Clinical and histological features of poor prognosis in cutaneous metastatic melanomas

Patients with melanoma metastatic to the skin show variable prognosis. Though some may survive for quite a long time, some die of disseminated disease within 1 year of removal of cutaneous metastases. The aim of this study was to find out whether there are any histological criteria indicating particular poor outcome. Clinical and histological features of 344 melanoma lesions metastatic to the skin were assessed and their prognostic relevance was investigated. H&E stained histological slides were scanned for the presence of morphological criteria expressing certain tumor cell - stroma interactions: capsule formation (CAPSULE), formation of intratumoral septa (NEWSEPTA), simple invasion between collagen of reticular dermis (DERM-SIMPLE), or subcutis (SCSIMPLE), preservation of preexistent collagen (PRECOLL) or fatty tissue (PREFAT) and, finally, histological site of metastasis. Additionally, anatomical location of the metastases, time between removal of primary tumor and metastases, age and sex of patients were recorded. The metastases were divided into two groups: lesions of patients who died within 1 year after resection (n=59) and lesions from patients with a longer survival (n=285). Metastases which were associated with death within one year were significantly more often found in male patients (54.2% versus 34.7%), in younger patients (mean age 51.1±14.1 years versus 58.8 ± 15.3 years), had developed earlier after the primary tumor (mean time of 21.7±19.9 months versus 43.3±27.4 months) and were more often found at distant sites than in localregional sites (45.7% versus 30.5%), and were more often involved in the subcutis (74.5% versus 56.1%). From a histological point of view, DERMSIMPLE (80% versus 46%; p<0.001) and PRECOLL (82.8% versus 57.6; p<0.01) were more frequent in metastases of poor outcome. The same was true for SCSIMPLE (50% versus 25.6%; p<0.01) and PREFAT (68.1% versus 46.8%; p<0.05) in lesion with subcutaneous growth, whereas CAPSULE (54.5% versus 75%) was less frequently seen. In conclusion, melanoma deposits metastatic to the skin with particular poor outcome differ clinically and histologically from other cutaneous melanoma metastases. This should be taken into account in the design of therapeutic clinical trials.     

载芬维A铵脂质体抑制A375黑素瘤细胞裸鼠皮下移植瘤的初步研究

目的 探讨载芬维A铵脂质体（4-HPR-L）对裸鼠皮下人恶性黑素瘤的抑制作用。方法 采用薄膜-超声分散法制备4-HPR-L。通过皮下接种黑素瘤A375细胞至BALB/c裸鼠右侧腋窝建立黑素瘤荷瘤裸鼠模型。取10只荷瘤裸鼠模型，随机等分为两组，分别给予尾静脉注射同浓度细胞膜近红外荧光探针（DiR）溶液和DiR脂质体（DiR-L），应用小动物活体成像仪观察给药后6、12、24 h药物在体内分布情况。取30只荷瘤裸鼠，随机等分为3组，即对照组、4-HPR组和4-HPR-L组，分别经尾静脉每次注射5%（质量分数）葡萄糖溶液0.2 ml、25 mg/kg 4-HPR和4-HPR-L溶液，于接种A375细胞后第8、10、12、14、16、18、20、22天给药，动态监测给药后各组裸鼠的体重和肿瘤体积，观察生存情况。于末次给药后第2天处死裸鼠，取心、肝、脾、肺、肾及肿瘤组织，HE染色和免疫组化染色观察黑素瘤体内转移情况，并用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测肿瘤细胞凋亡情况。计量资料采用单因素方差分析和独立样本t检验进行分析。结果 小动物活体成像仪显示，DiR-L能较长时间滞留于肿瘤组织，给药24 h后在肿瘤部位仍可观察到较强荧光；定量分析显示，肿瘤组织中DiR-L荧光强度（22.85 ± 1.66）显著高于DiR（8.45 ± 0.97，t = 12.957，P < 0.01）。与对照组和4-HPR组相比，4-HPR-L组末次给药后第2天离体瘤重明显降低（F = 27.055，t值分别为4.768、6.640，均P < 0.05）。HE染色显示，4-HPR-L组2只裸鼠发生肝脏转移，而对照组、4-HPR组全部裸鼠发生肝脏转移。荷瘤鼠的生存期观察显示，4-HPR-L组裸鼠于接种后76 d内全部死亡，对照组和4-HPR组分别于接种后56 d和59 d内全部死亡。对照组凋亡指数为（12.14 ± 1.33）‰，4-HPR组为（67.17 ± 15.18）‰，4-HPR-L组为（152.73 ± 11.27）‰，3组间差异有统计学意义（F = 167.588，P < 0.05），4-HPR-L组与对照组和4-HPR组相比，t值分别为18.162、11.075，均P < 0.05。结论 4-HPR-L能够有效抑制裸鼠皮下黑素瘤体积增长和黑素瘤细胞转移，并延长裸鼠生存期。     

