Prognostic value of RT-PCR tyrosinase detection in peripheral blood of melanoma patients

Malignant melanoma (MM) prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I-IV) were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p<0.05) and clinical stage III (p<0.05). Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p<0.02). These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence.     

Stem cell factor affects tumour progression markers in metastatic melanoma cells

Stem cell factor (SCF), next to various relevant biological effects exerted on many cell types, is able to keep melanocyte homeostasis through its receptor c-kit. Only a minority of metastatic melanoma cells (MMC) express c-kit receptor, but c-kit positive MMC move more slowly towards tumour progression and have a more natural tendency to undergo apoptosis. In our study c-kit positive MMC from human melanoma metastases and a c-kit positive human melanoma cell line—SK-MEL-28—showed a clear-cut reduction of cytokines normally up-regulated along melanoma progression after SCF stimulation. SCF was also able to maintain all MMC and SK-MEL-28 cells in a well differentiated status with an increase in organellogenesis and in particular of melanosomes in various degree of differentiation, but it did not induce apoptosis as observed in other in vitro models. The increase of melanosomes matched an increase of tyrosinase production. SCF did not modify the expression of NOS while it enhanced the expression of HLA-DR molecules on MMC membranes. Taken altogether these data stress the biological activity of SCF as a cytokine which is able to maintain MMC in a well differentiated status, and suggest a more in depth evaluation of possible effects of SCF on melanoma cells.     

复方中药含药血清对人A375黑素瘤细胞酪氨酸酶活性的影响

目的探讨复方中药含药血清对人A375黑素瘤细胞酪氨酸酶（TYR）活性的影响。方法体外培养人A375黑素瘤细胞,采用多巴速率氧化法研究复方中药含药血清对TYR活性的影响。结果低浓度（5%～10%）的Ⅰ方、Ⅱ方、Ⅲ方对酪氨酸酶活性无明显激活作用（P〉0.05）;高浓度（20%～30%）的Ⅰ方、Ⅲ方、Ⅵ方对TYR活性均有明显激活作用（P〈0.05）,以Ⅵ方作用最显著,且与其它各组相比,差异均有统计学意义（P〈0.01）。结论对TYR具有激活作用的中药组成复方并不一定对TYR有激活作用,具有祛风活血和补肾三重作用的Ⅵ方在促进TYR活性上优于祛风除湿Ⅰ方、养血活血Ⅱ方和滋补肝肾Ⅲ方。     

Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo

ABSTRACT: BACKGROUND: The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people. METHODS: The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens. RESULTS: ELISA test showed significantly (p<0.0000004 and p<0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls. Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alpha I (CD89) positive cells was determined in melanoma patients (p<0.002 and p<0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression. Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p<0.0007 and p<0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16+CD56+) natural killer (NK) cells (p<0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p<0.0007 and p<0.05 respectively). CONCLUSION: Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response. Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.     

Surface markers of NK cells in peripheral blood of patients with cirrhosis and hepatocellular carcinoma

The surface markers and NK activity of blood lymphocytes in 38 normal controls, 57 cirrhotic patients and 22 cirrhotics with hepatocellular carcinoma were studied by the flow cytofluorometry using monoclonal antibodies. A reduction of OKM1+ cells was remarkable in cirrhotics, and a further decrease in HNK-1+ cells as well as OKM1+ cells was observed in cirrhotics with hepatocellular carcinoma, accompanied by depression of NK activity. We postulate that the decreased NK activity in these patients may result from the disturbed maturation of NK cells.     

An N-glycosylated tyrosinase epitope associates with newly synthesized MHC class I molecules in melanoma cells

Endogenous antigenic epitopes are presented to CD8⁺ T cells by MHC class I molecules. Many endogenous antigens are glycoproteins, and it is not clear what effect the attachment of carbohydrate to potential immunogenic epitopes has on their processing and presentation (i.e., is the carbohydrate moiety removed prior to presentation, or is it presented along with the peptide to T cells?). A major question in this regard is whether natural antigenic epitopes that possess N-linked carbohydrate can associate with class I molecules during assembly in the endoplasmic reticulum (ER). One such antigenic epitope, corresponding to amino acids 369–377 of the enzyme tyrosinase, possesses an N-linked glycosylation site. We have studied the transport and loading of this epitope in streptolysin O-permeabilized melanoma cells. We show here that that the glycosylated epitope is capable of loading onto newly synthesized HLA-A2 molecules in the ER of two melanoma cell lines. The results are discussed in respect to the processing and presentation of the tyrosinase epitope.     

Whole genome methylation profiles as independent markers of survival in stage iiic melanoma patients

ABSTRACT: BACKGROUND: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM. METHODS: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses. RESULTS: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified. CONCLUSIONS: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.     

