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.

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.

Dysplastic naevi with moderate to severe histological dysplasia: a risk factor for melanoma.

BACKGROUND: The risk of malignant melanoma associated with histologically dysplastic naevi (HDN) has not been defined. While clinically atypical naevi appear to confer an independent risk of melanoma, no study has evaluated the extent to which HDN are predictive of melanoma. OBJECTIVES: To estimate the risk of melanoma associated with HDN. Secondarily, the risk associated with number of naevi and large naevi is estimated. METHODS: We enrolled 80 patients with newly diagnosed melanoma along with 80 spousal controls. After obtaining information on melanoma risk factors and performing a complete cutaneous examination, the most clinically atypical naevus was biopsied in both cases and controls. Histological dysplasia was then assessed independently by 13 dermatopathologists (0, no dysplasia; 1, mild dysplasia; 2, moderate dysplasia; 3, severe dysplasia). The dermatopathologists were blinded as to whether the naevi were from melanoma subjects or controls. Multivariate analyses were performed to determine if there was an independent association between the degree of histological dysplasia in naevi and a personal history of melanoma. RESULTS: In persons with naevi receiving an average score of > 1 (i.e. naevi considered to have greater than mild histological dysplasia), there was an increased risk of melanoma [odds ratio (OR) 2.60, 95% confidence interval (CI) 0.99-6.86] which persisted after adjustment for confounders (OR 3.99, 95% CI 1.02-15.71). Very few dermatopathologists reliably graded naevi of subjects with melanoma as being more dysplastic than naevi of control subjects. Among the entire group, the interobserver reliability associated with grading histological dysplasia in naevi was poor (weighted kappa 0.28). CONCLUSIONS: HDN do appear to confer an independent risk of melanoma. However, this result may add more to our biological understanding of melanoma risk than to clinical assessment of risk, because HDN assessed by a single pathologist generally cannot be used to assess risk of melanoma. Future studies should be directed at establishing reproducible, predictive criteria for grading naevi.

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.

The p75 neurotrophin receptor, relative to other Schwann cell and melanoma markers, is abundantly Expressed in spindled melanomas.

Seventeen cases of spindled melanomas and eleven cases of epithelioid melanomas were immunolabeled with various melanoma and Schwann cell markers. Standard melanoma markers included S100, HMB45, HMB50, tyrosinase, and Melan A. Schwann cell markers included the p75 neurotrophin receptor (p75NTR), glial fibrillary acidic protein (GFAP), and the L1 adhesion protein. The degree of immunocytochemical labeling was scored by levels of both intensity and pervasiveness. The results confirmed a distinct difference in labeling between epithelioid and spindled melanomas. The p75NTR was strongly expressed in spindled melanomas and weakly expressed in the epithelioid melanomas. The usual melanoma markers, including HMB45, HMB50, MelanA, and tyrosinase had the reverse pattern, being strongly expressed in virtually all epithelioid melanomas, but rarely expressed in the spindled variants. S100 was unique among the markers in being expressed by both epithelioid and spindled melanomas. Glial fibrillary acidic protein and L1 adhesion protein were expressed moderately, with preferential labeling of the spindled melanomas. The greatest immunophenotypic difference between spindled and epithelioid melanomas was the high abundance of p75NTR expression in spindled melanomas. The functional significance of the high level of p75 neurotrophin receptor expression may contribute to the high predisposition of perineural extension in the desmoplastic subset of spindled melanomas.

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.

The application of direct immunofluorescence to intraoperative neurosurgical diagnosis.

A diagnostic problem can occur at the time of intraoperative consultation of neurosurgical tumors as to whether the tumor is of neuroectodermal origin or whether it represents an epithelial metastasis from another site. Intraoperative diagnoses based on hematoxylin and eosin stained frozen sections are often later confirmed by immunocytochemical analysis of formalin-fixed, paraffin-embedded tissue sections that are not available at the time of surgery. The objective of the current study was to demonstrate that the application of direct immunofluorescence to the intraoperative diagnosis of neurosurgical tumors would provide unequivocal, and nearly immediate results. This report describes a new application of an existing technique for an optimized, rapid procedure utilizing direct immunocytochemistry with fluorescence-labeled primary antibodies to analyze surgical biopsies intraoperatively. The examination of five neurosurgical biopsies established a neuroectodermal origin of three tumors via immunolabeling for glial fibrillary acidic protein (GFAP) and lack of labeling with keratin markers, whereas several metastatic lung carcinomas were identified by immunostaining for keratin, but not GFAP, markers. The results of the direct immunolabeling method were unequivocal and required only minutes. The same diagnoses were confirmed by standard immunocytochemical labeling of formalin-fixed, paraffin-embedded sections, though it required several days to obtain the results. Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding whether a neurosurgical tumor is a primary glial or an epithelial metastatic tumor in origin. It is the first reported application of the technique for this aspect of rapid neurosurgical diagnosis.

