Melanoma Dissemination Research Articles

Saucerization biopsy of pigmented lesions

A variety of skin lesions can clinically resemble malignant melanoma, and bearlyQ melanoma often fails to demonstrate classic bABCDQ morphological features. Therefore, a definitive diagnosis of any pigmented lesion can only be made after histopathologic examination of an appropriate surgical specimen. If a physician is to bdo no harm,Q it is most important to maintain a high index of suspicion when viewing pigmented lesions and to sample enough lesions to encompass as many potentially lethal melanomas as possible without exposing patients to unnecessary surgery. Physicians should sacrifice some degree of diagnostic specificity to maintain a high degree of sensitivity. The decision to perform a biopsy vs a fullthickness excision of a pigmented lesion has a direct bearing on this issue and has been the topic of spirited debates. Biopsies of several types are commonly used because they are simpler to perform, quicker, less costly, and most often heal with excellent cosmetic results. Some physicians have, historically, worried that a biopsy may lead to hematogenous dissemination of melanoma or deeper implantation of tumor and suggest that all questionable lesions should be completely excised. 4 This approach is supported by those who caution that incisional biopsies may be more prone to sampling error that is avoided when a lesion is completely excised. In addition, complete removal of a lesion benefits patients who might be lost to follow-up. Finally, tangential excision specimens of suspicious pigmented lesions that

Lower Extremity Schwannoma: F-18 FDG PET and MR Demonstration

PET imaging with F-18 FDG is valuable in the staging of melanomas with helpful demonstration of high metabolic locoregional and distant metastases of this neoplasm. Dissemination of melanoma may be falsely diagnosed in the concomitant existence of benign soft tissue lesions taking up F-18 FDG as shown in the two following cases of lower extremity schwannomas. Epidemiology and imaging characteristics of schwannomas are briefly reviewed and discussed.

Presence and maturity of dendritic cells in melanoma lymph node metastases

Immune avoidance mechanisms play a key role in the successful dissemination of melanoma. One mechanism whereby this could be achieved is by interfering with dendritic cell (DC) presentation of tumour-associated antigens to naïve T cells. In particular, immature DCs characterized by the absence of accessory molecules are known to be immunosuppressive and to be involved in the induction of tolerance. The present study has investigated the presence and activation status of DCs within melanoma metastases in the regional lymph nodes. Using image analysis techniques, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA-DR and the morphological features of DCs were examined in paraffin sections from 26 lymph nodes containing melanoma metastases. DCs expressing FXIIIa were found in 70% of the lymph nodes. The number of DCs identified was generally small but there were more concentrated areas of DCs designated as hotspots. In these areas of high FXIIIa staining, the percentage area occupied by DCs varied between 0.1% and 10%. The majority of FXIIIa-positive cells did not express the DC maturation markers CD83 or CD40 and morphologically were rounded with few dendrites, indicating that they were immature. The cells did, however, express high levels of HLA-DR, suggesting that they have the ability to present antigen but lack the accessory molecules required to initiate an immune response. Immature DCs, characterized by phenotypic and morphological features, are therefore present within the tumour deposits in lymph nodes infiltrated by melanoma and may specifically modulate the anti-melanoma immune response.

Hepatocyte growth factor and melanoma: gene transfer studies in human melanocytes.

Hepatocyte growth factor (HGF), a fibroblast-derived protein that affects the growth, motility and differentiation of epithelial cells, is a mitogen for human melanocytes and has recently been implicated as an important factor for the development and dissemination of melanomas. To better define the possible role of HGF in the multi-step progression from melanocyte to melanoma cell, we used retrovirus-mediated gene transfer to introduce the gene encoding human HGF into normal human melanocytes, thus causing these cells to produce a growth factor they do not normally express and creating a new autocrine loop as sometimes occurs in melanoma cells. Cells were transduced with an efficiency of 6%, and the modified cells synthesized and secreted significant levels of HGF (6.6 ng/10(7) cells per 24 h) in vitro. Cells expressing HGF had a higher rate of proliferation when compared with unmodified cells and formed large, dense, melanin-positive colonies on a plastic surface. Immunostaining showed HGF-positive melanocytes with varying levels of expression, and HGF protein was detected throughout the whole cell. Although proliferation of HGF-expressing melanocytes was enhanced, they failed to form colonies in a soft agar assay. These results suggest that expression of HGF, by virtue of its ability to enhance proliferation and cell clustering, may play a role in the multi-step process of transformation, but an autocrine signal of HGF alone is not sufficient for malignant transformation.

