Skin biopsy utilization and melanoma incidence among Medicare beneficiaries.

BACKGROUND: Melanoma incidence has increased in recent decades in the U.S.A. Uncertainty remains regarding how much of this increase is attributable to greater melanoma screening activities, potential detection bias and overdiagnosis. OBJECTIVES: To use a cross-sectional ecological analysis to evaluate the relationship between skin biopsy and melanoma incidence rates over a more recent time period than prior reports. METHODS: Examination of the association of biopsy rates and melanoma incidence (invasive and in situ) in SEER-Medicare data (including 10 states) for 2002-2009. RESULTS: The skin biopsy rate increased by approximately 50% (6% per year) throughout this 8-year period, from 7012 biopsies per 100 000 persons in 2002 to 10 528 biopsies per 100 000 persons in 2009. The overall melanoma incidence rate increased approximately 4% (< 1% per year) over the same time period. The incidence of melanoma in situ increased approximately 10% (1% per year), while the incidence of invasive melanoma increased from 2002 to 2005 then decreased from 2006 to 2009. Regression models estimated that, on average, for every 1000 skin biopsies performed, an additional 5·2 (95% confidence interval 4·1-6·3) cases of melanoma in situ were diagnosed and 8·1 (95% confidence interval 6·7-9·5) cases of invasive melanoma were diagnosed. When considering individual states, some demonstrated a positive association between biopsy rate and invasive melanoma incidence, others an inverse association, and still others a more complex pattern. CONCLUSIONS: Increased skin biopsies over time are associated with increased diagnosis of in situ melanoma, but the association with invasive melanoma is more complex.

Melanocyte destruction after antigen-specific immunotherapy of melanoma: direct evidence of t cell-mediated vitiligo.

Current strategies for the immunotherapy of melanoma include augmentation of the immune response to tumor antigens represented by melanosomal proteins such as tyrosinase, gp100, and MART-1. The possibility that intentional targeting of tumor antigens representing normal proteins can result in autoimmune toxicity has been postulated but never demonstrated previously in humans. In this study, we describe a patient with metastatic melanoma who developed inflammatory lesions circumscribing pigmented areas of skin after an infusion of MART-1-specific CD8(+) T cell clones. Analysis of the infiltrating lymphocytes in skin and tumor biopsies using T cell-specific peptide-major histocompatibility complex tetramers demonstrated a localized predominance of MART-1-specific CD8(+) T cells (>28% of all CD8 T cells) that was identical to the infused clones (as confirmed by sequencing of the complementarity-determining region 3). In contrast to skin biopsies obtained from the patient before T cell infusion, postinfusion biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage, and the complete absence of melanocytes in affected regions of the skin. This study provides, for the first time, direct evidence in humans that antigen-specific immunotherapy can target not only antigen-positive tumor cells in vivo but also normal tissues expressing the shared tumor antigen.

Distinct inflammatory properties of late-activated macrophages in inflammatory myopathies.

Distinct mechanisms such as humeral immunity in dermatomyositis (DM) and T-cell-mediated cytotoxicity in polymyositis (PM) contribute to the pathology of inflammatory myopathies. In addition, different subsets of macrophages are present in both diseases. Herein, the characteristics of 25F9-positive macrophages in skeletal muscle inflammation are outlined. Muscle biopsies of subjects with DM and PM were studied by immunohistochemical multi-labelling using the late-activation marker 25F9, together with markers characterizing macrophage function including IFN-gamma, iNOS, and TGF-beta. In PM, a robust expression of IFN-gamma, iNOS, and TGF-beta was observed in inflammatory cells. Double- and serial-labelling revealed that a subset of 25F9-positive macrophages in the vicinity of injured muscle fibres expressed iNOS and TGF-beta, but not IFN-gamma. In DM, IFN-gamma, iNOS and TGF-beta were also expressed in inflammatory cells in the endomysium. Double- and serial-labelling studies in DM indicated that 25F9-positive macrophages expressed TGF-beta and to a lesser degree iNOS, but not IFN-gamma. In conclusion, our data suggest that late-activated macrophages contribute to the pathology of inflammatory myopathies.

