Confocal examination of untreated fresh specimens from basal cell carcinoma: implications for microscopically guided surgery

OBJECTIVE: To evaluate the diagnostic accuracy of confocal examination of basal cell carcinoma (BCC) in microscopy-guided surgery. DESIGN: Four independent observers with no previous experience in confocal laser scanning (CLS) microscopy received standardized instruction about diagnostic CLS microscopic features. Subsequently, 120 confocal images of fresh excisions from BCCs or normal skin were evaluated by each observer, imaged using a commercially available, near-infrared, reflectance CLS microscope. Logistic regression analysis was performed on a combination of all morphologic features using the forward-stepwise (Wald) method. Reliability (interobserver agreement) data were evaluated by kappa statistic. SETTING: Department of Dermatology, Medical University of Graz. PATIENTS: Twenty patients with histologically verified BCC. INTERVENTIONS: Evaluation of fresh BCC excisions by CLS microscopy. MAIN OUTCOME MEASURES: Diagnostic accuracy of the method was evaluated by chi2 test. Diagnostic impact and reliability of each morphologic feature were evaluated by logistic regression analysis and kappa statistic, respectively. RESULTS: Overall, high diagnostic accuracy was achieved by the 4 observers. Logistic regression analysis revealed that mainly tumor cell nuclei and tumor nests should be taken into account for diagnostic decisions, whereas disintegration of tumor cells, peripheral palisading, and retraction of stroma were rarely useful. However, most of the features were highly reliable. CONCLUSIONS: This diagnostic validation study of CLS microscopy in microscopy-guided surgery yielded promising results and opens avenues for further studies. In the future, CLS microscopy may guide microsurgery of any skin cancer.     

Contact sensitization to disperse dyes in children

From January 1996 to December 2000, 1098 children, including 667 subjects with suspected allergic contact dermatitis and 431 patients with atopic dermatitis (AD), were patch tested with seven disperse dyes: disperse blue 124 (DB124), disperse blue 106 (DB106), disperse red 1 (DR1), disperse yellow 3 (DY3), disperse orange 3 (DO3), p-aminoazobenzene (PAAB), and p-dimethylaminoazobenzene (PDAAB). Of these, 51 patients (4.6%; 34 girls and 17 boys) proved sensitized to disperse dyes. AD or history of AD was present in 30 patients (59%). The most common sensitizer was DY3 (17 patients), followed by DO3 (15 patients), and DB124 (14 patients). Among dye-positive patients, about 12% were sensitized to disperse dyes alone and only 14% reacted to para-phenylenediamine. In disperse dye-sensitive children not affected by AD, the feet, axillae, and groin appeared to be the most common localizations, whereas in those with AD, involvement of the face and the flexural areas of the limbs was more common. In conclusion, our study showed that in children with suspected contact sensitization, disperse dyes should be regarded as potential triggering allergens.     

Plasmacytoid dendritic cells are absent in skin lesions of polymorphic light eruption

BACKGROUND/PURPOSE: Polymorphic light eruption (PLE) is a common photodermatosis of potential autoimmune origin, and an overlap with lupus erythematosus (LE) has been described. Plasmacytoid dendritic cell (PDC)-induced expression of interferon (IFN)-alpha has been found to be present in LE skin lesions and plays a pivotal role in the pathogenesis of LE by promoting autoimmunity. We therefore asked whether PDCs may also be involved in the pathogenesis of PLE and searched for those cells [which can be identified by their high levels of interleukin (IL)-3 receptor alpha chain (CD123), combined with other cell markers such as CD68] in skin lesions. METHODS: Paraffin-embedded biopsy specimens from a total of 27 patients with clinically and histologically confirmed PLE (nine women, mean age 32.7 years, age range 18-43), LE (seven women, four men, CCLE: n=4, SCLE: n=2, lupus tumidus: n=5, mean age 48.5 years, age range 41-65) or psoriasis (four women, three men, mean age 43.3 years, age range 19-54) (as control group) were analyzed by immunohistochemical CD68/CD123 double staining. Quantification of the immunohistochemical staining was performed by visual cell counting of CD68-/CD123+, CD68+/123-, and CD68+/CD123+ cells separately in the epidermis and dermis of the samples in at least 10 random fields per sample at x 400 microscopic magnification by two of the investigators in a blinded fashion. RESULTS: Microscopic examination of the immunohistochemically stained sections revealed that CD68+/CD123+ cells were present in most specimens obtained from LE [10/11 (91%)] and psoriasis [6/7 (86%)] patients but not at all in those obtained from PLE patients. Quantification and statistical analysis of the dermal infiltrate revealed that CD68+/CD123+ cells were present at a mean+/-SEM field density of 5.6+/-1.3 in LE, 1.6+/-0.6 in psoriasis but totally absent in PLE (P=0.0010 vs. LE, P=0.0135 vs. psoriasis by an unpaired Student's t-test). CONCLUSION: The results confirm the potential significance of PDCs in LE and psoriasis, however the absence of PDCs in PLE contradicts the hypothesis that these cells might play a role in the latter disease.     

