are directly involved in the regulation of phospholipase A2 activity of human skin under physiologic conditions.

Effect of wetting the mouth on aortic blood pressure just before taking sublingual nitrates.

The absorption of sublingual nitrate tablets is sometimes variable. We performed a randomized controlled trial to determine whether wetting the mouth improved the decrease in aortic systolic blood pressure (BP) from sublingual nitrate tablet or spray. The 100 patients undergoing routine diagnostic cardiac catheterization were allocated to control (no nitrate), 0.3 mg sublingual nitrate tablet, 0.4 mg sublingual nitrate spray, water + 0.3 mg sublingual nitrate tablet, or water + 0.4 mg sublingual nitrate spray. Aortic systolic and diastolic BP were recorded using a fluid-filled catheter and measured off-line blind to the treatment group. Compared with control subjects, there were significant decreases in aortic systolic BP with both nitrate preparations (tablet = -7.1 mm Hg, 95% confidence interval [CI] = -12.5 to -1.6 mm Hg; spray = -8.0 mm Hg, 95% CI = -13.4 to -2.5 mm Hg). On average, water significantly increased the fall in aortic systolic BP with nitrate tablets (-5.5 mm Hg, 95% CI = -10.9 to -0.1 mm Hg, p = 0.044) but did not significantly enhance the effect of nitrate spray (-2.8 mm Hg, 95% CI = -8.3 to 2.6 mm Hg). Water significantly increased the fall in aortic diastolic BP with tablets only (-2.9 mm Hg, 95% CI = -5.5 to -0.2), and had no significant effect on heart rate. Water had a consistently larger influence on the hemodynamic effects of nitrate tablets than on the effects of nitrate spray. Patients with a dry mouth will have an increased effect from sublingual nitrate tablets if they wet their mouth before using sublingual nitrate tablets. Water does not appear to assist in the action of sublingual spray.

Abnormal cardiovascular regulation in the mitral valve prolapse syndrome.

Studies of patients with mitral valve prolapse syndrome have suggested autonomic nervous system dysfunction, but a precise definition of mechanisms is lacking. We measured supine and standing heart rate, blood pressure, cardiac output, oxygen consumption, plasma catecholamines, and blood volume in 23 symptomatic women with both echocardiographic and phonographic signs of MVP and in 17 normal control subjects. An analysis of the results revealed 2 distinct subgroups of patients: those with normal heart rates but increased vasoconstriction (Group I, n = 10) and those with orthostatic tachycardia (Group II, n = 13). Group II patients had heart rates at rest supine of 97 +/- 3 compared with 79 +/- 2 in Group I patients and 78 +/- 8 in control subjects. Estimated total blood volumes were lowest in Group I patients, intermediate in Group II patients, and highest in control subjects (p less than 0.05). Other measurements at rest supine were similar in patients and controls. After standing for 5 minutes, patients had a higher mean plasma epinephrine value, diastolic blood pressure (81 +/- 2 versus 74 +/- 3 mm Hg, p less than 0.05), and peripheral resistance (1,878 +/- 114 versus 1,414 +/- 92, dynes s cm-5, p less than 0.01), wider arteriovenous oxygen difference (6.7 +/- 0.4 versus 5.3 +/- 0.5 vol%), and lower stroke volume index (26 +/- 2 versus 33 +/- 2 ml/m2, p less than 0.01) than did the control subjects. Cardiac output was normal in Group II patients but reduced in Group I patients, who demonstrated marked vasoconstriction. No patient had evidence of a "hyperkinetic" circulatory state. A cycle of decreased forward stroke volume, vasoconstriction, and blood volume contraction appears to be present in at least some symptomatic patients with MVP.

Localization of annexins in normal and diseased human skin.

Annexins (AX) or lipocortins are a family of calcium and phospholipid binding proteins that have been implicated to play a role in the regulation of inflammation and cellular differentiation. To investigate a potential role of AX in skin disorders we studied the distribution of six different AX in normal human skin (NHS) and several inflammatory and hyperproliferative skin diseases. A distinct staining pattern could only be shown for AX-1 and AX-2. In NHS AX-1-antibody (Ab) displayed a very strong reactivity with eccrine sweat ducts. In the diseases investigated we found a highly increased expression of AX-1 in keratinocytes (KCs) in the vicinity of inflammatory processes such as psoriasis. Furthermore, the AX-1 expression was increased in differentiated squamous cell carcinoma (SCC) whereas undifferentiated SCC and basal cell carcinoma were negative. AX-3, -4, -5, and -6 showed no distinctive expression pattern. Our data demonstrate an abnormal distribution of AX-1 in association with proliferating KCs under inflammatory and neoplastic conditions. Its pattern of reactivity shows similarities to the known distribution of the EGF-receptor kinase, which has been demonstrated to phosphorylate AX-1 with high activity in various cellular systems. These results support the concept that the appearance of AX-1 is linked to a certain level of KC differentiation.

Autoantibodies to annexins: a diagnostic marker for cutaneous disorders?