Transgenic expression of the human amphiregulin gene induces a psoriasis-like phenotype.

Amphiregulin (AR) is a heparin-binding, heparin-inhibited member of the epidermal growth factor (EGF) family and an autocrine growth factor for human keratinocytes. Previous studies have shown that AR expression is increased in psoriatic epidermis. To test the hypothesis that aberrant AR expression is central to the development of psoriatic lesions, we constructed a transgene (K14-ARGE) encoding a human keratin 14 promoter-driven AR gene. Our results indicate that transgene integration and subsequent expression of AR in basal keratinocytes correlated with a psoriasis-like skin phenotype. Afflicted mice demonstrated shortened life spans, prominent scaling and erythematous skin with alopecia, and occasional papillomatous epidermal growths. Histologic examination revealed extensive areas of marked hyperkeratosis with focal parakeratosis, acanthosis, dermal and epidermal lymphocytic and neutrophilic infiltration, and dilated blood vessels within the papillary dermis. Our results reveal that AR exerts activity in the skin that is distinct from that of transgenic transforming growth factor-alpha or other cytokines, and induces skin pathology with striking similarities to psoriasis. Our observations also link the keratinocyte EGF receptor-ligand system to psoriatic inflammation, and suggest that aberrant expression of AR in the epidermis may represent a critical step in the development or propagation of psoriatic lesions.

Heparin effect on DNA synthesis in a murine fibrosarcoma cell line: influence of anionic density.

The effects of heparin subfractions on DNA synthesis in a murine cutaneous fibrosarcoma cell line were examined. Porcine mucosal heparin was preparatively fractionated for anionic charge density by DEAE-Sephadex chromatography and for molecular weight by Sephadex G-100 filtration. The cell line was plated from confluent monolayer cultures and grown in medium and fetal bovine serum, with or without a heparin fraction at a final concentration of 10 micrograms/ml. At intervals thereafter, the cells were pulsed with [3H]thymidine. A low-charge density heparin fraction stimulated [3H]thymidine incorporation (cpm/mg protein and cpm/cell) during the first 3 days of growth compared to control values without added heparin, whereas a high-charge density heparin fraction had little of this effect (186 +/- 35% of control vs. 101 +/- 14%, respectively; P less than .05). The augmentation of DNA synthesis observed with the low-charge density fraction correlated with increased proportions of cells in S and G2 phases compared with those of the controls, as determined by flow cytofluorometry. Low- and high-molecular-weight heparin fractions did not significantly alter DNA synthesis. Heparin subfractions are thus heterogeneous with respect to their effect on cellular DNA synthesis in this tumor line.

Inheritance of nevus number and size in melanoma and dysplastic nevus syndrome kindreds.

Previous studies of the genetics of melanoma have focused on the dysplastic nevus syndrome (DNS). The variability in clinical and histopathological expression of affected individuals, however, has made definition and diagnosis of the syndrome difficult and subjective. Independent of the DNS, case-control studies have demonstrated the total number of nevi to be a significant risk factor for melanoma. In this article, we report results of genetic analyses of two quantitative nevus phenotypes that can be measured objectively in all subjects: the total number of nevi on an individual (TNN) and total nevus density (TND), a derived phenotype which incorporates both number and size of nevi. Ten kindreds ascertained for multiple cases of DNS-melanoma (multiplex ascertainment) and 16 kindreds and 19 solitary cases ascertained from a sequential list of melanoma cases without regard for family history (simplex ascertainment) were studied. Both phenotypes exhibited increased levels in relatives of probands compared with those in spouse controls. While neither TNN nor TND exhibited evidence for a major factor in the simplex pedigrees, a major factor was strongly indicated in the multiplex kindreds for TND. When both phenotypes were examined in more detail in the multiplex kindreds, the phenotype incorporating nevus size, TND, fit a mendelian pattern of inheritance better than the TNN. Significant residual familial correlations were found for both phenotypes. Parameter estimates from the best fitting genetic model indicated that a major gene may be responsible for 55% of the phenotypic variability of TND in the multiplex kindreds.

