Comparison between cold plasma,electrochemotherapy and combined therapy in a melanoma mouse model

The study was undertaken to compare antitumor efficacy of electrochemotherapy (ECT) with cold plasma therapy (CP) in a melanoma mouse model. After melanoma implantation into the flank of C57BL/6N mice, CP by two different plasma sources (APPJ and DBD) was applied directly to the tumor surface. ECT was performed with bleomycin intravenously at a field strength of 1000 V/cm without or combined with CP. Primary endpoints were tumor growth acceleration (TGA), daily volume progression (DVP) and survival after treatment. Both plasma sources as single treatment showed a significant TGA delay, which proved less effective than ECT. CP (APPJ) combined with ECT (ECJ) significantly improved per cent mouse survival, with significant superiority compared with ECT. Plasma therapy alone albeit less effective seems a potential alternative to ECT in patients with melanoma and can be applied manifold in a session without general anaesthesia. Accordingly, CP alone and combined with ECT may serve as new option in palliative skin melanoma therapy.     

Synergistic effects of hyperthermia and peplomycin against human malignant melanoma xenografts

Two types of human malignant melanoma were treated either with peplomycin alone, hyperthermia alone, or a combination of peplomycin and hyperthermia. The antitumor effect of combination therapy was evaluated by tumor growth curves, maximal suppression rate, and histopathology. A marked antitumor effect of combination therapy with peplomycin (5 mg/kg) and hyperthermia (43°C, 30 min) was obtained for both types of melanoma. Histopathologic findings revealed advanced central necrosis and pyknotic nuclei in the tumor cells. Although peplomycin alone (20 mg/kg, 6 times) had no antitumor effect on C24 melanoma, the peplomycin (5 mg/kg, 6 times) + heat group showed complete inhibition of the tumor growth. These results suggest that hyperthermia enhances the antitumor effect of peplomycin even against peplomycin-resistant tumor cells.     

Thermochemotherapy for malignant melanoma: overcoming heterogeneity in drug sensitivity

Endo B (melanotic) and W (amelanotic) human malignant melanomas originated from the same tumor, both known to be heterogeneous in drug sensitivity to ACNU [( 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso- urea hydrochloride]), were treated experimentally with a combination therapy of ACNU and hyperthermia in mice. Whereas Endo W melanoma has no sensitivity, Endo B melanoma is sensitive to ACNU alone. However, in both types of melanomas, a marked synergistic effect of the combination therapy was noted. Histologically, marked degeneration of both tumor cells was detected. These results strongly suggest that thermochemotherapy may overcome the tumor heterogeneity in drug sensitivity.     

Pretreatment effects on the uptake/retention kinetics of L-dopa in Harding-Passey melanoma

Malignant melanoma cells possess a unique biochemical pathway that converts L-3,4-dihydroxyphenylalanine (L-dopa) to the biopigment melanin. Selective cytotoxic incorporation of exogenous L-dopa into melanoma cells in vivo may provide a means of designing specific chemotherapeutic agents useful in the treatment of this disease. Using the Harding-Passey murine melanotic tumor model, a preferential uptake of [3H]L-dopa by the tumor was characterized. Following pretreatment of the tumor-bearing mice with nonradioactive L-dopa, a significant enhancement (p less than 0.01) of [3H]L-dopa incorporation and retention into melanoma for a period of 24 h was observed, when compared with the concomitant tissue distribution and clearance of radioactivity in the control animals. This finding suggests that by initial pretreatment of melanoma with nonradioactive L-dopa, the subsequent selective accumulation of [3H]L-dopa in tumor may provide a useful tool in testing new modalities of therapy in malignant melanoma.     

Malignant melanoma--future prospects

As the incidence and already high mortality rates of malignant melanoma have been steadily increasing in recent decades, the early detection and excision of malignant melanoma have imposed as the most important task. Staging of malignant melanoma is determined according to the level of invasion (Clark level) and vertical thickness (Breslow scale). Besides operative therapy, which is the only effective treatment for malignant melanoma, postoperative adjuvant chemotherapy, immunotherapy, radiotherapy, and biologic therapy also are of great importance. In recent years, immunologic strategies including tumor vaccine and adjuvant therapy with interferon-alfa have been attempted to improve survival of patients with more advanced malignant melanoma. A recent melanoma research has focused on target therapy such as immunotherapy (vaccines, monoclonal antibodies, dendritic cells) and gene therapy. Genetic immunization has become an attractive strategy for the development of melanoma vaccines, because a number of antigens recognized by cellular components of the immune system have been identified at the molecular level. Numerous chemotherapeutic agents have shown activity in the treatment of metastatic malignant melanoma, such as dacarbazine (dimethyl triazene imidazole carboxamide); other agents have been used, however, with less success. However, a very modest effect was recorded in advanced malignant melanoma. There are many experimental trials using combined therapy for malignant melanoma, including chemotherapy (dimethyl triazene imidazole carboxamide) and biologic therapy (interleukin (IL)-2, interferon (IFN)-gamma, IFN-alfa). The results obtained open particularly interesting prospects in the field of malignant melanoma with high relevance for its development and progression. Molecular therapeutics and vaccine development will probably be an important focus for the future melanoma treatment.     