Novel tyramide-based tyrosinase assay for the detection of melanoma cells in cytological preparations

Fine-needle aspiration (FNA) has been used as a fast, minimally invasive, and reliable method for the evaluation of enlarged lymph nodes. However, there are some cases where the definitive diagnosis cannot be elicited with morphology alone, especially cases without a known primary lesion. Although immunocytochemical studies may be helpful in some situations, they are often complicated by nonspecific staining. Recently, a novel tyramide-based tyrosinase assay was developed. Since melanocytes, both benign and malignant, produce tyrosinase, we postulated that this assay could be useful as an in situ biochemical diagnostic test. We modified the Perkin Elmer TSA assay, a commercial assay based on tyramide, a tyrosine analog that is a substrate for tyrosinase, for use on air-dried cytological preparations. We validated the assay on cell lines, then tested a small series of melanoma and nonmelanoma cytology specimens. The YUGEN8 melanoma cell line was used to optimize the assay and it showed abundant reaction product, while HeLa cells served as a negative control. All melanoma cytology specimens were positive and all nonmelanoma specimens were negative. These results suggest that this simple, fast, and inexpensive assay is a sensitive and specific method for detection of melanoma cells in cytology specimens. This method may be a useful ancillary procedure for the resolution of challenging melanoma cases.     

DNA repair and replication proteins as prognostic markers in melanoma

Aims: Elevated expression of DNA repair and replication genes has been reported in thick, non‐fixed primary melanomas that subsequently went on to metastasize, when compared to non‐recurrent primary tumours. This increased expression could contribute to the extreme resistance shown by melanoma to DNA‐damaging chemotherapeutics. We have investigated the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in melanoma. Methods and results: We used a tissue microarray containing samples from all stages of melanomagenesis to investigate the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in a larger, more representative and readily available set of fixed primary melanomas. High expression of topoisomerase IIα (TOP2A), that relieves torsional stress during DNA replication, and XRCC5 (Ku80), required for DNA double‐strand break repair, were associated with significantly worse survival. Conclusions: Two (XRCC5 and TOP2A) of seven DNA repair and replication proteins studied were prognostic for melanoma.     

反义核酸抑制B16黑色素瘤细胞tyr基因的表达

目的：用反义核酸技术抑制小鼠B16黑色素瘤细胞酪氨酸酶基因（tyr）的表达。 方法： 构建tyr反义重组体pcDNA3.1(-)-tyr，用脂质体法导入B16细胞，用酪氨酸酶活性及黑色素含量测定，多巴染色及透射电镜等检测由反义重组体pcDNA3.1(-)-tyr转录产生的tyr反义核酸对B16细胞tyr表达的抑制情况。 结果： 成功构建了反义重组体pcDNA3.1(-)-tyr，转染了反义重组体的B16细胞其酪氨酸酶活性为0.0498±0.0036，显著低于对未转染组B16细胞的0.0916±0.0132(P<0.01);转染空质粒pcDNA3.1(-)及真核表达重组体pcDNA3.1(+)-tyr组B16细胞的氨酸酶活性分别为0.1015±0.0166和0.0948±0.0096,两者同对照组相比均无显著差异(P>0.05)。多巴染色及电镜观察结果表明转染了反义重组体的B16细胞其黑色素颗粒的含量明显低于对照组。 结论： 反义核酸可以显著抑制B16黑色素瘤细胞tyr的表达。     

Demonstration of carbohydrate structures in malignant melanoma tyrosinase

Summary Electrophoretic studies on malignant melanoma extracts before and after treatment with neuraminidase revealed that tyrosinase is a glycoprotein containing N-acetyl-neuraminic acid. Double diffusion tests usingConcanavalin A and the lectin fromRicinus communis show that the carbohydrate chain of tyrosinase contains D-mannose as a sugar unit located within the carbohydrate chain. The terminal neuraminic acid groups are linked to D-galactose. The enzymatic activity of tyrosinase is not inhibited byConcanavalin A.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Cysteine and indole derivatives as markers for malignant melanoma