The antiproliferative effects of enzymatic deglycosylation and metabolic undersulfation of proteoglycans from the cell surface.

Enzymatic deglycosylation of plasma membrane proteoglycans and metabolic inhibition of glycosaminoglycan sulfation were employed as complementary methods to evaluate the effects of reduced cell surface content of functionally intact proteoglycans on the proliferative potential of cells. A Flavobacter heparinum extract, possessing multiple glycosaminoglycan substrate specificities, markedly inhibited the time-dependent expansion of BALB/c 3T3 fibroblast and human squamous cell carcinoma monolayers in culture and concurrently reduced the proportion of subconfluent cell populations in S-cell cycle phase by DNA flow-cytometry analysis. This antiproliferative effect was partially reproduced by lyases with heparan sulfate or chondroitin sulfate monospecificity, alone and in combination. The observed lability of heparan sulfate lyases I and II in serum-containing medium possibly hampered full reproduction of the effects of the multifunctional reagent. Growth inhibition of comparable magnitude was observed when glycosaminoglycan sulfation was metabolically blocked with sodium chlorate. The chlorate anion had its expected effect of substantially reducing sulfated glycosaminoglycan synthesis by the cells. Following release from serum deprivation, analysis of the progression of synchronized cell populations past the G1 restriction point suggested that in situ digestion with the glycosaminoglycan lyases limited, but did not delay, the numbers of cells entering S phase. These data support the hypothesis that plasma membrane proteoglycans mediate some of the cell-growth-promoting effects of serum factors via their glycosaminoglycan side chains.

Topography of proteoglycan and glycosaminoglycan free chain expression in 3T3 fibroblasts and human keratinocytes.

Synthesis of heparan sulfate-free chains by human keratinocytes is upregulated during terminal differentiation. The cellular location of this product class and the significance of the differentiation effect are unknown. Differential plasma membrane shearing with cationized colloidal silica was used to evaluate the compartmentalization of the heparan and chondroitin sulfate free chains and their respective proteoglycans in 3T3 fibroblasts and human keratinocytes. The method exploits the topologic segregation of plasma membranes of adherent cells into ventral, dorsal, and intracellular domains and the selective binding of the silica to the dorsal membranes, which by shearing can be separated from ventral membranes adherent to the substratum. Analysis of membrane preparations from sheared cells that had been prelabeled with [35S]-sulfate revealed the proteoglycans to be predominantly ventral, at which location a matrix binding function could be accommodated. Proteoglycans were also recovered from dorsal and intracellular membranes, suggesting active trafficking between intra- and extra-cellular sites. In contrast, the major fraction of heparan and chondroitin sulfate free chains was either cytosolic or associated with intracellular membranes, with the remaining approximately 20% segregated to dorsal and ventral membranes. These results suggest different cellular functions for the proteoglycans and glycosaminoglycan free chains. The partial localization of the free chains to peripheral membranes is compatible with our prior hypothesis that they arise by processing of precursor proteoglycans on cell surfaces. Following this origin, the free glycosaminoglycan polymers could be available to bind ligands such as cytokines prior to transport to intracellular sites of action.

The intracutaneous growth of murine lymphomas: epidermal invasion is characteristic of multiple tumor phenotypes.