手术扩大切除联合小剂量干扰素α2b治疗侵袭性皮肤恶性黑素瘤32例

目的 回顾性分析手术扩大切除联合小剂量干扰素(IFN)α2b治疗皮肤恶性黑素瘤的疗效.方法 32例皮肤恶性黑素瘤患者经术前检查分期后(Ⅱ期21例、Ⅲ期11例)接受了手术扩大切除,手术范围距离皮损或组织病理取材后瘢痕边界1～2cm.手术方法包括:直接切除缝合4例、截指(趾)6例、皮肤游离移植15例、任意皮瓣转移3例和轴型皮瓣转移4例.有区域淋巴结转移行选择性淋巴结摘除者9例,腹股沟淋巴结清扫者2例.术后1周,给予IFN-α2b 300万IU皮下注射每周3次,连续1～3年.结果 所有患者的切口均甲级愈合.截止到2011年6月,2例患者失访,其余30例患者中,6例随访5年,生存4例,死亡2例(均为ⅢC期);7例随访3～5年,生存5例,死亡2例(均为ⅢC期);13例随访1～3年,生存12例,死亡1例(为ⅢB期);4例随访1年内的无1例死亡.现存的25例患者中,8例合并区域淋巴结转移,其中2例为手术后2年内新出现的区域淋巴结转移;8例完成了IFN-α2b治疗3年的疗程,11例已接受了1年以上的治疗,不良反应轻微.结论 手术扩大切除联合小剂量干扰素α2b治疗Ⅱ期和Ⅲ期皮肤恶性黑素瘤,能降低局部复发,患者生存率高.     

Cutaneous neoplasms composed of melanoma and carcinoma: A rare but important diagnostic pitfall and review of the literature

We report two cases of combined cutaneous tumors composed of melanoma and carcinoma. The first tumor presented as a 5-mm pink-blue macule over the right zygomatic arch in an 85-year-old man. Shave biopsy and immunohistochemical studies revealed that the tumor was composed of melanoma (highlighted by SOX10 and MART-1, with high Ki-67 proliferative index) intermixed with nodular basal cell carcinoma (highlighted by pan-cytokeratin and Ber-EP4). The neoplastic melanocytes were confined to the basal cell carcinoma nodules, and a diagnosis of combined melanoma in situ and basal cell carcinoma was rendered. After therapeutic excision, the patient was disease-free at 9 months after the initial diagnosis. The second tumor presented as a 6-mm pink-brown crusted papule on the right forehead in an 89-year-old man. Shave biopsy and immunohistochemical studies revealed that the tumor was composed of malignant melanoma (MM) (highlighted by S100 and MART-1) intermixed with squamous cell carcinoma (SCC) (highlighted by cytokeratin and p63), and a diagnosis of combined MM-SCC was rendered. These two cases highlight the importance of recognizing these rare types of melanocytic-epithelial cutaneous neoplasms to arrive at an accurate diagnosis that may inform appropriate disease stage and therapy.     

Comparison of a treatment strategy combining CCI-779 plus DTIC versus DTIC monotreatment in human melanoma in SCID mice

This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.     

大剂量重组人α-2b干扰素治疗晚期黑色素瘤的疗效及对血清LDH、TIL和PD-L1表达的影响

目的 探讨大剂量重组人α-2b干扰素联合常规化疗在晚期黑色素瘤患者中的应用安全性和有效性,以及对血清乳酸脱氢酶(LDH)、肿瘤浸润淋巴细胞(TIL)和抗程序性死亡因子配体1(PD-L1)表达的影响.方法 回顾性总结2016年8月—2018年8月入我院确诊为晚期黑色素瘤患者共86例(Ⅲ期51例和Ⅳ期35例),均接受大剂量...     