Characterization and longitudinal monitoring of melanoma growth in ret‐transgenic mice using a single‐sequence MRI protocol

Spontaneous melanoma models in transgenic mice are increasingly used in preclinical research as they most closely match the progression of melanoma in humans. While optical inspection only allows analysis of tumors located on the skin, the accurate measurement and growth of subcutaneous tumors have not been adequately assessed. To improve the measurement accuracy of melanoma tumors, we used a fast single‐sequence MRI protocol at 9.4 Tesla for longitudinal characterization of a ret‐transgenic mouse model. Repeated MRI (average acquisition time 30 min per animal) of the trunk (excluding head and distal limbs) in six siblings revealed an increase in the mean total tumor volume (TTV) from 102.0 ± 80.5 mm³ at 35 days of age to 434.8 ± 154.9 mm³ by 77 days. The main tumor load was located within the pelvis (>40%), followed by the proximal hind limbs and groins (>30%). The smallest detectable tumor measured 0.07 mm³. Inter‐rater reliability between a radiologist and a veterinarian analysing MRI data was 0.993 for TTV and 0.840 for number of tumors (both p < 0.001). We thus conclude that because of the high variance of TTV of same‐aged mice, MRI should be used (i) to establish treatment groups matched for TTV and (ii) for longitudinal examination of the TTV in mice over the course of treatments.     

In situ detection of supernumerary aberrations of chromosome-specific repetitive DNA targets in interphase nuclei in human melanoma cell lines and tissue sections.

The use of non-radioactive in situ hybridization (ISH) with chromosome-specific repetitive DNA probes to study genomic changes, aneuploidy, and heterogeneity during melanocytic tumor progression, relies on its applicability to non-mitotic interphase nuclei, present in cell suspensions and tissue sections. Therefore, we studied the feasibility of detecting numerical aberrations with respect to the (peri-) centromere regions of chromosomes 1 and 7 in intact nuclei of two human melanoma cell lines with different metastatic behavior in nude mice. In addition, we used paraffin sections from xenograft lesions, obtained by inoculation of these cell lines in nude mice (subcutaneous tumors and spontaneous lung metastases). Paraffin sections from the original primary cutaneous melanoma (with a subepidermal and a dermal part) and two loco-regional metastases were also studied, one of which was the source for the cell lines. These cells and tissues represent examples of materials used in different approaches to the study of melanocytic tumor progression. Regarding the targeted sequences, ISH analysis showed that both cell lines were heterogeneous and aneuploid. The results correlated well with those obtained by ISH on metaphase spreads. Differences between the lines, which could not be detected by flow-cytometric or conventional karyotyping analysis, included data suggestive of a polyploid subpopulation and an extra copy of chromosome 7 in the metastasizing cell line. The polyploid population could be detected also in the paraffin sections of the corresponding subcutaneous xenografts and lung metastases in the mice. Both areas in the patients' primary melanoma could be evaluated separately and showed similar supernumerary aberrations of the chromosome-specific targets. These abnormalities matched those found in both metastases. Our results demonstrate that ISH can be used to visualize genomic abnormalities at the single-cell level in melanocytic nuclei in their natural context, which makes it a promising tool in the histopathology of melanocytic lesions and in the study of melanocytic tumor progression.     