Affinity of lymphoid cells for the epidermis (epidermotropism) is characteristic of the cutaneous T-cell lymphomas, mycosis fungoides and the Sézary syndrome. Consistent with numerous studies indicating that mycosis fungoides is a neoplasm of OKT4+T8- ("helper/inducer") T lymphocytes is the possibility that epidermotropism is a phenotypic hallmark of this subset of malignant T cells. This proposal was investigated in mice using 8 phenotypically characterized lymphomas of BALB/c origin: 3 histiocytic (phagocytic, lysozyme-positive, FcR+, Ig-, Thy 1-), 1 B-cell (IgM+, FcR+, Thy 1-), and 4 T-cell (Ig-, Thy 1+) lines, including 1 with markers of mouse helper/inducer T cells (Lyt1+23-), 2 with suppressor/cytotoxic markers (Lyt1-23+), and 1 with markers of immature thymocytes (Lyt1+23+). The intracutaneous growth pattern of these lines was studied on hematoxylin and eosin-stained sections through the centers of tumors obtained at times after intradermal injection into parallel groups of syngeneic mice. All of these lymphomas manifested variable epidermotropism that followed a typical sequence. Following dermal growth and spread to the dermal-epidermal junction, tumor cells appeared within the stratum spinosum. Subsequently, collections of cells appeared in spaces within the epidermis (Pautrier-like microabscesses) in tumors greater than 2 cm in diameter, coincident with early epidermal necrosis. Thus, in this animal model it is clear that the intraepidermal invasion/growth does not correlate with the helper/inducer T-cell surface phenotype. These observations are nonetheless consistent with recent studies using monoclonal antibodies to cell surface antigens which have demonstrated a heterogeneity of lymphoid cell subsets within the epidermis in lesions of mycosis fungoides and of other malignant and benign dermatoses.

Glycosaminoglycan synthesis by human keratinocytes: cell growth and medium calcium effects.

The influences of cell density, differentiation, and medium calcium levels on glycosaminoglycan biosynthesis were evaluated in cultured human epidermal keratinocytes. Following metabolic labeling with [35S]-sulfate and [3H]-glucosamine under steady state conditions in "high" medium calcium (greater than 1.0 mMol), the majority of sulfated glycosaminoglycans remained associated with the cell layers, whereas hyaluronic acid, which was present in smaller amounts than the sulfated products, was about equally distributed between the medium and the cell layers. Of the sulfated glycosaminoglycans, heparan sulfate and chondroitin 4/6-sulfate were the major species and were present in roughly comparable amounts, whereas dermatan sulfate was quantitatively the lesser of the products. The effects of "low" medium calcium (0.3 and 0.025 mM) were complex, although a consistent decrease in the incorporation of the [3H]-glucosamine precursor was found at high cell density, probably reflecting a decrease in its intracellular specific activity. In "high" calcium cultures, there was a strong inverse correlation (r = -0.92) between keratinocyte cell number and cellular production of sulfated glycosaminoglycans, whereas no such relationship was evident in cultures grown in "low" calcium medium at comparable cell density. Because keratinocyte differentiation is inhibited in the low calcium conditions, the results suggest that the decrease in production of sulfated glycosaminoglycans by confluent keratinocytes may actually correlate with differentiation rather than with cell number.

Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture.

Glycosaminoglycan (GAG) synthesis and compartmentalization were studied in populations of human neonatal keratinocytes under conditions of proliferation and terminal differentiation in vitro. Following isotopic labeling with the precursors [3H]glucosamine and [35S]sulfate, GAGs were extracted from the keratinocytes into several operationally created compartments associated with the cells and extracellular matrix. Chondroitin sulfate and heparan sulfate accounted for the majority of the incorporated label in all preparations. Although total sulfated GAGs per culture increased from proliferative to differentiated conditions, GAG content normalized to the DNA content of the cultures demonstrated the reverse trend. This was particularly evident for the chondroitin sulfates, which declined 60-70% in the differentiated cultures. Furthermore, label incorporation into chondroitin and heparan sulfates revealed a relative compartmental shift to a trypsin-accessible site upon keratinocyte differentiation. An analysis of heparan sulfate structure by characterization of the oligosaccharide products resulting from low pH nitrous acid deaminative degradation provided evidence that the parent material from differentiated keratinocytes contains a larger region of N-sulfated glucosamine residues unassociated with ester sulfate groups. The correlation of variations in GAG content and compartmentalization with the growth condition of human keratinocytes constitutes evidence that this heterogeneous group of cell surface-associated carbohydrates is involved in some aspect of cell function associated with growth control or differentiation. Furthermore, the apparent differences in heparan sulfate primary structure indicate that there is structure-function specificity to this association.