Malignant melanoma is a skin tumour, which carries a very unfavourable prognosis. The early detection of a melanoma and even more its metastasis is of decisive importance for the survival prognosis of the patients. So there is always a desire for simple, economical and meaningful serological markers. From the cysteine- and indole-related derivatives, 5-S-cysteinyldopa (5-SCD) and 6-hydroxy-5-methoxy-indole-2-carboxylic acid (6H5MI2C) are the most important substances for this purpose. For 5-SCD, the sample pretreatment was carried out either by a manual extraction onto alumina, by an automated method onto boronic acid affinity gels or by an automated solid-phase extraction. For 6H5MI2C, liquid-liquid extractions or direct injection techniques were applied. The chromatographic analyses in the early years were mostly performed with GC-MS. Today HPLC is the nearly exclusively used separation technique. For HPLC, standard RP18 separating columns and usual compositions of eluents were applied. As detectors both the ECD and the FD showed a sufficient sensitivity and selectivity. 5-SCD and 6H5MI2C are very sensitive to light and oxidation. These properties must be taken into account in the complete analysis procedure, including the sample collection, otherwise false low values will result especially for plasma samples. For a critical discussion of the analytical methods and still more for the interpretation of the obtained results, the detailed analytical procedures must be considered. 5-SCD in plasma is one of the best markers of malignant melanoma. It shows an excellent specificity and also an adequate sensitivity in the metastatic melanoma stages. For the detection of primary melanomas and for urine instead of plasma samples, the sensitivity of 5-SCD is generally lower. Altogether, the sensitivity of this parameter is not yet sufficient. 6H5MI2C and other indole derivatives have been investigated far less than 5-SCD. 6H5MI2C correlates less clearly with the different stages of the melanoma and is therefore a less suitable marker. To improve the sensitivity of the findings, in future the investigations should be performed as multi-marker analysis with the simultaneous measurements of more than one marker substance in a given patient sample. Not only one measurement should be carried out per patient, it would be more meaningful to observe the patients with laboratory diagnostics in the follow-up.     

Quantitative real-time RT-PCR as a method for monitoring T lymphocyte reactivity to full-length tyrosinase protein in vaccinated melanoma patients

The major goal of therapeutic cancer vaccine trials is to mediate tumor regression. However, it is critically important to devise in vitro immunological assays that correlate with clinical outcome, for use as surrogate markers of vaccine efficacy. To date, clinical emphasis has been placed on peptide vaccines, but trends towards the use of more complex immunogens such as whole proteins require the development of efficient and sensitive methods for monitoring their immunological effects. In the context of a vaccination trial using full-length tyrosinase (Ty) to immunize patients with metastatic melanoma, a monitoring technique was developed in which autologous dendritic cells (DC) infected with a recombinant adenovirus encoding the Ty protein were used to assess the Ty-specific reactivity of fresh peripheral blood lymphocytes (PBL) collected from patients at different intervals during therapy. Quantitative real-time RT-PCR (qRT-PCR) was used to measure the production of cytokine mRNA by T cells following a 2.5-h incubation with Ty-expressing DC. Two out of ten patients studied demonstrated Ty protein-specific reactivity that increased during and after the period of vaccination. While one of these patients also reacted to an HLA-A1-compatible Ty peptide, the second did not recognize any of the known Ty epitopes, highlighting the importance of this technique for monitoring the effects of complex vaccines.     

2型糖尿病肾病患者外周血单个核细胞组织因子活性检测的意义

目的探讨2型糖尿病肾病患者外周血单个核细胞组织因子促凝活性（TF-PCA）的改变及其临床意义。方法 63名2型糖尿病患者按照尿白蛋白/肌酐比共分为三组：比值〈30mg/g为正常蛋白尿组（n=22）,30mg/g≤比值〈300mg/g为微量白蛋白尿组（n=26）,比值≥300mg/g为大量白蛋白尿组（n=15）;同时选择21名健康体检者作为对照组。采用一期凝固法测定外周血单个核细胞TF-PCA。常规检测空腹血糖、糖化血红蛋白、超敏CRP（Hs-CRP）、尿酸（UA）、胱抑素C（CYSC）,并进行相关分析。结果糖尿病组外周血TF-PCA随尿蛋白水平升高而升高,同时单个核细胞TF-PCA与空腹血糖、Hs-CRP、UA等呈正相关,相关系数分别为0.419、0.293、0.232（P〈0.05）。结论 2型糖尿病患者外周血单个核细胞TF-PCA明显升高,且与多种因素相关,TF-PCA升高可能与DN的发病机制、病程进展相关。     

自体细胞因子诱导的杀伤细胞降低恶黑患者外周血髓样抑制性细胞的数量研究

目的 探讨恶性黑色素瘤(MM)患者外周血中髓样抑制性细胞(MDSCs)的数量及自体细胞因子诱导的杀伤细胞(CIK)对MDSCs数量的影响.方法 采集20例MM患者CIK细胞治疗前后和18例健康志愿者外周血,用anti-CD33-mAb和anti-CD11b-mAb标记,应用流式细胞仪检测MDSCs比例.结果 MM患者外周血中MDSCs水平高于健康对照组,差异具有统计学意义(P＜0.05).MM患者经CIK治疗后,其MDSCs水平低于治疗前,差异有统计学意义(P＜0.05).结论 自体CIK可降低MM患者外周血MDSCs水平,这一结果揭示了CIK抗肿瘤作用的新机制.