Partial regression in thin primary cutaneous malignant melanomas clinical stage I

Summary 486 patients with primary cutaneous malignant melanoma clinical stage I were examined in order to evaluate the prognostic importance of partial regression in thin lesions. All the melanomas measured 1 mm or less in maximal thickness. The study showed that past regression with fibrotic scar tissue adversely affected survival in patients with thin melanomas. The 10 year survival was 95% for patients without regression in contrast to 79% for patients with past regression. It was, furthermore, demonstrated that active regression without fibrotic scar tissue did not influence survival significantly.The wider and the thicker the fibrotic area, the poorer the survival. Although the prognostic importance of this finding was not statistically significant, we suggest that the horizontal width of the fibrotic area in particular may be a valuable prognostic guide in thin melanomas with past regression.     

Biological behaviour of cutaneous malignant melanomas

The classification proposed by Clark et al. of cutaneous malignant melanoma into 3 groups: lentigo maligna melanoma (LMM), superficial spreading melanoma (SSM) and nodular melanoma (NM) was based on a correlation between histology and biological behaviour. Many studies have shown different growth rates, prognosis and incidence of antecedent benign nevi for each of these groups, confirming that each is a separate entity with its own biological behaviour. The recent proposal that acral lentiginous melanoma (ALM) is a distinct clinico-histological entity, however, is more difficult to accept. Many melanomas on acral locations are histologically intermediate between SSM and ALM and the epidemiological value of classifying ALM as an entity separate from SSM is doubtful.     

In situ identification of immune competent cells in gastrointestinal mucosa: An evaluation by immunoelectronmicroscopy

Summary Thein situ identification of lymphocyte subpopulations by means of immunopathological techniques using specific monoclonal antibodies provides a tool for the study of the gastrointestinal-associated lymphoid tissue (GALT) in health and disease. In this field, monoclonal antibodies have been applied previously using light microscopy and either immunofluorescence or immunoperoxidase; however, these techniques are not sensitive enough to allow precise evaluation of localization of labelling. We describe an immunoelectronmicroscopic method, which defines labelling specificity, since it allows the identification of cells by immunophenotype labelling and ultrastructural markers simultaneously. This in turn allows a better evaluation of the labelled cells and of the relationship between labelled and unlabelled cells.The main features of the method are the use of fresh tissue samples, fixing in paraformaldehyde CaCl₂, and the coupling of the immune reaction to an amplification system (avidin-biotin-peroxidase complex). The technique yields a good preservation of cellular ultrastructure, together with a strong and specific immunolabelling.Our results confirm the high specificity of monoclonal antibodies when applied to immunopathology techniques. We confirm the pattern of distribution of various lymphocyte subsets in the jejunal mucosa described by other authors by light microscopy.     

Flow cytometric determination of peripheral blood lymphocyte subpopulations and haematological values in the neonatal calf

Non-vaccinated Holstein female calves, 3–31 days old (n=106) were examined in order to determine peripheral blood lymphocyte subpopulations and haematological values. The relative populations of lymphocytes were determined using flow cytometry. Monoclonal antibodies were used to identify BoCD2⁺, BoCD4⁺, BoCD8⁺, B-cells,/ T-cells, and monocytes/neutrophils, respectively. Complete blood counts were also determined. Calves were stratified into four groups by age (days): group A = 3–7 days, group B = 8–14 days, group C = 15–21 days and group D = 22–31 days. Group A calves had 30%–70% lower numbers of all lymphocyte subtypes compared to older calves. Group B calves had significantly lower numbers of B-cells than calves in group D, but were not different from group C. Calves in groups B and C had significantly lower/ T-cell counts than calves in group D. All other group comparisons of lymphocyte subtypes and the ratios of BoCD4⁺ to BoCD8⁺ T-cells were not significantly different.Calves in group A had significantly lower white blood cell counts than older calves and had significantly lower lymphocyte numbers than group C and D calves. Calves in group D had significantly higher lymphocyte counts than younger calves. Groups A and B had significantly higher neutrophil to lymphocyte ratios than groups C and D. Absolute numbers of monocytes, eosinophils and basophils were not different among any of the groups. Group A calves had significantly lower red blood cell counts than older calves. Calves in groups A and B had significantly higher mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) values than calves in group C. Group C calves also had significantly higher MCH and MCV values than calves in group D. Significant differences in haemoglobin, haematocrit and mean corpuscular haemoglobin concentration values were not apparent among any of the four groups. In this cross-sectional study we observed that older calves consistently had higher numbers of all subtypes of peripheral blood lymphocytes, compared to younger calves.     