Melanoma genetics: an update with focus on the CDKN2A(p16)/ARF tumor suppressors.

UNLABELLED: Investigative interest in atypical nevi and familial melanoma has contributed to the identification of several candidate melanoma loci within the human genome. Molecular defects in both tumor suppressor genes and oncogenes have been pathogenically linked to melanoma in recent studies. Of the loci currently characterized, the major gene resides on chromosome 9p and encodes a tumor suppressor designated p16. This gene, which is also known as CDKN2A, is either mutated or deleted in a large majority of melanoma cell lines, as well as in many uncultured melanoma cells and in the germline of melanoma kindreds. A novel aspect of the p16 locus is that it encodes not just one but two separate gene products that are transcribed in alternative reading frames. Both products function as negative regulators of cell cycle progression. The p16 protein itself executes its effects by competitively inhibiting cyclin-dependent kinase 4, which is a factor necessary for cellular progression through a major regulatory transition of the cell division cycle. Inherited and acquired deletions or point mutations in the p16 gene increase the likelihood that potentially mutagenic DNA damage will escape repair before cell division. Notably, the second product of the locus, ARF (for alternative reading frame), regulates cell growth through independent effects on the p53 pathway. Although there is little evidence that ARF by itself is involved in the pathogenesis of melanoma, deletions at the p16 locus disable two separate pathways that control cell growth. These recent advances open up the possibility of genetic testing for melanoma susceptibility in the setting of familial melanoma and suggest novel therapeutic strategies for melanoma based on gene therapy or small molecule mimicry targeted to the correction of defects in the p16 regulatory pathway. (J Am Acad Dermatol 2000;42:705-22.) LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the historical aspects of melanoma genetics and should have a greater understanding of the CDKN2A(p16)/ARF tumor suppressor genes.

The expression of p16(INK4a), the product of a tumor suppressor gene for melanoma, is upregulated in human melanocytes by UVB irradiation.

BACKGROUND: The genetic locus CDKN2A has been linked to familial melanoma, and mutations or deletions in its coding sequence are seen in some cases of sporadic and familial melanomas. The protein encoded by CDKN2A, p16(INK4a), functions as a negative regulator of cell cycle progression and as a tumor suppressor, but the regulatory mechanisms involved in controlling its expression remain poorly defined. OBJECTIVE: This study tested the hypothesis that UVB irradiation, which transiently inhibits the growth of human melanocytes, is one of the regulators of p16(INK4a) expression. METHODS: Cultured human melanocytes were irradiated with UVB over a sublethal dosage range, and p16(INK4a) protein and mRNA levels were quantified at varying times thereafter by quantitative immunostaining and by Western and Northern blotting. RESULTS: Levels of p16(INK4a) protein in melanocytes increased significantly after sublethal UVB irradiation as compared with nonirradiated cells. Northern analysis indicated that p16(INK4a) messenger RNA coordinately increased in a dose-dependent manner more than 2-fold in irradiated cells at the tested doses. CONCLUSION: UVB irradiation transcriptionally activates the expression of p16(INK4a) in cultured human melanocytes. Therefore the growth arrest that occurs with irradiation of melanocytes could be mediated, in part, by upregulation of p16(INK4a). This transient arrest may allow repair of UVB-induced DNA damage before cell division. Conversely, hereditary or acquired defects in CDK4A that give rise to functional insufficiency of p16(INK4a) could permit the premature propagation of melanocytes harboring potentially carcinogenic DNA damage.

Angiogenesis and vascular growth factor receptor expression in malignant melanoma.

