Cutaneous Melanoma Metastases Research Articles

Angiotropism of Human Melanoma: Studies Involving In Transit and Other Cutaneous Metastases and the Chicken Chorioallantoic Membrane

Melanoma cell migration along the outside of vessels has been termed "extravascular migratory metastasis" (EVMM), as distinct from intravascular dissemination. Previous studies in both human and experimental melanoma models have shown angiotropism of melanoma cells, suggesting EVMM. Our objectives are to study the mechanism of dissemination of human melanoma cells in the chick chorioallantoic membrane (CAM) and to compare the histopathology in the CAM with that of patients with in transit and other cutaneous melanoma metastases. Human and murine melanoma cells were inoculated onto the CAM and observed over a 10-day period for tumor dissemination. Both human melanoma specimens from 26 patients and melanoma cells growing on the CAM showed the presence of tumor cell angiotropism at the invasive front of the tumor and at some distance from the tumor mass. In addition, a clear progression of melanoma cells spreading on the CAM was observed along the abluminal surface of vessels, where they occupied a perivascular location. By day 10 after injection, small micrometastases had developed along vessels, in a pattern similar to that in transit and other cutaneous melanoma metastases. In addition, the results suggested that the number of micrometastases directly correlated with increasing tumor volume. Taken together, these data suggest that the CAM is a relevant model for studying tumor cell dissemination, and that EVMM may be a mechanism by which some melanoma cells spread to nearby and even distant sites.

Diagnosis of lymphoma in the draining lymph node basin at the time of in-transit metastasis of cutaneous melanoma

The association between cutaneous melanoma and non-Hodgkin's lymphoma (NHL) has been described. Herein we describe two cases in which lymphoma was detected in the draining lymph node basin at the time of the finding of in-transit metastasis of cutaneous melanoma. In both of these cases, sentinel lymph node biopsy at the time of the excision of the primary melanoma showed no evidence of metastatic melanoma or lymphoma. The temporal and spatial correlation of melanoma and NHL in these two cases suggests the possibility of a local failure in immune surveillance.

The correlation of angiogenesis with metastasis in primary cutaneous melanoma: a comparative analysis of microvessel density, expression of vascular endothelial growth factor and basic fibroblastic growth factor

To establish whether there is a correlation between angiogenesis and metastasis in primary cutaneous melanoma (PCMM). We studied the microvessel density and the expression of vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) in 22 cases of PCMM with metastasis at presentation (metastatic group) and 28 cases of PCMM without metastasis for 24 months or more (non-metastatic group). Microvessels were stained with CD31/PECAM-1 antibody and counted. We assessed the proportion of VEGF expression in tumour cells, lymphocytes infiltrating the tumour (TIL) and lymphocytes at the periphery of the tumour, as well as the proportion of bFGF expression in tumour cell cytoplasms, nuclei and intra- and peritumoral vessels. An increased microvessel density was detected in the metastatic group (15-33 [24.09 +/- 5.55] versus 2-24 [12.96 +/- 6.02]). Moreover, enhanced expression of VEGF in tumour cells and peritumoral lymphocytes (Chi-square p = 0.038 and p = 0.018) and bFGF in peritumoral vessels (chi(2) p = 0.013) correlated with the simultaneous presence of melanoma metastasis in PCMM. Furthermore, microvessel density was correlated with the expression of bFGF in peritumoral vessels (rs = 0.53, p = 0.049) and VEGF in tumour cells (rs = 0.37, p = 0.019). Microvessel density as well as the expression of both VEGF and bFGF might be informative concerning the progression of melanoma.

