Expression of the Tumor Metastatic Suppressor Gene in Mouse Melanoma Model: Inverse Association to Metastatic Potential

Data obtained in experimental cutaneous melanomas have suggested that the nm23 gene may function as a metastasis suppressor gene. The nm23 level in 8 human cutaneous melanoma cell lines and 2 murine melanoma cell lines were examined. Each melanoma cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting lung tumor fool and by determining host survival time. It was found that expression of 'm23 mRNA in human melanomas is correlated closely with reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of malignancy and survival in patients with melanomas.

Antitumor Effect of Cationic INKKI Peptide from Bovine <i>β</i>-Casein on Melanoma B16F10

Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo, antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci. In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.

Growth of Circulating Tumor Cell-Derived Colonies from Peripheral Blood of Melanoma Patients: Preliminary Characterization of Colony Composition

Circulating tumor cells (CTC) are rarely detected in the blood of cancer patients, even though they are a direct harbinger of eventual patient demise. We developed an innovative CTC culture technology to allow more sensitive isolation, expansion, and characterization of viable colonies from patient blood. In this assay, the entire leukocyte fraction from 10 ml of anticoagulated patient blood is placed into culture medium without any pre-selection. After 16 days in culture, CTC derived colonies are counted. As a proof-of-principle, blood samples from 58 Stage IIa-IV melanoma patients were tested. Ninety percent of these samples grew colonies. The colony numbers ranged from 0 - 308 (mean 63 ± 9.5 SEM). Ten normal volunteers had virtually no growth (mean 0.5 ± 1.4 colonies). Colonies were harvested using a micropipette for characterization. Tumor-cell containing spheroids were embedded in paraffin, sectioned, and stained with melanoma-specific mAb for histologic characterization. MITF proved to be the most consistent immunostain that identified melanoma cells in these colonies. A host-cell component in colonies was also identified using CD68 and CD43 mAb staining. Following enzymatic dissociation of colonies, a variety of immunostains were tested. Papanicolau staining proved most useful for identifying the abnormal nuclei of tumor cells. Flow cytometry could readily distinguish host and tumor cell populations based on DNA content and forward/side scatter in dissociated colonies. The stem cell marker ALDH1A1 associated with the aneuploid population, but CD45 was expressed on both diploid and aneuploid cells. The ability to repeatedly isolate CTC derived colonies from cancer patient blood samples opens the door to a novel type of long-term clinical monitoring. This novel CTC culture technology may prove useful to perform molecular characterization, assessment of treatment response, and testing of drug sensitivity and resistance in patients during treatment.

Melanoma Immunotherapy: Overcoming Obstacles to Augment Anti-Tumor Immune Responses

Melanoma is the most aggressive form of skin cancer and accounts for the vast majority of skin cancer-related deaths. Its ability to metastasize quickly, often before diagnosis, makes this cancer difficult to treat with traditional therapies. The identification of anti-melanoma immune responses in patients and the discovery of tumor antigens targeted by these immune responses have paved the way for immunotherapy as a novel approach to treating this cancer. In this review, the major immunotherapies targeting these melanoma tumor antigens are discussed. The advantages and limitations of peptide-, protein-, and gene-based vaccination maneuvers and adoptive cell transfer therapies are emphasized. Recent insights into melanoma immune evasion strategies are also highlighted, with particular focus on how our increasing knowledge of tumor/immune cell interactions is driving the development of novel immunotherapeutic strategies for the treatment of melanoma.

Myometrium and Sigmoid Colon Metastatic Melanoma Simulating a Ewing Tumor: A Case Report

Malignant melanomas or lymphoma of the skin are malignant tumors of the skin and/or the mucous membranes whose uterine metastases are rare. The secondary uterine localizations, although rare, must be evoked in front of a pelvic tumoral syndrome, or diffuse metastases and a personal past history of melanoma, even after a long time of remission. In our observation, the evolution of the tumor in the pelvis extended to the muscular structures of the uterus and the sigmoid colon of a 72 year old patient, what made it an exceptional case. The diagnosis of these secondary localizations is a diagnosis of elimination, almost always post-operative, made on the histopathological and immunocytochemical study of the surgical specimen, supported by cytogenetics, even molecular biology. The treatment is based on chemotherapy.