Reflectance confocal microscopy of facial lentigo maligna and lentigo maligna melanoma: a preliminary study

Background Facial lentigo maligna (LM) and lentigo maligna melanoma (LMM) may be difficult to diagnose clinically and dermoscopically. Reflectance confocal microscopy (RCM) enables the in vivo assessment of equivocal skin lesions at a cellular level. Objectives To assess cytomorphological and architectural RCM features of facial LM/LMM. Methods Four women and eight men aged 58–88 years presenting with facial skin lesions suspicious of LM/LMM were included. In total, 17 lesion areas were imaged by RCM before biopsy. The histopathological diagnosis of LM was made in 15 areas; the other two were diagnosed as early LMM. Results A focal increase of atypical melanocytes and nests surrounding adnexal openings, sheets of mainly dendritic melanocytes, cord‐like rete ridges at the dermoepidermal junction (DEJ) and an infiltration of adnexal structures by atypical melanocytes were found to be characteristic RCM features of facial LM/LMM. Areas with a focal increase of atypical melanocytes and nests surrounding adnexal openings were observed at the basal layer in three cases. The remaining cases displayed these changes at suprabasal layers above sheets of mainly dendritic melanocytes. Cord‐like rete ridges at the DEJ and an infiltration of adnexal structures by atypical melanocytes were observed in all cases. Previously described criteria for RCM diagnosis of melanoma, such as epidermal disarray, pleomorphism of melanocytes and pagetoid spreading of atypical melanocytes, were additionally observed. Conclusions We observed a reproducible set of RCM criteria in this case series of facial LM/LMM.     

Rimming of adipocytes by neoplastic lymphocytes: a histopathologic feature not restricted to subcutaneous T-cell lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic T-cell lymphoma of the skin presenting with histopathologic features simulating those of a lobular panniculitis. The presence of neoplastic T-lymphocytes forming a rim around the individual fat cells in the subcutaneous lobules, so-called "rimming" of adipocytes, is considered a characteristic morphologic feature of this type of cutaneous lymphoma. In this study we reviewed a series of 45 biopsy specimens of primary and secondary cutaneous B- and T-cell lymphomas and one of myeloid leukemia involving the subcutaneous tissues and showing rimming of adipocytes (subcutaneous panniculitis-like T-cell lymphoma: n = 16; mycosis fungoides, tumor stage: n = 3; aggressive epidermotropic CD8(+) T-cell lymphoma: n = 2; cutaneous gamma/delta T-cell lymphoma: n = 4; extranodal NK/T-cell lymphoma, nasal type: n = 4; cutaneous medium-large pleomorphic T-cell lymphoma, NOS: n = 5; CD4(+)/CD56(+) hematodermic neoplasm (blastic NK-cell lymphoma): n = 7; secondary cutaneous large B-cell lymphoma: n = 3; secondary cutaneous lymphoplasmacytic lymphoma: n = 1; specific cutaneous manifestations of acute myelogenous leukemia: n = 1). We could demonstrate that rimming of adipocytes by neoplastic cells can be recognized not only in subcutaneous panniculitis-like T-cell lymphoma, but also in several different entities of malignant lymphoma with skin involvement. Precise classification of cases with prominent involvement of the subcutaneous tissues can only be achieved upon precise correlation of clinicopathologic and phenotypic features. Rimming of adipocytes should not be considered specific of subcutaneous panniculitis-like T-cell lymphoma.     

Cutaneous B-cell pseudolymphoma at the site of vaccination

Pseudolymphomas are a rare complication of vaccination, presenting with dense lymphoid infiltrates and prominent follicular pattern. We report our observations on 4 patients with vaccination-induced B-cell pseudolymphoma (all females; age range 19 to 60 years; median: 34.5 years). Clinically 3 patients presented with subcutaneous nodules and 1 presented with a large, indurated, erythematous plaque. Histology revealed in all cases dense lymphoid infiltrates in the subcutaneous fat with prominent follicular pattern. The follicles displayed features of reactive germinal centers (normal mantle zone, presence of tingible body macrophages, normal proliferation). Necrotic areas surrounded by palisaded histiocytes were seen in 3 biopsies from 2 patients. A mixed-cell infiltrate with eosinophils and plasma cells was present in all cases. In addition, histiocytes with granular basophilic cytoplasm could be observed around the focal area of necrosis or within the inflammatory infiltrate. Follow-up was available for 3 patients. One patient was alive with persistent disease 6 months after the first observation. Two patients were treated with local radiotherapy and are alive and free of disease after 12 and 72 months, respectively. One of these two patients had a second pseudolymphoma on the contralateral arm after a new injection of vaccine. Cutaneous pseudolymphoma after vaccination should be distinguished histopathologically from low-grade cutaneous B-cell lymphomas (follicle center cell lymphoma, marginal zone lymphoma) and from other B-cell pseudolymphomas with prominent follicular pattern requiring different treatment (eg, Borrelia burgdorferi-induced lymphocytoma cutis).     

Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1- dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post- labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.