Annexins/lipocortins are a group of structurally related calcium and lipid binding proteins which have been implicated as mediators of the anti-inflammatory action of corticosteroids. Autoantibodies against annexin-1 have been reported in association with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and their presence has been hypothesized as the reason for the steroid resistance phenomenon. In this study we investigated IgG- and IgM-autoantibodies against annexin-1,-2,-3,-4,-5 and -6 in sera of 221 patients with skin disorders and 114 healthy blood donors with newly established ELISAs. Patients were clustered into 5 groups according to their diagnosis: autoimmune diseases, psoriasis, leg ulcer, malignant melanoma, and miscellaneous diseases. Autoantibodies directed against each annexin were detectable in all investigated groups, in the control group as well as in the disease groups, without displaying any significant correlation to any of the disease states. The homogenous distribution of annexin-autoantibodies throughout the control group and all the disease groups studied, do not support the implication of annexin-autoantibodies in pathophysiological states and make them an unlikely candidate for use as a diagnostic marker.

Preoperative characterization of pigmented skin lesions by epiluminescence microscopy and high-frequency ultrasound.

BACKGROUND AND DESIGN: Previous studies have referred to the value of epiluminescence microscopy in the differential diagnosis of pigmented skin lesions and to the possibility of preoperative tumor thickness measurement in malignant melanoma by high-frequency ultrasound. Both noninvasive methods have been combined in this study. The question of improved diagnostic accuracy was discussed. Previously proposed epiluminescence microscopic characteristics of 508 melanocytic lesions and sonographic characteristics of 792 skin tumors were investigated for their sensitivity and specificity. The tumor thickness of 108 malignant melanomas was measured sonographically. RESULTS: Black dots, irregular pigment network, and grayish-blue areas have been shown to be the most sensitive characteristics, whereas pseudopods, grayish-blue areas, and a whitish veil have been shown to be the most specific epiluminescence microscopic features for malignant melanoma. Sonography alone cannot reliably distinguish between different skin tumors. Preoperatively, the tumor thickness of 85% of the melanomas was assessed correctly concerning the pT stage. CONCLUSIONS: A 20-MHz ultrasound, in addition to epiluminescence microscopy, may improve the diagnostic accuracy by delivering information about depth and topographic location of skin tumors, but cannot give highly specific information about tissue dignity. It is a reliable tool for tumor thickness measurement for surgical planning.

Phospholipase A2 is secreted by murine keratinocytes after stimulation with IL-1 alpha and TNF-alpha.

Phospholipase A2 (PLA2)-catalysed liberation of arachidonic acid is the rate-limiting step in the generation of the lipid mediators prostaglandins and leukotrienes. PLA2 regulation thus represents a pivotal mechanism in the pathogenesis of inflammation. In this study we investigated the effects of TNF alpha and IL-1 alpha on PLA2 activity in cultured murine keratinocytes. Starting 18 h after stimulation, PLA2 activity increased significantly by about 250-320%) in the supernatants and in the cell pellets. This effect was completely inhibited either by preincubation of the cells with dexamethasone 48 h before stimulation or by coincubation with actinomycin D. PLA2 activity detected in the supernatants was blocked by reduction with dithiothreitol, whereas the PLA2 activity in the pellets was dithiothreitol-resistant. We conclude that in murine keratinocytes IL-1 alpha induce de novo synthesis and release of a secretory PLA2 and the induction of a different PLA2 activity in the cytosol. These findings indicate a crucial link between early cytokine effects and the initiation of the lipid mediator cascade in keratinocytes. The observation that PLA2 induction could be completely inhibited by preincubation with dexamethasone allows new insights into the mechanism of steroid effects on epidermal inflammation and renders PLA2 regulation an interesting therapeutic target.

Paraneoplastic pemphigus treated with dexamethasone/ cyclophosphamide pulse therapy.

Paraneoplastic pemphigus (PNP) is an autoimmune, mucocutaneous bullous disease associated with underlying malignancies. We report a patient with Waldenström's macroglobulinemia who developed clinical, histological and immunopathological features typical of PNP. The patient was treated twice with i.v. dexamethasone and cyclophosphamide pulse therapy (day 1: cyclophosphamide 500 mg i.v.; day 1-3: dexamethasone 100 mg i.v.) at 3-week intervals. Therapy was continued with oral cyclophosphamide (50 mg/d). Two weeks after initiation of treatment, significant improvement of the cutaneous and mucosal lesions was noted. The therapy also had beneficial effects on the macroglobulinemia in terms of a marked reduction of the IgM lambda serum level. Three months after the second pulse, severe stomatitis recurred but the patient rejected any further systemic therapy. The initial response of the usually recalcitrant mucosal and skin lesions of PNP makes dexamethasone/cyclophosphamide pulse therapy an interesting therapeutic option.

Giant lichenification of the vulva with marked ulcerations. A case report.

A case of vulvar giant lichenification of Pautrier showed uncommon ulcerations in the center of hyperplastic verrucoid plaques. The diagnosis was made from the history, clinical findings and characteristic features on the skin biopsy. Other causes of vulvar ulcers, such as venereal disease, chronic bullous and autoimmune diseases, and neoplastic conditions were excluded. We treated the patient with systemic antihistamines, topical antiseptics and corticosteroids, and sublesional injections of triamcinolone. The itch-scratch cycle was interrupted by the patient's wearing cotton gloves at night. Complete healing of the ulcers and an improvement in the pruritus was achieved within 14 weeks.