Detection of neoplastic cells in blood of miniature pigs with hereditary melanoma

Tyrosine, a key enzyme of melanin biosynthesis, is widely used as a specific marker for the detection of dissemination or metastatic melanoma cells in peripheral blood and other tissues like lymph node or bone marrow, which are normally tyrosinase negative. The amplification of tyrosinase-specific mRNA by means of RT-PCR is a sensitive technique capable of detecting a single tumour cell in 5–10 ml of whole blood. We have utilised this method to analyse the peripheral blood of laboratory miniature pigs with advanced cutaneous melanoma for the presence of tumor cells. This highly invasive hereditary malignancy can serve as an experimental model for the study of melanoma development and dissemination. For amplification of porcine gene, oligonucleotide primers derived from the sequence of human tyrosinase were used. These primers amplified fragment of the predicted length and restriction enzyme digestion confirmed their homology with the sequences of human tyrosinase gene. After the second round of amplification, tyrosinase could be detected up to the amount of 1 × 10<sup>–5</sup> µg of total RNA isolated from porcine melanoma per 1 µg of control RNA. Blood samples from eight animals with advanced melanoma and from five non-melanoma control animals were examined for tyrosinase expression. Tyrosinase mRNA was detected in five samples from animals with malignant melanoma. Non-melanoma control animals gave negative results.

Cathepsin A activity in primary and metastatic human melanocytic tumors.

Several lysosomal proteases including cathepsins B, D, H and L have been found to play a role in the metastasis of tumor cells. However, up to now no information on the role of cathepsin A, a lysosomal multifunctional peptidase, in the proliferative, invasive, and metastatic potential of malignant tumors has been available. In the present study we compared the activity of cathepsin A in lysates of 34 human melanocytic tumors: primary (n = 12) and metastatic (n = 5) malignant melanoma, dysplastic pigmented nevi (n = 6) and pigmented nevi without evidence of dysplastic melanocytes (n = 11). The carboxypeptidase activity of cathepsin A was assayed at pH 5.0 with its specific substrate Cbz-Phe-Ala. The amount of released C-terminal alanine was measured by the ninhydrin method. We found that lysates of primary malignant melanoma lesions exhibited significantly higher cathepsin A activity than lysates of dysplastic and normal pigmented nevi. The cathepsin A activity in lysates of metastatic lesions of malignant melanoma was significantly higher than in primary focus lysates. It seems that cathepsin A may play a role in malignant transformation and metastatic dissemination of malignant melanoma.

Expression of angiogenic and immunosuppressive factors by uveal melanoma cell lines.

Dissemination of uveal melanomas is almost exclusively haematogenous, making angiogenesis of the tumour a prerequisite for the formation of metastases. Uveal melanomas must employ strategies to evade the immune system in order to escape immune surveillance. We therefore determined the expression of the following angiogenic and immunosuppressive factors in seven human uveal melanoma cell lines using reverse transcriptase-polymerase chain reaction (RT-PCR): secreted interleukin-1 receptor antagonist (sIL-1ra), interleukin (IL)-6, IL-8, IL-10, transforming growth factor (TGF)-alpha, TGFbeta, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and angiopoietin-2. In addition, the secretion of sIL-1ra, IL-6, IL-8, IL-10, TGFbeta and VEGF was assayed by enzyme linked immunosorbent assay (ELISA). The potential of uveal melanoma cell lines to convert plasminogen to angiostatin was tested in an in vitro assay. All the factors except angiopoietin-1 were determined in one or more cell lines using RT-PCR, although these results were not necessarily confirmed by ELISA. Expression of VEGF and angiopoietin-2 was found in all seven cell lines. Production of angiostatin was observed in one cell line. All seven cell lines examined expressed angiogenic factors and most cell lines expressed immunosuppressive factors. The expression of VEGF and angiopoietin-2 in combination with a lack of angiopoietin-1 expression suggest high vascular remodelling capacity and could be of great relevance for the metastatic potential of uveal melanoma.