Effects of heat shock protein-antigen peptide complexes (HACs) in melanoma B16 cell line on transplanted tumor development in mice

目的 :建立黑色素瘤 B16细胞热休克蛋白 -抗原肽复合物 (HACs)的制备方法并测其抑瘤效应。方法 :应用 Sephacryl S- 2 0 0凝胶过滤制备 HAC粗提物 ,应用 SDS- PAGE纯化 HACs,并测其抑瘤效应。结果 :应用 SDS- PAGE纯化的 HAC6 0、 HAC75和 HAC97不同程度地降低肿瘤发生率 ,延迟肿瘤发生时间和减慢肿瘤生长速度。结论 :6 0 0 0 0～ 970 0 0

Improving recognition of hepatic perivascular epithelioid cell tumor:Case report and literature review

A 58-year-old man presented with the chief complaint of abdominal bloating and was incidentally found to have a liver tumor.As diagnostic imaging studies could not rule out malignancy,the patient underwent partial resection of segment 3 of the liver.The lesion pathologically showed eosinophilic proliferation,in addition to immunohistochemical positivity for human melanoma black 45 and Melan-A,thereby leading to the diagnosis of a hepatic perivascular epithelioid cell tumor(PEComa).A PEComa arising from the liver is relatively rare.Moreover,the name ‘PEComa' has not yet been widely recognized,and the same disease entity has been called epithelioid angiomyolipoma(EAML),further diminishing the recognition of PEComa.In addition,PEComa imaging findings mimic those of malignant liver tumors,and clinically,this tumor tends to enlarge.Therefore,a PEComa is difficult to diagnose.We conducted a systematic review of PEComa and EAML cases and discuss the results,including findings useful for differentiating perivascular epithelioid cell tumors from malignant liver tumors.

Primary gastric melanoma: A case report with imaging findings and 5-year follow-up

BACKGROUND Most melanomas identified in the stomach are metastatic;primary gastric melanoma(PGM)is extremely rare,and the relevant studies are relatively scarce.PGM may be incorrectly diagnosed as other gastric malignant tumor types.CASE SUMMARY We describe a rare case of PGM confirmed through long-term clinical observation and pathological diagnosis.A 67-year-old woman presented to our hospital with recurrent chest tightness and chest pain.Digital gastrointestinal radiography revealed a circular shadow in the gastric cardia.Computed tomography(CT)revealed a heterogeneous tumor with uneven enhancement.Enlarged lymph nodes were noted in the lesser curvature of the stomach.On magnetic resonance imaging(MRI),T1-and T2-weighted imaging revealed hyperintensity in and hypointensity in the tumor,respectively,both of which increased substantially after uneven enhancement.Near total gastrectomy was performed,and the tumor was pathologically confirmed to be a gastric melanoma.Because no other possible primary site of malignant melanoma was suspected,a clinical diagnosis of PGM was made.The patient was followed for nearly 5 years,during which she received CT reexamination,but no recurrence or metastasis was observed.CONCLUSION Certain imaging characteristics could be revealed in PGM.Imaging examination can be of great value in preoperative diagnosis,differential diagnosis,and followup of patients with PGM.

Comparative Study on the Inhibitory Effect of RecombinantFN Polypeptide CH50 and CH56 on the Metastasis ofMelanoma Cells

On the basis of preparation of anti-metastatic recombinant FN polypeptides, CH50 and CH56, we further studied the function of these polypeptides.The capacity of CH50 binding with melanoma cells (ED50 30 mM) was higher than that of CH56 (ED50 45 mM). Both of the polypeptides could significantly suppress the binding of melanoma B16 cells to laminin. There was no significant difference in the inhibitory effect between two polypeptides. In the experimental metastasis of melanoma cells, both of CH50 and CH56 could significantly inhibit the metastasis of the tumor cells, and reduce the number of lung metastasis by about 80%. Our results suggest that Ⅲ-11 and ED-A repeats influenced, to some extent, the binding capacity of bifunctional-domain polypeptide to cells, but did not affect the inhibition of the polypeptide on the metastasis of melanoma cells. The presence and connection of cell Ⅰ and Hep Ⅱ domains are the elements which determine the ability of recoinbinant FN polypeptides to inhibit the metastasis of tumor cells.