Radioiodine-labelled alpha-methyl-tyrosine in malignant melanoma:cell culture studies and results in patients

Tyrosine is a precursor of melanin synthesis and might thus present a valuable marker for melanoma. The aim of this study was to evaluate the uptake of alpha-methyl-tyrosine(AMT) in melanoma cell cultures and to assess its usefulness as a radiopharmaceutical for staging melanoma patients with whole-body scintigraphy. Melanoma (M19-cell lines) and fibroblast (negative control) cell cultures were incubated with¹²⁵I-AMT and the radioactive uptake in the cell lines was measured in a gamma-counter over 24h. For in vivo studies, planar whole-body scintigraphy and single photon emission computed tomography (SPECT) of the tumour region was performed following injection of 250–350 MBq¹²³I-AMT in six patients with known melanoma metastases. Findings were compared with results of whole-body positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) as a standard of reference. Fibroblasts showed an unchanged uptake of (mean±SD)0.56±0.09% 15min and 0.066±0.09% 24h, respectively, after incubation of ¹²⁵I-AMT, whereas there was an increased uptake in melanoma cell cultures over time from 0.9±0.05% to 7.5±1.6%. In staging melanoma patients, the sensitivity of whole-body AMT-scintigraphy compared with FDG-PET was 37%(10 of 27 metastases). AMT is transported and metabolized to a high extent in melanoma cells and ¹²³I-AMT is accumulated in melanoma metastases. Owing to its low sensitivity, however, the clinical use of whole-body AMT scintigraphy cannot be recommended.     

可溶性PD-1及联合Hsp70-B16抗原肽复合物对小鼠黑素瘤肺转移的抑制作用

目的 探讨黑素瘤肺转移小鼠模型体内表达可溶性PD-1（sPD-1）对B7-H1/PD-1信号传导的阻断作用，及联合Hsp70-B16抗原肽对小鼠黑素瘤肺转移的抑制作用。方法 建立小鼠黑素瘤肺转移模型，应用免疫组织化学染色和流式细胞术检测肺转移灶PD-1及B7-H1的表达情况，从小鼠尾静脉注射sPD-1表达质粒，并联合应用Hsp70-B16抗原肽皮下免疫小鼠，于接种瘤细胞后第17天解剖小鼠，观察黑素瘤肺转移的情况，检测各组小鼠肺转移灶局部淋巴细胞浸润及部分免疫学指标。结果 小鼠黑素瘤肺转移模型成功建立，肺转移灶局部有大量的PD-1阳性细胞，转移瘤B16细胞表面表达较多B7-H1分子，静脉注射sPD-1表达质粒联合抗原肽免疫治疗组小鼠肺转移明显受抑制，抑制率达95％，显著高于对照组，其对应组小鼠肺部CD8+ T淋巴细胞的数量、脾淋巴细胞的细胞毒活性及血清中的IL-2和IFN-γ的浓度均明显高于对照组（P < 0.01）。结论 小鼠体内转染表达sPD-1可阻断B7-H1/PD-1信号传导，抑制小鼠黑素瘤肺转移，联合应用Hsp70-B16抗原肽具有明显的协同作用。     

基质金属蛋白酶7在恶性黑素瘤中的表达

目的 探讨基质金属蛋白酶7（MMP7）的表达与恶性黑素瘤浸润、转移的关系。方法 采用免疫组化SP法研究MMP7在恶性黑素瘤（30例）、交界痣（30例）、正常人皮肤（15例）组织中的表达，并观察其在恶性黑素瘤A375细胞株和不同浓度乙酰肝素酶反义寡核苷酸转染的裸鼠体内恶性黑素瘤移植瘤中的表达情况。结果 MMP7在恶性黑素瘤组、交界痣组及正常人皮肤组中的阳性表达率分别为83.33%（25/30）、6.67%（2/30）和0，恶性黑素瘤组与交界痣组MMP7的阳性表达率比较，χ2 = 35.62，P < 0.01；交界痣组与正常人皮肤组比较，差异无统计学意义。10、20、30 μmol/L乙酰肝素酶反义寡核苷酸对裸鼠体内恶性黑素瘤移植瘤MMP7表达表现出不同程度的抑制作用，30 μmol/L组的抑制作用最强，与其他2个组相比，差异均有统计学意义（P < 0.05）。MMP7在A375细胞株中呈强阳性表达。结论 MMP7在恶性黑素瘤中的表达明显高于其在交界痣和正常人皮肤；乙酰肝素酶反义寡核苷酸对裸鼠体内恶性黑素瘤移植瘤中MMP7的表达有显著抑制效应；MMP7在A375细胞株中呈强阳性表达。     

Dacarbazine, nimustine hydrochloride, cisplatin and tamoxifen combination chemotherapy for advanced malignant melanoma.