Autocrine regulation of keratinocytes: the emerging role of heparin-binding, epidermal growth factor-related growth factors.

Although originally conceived as a basis for malignant cell growth, autocrine signaling networks are currently known to be activated during tissue repair and with in vitro cultivation. In human epidermal keratinocytes, activation of the epidermal growth factor receptor by cognate ligands mediates the majority of the autonomous replicative capacity of these cells and is necessary to inhibit differentiation and apoptosis. The importance of heparin-binding growth factors in activation of this receptor was first suggested by the strong anti-proliferative effects of soluble heparin-like molecules on keratinocyte growth. This and related evidence led to the identification of amphiregulin as a major autocrine factor for keratinocytes. The binding of amphiregulin and its homolog, heparin-binding epidermal growth factor-like growth factor, to the receptor is potentially amplified by autoinduction and cross-signaling through epidermal growth factor-related polypeptides and by transmodulation of other ErbB-family receptors (HER-2, -3, -4) in cells expressing these receptors. Heparan sulfate proteoglycans and the tetraspanin family of membrane-associated proteins appear to act as cofactors in amphiregulin-driven mitogenesis mediated by the epidermal growth factor receptor, but amphiregulin's immunolocalization to keratinocyte nuclei and to filopodia may indicate other potentially novel effects. Following from the observation that amphiregulin is overexpressed in lesional psoriatic epidermis, the importance of amphiregulin in hyperproliferative skin diseases has been further supported by recent studies of the targeted expression of a transgene encoding keratin 14 promoter-driven human amphiregulin to the basal epidermis of mice. Founder transgenic mice displayed a morphologic and microscopic cutaneous phenotype that shares characteristics with psoriasis. Pharmacologic regulation of amphiregulin's expression and receptor signaling may eventually prove to be an effective strategy in the treatment of hyperproliferative skin diseases.

Expression of the tumor suppressor gene product p16INK4 in benign and malignant melanocytic lesions.

The gene MTS1 encodes p16INK4, an inhibitor of cyclin-dependent kinase 4, and is frequently deleted, mutated, or silenced by promoter methylation in melanoma cells and in the germline of familial melanoma patients. Although MTS1 may thus be the candidate melanoma suppressor gene that maps to chromosome 9p21, it is not clear how dysfunction at that locus temporally relates to melanoma progression. To further test its role in sporadic melanoma, the expression of p16INK4-protein and -mRNA was characterized in melanomas and melanocytic nevi by immunocytochemistry and in situ reverse transcriptase-polymerase chain reaction. Histologic tissue sections were immunolabeled with anti-p16INK4 antibody for 108 melanocytic lesions, including common and atypical nevi, in situ melanomas, primary invasive melanomas, and metastatic tumors. A subset of the lesions was analyzed for expression of p16INK4-mRNA, employing forward and reverse intron-bridging primers for reverse transcriptase-polymerase chain reaction amplification of the transcript corresponding to exons 1 and 2 of MTS1. Strong immunolabeling was detected in the melanocytes of common nevi and of nevi with architectural disorder and cytologic atypia. By digital image analysis, in contrast, labeling intensity decreased significantly and progressively in the melanocytes of in situ, invasive, and metastatic melanomas. Results from the in situ reverse transcriptase-polymerase chain reaction analysis were confirmatory, showing a strong signal in the melanocytic nevi but progressive signal attenuation with increasing stage of melanoma. These data indicate correlation between gradual loss of expression of the MTS1 locus and progression of melanoma, further supporting an emerging role for the gene in the malignant transformation of melanocytes. The failure to demonstrate reduced expression in nevi suggests either that these lesions are not an early stage in melanoma development, in contrast to prevailing assumptions, or that loss of p16INK4 function is not an initiating event in melanocyte transformation.