Novel anti-melanoma treatment:focus on immunotherapy

Melanoma is an intractable cancer that is aggressive, lethal, and metastatic. The prognosis of advanced melanoma is very poor because it is insensitive to chemotherapy and radiotherapy. The incidence of melanoma has been ascending stably for years worldwide, accompanied by increasing mortality. New approaches to managing this deadly disease are much anticipated to enhance the cure rate and to extend clinical benefits to patients with metastatic melanoma. Due to its high degree of immunogenicity, melanoma could be a good target for immunotherapy, which has been developed for decades and has achieved certain progress. This article provides an overview of immunotherapy for melanoma.

Complete resection of isolated pancreatic metastatic melanoma:A case report and review of the literature

Isolated metastatic melanoma of the pancreas is very rare.Currently,there is very limited experience with surgical resection of pancreatic metastasis.The potential benefit of metastasectomy can improve the quality of life and survival time of patients.We present a case of a 39-year-old Chinese male with a solitary pancreatic tumor which was considered a cystic benign lesion for years.Pathology and immunohistochemistry showed that the tumor in pancreatic tail was a metastasis from a malignant melanoma of the eyeball.No other metastastic foci were found in abdomen.The tumor was completely resected with combined distal pancreatectomy and splenectomy.The patient has survived 25 mo without any signs of local recurrence or other metastatic lesions after operation,indicating that complete surgical resection of a solitary metastatic melanoma of the pancreas can prolong the survival time of patients.

Expression of Phosphorylated-STAT3 and Osteopontin and Their Correlation in Melanoma

The expressions of p-STAT3 and osteopontin in 22 cases of normal nevi and 43 cases of malignant melanoma were immunohistochemically detected, and the correlation between p-STAT3 and osteopontin in malignant melanoma and the correlations of p-STAT3 （or osteopontin） with invasion, metastasis and thickness of malignant melanoma were examined. The results showed p-STAT3 was expressed in 2 of 22 cases of normal nevi and 30 of 43 cases of malignant melanoma, while osteopontin was expressed in 3 cases of normal nevi and 29 cases of malignant melanoma. The expressions of p-STAT3 and osteopontin in melanoma were significantly higher than that in benign nevi. There existed significant correlations between the expression of p-STAT3 and that of osteopontin in melanoma. Furthermore, the expression rates of p-STAT3 were significantly higher in invasive or metastatic melanomas than that their non-invasive or non-metastatic counterparts, and the expression rates of osteopontin were significantly higher in invasive melanomas than that in non-invasive ones. It is concluded that p-STAT3 and osteopontin may play important roles in the pathogenesis of malignant melanoma.

Apoptotic B16-F1 Cells Coated with Recombinant Calreticulin Mediated Anti-tumor Immune Response in Mice

Objective： To investigate the recombinant calreticulin （rCRT） mediated antitumor immune response. Methods： Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expression and cell localization were assayed by western blotting, QT-RT-PCR and immunofluorescence assays. The mouse melanoma cell line B16-F1 was treated with polyamine analogue BENS to induce apoptosis and incubated with rCRT to get rCRT coated on the membrane, and then the cells were used to immune BALB/c mice as a cell-antigen. Immunized animals were rechallenged by live B16-F1 cells and then tumor generation ratio and the lactate dehydrogenase release assay were used to evaluate antitumor effects of rCRT-mediated immunity. Results： BENS induced apoptosis of B16-F1 cells without the redistribution of CRT within the cells. When B16-F1 cells coated with rCRT were used as cell-antigen to inoculate the animals, the mice obtained the ability in inhibiting proliferation of homologous tumor cells in vivo. Comparing with the positive control group, the splenocytes from those inoculated mice have an obvious enhancement on their cytolytic effects specifically against B16-F1 cells. Conclusion： rCRT coated on the cell surface can enhance immunogenicity of apoptotic tumor cells and mediated effective anti-tumor immunoresponse in mice.

Inhibitory Effect of Recombinant Fibronectin Polypeptide CH50 on Invasion and Metastasis of Melanoma B16 Cells

In order to investigate the inhibitory effect and mechanism of recombinant polypeptide CH50 on invasion and metastasis of melanoma B16 cells, the recombinant polypeptide CH50 was separated and purified by ion exchange chromatographic technique. The melanoma B 16 cells treated with purified CH50 were cultured in vitro, the number was counted at 4, 24, 48 and 72 h and their morphological changes were observed in order to detect their adhesion and spreading abilities. In in vivo study, the melanoma B16 cells were labeled with CFSE and treated with CH50 and then they were injected into mice via mouse-tail veins. After 5 h, the lung tissues were fixed by frozen section. Accumulation and invasion abilities of B 16 cells on lung tissues were observed under the fluorescent microscopy. The results showed that the morphological character of B16 cells treated with CH50 changed greatly and the number of B16 cells treated with CH50 decreased significantly （P〈0.05）. The adhesion and spreading abilities of B16 cells treated with CH50 were weakened obviously and the metastasis foci on lung tissues reduced. It was concluded that the recombinant polypeptide CH50 inhibited invasion and metastasis of melanoma B16 cells on tissues and could be a prospective bio-product in tumor general therapy.