The growth and metastases of many solid tumors are dependent on the recruitment of new blood vessels. Tumor angiogenesis is most likely initiated by paracrine release of growth factors that bind to their corresponding endothelial cell surface receptors. To determine whether angiogenesis and growth factor receptor expression are consistent findings in malignant melanoma, primary human melanomas were examined for mRNA expression of receptors for fibroblast growth factors (FGFR-1, FGFR-2), vascular endothelial growth factor (VEGFR-1, VEGFR-2), and the receptors Tiel and Tie2. Charts were reviewed and archival formalin-fixed, paraffin-embedded primary tumors were obtained from patients with thin (<1 mm; n = 10), intermediate (1 to 4 mm; n = 10), or thick malignant melanoma (>4 mm; n = 8). Also examined was whether melanoma cell lines could induce endothelial growth factor receptor synthesis by metabolic labeling. It was found that tumor vascularity did not correlate with clinical stage, melanoma thickness, or clinical outcome. It was also found that melanoma cell lines were not capable of directly regulating endothelial cell synthesis of growth factor receptors. However, expression of Tiel and VEGFR-2 mRNA by the tumor vasculature in select stage IA-IIB patients, and FGFR-1 mRNA expression by the tumor cells in the same clinical stages was found. The expression of these growth factor receptors did not correlate with clinical outcome. These data suggest that angiogenesis is not a prominent characteristic of primary malignant melanoma lesions and that the endothelial cell expression of Tiel and VEGFR-2 in vivo is probably not directly induced by the tumor.

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.

Heparan sulfate composition of alternatively spliced CD44 fusion proteins.

Prior analyses of recombinant CD44 fusion proteins have indicated that combinatorial splicing of variant exons exerts distal effects on chondroitin sulfate content and structure, which may regulate the biological properties of the respective CD44 isoforms. The consequences of splicing of variant exons V4-7 on the heparan sulfate moieties were therefore examined, utilizing recombinant chimeras containing exons V3 and V8-10, engineered with or without exons V4-7 and expressed as Ig fusion proteins in COS cells. Splicing of exons V4-7, though they contain no consensus motifs for glycosaminoglycan assembly, resulted in markedly increased polymer sulfation levels of the heparan sulfates. The sulfate groups of both the CD44 V3-10 and V3,8-10 isoforms occurred as di- and tri-sulfated dissacharide units and were restricted to one N-sulfated block domain within the polymers. Compared to native human keratinocyte CD44, the recombinant heparan sulfates were relatively low in sulfate content. Our data indicate that variant exon V4-7 splicing exerts distal effects on the composition of this glycosaminoglycan. These effects may regulate those functions that are mediated through the heparan sulfate moieties, such as the binding of growth factors.

Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44.

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.

Trichodysplasia spinulosa--a newly described folliculocentric viral infection in an immunocompromised host.

This is a case report of an immunocompromised individual who presented with progressive alopecia, friable follicular spinous processes, and erythematous, indurated papules. Examination of skin biopsies using light microscopy and immunohistochemistry revealed pathologic changes of the follicular inner root sheath epithelium with dystrophic trichohyaline granules. Electron microscopy of thin sections of tissue revealed intracellular viral particles with a size and appearance consistent with those in the Papovaviridae family. Electron microscopy of negatively stained extract from a homogenized lesion also demonstrated icosahedral viruses with papovavirus morphology. We believe this is a previously unreported folliculocentric viral infection in an immunosuppressed human host and have termed this entity "trichodysplasia spinulosa".

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.

Tumor vascularity, proliferation, and apoptosis in human melanoma micrometastases and macrometastases.

BACKGROUND: Clinically undetectable or dormant metastases (micrometastases) probably account for disease recurrence, ie, clinically evident metastases, in patients after disease-free intervals of variable length. Recently developed animal models have shown that dormancy may potentially be explained by the fact that these micrometastases are not vascularized and have comparable rates of cellular proliferation and programmed cell death (apoptosis), enabling them to remain viable indefinitely but not to show progressive growth. OBSERVATION: We report for the first time that melanoma micrometastases from humans are similarly not vascularized (mean number of microvessels, 10.2), have significantly lower rates of tumor cell proliferation (mean, 2.4%), comparable rates of proliferation and apoptosis (means, 2.4.% and 0.2%, respectively), compared with melanoma macrometastases, which have significantly greater tumor vascularity (mean number of microvessels, 18.7), higher rates of proliferation (mean, 18%), and higher rates of proliferation relative to apoptosis (means, 18% vs 1.6%). Tumor vascularity was quantified using the lectin Ulex europaeus agglutinin I to identify the number of microvessels per unit area (microscope ocular grid with an area of 7.84 x 10(-2) mm2 at x400 magnification). Melanoma cell proliferation rate was assessed with the MIB-1 antibody (Ki-67) as the number of positive nuclei per total number of tumor nuclei counted at x400 magnification. Apoptosis was quantified using the method of terminal deoxynucleotidyl transferase-medicated deoxyuridine triphosphate-biotin nick end labeling. The number of positive nuclei were quantified per total number of tumor nuclei; usually 200 tumor nuclei were counted at x400 magnification. CONCLUSION: We report, for the first time, that human micrometastases demonstrate attributes, ie, the lack of significant tumor vascularity and low but comparable rates of proliferation and apoptosis, that may explain the dormant state.