The distinctive pattern of proteoglycan and glycosaminoglycan free chain synthesis by cultured human epidermal keratinocytes.

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Expression of amphiregulin is regulated in cultured human keratinocytes and in developing fetal skin.

Previous studies have indicated that amphiregulin is a major autocrine factor for human keratinocytes. To evaluate the possibilities that amphiregulin could function in fetal skin morphogenesis and contribute to the growth regulation of epidermis, immunostaining with a specific anti-amphiregulin monoclonal antibody was observed at different stages of fetal skin development, and the results were compared with neonatal and adult skin specimens and cultured neonatal keratinocytes. Immunoreactive amphiregulin was readily detected in the periderm and basal epidermal layers of embryonic epidermis but became gradually less detectable in the periderm concurrent with an increase in staining of the spinous layer as it developed during the fetal period. Basal and spinous keratinocyte expression of amphiregulin was predominantly cytoplasmic, but with punctate nuclear foci, and this pattern persisted into the neonatal period. At all developmental stages, epithelial and mesenchymal cells of the follicle were reactive, often in a nuclear pattern. Dermal mesenchymal cells were increasingly reactive in late fetal skin, but the staining decreased postnatally. In adult skin only randomly scattered nuclei of spinous keratinocytes and follicular structures such as the inner root sheath were stained. Examination by scanning laser confocal microscopy of cultured neonatal keratinocytes showed a nonrandom distribution of amphiregulin to the peripheral cytoplasm and plasma membranes at the outer perimeter of cell colonies, with much less reactivity of apposed keratinocyte membranes at interior sites. Nuclei were heterogeneously stained. Amphiregulin reactivity declined at higher cell densities. These data indicate that expression of amphiregulin is regulated in vitro and developmentally during cutaneous morphogenesis.

Influence of genes of the major histocompatibility complex on ulcerative dermal necrosis induced in mice by Mycoplasma arthritidis.

All mouse strains injected s.c. with Mycoplasma arthritidis developed severe abscesses in the subdermal tissues. However, M. arthritidis strain 14124 P10 also induced an ulcerative dermal coagulation necrosis in mouse strains expressing the k and d haplotypes but not in those expressing the b, q, or s haplotypes. The use of inbred and congenic mouse strains established that the ulcerative necrosis was associated with the haplotypes expressed at the H2 major histocompatibility complex (MHC). The gene restriction seen could be partially overcome by using a more virulent mouse-passaged strain of M. arthritidis (158 P10P9). The data suggest that genes of the MHC function by rendering certain mouse strains more susceptible to an as yet unidentified necrotizing moiety. The close histologic resemblance of the dermal necrosis induced by M. arthritidis to certain human diseases such as necrotizing fasciitis, the ulcerative lesions induced by Mycobacterium ulcerans, and the crepitant and gangrenous cellulitides may therefore provide a unique model to study the genetic factors and mechanisms of pathogenesis in these latter human conditions.

The repertoire of T cell antigen receptor beta-chain variable regions associated with psoriasis vulgaris.

We investigated whether the pattern of T-cell receptors expressed by T cells in inflamed psoriatic skin differed substantially from the pattern seen in T cells from the peripheral blood. A bias or restriction in the repertoire of T-cell receptors found in the lesional skin of different patients might imply that specific subsets of T cells were causally associated with initiating or maintaining the lesions. By using a polymerase chain reaction-based assay of T-cell receptor beta-chain variable region mRNA, we found that the patterns of beta-chain mRNAs displayed in 14 samples of lesional skin or six samples of noninvolved skin were not significantly less diverse than the patterns found in matched peripheral blood samples. There was no evidence that the active lesions of multiple patients showed overexpression of T cells expressing one or a few T-cell receptor forms. The pattern of T-cell receptors displayed in clinically normal skin from normal control individuals showed about the same diversity as normal blood. While these results may not exclude either classical antigen or superantigen-based T-cell activation mechanisms in active plaques, the absence of a simple pattern of Vbeta usage in different patients suggests than other aspects of T-cell biology including trafficking, proliferation, co-stimulation, or responses to cytokines must also be considered.