Bcl-2 and bcl-xL antisense oligonucleotides induce apoptosis in melanoma cells of different clinical stages

Recent clinical studies have shown the promise of bcl-2 antisense therapy in patients with melanoma. To further demonstrate the importance of bcl-2 and validate the related antiapoptotic protein bcl-xL as targets for antisense therapy in melanoma, their implication as survival factors in melanoma cells of different clinical stages as well as in normal melanocytes was investigated. Primary cell cultures derived from 17 melanomas, the cell line A375, and normal melanocytes from healthy donors were treated with antisense oligonucleotides targeting either the bcl-xL mRNA or the bcl-2 and the bcl-xL mRNAs simultaneously. Bcl-2 and bcl-xL expression in cells was analyzed by real-time polymerase chain reaction and Western blotting. Cell viability was assessed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and apoptosis assays. Bcl-2 expression was low in melanoma cells of stages I, II, and III, hardly detectable in A375 cells, but high in normal melanocytes. Bcl-xL expression was high in all cell types tested. As shown in A375 cells and the stage III melanoma cells 0513, both the bcl-xL monospecific oligonucleotide 4259 and the bcl-2/bcl-xL bispecific oligonucleotide 4625 effectively reduced tumor cell viability by induction of apoptosis with IC50 values ranging from 200 to 350 nM. Oligonucleotide 4625 proved to be superior to 4259, as it significantly reduced the viability of cells from all melanoma stages. Both oligonucleotides reduced also the viability of normal melanocytes. Our data suggest that bcl-2 and bcl-xL are promising targets for antisense therapy of melanoma, and that the simultaneous downregulation of their expression may provide additional clinical benefit.     

In situ detection of cutaneous melanoma by prompt gamma-ray spectrometry using melanoma-seeking 10B-dopa analogue

10B1-para-boronophenylalanine (10B1-BPA), one of our boronated dopa analogues developed for thermal neutron capture therapy, has been found to have a selective affinity for malignant melanoma. We have established a method of 'in situ' detection of subcutaneous melanoma lesions, using this melanoma-seeking 10B-labeled compound. In this study, we applied an 'in situ' 10B microanalysis system via detection of the prompt gamma-ray from the 10B(n, alpha)7Li reaction triggered by irradiating the 10B-containing target with pure thermal neutrons, called prompt gamma-ray spectrometry, to hamsters bearing Greene's melanoma in subcutis and to a human patient whose occipital subcutaneous tumor was suspected of being a metastatic melanoma. In the hamsters, the time-dependent 10B dynamics showed increased 10B accumulation in melanoma, after 10B1-BPA administration, in contrast to that in non-melanoma normal skin. In the human patient, after subcutaneous injection of 10B1-BPA into perilesional sites 4 cm distant from the tumor margin, the average 10B concentration in the tumor was determined to be 24 ppm (microgram/g), in contrast to 3 ppm in skin covering the tumor and 1.1 ppm in blood, indicative of selectively high 10B1-BPA uptake by the tumor.     

Randomized controlled study of electrochemotherapy in the local treatment of skin metastases of melanoma

BACKGROUND: Electrochemotherapy (ECT) combines intralesional injections of bleomycin with electroporation (EP), which permeabilizes tumor cells and thus increases the bleomycin efficacy at the tumor site. OBJECTIVE: To assess whether EP therapy improves the local control of skin metastases of melanoma by intralesional bleomycin. The secondary objective was to evaluate tolerance of the treatments. PATIENTS: Patients with at least two measurable skin metastases of melanoma that were previously untreated, either in stage III with in-transit melanoma skin metastases or stage IV with no efficacy of systemic chemotherapy on these metastases. DESIGN: A prospective internally controlled study with randomization of melanoma skin metastases in each individual to intralesional injections of bleomycin alone or to intralesional injections of bleomycin with EP. The primary end point was the rate of complete local response per treated melanoma skin metastasis at week 12, and the secondary end point was tolerance. RESULTS: Fifty-four melanoma skin metastases were treated in 12 patients (8 stage IV patients under chemotherapy and 4 stage III patients free of other treatment). A local complete response was obtained in 36% (11 of 30) of melanoma skin metastases treated with bleomycin + EP and only in 8% (2 of 24) of melanoma skin metastases treated with bleomycin alone (p = .016). In the per protocol population, complete response was obtained in 74% (17 of 23) and 13% (2 of 15) of the lesions treated, respectively (p = .017). All patients (12 of 12) reported discomfort during the EP procedure, including local pain for 9 patients (75%) at the treatment site and muscle spasm with myoclonia in 3 cases (25%). No clinical or biologic systemic toxicity was noticed. CONCLUSIONS: EP increases the effect of intralesional bleomycin and improves the rate of local control in melanoma skin metastases without inducing a more systemic effect. This local treatment could be useful in a palliative strategy in patients with melanoma skin metastases.     