Heterogeneity of T-Cell Clones Infiltrating Primary Malignant Melanomas

It is established that primary malignant melanomas (pMM) can be infiltrated by T-cell populations with predominantly one T-cell clone. As pMM generally express multiple tumor-associated antigens (TAA), here we used laser-capture microdissection (LCM) to isolate different tumor-infiltrating lymphocyte (TIL) clusters in order to determine whether pMM are infiltrated only by one single clone or whether the TAA may attract various T-cell populations. As T-cell receptor (TCR) clonality is a useful tool for the demonstration of specific T-cell clones, we analyzed 56 pMM, three cutaneous melanoma metastases, and 15 pairs of pMM with a sentinel lymph node (SLN) for clonal rearrangements of the (TCR) gamma chain gene. We detected the clonality of TCR gamma chain gene in 25 of 56 pMM, and in 10 of 17 SLN studied. In four of the 15 pairs of primary tumor and SLN, we found clonal TCR gamma in both the melanoma and the SLN, with two pairs harboring the identical clone. As we detected different clones in pMM and the corresponding SLN, we subsequently performed LCM in 21 malignant melanomas with multiple lymphocytic clusters for the presence of focal clonal T cells in different regions of the melanoma. In seven melanomas, both clusters of TILs showed the same rearranged TCR gamma chain gene and in five of the seven biopsies the clonal rearrangement occurred in different variable (V) regions of the TCR gamma chain gene. These tumors showed infiltration by more than one clone. In 10 biopsies TCR clonality was restricted to one cluster, while the second microdissected sample of the infiltrate was polyclonal. In conclusion, within one primary malignant melanoma several T-cell clones with different rearrangements may occur. The balance between these clones may decide on the progress of melanoma.

Expression of stromal cell markers in distinct compartments of human skin cancers

The importance of changes in the supporting tumor stroma for cancer initiation and progression is well established. The characteristics of an activated tumor stroma, however, are not completely understood. In an effort to better characterize the desmoplastic response to human skin tumors, we evaluated the expression pattern of three stromal cell markers, fibroblast-activation protein (FAP), endoglyx-1, and endosialin, in a series of melanocytic and epithelial skin tumors. Immunohistochemistry using antibodies against FAP, endoglyx-1, and endosialin was carried out in skin samples obtained from 43 patients. Furthermore, microarray data from an independent set of human skin cancers were analyzed. FAP-positive fibroblasts were detected in all tumor tissues tested, including cases of melanocytic nevi, melanoma metastases, basal cell carcinomas, and squamous cell carcinomas. In cutaneous melanoma metastases, we identified different compartments within the stromal response on the basis of the regions of FAP expression. Endoglyx-1 expression was confined to normal and tumor blood vessel endothelium including 'hot spots' of neoangiogenesis within the cutaneous melanoma metastases. Endosialin was selectively induced in subsets of small- and medium-sized tumor blood vessels in melanoma metastases and squamous cell carcinomas. These data describe novel aspects of stromal marker expression in distinct compartments of human skin tumors and may point to potential targets for novel therapeutic strategies aimed at the tumor stroma.

Expression pattern of the lymphatic and vascular markers VEGFR-3 and CD31 does not predict regional lymph node metastasis in cutaneous melanoma

Malignant melanoma of the skin preferentially metastasises via the lymphatic system. Novel molecular biomarkers, which are involved in malignant transformation, proliferation, angiogenesis and lymphangiogenesis, are currently under investigation to elucidate the risk for lymph node metastasis. To this end, the vascular endothelial growth factors VEGF-C and VEGF-D have been identified to promote lymphangiogenesis and lymphatic spread through activation of its receptor, Vascular endothelial growth factor receptor-3 (VEGFR-3). Prompted by this assumption, we estimated the degree of lymphangiogenesis by semiquantitative immunohistochemical analysis of the expression of VEGFR-3 and the panvascular marker CD31 in primary cutaneous melanoma (n=26) and correlated these findings with the sentinel lymph node (SLN) status. The cohort was selected for matched prognostic markers in SLN-positive and SLN-negative patients. In contrast to other studies, we observed an inverse correlation between expression of these markers with lymph node metastases. Additionally, no difference between intratumoral versus peritumoral CD31- or VEGFR-3 expression on blood vessels versus lymphatic capillaries could be detected. Interestingly, VEGFR-3 upregulation was not restrained to vascular structures but also appeared on tumor cells. In summary, in our series VEGFR-3/CD31 immunohistochemical staining of primary melanoma does not serve as a valid marker to predict lymph node involvement. As lymphatic spread is a complex, multi-step process, several different biomarkers have to be combined to define new prognostic subgroups in cutaneous melanoma.

Carbon dioxide laser ablation as an alternative treatment for cutaneous metastases from malignant melanoma.