中性内肽酶及移动相关蛋白-1在恶性黑色素瘤中的表达及意义

目的探讨中性内肽酶（CD10）及移动相关蛋白-1（CD9）在恶性黑色素瘤中的表达及其临床意义。方法采用免疫组织化学SP法检测48例原发性皮肤恶性黑色素瘤（CMM）及23例转移性恶性黑色素瘤（转移性MM）中CD10、CD9蛋白表达水平,并以23例色素痣作为对照。结果（1）CD10在转移性MM、CMM及色素痣中的表达依次减弱,差异均有统计学意义（P〈0．01）,CD9在转移性MM中的表达较CMM显著降低（P〈0．05）;（2）CD9和CD10在恶性黑色素瘤中的表达水平呈负相关（CMM：r=-0.40,P=0.005;转移性MM：r=-0．44,P=0．034）;（3）CD10和CD9在CMM中的表达与病理学类型、淋巴结转移和浸润深度相关;（4）CD10、CD9在CMM间质成纤维细胞中的表达呈负相关（r=-0．43,P=0．007）,且与肿瘤浸润深度明显相关,与淋巴结转移有一定相关性。结论（1）CD10及CD9与恶性黑色素瘤的浸润、转移密切相关,两者在此过程中可能起相互拮抗作用;（2）肿瘤细胞CD10、CD9的表达对CMM的辅助诊断及预后评估具有重要的临床意义;（3）肿瘤间质成纤维细胞中CD10、CD9的表达在CMM的发展过程中亦可能扮演着重要角色。     

Poly(adenosine diphosphate-ribose) polymerase 1 expression in malignant melanomas from photoexposed areas of the head and neck region

The family of the poly(adenosine diphosphate-ribose) polymerase (PARP) proteins is directly involved in genomic stability, DNA repair, and apoptosis by DNA damage. In this study, we evaluated the role of PARP-1 in melanoma and its prognostic importance. We studied by immunohistochemistry and Western blot analysis PARP-1 expression in a selected series of 80 primary melanoma of the head and neck region. The results were correlated with tumor thickness and patient's outcome. A follow-up of at least 3 years was available. Fifteen cases of benign melanocytic nevi were used as controls. Normal melanocytes showed only scattered, focal nuclear positivity and were considered as negative for PARP-1 expression by immunohistochemistry (score, 0). Thirty cases of melanoma (37.5%) showed nuclear expression of PARP-1 in both radial and vertical growth phases. Western blot analysis showed the presence of a high signal for full-length PARP-1 only in the cases with high immunohistochemical (nuclear) expression of protein (score, ++/+++) in both radial and vertical growth phase. A significant correlation was present between PARP-1 expression in vertical growth phase and the thickness of tumor lesion (P = .014); all but one tumor measuring less than 0.75 mm showed no or low PARP-1 expression. No correlation was found between PARP-1 expression in radial growth phase and tumor thickness (P = .38, data not shown). These data suggest that PARP-1 overexpression is a potential novel molecular marker of aggressive cutaneous malignant melanoma and a direct correlation between PARP-1-mediated inhibition of the apoptosis and biologic behavior of cutaneous malignant melanoma.     

Immuno-architecture of human fetal lymphoid tissues

Summary Spleen, thymus and lymph node of human fetuses from the 12th to the 38rd week (spleen from 9 weeks) were investigated in an immunohistological study on B5-fixed paraffin embedded tissues, employing a panel of recently developed monoclonal antibodies, reactive with antigens resistant against fixation and paraffin embedment. The monoclonal antibodies included were MT1, MT2, MB1, MB2, MB3, LN1, LN2, LN3, LeuM1, Leu7, VIE-G4, together with polyclonal antibodies reactive with immunoglobulin heavy and light chains, and with lysozyme and S100-protein. The preservation of morphological detail together with immunoperoxidase staining of cellular subsets, allowed an accurate determination of the ontogenic development of the different cell types in situ, in relation to their micro-environment. The use of paraffin tissue reactive (monoclonal) antibodies gives an extra dimension to the study of fetal lymphoid tissues. This is of particular advantage in studies on very fragile tissues as in early embryonal and fetal ontogeny.     