Interobserver reproducibility of ulceration assessment in primary cutaneous melanomas.

In the recently revised melanoma staging system proposed by the American Joint Committee on Cancer (AJCC), ulceration assessment by the pathologist is a pivotal parameter. Patients upstaged because of ulceration might be included in adjuvant trials conducted in AJCC stage II melanoma patients. Therefore, accuracy based on interobserver reproducibility for melanoma ulceration assessment is crucial for proper clinical management. In some cases, it is extremely difficult, even for an experienced pathologist, to distinguish between trauma-induced ulceration, artifact and tumoral ulceration. Whether this difficulty may be resolved by the use of a more precise definition of ulceration has not been evaluated. Therefore, we have proposed a refined definition of melanoma ulceration and we tested whether this definition might improve the interobserver interpretative reproducibility of ulceration in primary cutaneous melanomas. The results of this study support the need for a more precise definition of melanoma ulceration that rules out biopsy trauma or processing artifact and could be incorporated into a standardised pathology worksheet for reporting primary melanomas.

Infective endocarditis associated with cell wall-deficient bacteria. Electron microscopic findings in four cases.

Although cell wall-deficient bacteria have been isolated in vitro from cases of endocarditis, no pathogenic role has been established for these forms in human disease. One criterion difficult to satisfy is the demonstration of these variants in human tissue, and electron microscopic documentation has not been reported. Cardiac valvular vegetations from four cases of endocarditis were examined by electron microscopy because of unusual histologic features of minimal inflammation and organization and small organisms that stained poorly by Gram stain. Although cell wall-complete bacteria were identified in the specimens, each showed the presence of cell wall-deficient forms within the vegetations; these variants predominated in three cases. Since manifestations of infective endocarditis were present in three cases and conventional cultures were negative, the evidence indirectly suggests a pathogenic role for these aberrant bacteria in human disease.

Atypical Spitz nevi/tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome.

The biological nature of Spitz nevi/tumors and their diagnostic distinction from, or relationship to, melanoma remain unresolved issues. In this report, a series of 30 melanocytic lesions removed from 28 patients, including atypical Spitz nevi/tumors and metastasizing Spitzoid tumors/melanomas, were evaluated by a panel of dermatopathologists to evaluate interobserver diagnostic concordance and to assess the prognostic power of histological criteria. For inclusion in the study, each lesion had to display some criteria for the Spitz nevus, and in addition one of the following was required: (1) definitive clinical outcome such as metastasis or death of disease, or (2) long-term follow-up if the patient remained disease free. Each lesion was reviewed independently and blinded as to the clinical data by 10 pathologists, who categorized them as (1) typical Spitz nevus/tumor, (2) atypical Spitz nevus/tumor, (3) melanoma, (4) tumor with unknown biological potential, or (5) other melanocytic lesion. There was limited discussion of criteria before the review. Evaluation of 17 Spitzoid lesions yielded no clear consensus as to diagnosis; in only one case did six or more pathologists agree on a single category, regardless of clinical outcome. Notably, however, some lesions that proved fatal were categorized by most observers as either Spitz nevi or atypical Spitz tumors. Conversely, seven or more pathologists scored 13 lesions as melanoma. These results illustrate (1) substantial diagnostic difficulties posed by many Spitz tumors, especially those with atypical features, even among experts, and (2) the lack of objective criteria for their distinction from melanoma and for gauging their malignant potential. Nevertheless, our observations do suggest that a biological relationship exists between the Spitz nevus/tumor and melanoma.