Surgical excision of cutaneous malignant melanoma metastases is practical only when the number of lesions is small. In some patients isolated limb perfusion is not possible or fails to achieve control, and carbon dioxide laser ablation is then an alternative treatment. Between September 1992 and September 1994, 19 patients aged 45-94 years were treated with carbon dioxide laser. Two patients had received no previous limb perfusion, while the remaining 17 had up to three previous perfusions. The number of lesions per patient ranged from three to 40 nodules. A Sharplan 1030 portable carbon dioxide laser was used, which generates a beam wavelength of 10.6 microns and has a maximum power output of 80 W. All procedures were carried out under general anaesthesia. Each lesion was destroyed with a focused laser beam of 10-20 W with a spot size of 0.5-1.0 mm. At a mean follow-up of 15 months, five patients have died from the disease. Among the 14 survivors, eight have had no limb recurrence of the disease, three have had one further treatment and three a further two treatments to control cutaneous metastases at new sites. Early experience suggests that carbon dioxide laser ablation of cutaneous metastases is an effective palliative treatment after failed isolated limb perfusion, and there may be a group of patients in whom laser ablation should be the initial treatment of choice.

Signaling Networks in Cutaneous Melanoma Metastasis Identified by cDNA Microarrays

Signaling Networks in Cutaneous Melanoma Metastasis Identified by cDNA Microarrays

Sentinel lymph node biopsy in oral malignant melanoma—a possible means of investigating the clinically node-negative neck

Sentinel node biopsy has been used to determine the presence of nodal metastases in cutaneous melanoma and is being investigated as a tool in patients with oral squamous cell carcinomas. Two patients at Canniesburn Plastic Surgery Unit with oral mucosa malignant melanoma underwent sentinel node biopsy, using a combination of preoperative lymphoscintigraphy to identify lymphatic drainage, the preoperative use of blue dye injection (in one case) and localisation of radioactive nodes with a hand-held gamma probe (in both cases). The first patient presented with a well lateralised anterior tongue melanoma and was found to have “sentinel” nodes on both sides of the neck, all of which were clear of tumour; the second patient had diffuse melanosis of the palate with an invasive component on the left side and was found to have a sentinel node on the right side containing melanin. Our experience suggests that sentinel node biopsy is technically possible for oral melanoma and may be used to investigate the neck of patients with oral melanoma.

A Preliminary Study of Imiquimod Treatment in Variants of Basal Cell Carcinoma

Imiquimod is a topical immune response modifier that has proved efficacious in the treatment of the superficial variant of basal cell carcinoma. The nodular variant of basal cell carcinoma has shown moderate response to imiquimod; other variants have not been tested. The mechanism of action is largely unknown; however, studies indicate the mechanism involves alteration of local cytokine production. The objective of this study is to evaluate the cytokine response of imiquimod in all variants of basal cell carcinoma. Ten patients were selected who had clinically and histologically proven basal cell carcinoma. All lesions were treated with imiquimod once a day, 5 days a week, for 3 weeks. After a 3-week rest period, the lesions were rebiopsied. All biopsy specimens were analyzed via polymerase chain reaction (PCR) for various cytokines. Nine of 10 lesions resolved clinically, which included nodular, superficial, infiltrative, adenoid, and micronodular variants. The cytokine with the greatest change pre- and post-treatment was IL-8, which decreased an average of 44 per cent (P = 0.06). We concluded that topical 5 per cent imiquimod is an effective treatment of various subtypes of basal cell carcinoma. IL-8, which plays an important role in the development and metastasis of melanoma, may be involved in the mechanism of action of imiquimod on cutaneous malignancies. Larger studies are needed to prove the efficacy of imiquimod on nonsuperficial variants of basal cell carcinoma and cutaneous melanoma metastasis.

Prognostic factors after cervical lymph node dissection for cutaneous melanoma metastases

Cervical lymph node dissection (CLND) is the surgical therapy used for the local control of regionally metastasized cutaneous head and neck melanoma. This study evaluated the outcome of patients undergoing CLND at our institution in order to determine the prognostic factors for recurrence-free survival and overall survival after this procedure. The hospital records of 66 patients with histologically proven lymph node metastases who underwent curative or palliative CLND between 1982 and 2004 were analysed. The characteristics of the patients, the primary tumour and the surgical procedure were recorded. During follow-up, the incidence of local or distant recurrences was recorded and the survival was determined. Of the 66 patients, a (modified) radical neck dissection was performed in 20 and a selective procedure in 46. The 5-year actuarial overall survival was 26% and the recurrence-free survival was 22%. Neither the primary tumour characteristics nor the extent of surgery was of prognostic value; the number of positive nodes affected both the overall survival (P=0.046) and overall recurrence-free survival (P<0.001). Selective CLND is the recommended procedure for patients with cervical metastases of cutaneous melanoma. The number of positive lymph nodes significantly affects the outcome of the patients.