Preliminary Study on the Cooperation of IL-6 and mB7-1 in the Induction of Effective Antitumor Immunity In Vitro

To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7 1 costimulation alone, or combined with IL 6, in inducing antitumor immunity in vitro was investigated. It was found that mB7 1 cDNA transfected B16 cells (B16 mB7 1) induced the expansion of effector lymphocytes and the generation of specific lytic activity more effectively than wild type B16 melanoma cells (B16 wt) or mock transfected B16 cells (B16 neo) did. ( P <0.01), IL 6 could effectively stimulate lymphocytes proliferation, but failed to enhance its cytotoxicity, while the combination of mB7 1 and IL 6 increased both lymphocyte proliferative response and T cell mediated cytotoxicity more significantly than B7 1 or IL 6 did alone ( P <0.01) . It was inferred that the costimulatory molecule B7 1 is required for the activation and proliferation of T lymphocytes; the expression of mB7 1 in tumor cells could increase their immunogenicity and induce effective antitumor immune response, and the combination of B7 1 and IL 6 could induce more effective antitumor immunity, indicating that cooperation of IL 6 and mB7 1 plays a role in T lymphocyte activation.

The Application and Indications for Sentinel Lymph Node Biopsy in Pediatric Solid Tumors: Current Status and Future Directions

Oncologic staging of many solid organ malignancies involves an understanding of how cancers spread via the lymphatic system, and ultimately may involve evaluation of the primary nodal drainage basin by performing a sentinel lymph node biopsy. In the adult population, there are well established indications for sentinel lymph node biopsy in diseases like melanoma and breast cancer. However, its use and relevance in the pediatric population is less defined. This review details the history and development of sentinel lymph node biopsy technique, advanced lymph node mapping techniques currently under investigation, and the applications of sentinel node biopsy towards childhood cancers.

Surgical resection plus biotherapy/chemotherapy improves survival of hepatic metastatic melanoma

AIM: To analyze the correlation of treatment method with the outcome of all the hepatic metastatic mela-noma (HMM) patients from our hospital.METHODS: There were altogether nine cases of HMM that had been treated in the PUMCH hospital during the past 25 years, from December 1984 to February 2010. All of the cases developed hepatic metastasis from primary cutaneous melanoma. A retrospective review was performed on all the cases in order to draw informative conclusion on diagnosis and treatment in correlation with the prognosis. Clinical features including symptoms, signs, blood test results, Bultrasound and computed tomography (CT) imaging characteristics, and pathological data were analyzed in each case individually. A simple comparison was made on case by case basis instead of performing statistical analysis since the case numbers are low and patients were much diversified in each item that has been analyzed. Literatures on this subject were reviewed in order to draw a safe conclusion and found to be supportive to our finding in a much broad scope. RESULTS: There are six males and three females whose ages ranged 39-74 years old with an average of 58.8. Patients were either with or without symptoms at the time of diagnosis. The liver function and tumor marker exam were normal in all but one patient. The incidence of HMM does not affect liver function and was not related to virus infection status in the liver. Most of these HMM patients were also accompanied by the metastases of other locations, including lung, abdominal cavity, and cervical lymph nodes. Ultra-sound examinations showed lesions ranging 2-12 cm in diameter, with noor low-echo peripheral areola. Doppler showed blood flow appeared inside some tumors as well as in the surrounding area. CT image demonstrated low density without uniformed lesions, characterized with calcification in periphery, and en-hanced in the arterial phase. Contrast phase showed heterogeneous enhancement, with a density higher than normal liver tissue, which was especially appar-ent at the edge. Patients were treated differently with following procedures: patients #1, #6 and #8 were operated with hepatectomy with or without removal of primary lesion, and followed by comprehensive biotherapy/chemotherapy; patient #9 received hepatectomy only; patient #2 received bacille calmette-guerin treatment only; patient #7 had Mile’s surgery but no hepatectomy; and patients #3, #4 and #5 had supportive treatment without specific measurement. The patients who had resections of metastatic lesions followed by post-operative comprehensive therapy have an average survival time of 30.7 mo, which is much longer than those did not receive surgery treatment (4.6 mo). Even for the patient receiving a resection of HMM only, the post-operative survival time was 18 mo at the time we reviewed the data. This patient and the patient #6 are still alive currently and subjected to continue following up. CONCLUSION: Surgical operation should be first choice for HMM treatment, and together with biothera-py/chemotherapy, hepatectomy is likely to bring better prognosis.