Low-doses of ionising radiation induce melanoma metastases and trigger the immune system—adrenal axis feedback loop

Low-doses of ionising radiation are frequently implicated in triggering and/or accelerating the growth of skin and other malignancies. It seemed probable that the radiation at similar dose levels might initiate metastasis from already existing tumours. Highly pigmented human melanoma xenograft that had lost its ability for a spontaneous metastasising and grown subcutaneously in athymic mice was exposed to very low and well-defined doses of ionising radiation to determine whether low linear energy transfer radiation can restore metastatic potential of the tumour. To ensure that all effects derived from radiation-activated neoplastic cells only, 131I was delivered selectively to the cutaneous melanoma instead of using the external beam. The direct response of these tumours to radiation was monitored by determining the growth rate of the lesions. Histopathological methods were employed to detect metastases. The lowest radiation dose of approximately 6 cGy deposited in the tumours initiated metastatic spread in all animals. Gradual increase of the radiation doses diminished both the frequency of the appearance of metastases and their distance from the primary lesions. There were no metastases from non-irradiated melanomas. The highest dose used (60 cGy) did not affect significantly the growth of cutaneous (primary) tumours, but lower doses that enhanced inflammatory infiltration of the lesions reduced tumour growth. Such radiation-stimulated immune responses were accompanied by increased pigmentation in cutaneous lesions and activation of the adrenal cortex indicating that the immune system–adrenal axis feedback loop had been triggered. The results demonstrate that very low-doses of ionising radiation induce melanoma metastases. The phenomenon is accompanied by the stimulation of the immune system–adrenal axis feedback loop that regulates eicosanoid synthesis, thereby suggesting an involvement of these molecules in the process. Radiation doses approaching the therapeutic level do not initiate melanoma dissemination.

Development of metastases in malignant melanoma is associated with an increase in the plasma L-dopa/L-tyrosine ratio.

In this prospective study we evaluated a new biochemical approach in which the plasma ratio of the melanin precursors L-dopa and L-tyrosine serves as a marker of metastatic dissemination in malignant melanoma. Control values (11.20 x 10(-5) +/- 2.92 x 10(-5)) were determined. The L-dopa/L-tyrosine ratio was evaluated in the plasma of 90 patients with malignant melanoma (stage I/II, n = 33; stage III, n = 33; stage IV, n = 24) classified according to the tumour/node/metastasis (pTNM) classification. A total of 106 samples were studied. Serial measurements were performed in eight stage III-IV patients. The L-dopa/L-tyrosine ratio was significantly elevated in melanoma patients with clinical stage III (15.23 x 10(-5) +/- 3.34 x 10(-5)) compared with stage I (10.88 x 10(-5) +/- 2.52 x 10(-5)). Stage IV patients showed a significant increase in the plasma L-dopa/L-tyrosine ratio (45.73 x 10(-5) +/- 61.75 x 10(-5)) compared with the other groups. The ratio was higher for those with two rather than one metastatic site and markedly higher for those with widespread metastases. The development of metastases was associated with an increase in plasma L-dopa, a decrease in plasma L-tyrosine and a significant increase in the plasma L-dopa/L-tyrosine ratio. These data suggest that the plasma L-dopa/L-tyrosine ratio reflects the tumour burden and correlates with the progression of malignant melanoma.

Targeting melanoma with 211At/131I-methylene blue: preclinical and clinical experience.

The increasing incidence of melanoma and a lack of effective therapy have stimulated a search for new methods of early detection and treatment of the disease. Melanin synthesized in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognized by a suitable drug. Methylene blue possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumor, methylene blue serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localized source of radiation. Hence, radioderivatives of the compound can be used for diagnosis and therapy of disseminated melanoma. 131I-methylene blue in conjunction with gamma camera imaging has already proved in clinical studies to be a useful tool for the detection of early melanoma dissemination. 211At-methylene blue exceptional efficacy in treating melanoma and preventing its metastatic spread without damaging normal structures when administered systemically to human melanoma-bearing mice led to the approval of this alpha-particle emitting methylene blue derivative for the Phase I clinical trial.

Prognostic value of MMP‐2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (≥34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P<0·05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P<0·05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4·5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis. © 1998 John Wiley & Sons, Ltd.

Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma.

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (> or = 34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P < 0.05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P < 0.05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4.5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis.

Early detection of melanoma metastases with radioiodinated methylene blue.