Enhanced type I interferon signaling and recruitment of chemokine receptor CXCR3‐expressing lymphocytes into the skin following treatment with the TLR7‐agonist imiquimod

Imiquimod (Aldara) is an immune response modifier approved for the topical treatment of external genital and perianal warts which can mediate regression of several cutaneous malignancies [basal cell carcinoma (BCC), Bowen's disease, actinic keratosis, and metastasis of malignant melanoma]. Recently, it was discovered that imiquimod acts through the toll-like receptor (TLR) 7. We hypothesize that TLR7-signaling strongly induces the production of interferon (IFN) alpha, which is able to enhance Th1-mediated cellular antiviral and antitumor immunity. In the present study we analyzed the expression of MxA, a protein specifically induced by type I IFNs during topical imiquimod treatment in several patients suffering from different cutaneous malignancies (BCC, cutaneous metastasis of melanoma, and breast cancer), and characterized the inflammatory infiltrate, along with the expression of chemokine receptor CXCR3, by immunohistochemistry. Treatment with the TLR7-agonist imiquimod induced a significant lesional lymphocytic inflammation, associated with strong expression of MxA, indicating the induction of type I IFN signaling. The extent of lesional MxA staining closely correlated with the number of infiltrating T lymphocytes and the expression of the chemokine receptor CXCR3, characteristic for Th1-biased immune responses. Our in vivo results suggest an important role for TLR7-induced production of type I IFN, which links innate and adaptive immunity and promotes specific Th1-biased cellular immune response capable of eliminating cutaneous malignancies. MxA appears to be a valuable parameter to demonstrate IFN-type I expression in imiquimod therapy.

Signaling Networks in Cutaneous Melanoma Metastasis Identified by Complementary DNA Microarrays

Melanoma is a complex multigenic disease, susceptibility to which is determined by several parallel and stepwise progressive pathways affecting growth control, differentiation, cell adhesion, and survival. Melanoma and human cancers in general undergo a continuous development from benign to malignant states, as most thoroughly documented in the multistep mole-to-melanoma transition. To examine how high-throughput microarrays are being used in expression profiling to identify regulated genes, patterns, and pathways that may lead to functional characterization and tumor subclassification. Ten melanoma metastases were analyzed by DNA array technology for important regulated candidate genes, with subsequent confirmation by real-time reverse transcription polymerase chain reaction. Hepatocyte growth factor receptor c-met, growth factor receptor-bound protein 10, B-raf proto-oncogene, and several mitogen-activated protein kinase kinase genes were significantly up-regulated in melanoma metastases and several melanoma cell lines relative to normal human melanocytes (P = .03). Among the up-regulated genes, phosphorylated growth factor receptor-bound protein 10 is known to serve a molecular switch turning on the mitogen-activated protein kinase pathway in response to hepatocyte growth factor receptor binding. As suggested by the DNA arrays, we found the mitogen-activated protein kinase kinase/extracellular-regulated kinase pathway to be activated in most of the cutaneous melanoma metastasis specimens. These findings are in the context of the current microarray technology in melanoma research. Additional steps are needed to gain insights into the pluralistic signaling milieu of this malignancy as we enter the postgenomic era.

Loco-regional control of cutaneous metastases of malignant melanoma by treatment with miltefosine (Miltex®)

Malignant melanoma is a very heterogeneous malignant disease that can invade adjacent tissues and metastasize to any distant tissue and organ. The treatment of primary melanoma is predominantly sur...

The developmentally regulated neural crest-associated glycotope HNK-1 predicts metastasis in cutaneous malignant melanoma.

Aberrant glycosylation is a common feature of metastatic sub-clones of malignant tumours and in uveal melanoma in particular, the HNK-1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK-1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10-years' follow-up. Furthermore, HNK-1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan-Meier analysis revealed a positive association between HNK-1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK-1 expression as an independent prognostic marker. HNK-1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK-1 immunoreactivity. None of the benign pigmented lesions exhibited HNK-1 immunoreactivity. Expression of the HNK-1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK-1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK-1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma.