Treatment of High Risk Resected Melanoma in Australia: Current Landscape and Practises

Stage III melanoma involves regional lymph nodes and/or in-transit or satellite disease, without spread to distant metastatic sites. Stage IIIA melanoma includes a T1a-T2a primary lesion with N1a or N2a nodal involvement, whilst stage IIID melanoma includes a T4b primary lesion with N3a-N3c nodal involvement. With surgery alone, patients with stage IIIA melanoma have 10-year survival rates of ~88%; however, patients with stage IIID melanoma have 10-year survival rates of only ~24%. Targeted therapy and immunotherapy are being explored in stage III disease as adjuvant therapy after surgical resection, to eliminate micro-metastatic disease and thereby prevent relapse of melanoma and increase patient survival. A number of pivotal trials published in the last two years have shown improved relapse-free survival (RFS) and overall survival in patients with stage III melanoma treated with adjuvant therapy. COMBI-AD showed adjuvant dabrafenib and trametinib improving RFS compared with placebo (HR 0.49; 95% CI 0.40–0.59). Checkmate-238 demonstrated an improvement in RFS of adjuvant nivolumab over ipilimumab (HR 0.68, P < 0.001) whilst Keynote-054 demonstrated an improvement in RFS with adjuvant pembrolizumab over placebo (HR 0.57, P < 0.001). Many nuances need to be considered when interpreting this data, including implications of an updated staging system, which patients are suitable for adjuvant therapy and the choice between adjuvant targeted therapy and immunotherapy in BRAF mutant patients. This review article summaries the currently available literature on adjuvant targeted therapy and provides a guide on applying this data in everyday practise.     

黑素瘤相关通路及治疗的新进展

黑素瘤的发病机制与遗传、环境因素如紫外线长期照射有关.研究表明,黑素瘤的发生发展主要与细胞外调节蛋白激酶通路和磷酸酰肌醇3激酶/蛋白激酶/哺乳动物雷帕霉素靶蛋白信号通路改变相关.相关的基因改变有:BRAF基因、NRAS基因、GNAQ基因和GNA 11基因、C-kit基因、苏氨酸蛋白激酶基因等.黑素瘤是皮肤侵袭性肿瘤,对于放疗和化疗均不敏感.针对黑素瘤发病机制的分子靶向治疗发挥了重要的作用,包括免疫治疗、基因治疗以及针对肿瘤血供的治疗.生物治疗比传统治疗有更好的靶向性和特异性.     

Photodynamic therapy with chlorin e(6) for skin metastases of melanoma

BACKGROUND: Photodynamic therapy (PDT) has been successfully applied in clinical settings to destroy neoplasms, but the efficacy of such a treatment is dependent on the type of neoplasm and the photosynthesizer used. Here, we perform a clinical assessment of PDT for skin metastases of pigmented melanoma using chlorin e(6). STUDY DESIGN/MATERIALS AND METHODS: PDT with chlorin e(6) photosensitizer was administered to 14 patients with skin metastases from melanoma (10 females, four males, mean age 49.6 years). Chlorin e(6) at a dose of 5 mg/kg of patient's weight was intravenously injected. The treatment course consisted of two courses of PDT exposure 1 h after intravenous chlorin e(6) injection and 24 h post-injection. The light energy density for each skin tumor was 80-120 J/cm(2) per treatment, with a light power density of 250-300 mW/cm(2). RESULTS: All skin melanoma metastases that received PDT showed complete regression with no recurrence during the study period. The complete response of all skin metastases from melanoma occurred in eight cases after one PDT treatment. In the remaining six individuals, tumors required multiple PDT courses prior to complete regression. No cases of photodermatitis were registered. The Karnofsky performance scale score of the patients with skin metastases from melanoma showed no significant difference before and after PDT. No patients had significant changes in blood cell counts that would indicate chlorin e(6) systemic toxic effect. Blood chemistry and urinalysis did not show any evidence of chlorin e(6) renal and hepatic injury. CONCLUSIONS: PDT with chlorin e(6) for skin metastases from melanoma is effective and well tolerated. Further clinical investigation of PDT with chlorin e(6) is warranted.     