Melanin synthesised in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognised by a suitable drug. Methylene blue (MTB) possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumour, MTB serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localised source of radiation. Hence, radioderivatives of the compound can be used for both diagnosis and therapy of disseminated melanoma. Eleven patients with confirmed metastatic melanoma and one with a recent local recurrence were studied using radioiodinated (iodine-123 or iodine-131) MTB and a gamma camera. Biopsies of cutaneous lesions were taken to determine directly the compound uptake in tumours. This first clinical investigation concerning the diagnostic potential of radioiodinated MTB in patients with disseminated melanoma confirmed the existence of approximately 80% of internal lesions previously identified by routine methods and, additionally, enabled detection of unknown secondaries in 6 of 12 patients studied. There were no false-positive gamma camera images regardless of whether 123I or 131I was used. 131I proved to be more suitable than 123I for detecting melanoma metastases with radioiodinated MTB. Hazy images of the lesions treated with external beam radiation and/or some drugs suggest that initial radio- and chemotherapy might affect MTB uptake in melanoma metastases and reduce the clarity of the scintigrams obtained from a gamma camera. However, small, untreated internal lesions that cannot be visualised easily with the standard diagnostic methods are revealed with 131I-MTB regardless of their localisation. It is concluded that use of radioiodinated MTB in conjunction with gamma camera or positron emission tomographic imaging might prove to be a useful and accessible tool for the detection of early melanoma dissemination.

Enhanced expression of vascular endothelial growth factor in metastatic melanoma.

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.

MIB-1 immunoreactivity correlates with metastatic dissemination in primary thick cutaneous melanoma

Background: The proliferation rate of transformed cells is a putative prognostic indicator. MIB-1 is a murine monoclonal antibody to a Ki-67 epitope that detects a nuclear antigen found only in proliferating cells. Objective: The aim of this study was to test for a correlation between MIB-1 immunoreactivity and the metastatic potential of malignant melanoma. Methods: MIB-1 reactivity (% total tumor nuclei) was assessed in 34 formalin-fixed, paraffin-embedded primary cutaneous melanomas and correlated with metastatic potential and overall survival (follow-up, 10.5 ± 1.8 years). Results: Whereas no correlation was found between MIB-1 reactivity and metastases in primary thin cutaneous melanoma (Breslow thickness, <0.75 mm; mean thickness, 0.39 ± 0.16 mm), good correlation was found ( p = 0.0001) in primary thick cutaneous melanoma (Breslow thickness, >1.5 mm; mean thickness, 3.0 ± 1.3 mm). MIB-1 reactivity was 12.3% ± 7.7% and 0.7% ± 1.3% with and without metastases, respectively, and was highest in the primary melanomas that later metastasized. However, overall survival in patients with thick cutaneous melanoma and metastases did not correlate with MIB-1 reactivity. Conclusion: MIB-1 proliferative activity is a useful prognostic indicator in primary cutaneous melanomas thicker than 1.5 mm and may predict the development of metastases.

Reverse transcriptase-polymerase chain reaction for expression of tyrosinase to identify malignant melanoma cells in peripheral blood

Circulating tumour cells in the peripheral blood may be important for haematogenous spread of disease. The detection of these cells may therefore be a poor prognostic indicator. Reverse-transcriptase polymerase chain reaction (RT-PCR) of target tumour-specific protein expression has been used as a sensitive and specific method for the detection of these tumour cells. Initial reports by our laboratory and other suggested RT-PCR amplification of the enzyme tyrosinase is a useful method for detection of melanoma cells in peripheral blood [1-3]. In this report, we have evaluated the application of RT-PCR for tyrosinase mRNA as a detection method for melanoma cells in a series of 24 patients with advanced, metastatic malignant melanoma. A single round RT-PCR method is described. The single round RT-PCR was as sensitive as previously described nested PCR methods, and had the advantage of reduced contamination risks. Blood samples from three out of the twenty-four patients were positive. The frequency of tumour cell detection in peripheral blood from patients with advanced disease was lower than previously reported. It may be only small numbers of circulating tumour cells are present at any one time in the peripheral blood of patients with malignant melanoma. If this is the case increased sampling will improve detection frequency. Alternatively, dissemination of melanoma through peripheral blood may be a rare event. In our experience, RT-PCR for tyrosinase mRNA as a staging test for melanoma patients must be interpreted cautiously.