Immune modulation and apoptosis induction: two sides of the antitumoral activity of imiquimod.

Imiquimod, the first member of the imidazoquinoline family of immune response modifiers, has proven good clinical efficacy against basal cell carcinomas and actinic keratoses in several independent studies. In addition, there is recent evidence that imiquimod is also efficacious against other tumors such as cutaneous metastases of malignant melanoma or vascular tumors. Imiquimod exerts its antitumoral effect, at least in part, through binding to TLR-7 and TLR-8 on dendritic cells followed by secretion of a multitude of proinflammatory cytokines. The net result of this proinflammatory activity is a profound tumor-directed cellular immune response. However, recent experimental and clinical data indicate that imiquimod also possesses considerable direct pro-apoptotic activity against tumor cells both in vitro and in vivo. This novel mode of action appears to be independent of membrane bound death receptors, but involves caspase activation. Induction of apoptosis by imiquimod is, at least in part, presumably mediated through Bcl-2-dependent release of mitochondrial cytochrome c and subsequent activation of caspase-9. The structural analogue, resiquimod, exhibited very limited, if any, such pro-apoptotic activity, possibly due to its lacking ability to enter the cell. Bypassing molecular mechanisms of apoptosis deficiency by a topical compound may be of great utility for treating certain cutaneous tumors.

Death Receptor-Independent Apoptosis in Malignant Melanoma Induced by the Small-Molecule Immune Response Modifier Imiquimod

Bypassing molecular mechanisms of apoptosis deficiency may be of great utility for the successful treatment of malignant tumors. We have discovered that imiquimod, a small-molecule immunomodulator, exerts rather tumor-selective direct pro-apoptotic activity in vivo and in vitro towards cutaneous metastases of malignant melanoma, an aggressive skin tumor. This pro-apoptotic activity was not detectable with resiquimod, a closely related structural analogue whose pro-inflammatory activity is even greater than that of imiquimod. Unresponsiveness of some melanoma metastases to imiquimod in vivo corresponded to resistance towards imiquimod-induced apoptosis in vivo and in vitro. At the molecular level, the pro-apoptotic activity of imiquimod was independent of membrane-bound death receptors, but depended on Bcl-2 expression as demonstrated by overexpression of Bcl-2 in melanoma cells. Imiquimod is the first topical compound with the potential to bypass molecular mechanisms of apoptosis deficiency, a concept that may be relevant for other tumors as well.

Cutaneous amelanotic melanoma metastasis and dermatofibromas dermoscopically characterized by a dotted vascular pattern

Dermoscopic patterns specific for hypopigmented and amelanotic cutaneous lesions have not yet been truly characterized, making dermoscopic diagnosis of these lesions difficult. We report a dotted vascular pattern in three amelanotic lesions, including a cutaneous local recurrent melanoma and a dermatofibroma of a middle-aged woman, as well as a dermatofibroma of a young man with no history of melanoma. These findings suggest that the presence of a dotted vascular pattern in the absence of dermoscopic features typical of melanocytic lesions, does not lead to a definite diagnosis. The dermoscopic similarity of the malignant and non-malignant amelanotic lesions underscores the importance of considering clinical examination and patient history when amelanotic lesions are evaluated.

Locoregional cutaneous metastases of malignant melanoma and their management.

The correct classification of locoregional metastases of malignant melanoma to skin is central to the planning of treatment. Local recurrence means persistence of neoplastic cells at the local site by virtue of incomplete excision of the primary melanoma. Standard treatment is excisional surgery. In contrast, locoregional metastases of malignant melanoma (satellites, in-transit metastases) are metastases around a primary melanoma or between a primary melanoma and regional lymph nodes. They represent intralymphatic or hematogenous spread of neoplastic cells. We present a variety of available treatment options and discuss especially topical imiquimod as a novel approach for the palliative treatment of locoregional cutaneous melanoma metastases in selected patients.

Locoregional Cutaneous Metastases of Malignant Melanoma and their Management

Locoregional Cutaneous Metastases of Malignant Melanoma and their Management