Melanoma is an uncommon disease in Japan. The incidence, however, has been gradually increasing in the last two decades, as in many other countries worldwide. Ten patients with metastatic malignant melanoma were treated between March of 1997 and April of 1998 in the Department of Dermatology, National Cancer Center Hospital, with a combination chemotherapy consisting of dacarbazine (DTIC), nimustine hydrochloride (ACNU), cisplatin (CDDP), and tamoxifen (TAM). The patients characteristics were as follows: four were males and six females; the age range was 33-70 years; all were Japanese; sites of primary disease: extremities 4, primary unknown 3, nasal cavity 1, anus 1, scalp 1; sites of metastases: lymph nodes 6, pulmonary system 5, skin 2, liver 3, gall bladder 1, adrenal gland 1. The chemotherapy regimen included DTIC 220 mg/m2/i.v. on days 1 through 3, ACNU 60 mg/m2/i.v. on day 1, cisplatin 25 mg/m2/i.v. on days 1 through 3, and tamoxifen 10 mg p.o. twice daily. One patient achieved a complete response and 3 showed partial responses. The response rate was 40%. The four responders included those with metastases to the nodes, lung, and liver. The main toxicities were nausea, vomiting, leucopenia, anemia, and thrombocytopenia. This regimen is a fairly effective combination against metastatic melanoma.     

Time-dependent suppression of melanoma metastases and natural killer cell activation by interferon

Summary The effect of murine / interferon (IFN) on experimental metastasis was investigated using B16-F10 melanoma cells. Since the outcome of metastasis of blood-borne tumor cells is mainly determined within the first 24 h after i.v. inoculation of tumor cells, i.p. injection of IFN was focused on this critical early phase. The inhibition of pulmonary metastases by IFN was found to be maximal when given 3 h prior to tumor cell inoculation, while mice with 24-h and 12-h pretreatment and simultaneous IFN treatment also showed a reduction in metastases, but to a lesser extent. However, mice receiving IFN 2 h after tumor cell inoculation did not show any reduction. Tumor cells cultured for 24 h in IFN-containing medium showed no reduction in metastases. Administration of anti-asialo GMl prior to IFN treatment was found to eliminate the inhibitory effect of IFN 3 h pretreatment. However, natural killer (NK) cell activity in vitro measured at 3 h, 13 h and 24 h after IFN administration was enhanced to the same extent, not paralleling the inhibitory effect on pulmonary metastases. These data indicate that prepared host status against blood-borne tumor cells is established by IFN pretreatment, being maximal when injected several hours prior to tumor cell inoculation, and that this effect is substantially dependent on NK cell activity, though the implication of other factors is not excluded.     

Ultraviolet A exposure might increase metastasis of mouse melanoma: a pilot study

BACKGROUND: The major sources of long-wave ultraviolet A radiation (UVA; 320-400 nm) exposure are extensive sunbathing and tanning in solaria. While the carcinogenic effects of mid-wave ultraviolet B radiation (UVB; 280-320 nm) are well recognized, the potentially hazardous effects of UVA are less understood. Several studies have shown that a variety of physiological processes in the cell are modified by UVA exposure, some of which might be involved in the regulation of tumor metastasis. In this study we suggest that UVA radiation could lead to the increase of metastatic capability of melanoma cells in mice. METHOD/RESULT: A pilot in vivo study was executed using C57BL/6 mice and syngeneic B16 melanoma cell lines. Mice were intravenously (i.v.) injected with either B16-F1 or B16-F10 melanoma cells into the tail vein and then immediately exposed to UVA. Fourteen days after melanoma injection, lungs were collected and the quantity and quality of metastases were determined under a dissecting microscope. As an outcome of the pilot study we observed that i.v. injected melanoma cells formed more lung metastases in the UVA-exposed mice in comparison with the control mice. CONCLUSION: This result suggests that the UVA exposure of mice, with melanoma cells present in blood circulation, increases the formation of melanoma metastases in lungs. Further studies should determine whether a similar pro-metastatic effect, as observed in mice, could occur in humans and whether other than melanoma tumors might be susceptible.     