Osteonectin expression correlates with clinical outcome in thin cutaneous malignant melanomas

Osteonectin, also termed BM40 or SPARC (secreted protein, acidic and rich in cysteine) is a multifunctional glycoprotein involved in tissue mineralization, cell-extracellular matrix interactions as well as angiogenesis. It has been suggested that osteonectin may play a key role in the process of tumoral invasion and metastasis in certain malignancies. In this study, we reviewed the clinical records and the histopathologic slides of 188 thin cutaneous malignant melanomas (< or = 0.75 mm). Among them, 12 cases underwent progression and were selected for the study. Osteonectin expression was investigated by immunohistochemistry in these 12 patients and 24 matched controls who did not undergo progression. Osteonectin staining was correlated with clinical outcome and other clinicopathologic parameters. Progression-free and disease-specific survival rates were calculated with the Kaplan-Meier method and their differences were evaluated by the log rank test. Overall, immunoreactivity for osteonectin was found in 23 (63.8%) cases. Eighteen cases (50%) displayed staining in 1% to 50% of neoplastic cells whereas five cases (13.8%) showed a diffuse positivity in more than 50% of the tumor cells. Osteonectin expression was significantly correlated with risk of progression (P = .01), incidence of distant metastases (P = .005) and survival (P = .03). There was a higher incidence of osteonectin-positive tumors in cases that did experience regional lymph node metastases versus those cases that did not, but that difference did not reach statistical significance (P = .06). No significant correlation was found between osteonectin expression and other clinicopathologic features, including age, sex, site, histotype, Clark's level, presence of regression, presence of inflammatory response, and tumor growth phase. Our data showed that osteonectin expression is a predictor of clinical outcome in thin cutaneous melanomas.     

Mitotic indices, anti-PCNA immunostaining, and AgNORs in thick cutaneous melanomas displaying paradoxical behaviour.

Those melanomas which fail to behave as expected from their Breslow thickness provide interesting material for study. In an attempt to explain differences in behaviour, we have evaluated three distinct proliferative markers in 23 thick melanomas which failed to metastasize and in 20 well-matched control tumours with documented metastasis. The test group demonstrated significantly greater numbers of mitoses when expressed as an index (mitoses per 1000 cells), whilst no difference was found when the results were expressed in terms of mitoses per unit area. Tumours showing epidermal ulceration possessed higher mitotic indices than those of non-ulcerated lesions. High fractions of PCNA immunolabelling combined with low mitotic indices were observed frequently in the non-metastasizing group. This result and its possible relation to survival advantage are discussed in detail. Both AgNOR numbers and patterns failed to act as prognostic variables--indeed, AgNORs failed to correlate with the other proliferative indices, suggesting that their value as a marker of tumour growth is severely limited.     

Survival with primary cutaneous malignant melanoma,evaluated from 2012 cases

Summary Cox's multivariate regression model for survival data was applied to 2,012 patients with primary cutaneous melanoma in order to evaluate the relative prognostic value of numerous clinical and histological variables and to establish their prognostically most efficient combination. The material was divided into 4 groups according to the size of resection margin of the primary lesion (<2.0 cm, 2.0 cm, 2.1–4.9 cm, and 5.0 cm). Data were analysed separately in these 4 groups and equivalent results were obtained.The risk factors were clinical stage, site of tumour, tumour thickness, level of invasion, mitotic activity, ulceration, lymphocytic reaction, predominant type of invasive tumour cell and partial regression. When accounting for these factors, histological type, nuclear pleomorphism, nucleolar size, vascular invasion, pigmentation, verrucous growth pattern, and dermal elastosis were without prognostic influence. The effect of sex and age of patient was uncertain and both variables, therefore, were retained in the model. By using Cox's method it is possible to make a qualified estimate of the survival for the individual patient.     