Effects of Ganoderma lucidum polysaccharides peptide on tumor metastasis in mice with sleep fragmentation

In recent years,studies have shown that there is a correlation between sleep disorders and cancer,but at the present stage,the research on sleep disorders and tumor related animal models is relatively insufficient.Our research will focus on mice bearing B16F10-luc-G5 melanoma tumor with sleep fragmentation,detecting promoting effect of sleep fragmentation(SF)on the metastasis of melanoma.At the same time,we used Ganoderma lucidum poly⁃saccharides peptide(GL-pp,80 mg·kg-1),a component of traditional Chinese medicine Ganoderma lucidum,which has long enjoyed a good reputation at home and abroad,to observe its anti-tumor metastasis effects on B16F10-luc-G5 mice with SF.Then we used whole proteomics to analyze the difference proteins expressed in lung tissue and compared between groups,includes mice bearing B16F10-luc-G5,mice bearing B16F10-luc-G5 with SF and GL-pp administered mice bearing B16F10-luc-G5 with SF.With the analysis using bioinformatics,we found several key proteins,their genes name are Adcy9,ptk2,Yap1 and Lpin2,Per1 and Tim.And several important clusters,they are,immune system,platelet aggres⁃sion,energy metabolism,cell cytoskeleton,cell adhesion and circadian rhythms.Moreover,we detected the TLR4 signal pathway and macrophage differentiation to reconfirm the results of proteomics and trying to elucidate the mechanism of SF on tumor growth and metastasis and the effects of GL-pp.

ANTI-METASTATIC EFFECT OF ONCOLYSATES FROM MURINE MELANOMA CELLS TRANSFECTED WITH RECOMBINANT VACCINIA VIRUS ENCODING HUMAN IL-2

Oncolysates, debris of tumor cells, have been proven to be effective in active immunotherapy of cancer. In this experiment, the oncolysates from murine melanoma cells B16F10 transfected by recombinant vaccinia viruses encoding human IL2(IL2VBO) were used as vaccine. After treatment of tumor bearing mice with pulmonary metastases by intravenous injection of IL2VBO or rVVIL2, higher level IL2 activity was detected in the serum of IL2VBO or rVVIL2 treated mice at 8h. Further experiment results demonstrated that IL2VBO significantly reduced the number of pulmonary metastases and prolonged the survival time of tumorbearing mice when compared with other preparations. Fresh peripheral blood lymphocytes from IL2VBO treated mice showed potent cytotoxicity to B16F10 cells and YAC1 cells. But only cytotoxicity to B16F10 cells is more marked than that in rVVIL2 group, indicating that the IL2VBO could induce specific and non specific antitumor immunity. Because IL2 expression was at the same level in the serum of IL2VBO or rVVIL2 treated mice, the results suggested that the specific antitumor immunity induced by IL2VBO might contribute to the enhanced therapeutic effect of IL2VBO.

Melanoma involving the ileum: Report of a case and review of literature

Malignant melanoma accounts for less than 3% of all gastrointestinal tract malignancies, and it is mainly metastatic in origin. In contrast, primary melanoma of the small bowel is an extremely rare entity. We report the case of a 34-year-old male who was diagnosed with melanoma involving the ileum most likely secondary in origin. Histologically, the neoplastic cells involved the entire wall thickness, showed nuclear pleomorphism, nuclear pseudoinclusions, abundant eosinophilic cytoplasm, and were immunoreactive for S100, HMB45, and Melan-A. Electron microscopy showed electron-dense complexes that suggested aggregates of melanin. The final diagnosis was given based on the clinical, radiologic and histopathologic correlation. This case demonstrates the need of clinical and histological immunohistochemistry, electron microscopy and molecular studies together to make a correct diagnosis of spindle cell and epithelioid tumors of the gastrointestinal tract.