Evaluation of twenty-one human adenovirus types and one infectivity-enhanced adenovirus for the treatment of malignant melanoma

Advanced melanoma is associated with poor prognosis warranting the development of new therapeutics, such as oncolytic adenoviruses for immunovirotherapy. Since this approach critically depends on efficient transduction of targeted tumor cells, we screened a panel of 22 different adenovirus types for their internalization efficiency in melanoma cells. We demonstrated that the virions of Ad35, Ad38, and Ad3 have significantly higher internalization efficiency in melanoma cells than Ad5, so far the only adenovirus type used in clinical trials for melanoma. Therefore, we developed a conditionally replication-competent Ad5-based vector with the Ad35 fiber shaft and knob domains (Ad5/35) and compared its therapeutic efficacy with the homologous vector carrying the native Ad5 fiber. To further enhance virotherapy, we combined the oncolytic adenovirus vectors with intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-H/F) and dacarbazine chemotherapy. In a human melanoma xenograft model, established from a short-term culture of primary melanoma cells, we demonstrated that the Ad5/35-based therapy had a significantly greater anti-neoplastic effect than the homologous Ad5-based therapy. Furthermore, the combination of virotherapy, intratumoral expression of MV-H/F, and chemotherapy was clearly superior to single- or double-agent therapy. In conclusion, Ad35-based vectors are promising for the treatment of melanoma.     

mRNA expression of tumor-associated antigens in melanoma tissues and cell lines

Tumor-associated antigens (TAA) are increasingly used as specific targets for immune therapy of malignant melanoma. The aim of the present study was to provide a basis for selecting the most suitable TAA by analyzing the mRNA expression of a large panel of TAA by RT-PCR and Northern blotting. We have chosen primers differentiating four groups of TAA (MAGE-A, MAGE-B, and two groups of GAGE-genes) and 13 individual TAA (MAGE-A2 and -A3, RAGE-1, -2, -3, and -4, LAGE-1a and -1b, NY-ESO-1, GAGE-1, SSX-2, SCP-1, and cTAGE-1) based on most recent sequence data. In addition, the RAGE-gene family has been separated into its four members by a novel designed nested PCR, which was confirmed by Northern analysis. Furthermore, the chromosomal organization and relationship between the RAGE-family and MOK was analyzed. RAGE-4 mRNA could be shown for the first time to be present in testis tissue. The most frequently expressed TAA were the MAGE-A and the GAGE-3,-4,-5,-6,-8 group, whereas among individual TAA MAGE-A2, -A3, RAGE-1, -3, and LAGE-1a/b were found within most specimens and are thus promising candidates for immune therapy. In comparison, melanoma metastatic specimens and cell lines show similar profiles of TAA expression, but individual TAA differ notably between both types of samples indicating that results from cell lines are not always applicable to tumor specimen.     

Radioimaging of malignant melanoma xenografts in nude mice using 111In-labeled monoclonal antibody against human malignant melanoma

By using ¹¹¹In-labeled monoclonal antibody (ZME-018) against human malignant melanoma, we examined its usefulness in radioimmunodetection of human melanoma xenografts in nude mice. Two human malignant melanoma cell lines were used in this study, KHm-1/4; and KHm-3/ps. KHm-1/4 cells express melanoma-associated antigen which is reactive with ZME-018, whereas KHm-3/ps cells do not. ZME-018 was conjugated with DTPA (diethylene triamine pentaacetic acid) first; then the conjugate was labeled with ¹¹¹In. After these procedures, labeled ZME-018 retained its binding activity against KHm-1/4 cells in vitro. Nude mice, bearing melenoma xenografts started from injections into the right hind legs, were i.p. or i.v. injected with ∽300 μCi (60 μg) of labeled ZME-018. Tumor images were taken with gamma camera at 24 hr intervals. Clear tumor images were obtained by 24 hrs after injection. The best imaging was obtained at 72 hrs with no background of internal organs. Specific localization was confirmed by the absence of imaging in the mice which received free ¹¹¹In only and also by using control mice bearing antigen-negative melanoma cells (KHm-3/PS). An example of tissue distribution of the labeled antibody in terms of tumor to tissue ratio at day 3 is as follows: tumor/intestine; 20/1, tumor/liver; 10/1, tumor/blood; 8/1, tumor/muscle; 8/1, tumor/heart; 3/1, tumor/kidney; 2/1. This study demonstrates the future applicability of ¹¹¹In-labeled monoclonal anti-melanoma antibody for radioimmunodetection of metastatic lesions in melanoma patients.     

Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft

Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.