Immunocytochemical and immunohistochemical methods as auxiliary techniques for histopathological diagnosis of cutaneous leishmaniasis

A significant increase in cutaneous leishmaniasis (CL) and its geographic expansion has motivated the development of techniques to help with diagnosis of the disease. Here we describe immunocytochemical (ICC) and immunohistochemical (IHC) techniques for the diagnosis of CL in the laboratory. Polyclonal antibodies and a modified avidin-biotin complex (Ultra Streptavidin^®) for Leishmania (V.) braziliensis or Leishmania (L.) amazonensis were developed for the present study. In vitro culture and histological sections from experimentally infected tissues were submitted to ICC/IHC techniques. The polyclonal antibody specificity, stability and immunostaining were evaluated. The polyclonal antibodies purified by chromatography (Sephadex^®) and obtained from L. (V.) braziliensis and L. (L.) amazonensis insoluble antigens presented 83.3% sensitivity, when the presence of antigens was evaluated, i.e., higher than histopathology or any equivalent method (in vitro culture). The polyclonal antibody presented 100% specificity when used against species frequently found in CL lesions. The ICC/IHC techniques developed in the current study were able to recognize amastigotes and antigens from in vivo and in vitro cultures and from biopsies, offering additional help in the diagnosis of CL. This methodology could be beneficially adopted in public health laboratories.     

Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis

Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.     

Immunohistological characterization of proximal colonic lymphoid tissue in the rat

Proximal colonic lymphoid tissue (PCLT) is a lymphoid structure located in the proximal colon of the mouse and the rat. In the present investigation we studied the immunomorphology and cytology of PCLT in the rat. We also studied sites of lymphocyte proliferation using the BrdU-anti BrdU technique. Results demonstrated no evident phenotypical differences between the lymphocyte populations of PCLT and either jejunal or ileal Peyer's patches (PP). The majority of the lymphocytes within PCLT were B cells localized in follicles, which were separated from each other by interfollicular T cell areas. Germinal centers (GC), containing ED5⁺ follicular dendritic cells, are found within PCLT follicles. The T cell areas contained both MHC Class II⁺ interdigitating cells and high endothelial venules. Studies using BrdU-anti BrdU indicated that lymphocyte proliferation within PCLT taken place mainly in germinal centers. Together the data show that the organization, lymphoid constituents, and sites of lymphocyte production are very similar in PCLT and PP. We therefore conclude that PCLT in the rats is not a Bursa equivalent, but more likely a PP with some special characteristics. © 1992 Wiley-Liss, Inc.     

Therapeutic and clinico-pathological factors in the survival of 1,469 patients with primary cutaneous malignant melanoma in clinical stage I

Summary Therapeutic and clinico-pathological data of 1,469 patients with clinical stage I malignant melanoma of the skin without histological evidence of fibrotic areas of regression were examined by multivariate regression analysis. In accordance with a previous analysis anatomical site of tumour, tumour thickness, level of invasion, mitotic rate, ulceration, lymphhocytic reaction, dominant type of invasive tumour cell, and sex were found to act as independent risk factors. The present analysis, furthermore, showed that size of resection margin, diagnostic biopsy, removal of the deep fascia, age at surgery, as well as presence and depth of nevus cells did not influence prognosis when adjusting for the independent risk factors.     

Comparison of cutaneous sarcoidosis with systemic sarcoidosis: a retrospective analysis

Objective: To investigate clinical characteristics and treatment effect of sarcoidosis with cutaneous lesions in Chinese patients, and to compare them with previous works. Methods: Retrospective analysis was conducted based on clinical manifestations, systemic examinations and treatment of biopsy-proved 36 patients with sarcoidosis with cutaneous lesions in our hospital since 2000. Patients were divided into cutaneous sarcoidosis (CS) group without systemic involvement and systemic sarcoidosis (SS) group with systemic involvement according to whether extracutaneous systems were involved. Results: Male to female ratio was 1:4.1 in total 36 patients. Average age of onset was (43.6±15.8) years old in CS group and (54.4±11.5) years old in SS group. The most common cutaneous lesions were papulonodules (41.7%) and frequently found in limbs (61.1%). There were 26 patients in SS group, and lung was the most common organ with systemic involvement, followed by lymph nodes. In SS group, elevation of inflammatory parameters and evident changes of chest radiologic examination were often observed. 72.2% patients were treated with glucocorticoid and the overall therapeutic efficacy rate was 48.4%. The therapeutic efficacy in CS group (80%) was significantly higher than SS group (33.3%). Papulonodules type had better response to therapy and usually resolved after treatment. Lupus pernio type was resistant to treatment. Conclusion: Sarcoidosis occurs more frequently in females. Lung is the most commonly affected extracutaneous organ in SS patients. CS patients have better response to therapy than SS patients. Types of cutaneous lesions and existence of systemic involvement are related to prognosis of cutaneous lesions.