The detection of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction

Both cutaneous and uveal melanoma undergo haematogenous dissemination. Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) has been described as an extremely sensitive way of detecting circulating viable melanoma cells in the peripheral venous blood, and this technique may be of value in the early detection of dissemination. Also, it has been suggested that surgical manipulation of the eye, such as occurs during enucleation, can provoke uveal melanoma dissemination. The purpose of this study was to evaluate whether tyrosinase mRNA is detectable in the peripheral blood of patients with uveal and cutaneous melanoma and in patients with uveal melanoma undergoing surgical procedures on the eye harbouring the tumour. Venous blood samples from 36 patients diagnosed as having active uveal melanoma and from six patients with advanced metastatic cutaneous melanoma were analysed. In addition, blood samples were spiked with known numbers of cells from three cell lines and four primary uveal melanoma cultures. The reported sensitivity of the technique was confirmed, with an ability to detect down to one cell per ml of blood. All 51 blood samples from the 36 patients with uveal melanoma were negative, and this included 20 perioperative blood samples. The test was also negative for the six patients with advanced cutaneous melanoma. There were two positives among 31 control samples analysed. This study demonstrates that there are far fewer circulating viable melanocytes than has been previously supposed in patients with melanoma and that the RT-PCR is of no clinical value in detecting metastatic melanoma disease. There was no evidence for surgery causing a bolus of melanoma cells to enter the peripheral circulation.

Differential Expression and Mutation of nme Genes in Autologous Cultured Human Melanoma Cells with Different Metastatic Potentials

The putative metastasis suppressor genes, NME1(nm23-1) and NME2(nm23-2),were examined in a model system we developed to approximate the dissemination of melanoma from a primary skin tumor. We utilized two autologous human melanoma cell lines, IV Cl 1 and IV Cl 3, which displayed qualitatively different metastatic phenotypes following subdermal inoculation into nude mice. Highly metastatic IV Cl 1 cells expressed approximately 5 fold lower levels of proteins encoded by NME genes than non-metastatic IV Cl 3 cells. Similar differences in NME protein levels were observed in tumors induced by the two cell lines in nude mice. There were no differences in NME mRNA levels between these two cell lines, suggesting that expression of these proteins is regulated at a post-transcriptional level. We found a ser 122-pro mutation in the NME2 gene of metastatic IV Cl 1 cells. A similar ser 120-gly mutation in NME1 has been found in human neuroblastoma, suggesting that mutation in this region may be a general phenomenon related to tumor progression. These mutations may have functional consequences since they eliminate potential phosphorylation sites and may affect the tertiary structure of mature protein complexes.

Continuous hyperthermic peritoneal perfusion with cisplatin in mice with peritoneal dissemination of B16 melanoma.

We designed a model of peritoneal dissemination of gastric cancer in mice. B16 melanoma cells were injected into the peritoneal cavity of C57 BL mice, the object being to search for an effective treatment for peritoneal dissemination, using hyperthermia combined with cisplatin. Following the intraperitoneal administration of tumor cells, each mouse was subjected to continuous hyperthermic peritoneal perfusion (CHPP) using the peritoneal cavity expander (PCE), with or without cisplatin in the perfusate under conditions of laparotomy. Effects of the therapy were evaluated, based on comparison of survival time between the control and treated groups. There was no prolongation of mean survival periods for the groups given CHPP, with or without cisplatin, compared to findings in the no treatment control group. Optimal timing and doses of hyperthermia and cisplatin, or adding biochemical modulators to enhance the effects of cisplatin, will need to be determined to increase the efficacy of CHPP, with or without the concomitant administration of cisplatin.

Granulocyte-macrophage colony-stimulating factor-induced antibody- dependent cellular cytotoxicity in bone marrow macrophages: application in bone marrow transplantation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to induce antitumor activity in peripheral blood monocytes. We examined the role of GM-CSF on bone marrow (BM) macrophages in inducing antibody-dependent cellular cytotoxicity (ADCC) against murine and human tumor cells in vitro and in vivo with the aim of applying this approach in an autologous bone marrow transplantation (BMT) setting. GM- CSF induced a potent ADCC in BM macrophages against a murine melanoma in vitro. Treatment with GM-CSF alone or with antibody alone had no effect, whereas therapy with combination of both these agents resulted in a significant reduction in dissemination of melanoma both in a nontransplant as well as in BMT settings, with results being more optimal in the latter setting. Adoptive transfer of BM macrophages harvested from mice undergoing therapy with GM-CSF plus antibody significantly reduced the dissemination of melanoma in secondary recipients but only after irradiation, not in intact mice. GM-CSF also induced significant ADCC in human BM macrophages against a melanoma and a lymphoma in vitro and against a lymphoma implanted in nude mice in vivo. Again, these effects were more optimal after chemotherapy. These data suggest that treatment with GM-CSF plus tumor-specific monoclonal antibodies after BMT may induce an antitumor effect and help eradicate the minimal residual disease.