Diagnostic modalities of visceral metastases during follow-up of patients with stage I-II melanoma

INTRODUCTION: Although not recommended in France at the consensus conference of 1994, routine monitoring of patients with stage I melanoma using imaging techniques is commonly carried out. The aim of this retrospective regional study was to define methods for diagnosing transition to the metastatic stage of melanoma. PATIENTS AND METHODS: This was a retrospective study based on questionnaires among dermatologists in the Champagne-Ardenne and southern Aisne regions of France. For each patient with stage IV melanoma between 1987 and 2002, data were collected concerning the primary melanoma (date of diagnosis, clinical picture, histopathologic features), stage of melanoma prior to diagnosis of metastatic melanoma and characteristics of the metastases (date, number, type, site and modern discovery: clinical signs or routine imaging). RESULTS: One hundred and eight patients (63 men and 45 women; mean age: 59 years) were included in the study. The predominant site of the primary melanoma was the trunk for men (n=31) and the lower limbs for women (n=16) and the mean Breslow index was 4.31 mm (SD=4.22), with histologic ulceration being present in 40% of cases. The mean time to transition to stage IV after discovery of the primary tumour was 2.8 years (SD=2.95). The modes of discovery of metastases comprised clinical examination (functional signs or physical examination) in 58 cases and routine imaging in 50 cases, with no significant differences based on whether patients were initially in stage I-II or in stage III. DISCUSSION: This study shows that over half of patients progressing to stage IV melanoma had a suspicious sign or clinical symptom, once again highlighting the importance of clinical monitoring. In contrast, many organ metastases, particularly pulmonary, were discovered by routine imaging examinations carried out as part of patient follow-up, although this is not currently recommended practice in France. CONCLUSION: The role of powerful imaging examinations such as scans, with constantly improving resolution, still remains to be defined in the follow-up of patients with stage I-II melanoma, and further prospective studies are thus required.     

荷载白介素24的溶瘤腺病毒联合达卡巴嗪对裸鼠黑素瘤的抑制作用

目的 研究荷载白介素24（IL-24）的溶瘤腺病毒ZD55-IL-24联合达卡巴嗪抑制裸鼠黑素瘤的作用。方法 建立裸鼠恶性黑素瘤A375细胞移植瘤模型后,分别给予ZD55-IL-24联合达卡巴嗪、ZD55-IL-24、达卡巴嗪、磷酸盐缓冲液（PBS）干预。Western印迹法检测A375细胞移植瘤组织IL-24、E1A蛋白表达。测量裸鼠肿瘤生长体积。结果 Western印迹结果表明,ZD55-IL-24联合达卡巴嗪作用的裸鼠黑素瘤高效表达IL-24、E1A蛋白。接种30 d后,ZD55-IL-24联合达卡巴嗪组肿瘤平均体积为（2346.5 ± 576.0） mm3,ZD55-IL-24组为（4141.6 ± 1348.2） mm3,达卡巴嗪组为（5230.1 ± 922.8） mm3,PBS组为（7135.1 ± 1002.3）mm3,ZD55-IL-24联合达卡巴嗪组与各组之间差异均有统计学意义（P ＜ 0.05）。结论 荷载IL-24基因的溶瘤腺病毒ZD55-IL-24联合达卡巴嗪有抑制黑素瘤增殖的作用。     

IP-10-encoding plasmid DNA therapy exhibits anti-tumor and anti-metastatic efficiency

We report here that the interferon-induced protein of 10 kDa (IP-10 or CXCL10) elicits strong anti-tumor and anti-metastatic responses in mice when administered by plasmid DNA. Intratumoral but not intramuscular IP-10 DNA inoculation resulted in reduced tumor formation of malignant melanoma (B16F10) and Lewis lung carcinoma (LL/2) in C57BL/6 mice. In addition, plasmid DNA-encoding IP-10 substantially reduced the establishment of metastases when injected systemically by the intramuscular route. In contrast to the primary tumor model, the anti-metastatic effect of DNA-encoding IP-10 was primarily mediated by NK cells. Compared to DNA-encoding interleukin-12 (IL-12), therapy with DNA-encoding IP-10 exhibits lower efficacy against primary melanoma tumors but equivalent efficacy against primary Lewis lung tumors and against B16F10 lung metastasis formation. Co-administration of DNA-encoding IP-10 and IL-12 enhanced the anti-tumor activity of IL-12 in the lung metastasis model but had little effect in the local treatment of established subcutaneous tumors. Interestingly, treatment of nude mice lacking T lymphocytes with DNA-encoding IP-10 or IL-12 still resulted in a pronounced reduction of tumor growth or metastasis formation.     

The roles of P- and E-selectins and P-selectin glycoprotein ligand-1 in primary and metastatic mouse melanomas

Background

Malignant melanoma is often accompanied by a host response of inflammatory cell infiltration that is highly regulated by multiple adhesion molecules.

Objective

To evaluate the role of adhesion molecules, including P-selectin glycoprotein ligand-1 (PSGL-1), P-selectin, and E-selectin.

Methods

Subcutaneous primary growth and metastasis to the lung of B16 melanoma cells were examined in mice lacking PSGL-1, P-selectin, or E-selectin.

Results

Primary subcutaneous growth of B16 melanoma was augmented by loss of PSGL-1, P-selectin, or E-selectin, while pulmonary metastasis was reduced by the loss of E-selectin. The enhancement of subcutaneous tumor growth was associated with a reduced accumulation of natural killer cells, CD4⁺ T cells and CD8⁺ T cells, while the attenuation of pulmonary metastasis was related to the numbers of CD8⁺ T cells. The expressions of transforming growth factor (TGF)-β and interleukin (IL)-6 were correlated with primary subcutaneous growth; TGF-β, IL-6, and interferon-γ were related to number of metastatic lung nodules. Cytotoxicity against melanoma cells in splenocytes and in tumor-draining lymph node cells were not defective by the absence of adhesion molecules, suggesting that the enhancement of tumor growth and metastasis caused by the loss of selectins results from an impaired migration of effector cells into the tissue.

Conclusions

The results indicate the complexity of anti-tumor responses mediated by adhesion molecules in primary subcutaneous tumors and pulmonary metastasis of murine experimental melanoma.     

Open trial of ciclosporin treatment for Stevens–Johnson syndrome and toxic epidermal necrolysis

Background Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute mucocutaneous reactions associated with poor prognosis. The treatment is mainly symptomatic, based on supportive care. Until now, several curative treatments have been proposed without evidence of effectiveness. Objectives To evaluate the effect of ciclosporin on SJS and TEN after a short series had suggested a benefit. Methods We conducted an open, phase II trial to determine the safety and possible benefit of ciclosporin. Among the 45 consecutive patients admitted for SJS/TEN from March 2005 to September 2007, 29 fulfilled inclusion criteria. Ciclosporin was administered orally (3 mg kg^?1 daily for 10 days) and tapered over a month. Clinical and biological evaluations were performed sequentially. Predicted death rate was estimated with a validated prognostic score (SCORTEN). Results Twenty‐nine patients were included at a mean ± SD of 2·8 ± 1·8 days after onset. The final diagnosis was SJS (n = 10), SJS/TEN overlap (n = 12) and TEN (n = 7). One month of treatment was completed in 26. Ciclosporin was stopped after more than 10 days in three cases for side‐effects including posterior leucoencephalopathy (n = 1), neutropenia (n = 1) and nosocomial pneumopathy (n = 1). Ciclosporin dosage was tapered earlier than scheduled in two cases for alteration in renal function. The prognostic score predicted 2·75 deaths; none occurred (P = 0·1). Mean epidermal detachment remained stable in 18 of 29 cases (62%). The mean ± SD hospital stay was 16·2 ± 9·1 days. Conclusions Both the death rate and the progression of detachment seemed lower than expected, suggesting a possible usefulness of ciclosporin in SJS and TEN that needs to be confirmed.     

CD26/dipeptidyl-peptidase IV and adenosine deaminase serum levels in psoriatic patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet A phototherapy

Objective The aim of this study is to determine serum levels of soluble forms of CD26/dipeptidyl‐peptidase IV (DPP‐IV) and adenosine deaminase (ADA), thought to be markers of T‐cell activation, and changes in their levels in response to cyclosporine, etanercept, and psoralen plus ultraviolet A (PUVA) treatments with respect to disease activity. Methods This study is designed as a prospective clinical study with a control group and three months of follow‐up. The study included 41 patients with psoriasis and 41 healthy controls that were older than 18 years of age. There were three different treatment groups: PUVA (n = 15), cyclosporine (n = 15), and etanercept (n = 11). To determine disease severity of patients with psoriasis, psoriasis area and severity index (PASI) scores were calculated. Results Only mean serum ADA levels were different between patients with psoriasis [mean ± standard deviation (SD) = 13.9 ± 3.3 U/ml] and control group (mean ± SD = 12 ± 3.5 U/ml). Mean serum ADA levels were significantly higher before treatment than after treatment (mean ± SD = 12.4 ± 3.4 U/ml). Contrarily, following three months of therapy, mean serum CD26 levels increased significantly from 777.7 ± 214.6 to 835.3 ± 203 ng/ml (P < 0.05) and mean serum DPP‐IV activity increased significantly from 12.1 ± 4 to 15.9 ± 4.2 nmol/min (P < 0.05). There was no correlation between ADA and CD 26/DPP‐IV with PASI values. Conclusions The results show that ADA might be a useful marker indicating disease activity and T‐cell activation. As significant changes were observed in serum CD26/DPP‐IV before and after treatment, we think CD26/DPP‐IV might play a role in psoriasis pathogenesis, which should be clarified by further studies.     

Postoperative DAV‐IFN‐β therapy does not improve survival rates of stage II and stage III melanoma patients significantly

Background DAV‐interferon (IFN)‐β therapy is a combination chemotherapy of dacarbazine (D TIC), nimustine (A CNU) and vincristine (V CR) with local subcutaneous injection of IFN‐β that is widely employed as postoperative adjuvant chemotherapy to treat malignant melanoma in Japan. However, the efficacy of DAV‐IFN‐β therapy has not been confirmed by randomized controlled trials and the benefit of DAV‐IFN‐β therapy has not been established yet. This study evaluated the contribution of DAV‐IFN‐β therapy to improve survival of postoperative patients with cutaneous melanoma. Methods Patients with stage II or III cutaneous melanoma seen at Nagoya University Hospital from January 1998 to December 2009 were eligible for this study. Disease‐free survival rates and melanoma‐specific survival rates were evaluated. A propensity score was calculated to control for the effects of variables related to decisions regarding the application of DAV‐IFN‐β therapy. Results Eighty‐two stage II and 60 stage III melanoma patients were included. In the post‐matched stage II patients (17 matched pairs), the mean (±SE) disease‐free survival rates were 39.9±13.7% for DAV‐IFN‐β therapy and 73.1±11.7% for non‐use (hazard ratio for recurrence, 2.06; 95% CI, 0.63–6.69; P = 0.23), and the melanoma‐specific survival rates were 66.2±20.0% for DAV‐IFN‐β therapy and 86.2±9.1% for non‐use (hazard ratio for death, 1.09; 95% CI, 0.17–6.82; P = 0.93). In the post‐matched stage III patients (nine matched pairs), the disease‐free survival rates were 29.6±16.4% for DAV‐IFN‐β therapy and 33.3±15.7% for non‐use (0.69; 95% CI, 0.22–2.17; P = 0.53), and the melanoma‐specific survival rates were 55.6±16.6% for DAV‐IFN‐β therapy and 44.4±16.6% for non‐use (0.67; 95% CI, 0.18–2.50; P = 0.55). Conclusions DAV‐IFN‐β therapy brought no significant improvement in either disease‐free survival rates or melanoma‐specific survival rates of patients with stage II or III cutaneous melanoma. A randomized controlled trial would be required to further evaluate the efficacy of DAV‐IFN‐β therapy as an adjuvant chemotherapy.     

Diagnostic utility of 5‐hydroxymethylcytosine immunohistochemistry in melanocytic proliferations

Decreased hydroxymethylated cytosine (5‐hydroxymethycytosine, 5‐hmC) is reported to correlate with melanocyte dysplasia. The purpose of this study was to assess the diagnostic utility of this observation. 5‐hmC immunohistochemistry was performed on tissue microarrays containing 171‐melanocytic lesions from two different institutions. An immunohistochemical staining score representing the percentage and intensity of nuclear staining was assigned. The performance characteristics of 5‐hmC immunohistochemistry for discriminating between a nevus and melanoma were determined. Additional cases of melanoma arising in a nevus (n = 8), nodal nevi (n = 5) and melanoma micrometastases to a lymph node (n = 6) were also assessed. Pronounced 5‐hmC loss was observed in melanomas when compared with nevi (mean ± standard deviation = 6.71 ± 11.78 and 55.19 ± 23.66, respectively, p < 0.0001). While the mean immunohistochemical staining score values for melanocytic nevi and melanoma were distinct, there was considerable variability in immunohistochemical staining score within a single diagnostic category. The sensitivity and specificity of this assay for nevus vs. melanoma is 92.74 and 97.78%, respectively. Distinct biphasic staining patterns were observed in cases of melanoma arising in association with a nevus. Relative changes of 5‐hmC expression within a single lesion may be more informative than absolute values when using 5‐hmC as a diagnostic adjunct.     

Tumorigenicity and metastatic behavior in nude mice of two human squamous cell carcinoma lines that differ in production of the cytokine ETAF/IL-1

The purpose of these studies was to determine whether the production of the cytokine epidermal cell thymocyte-activating factor (ETAF) by human squamous cell carcinoma (SCC) cells correlated with their tumorigenicity and metastatic potential in athymic nude mice. Cells of the human SCC line A431 produced rapidly growing subcutaneous tumors, few experimental lung metastases, and low levels of ETAF activity in vitro. In contrast, cells of the SCC Colo-16 line produced slower growing subcutaneous tumors, high numbers of experimental lung metastases, and a high level of ETAF activity in culture supernatants. The apparent relationship between production of ETAF and experimental metastasis formation was not consistent. Clonal populations of the SCC A431 and Colo-16 were isolated in vitro. The clones of Colo-16 varied in their ability to produce experimental metastases and in production of ETAF in vitro. However, the levels of ETAF production did not correlate with the propensity of the SCC cells to produce experimental metastases. We conclude that while the growth and metastasis of human SCC in nude mice may benefit from production of the cytokine ETAF, the ETAF production per se is not invariably linked with the capability of the SCC cells to metastasize.     

Evidence for vitamin D deficiency and increased prevalence of fractures in autoimmune bullous skin diseases

Background Vitamin D deficiency plays a role in autoimmune diseases and risk of fractures. No data are available on vitamin D levels and vertebral fractures in autoimmune bullous skin diseases. Objectives To assess serum vitamin D levels and the prevalence of vertebral fractures in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), potentially fatal autoimmune bullous disorders. Methods We studied 13 consecutive inpatients with untreated active PV (six men and seven women, mean ± SD age 53·5 ± 14·3 years), 15 with BP (seven men and eight women, mean ± SD age 76·9 ± 12·4 years) and 28 age‐, body mass index‐ and sex‐matched controls. The 25‐hydroxyvitamin D (25‐OHD) levels and presence of vertebral fractures on spinal X‐ray were assessed in all subjects. Results In patients with PV, 25‐OHD levels were lower (mean ± SD 12 ± 4·4 ng mL^?1) and prevalence of severe hypovitaminosis D higher (62%) than in controls (mean ± SD 22·2 ± 11·7 ng mL^?1, P = 0·012; 23%, P = 0·0047, respectively). The prevalence of fractures was 54% and 31% in patients with PV and controls, respectively. Patients with BP showed lower 25‐OHD levels (mean ± SD 9·6 ± 7·2 ng mL^?1) and higher prevalence of severe hypovitaminosis D (73%) than controls (mean ± SD 22·6 ± 18·7 ng mL^?1, P = 0·022; 27%, P = 0·01, respectively). The prevalence of fractures tended to be higher in patients with BP than in controls (67% vs. 33%, respectively, P = 0·068). Conclusions The low 25‐OHD levels found in PV and BP may suggest a role for this agent in their pathogenesis. The increased prevalence of fractures should be taken into consideration in patients who must be given corticosteroids.