Melanoma Micrometastases Research Articles

Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma

Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma

Dipyrimidine Amines: A Novel Class of Chemokine Receptor Type 4 Antagonists with High Specificity

The C-X-C chemokine receptor type 4 (CXCR4)/stromal cell derived factor-1 (SDF-1 or CXCL12) interaction and the resulting cell signaling cascade play a key role in metastasis and inflammation. On the basis of the previously published CXCR4 antagonist 5 (WZ811), a series of novel nonpeptidic anti-CXCR4 small molecules have been designed and synthesized to improve potency. Following a structure-activity profile around 5, more advanced compounds in the N,N'-(1, 4-phenylenebis(methylene)) dipyrimidin-2-amines series were discovered and shown to possess higher CXCR4 binding potential and specificity than 5. Compound 26 (508MCl) is the lead compound and exhibits subnanomolar potency in three in vitro assays including competitive binding, Matrigel invasion and Gα(i) cyclic adenosine monophosphate (cAMP) modulation signaling. Furthermore, compound 26 displays promising effects by interfering with CXCR4 function in three mouse models: paw inflammation, Matrigel plug angiogenesis, and uveal melanoma micrometastasis. These data demonstrate that dipyrimidine amines are unique CXCR4 antagonists with high potency and specificity.

Transmission of donor melanoma by organ transplantation

Transplant-related malignancies are a major contributor to morbidity and mortality in the organ-recipient population, and most often develop de novo in the immunosuppressed recipient or as recurrent malignancy after transplantation. The least common scenario, and a rare event, is a recipient malignancy derived from the donor organ. Melanoma is one of the most often reported and lethal donor-derived malignancies with a high transmission rate. Donor transmission of melanoma might be related to the biology of melanoma, with regard to tumour dormancy, late recurrence, circulating tumour cells, and the destiny of some micrometastases. Melanoma-cell dormancy explains the late recurrence that can occur after the initial treatment of melanoma, and may be relevant to our understanding and management of some melanoma micrometastasis in the sentinel node. The high incidence of circulating tumour cells in early melanoma should be considered in the context of the transmission of melanoma by apparent disease-free organ donors following removal of a primary melanoma up to 32 years before. This scenario suggests that melanoma cells can remain dormant at distant sites for decades (and possibly forever) in immunocompetent patients, only to reactivate after transplantation into an immunosuppressed recipient. Potential organ donors should be carefully screened for a history of melanoma, and excluded. The current recommendation for treatment of donor-related melanoma includes withdrawal of immunosuppression, graft rejection, and explantation of the allograft after rejection has been established. In non-renal transplant patients with life-sustaining organs, withdrawal of immunosuppression and graft rejection is not feasible, and reduction of immunosuppression or urgent retransplantation are the only possible salvage strategies. The transmission of malignancy by organ donation could be considered "nature's own experiment", but raises questions that our current understanding of the biology of melanoma cannot answer.

Bevacizumab suppression of establishment of micrometastases in experimental ocular melanoma.

This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastases. The human uveal melanoma cell lines Mel290 and Mel 270, HUVECs, mouse B16LS9 melanoma cells, and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture medium was measured by ELISA. In vitro angiogenesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mouse eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosage experiments were performed by using 50 or 250 microg bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis, and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining. Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. It also inhibited cell tube formation and decreased in vitro invasion of tumor cells. In the mouse model, bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab. Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.

Photoacoustic Detection of Melanoma Micrometastasis in Sentinel Lymph Nodes

Melanoma is the deadliest form of skin cancer and has the fastest growth rate of all cancer types. Proper staging of melanoma is required for clinical management. One method of staging melanoma is performed by taking a sentinel node biopsy, in which the first node in the lymphatic drainage path of the primary lesion is removed and tested for the presence of melanoma cells. Current standard of care typically involves taking fewer than ten histologic sections of the node out of the hundreds of possible sections available in the tissue. We have developed a photoacoustic method that probes the entire intact node. We acquired a lymph node from a healthy canine subject. We cultured a malignant human melanoma cell line HS 936. Approximately 1 x 10(6) cells were separated and injected into the lymph node. We also had a healthy lymph node in which no melanoma cells were implanted. We used a tunable laser system set at 532 nm to irradiate the lymph nodes. Three piezoelectric acoustic detectors were positioned near the lymph node to detect photoacoustic pulses generated within the lymph nodes. We also acquired lymph nodes from pigs and repeated the experiments with increased amplification and improved sensors. We detected photoacoustic responses from a lymph node with as few as 500 melanoma cells injected into the tissue, while normal lymph nodes showed no response. Photoacoustic generation can be used to detect melanoma micrometastasis in sentinel lymph nodes. This detection can be used to guide further histologic study of the node, increasing the accuracy of the sentinel lymph node biopsy.

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFN gamma and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.

The detection and significance of melanoma micrometastases in sentinel nodes

Sentinel node (SN) biopsy in melanoma patients is an accurate and minimally invasive method of staging and determining prognosis in patients with early-stage disease, and of identifying those patients who may benefit from complete regional lymph node dissection. Careful identification, removal and pathological assessment of SNs is critical to the accuracy of the technique and deficiencies in any of these steps may result in a false-negative biopsy. Pathologists should examine multiple sections from each SN, stained routinely with haematoxylin-eosin and immunohistochemically for melanoma-associated antigens. However, the most appropriate staining and sectioning protocol is not clear from the available evidence. While it appears that more extensive sectioning protocols detect more melanoma, failure rates and false-negative rates of the procedure do not appear to be significantly worse than with less extensive sampling protocols. Micromorphometrical parameters of melanoma deposits in SNs (such as their size, maximal tumour penetrative depth, microanatomical location and percentage nodal cross-sectional area) have been shown to be predictive of regional non-SN involvement and of clinical outcome. Classifying and measuring these parameters can be difficult, depending on the nature and distribution of the tumour deposits in the SN. Because the positive SNs have been removed in all patients included in studies assessing the clinical significance of tiny SN melanoma micrometastases, the likely therapeutic effect of the SN biopsy itself and/or any complete lymph node dissection, as well as lead time bias, are important confounding factors that must be considered when interpreting these studies. Limited data from some retrospective studies with relatively short follow-up suggest that some melanoma metastases may not be clinically relevant, but other studies imply a likely clinical significance of even very small SN metastases. Until there is unequivocal evidence from prospective randomised clinical trials with long term follow-up for the prognostic significance (or lack thereof) of very small SN tumour deposits, it is probably prudent to treat patients with such deposits as SN-positive.

Melanoma patients with melanoma micrometastases in sentinel node that refused completion lymphadenectomy

Journal of the European Academy of Dermatology and VenereologyVolume 22, Issue 8 p. 1008-1009 Melanoma patients with melanoma micrometastases in sentinel node that refused completion lymphadenectomy AM Manganoni, Corresponding Author AM Manganoni Department of Dermatology, *Corresponding author, Department of Dermatology, University Hospital Spedali Civili, P.le Spedali Civili 1, 25123 Brescia, Italy, tel. +390303995300; fax +390303995016; E-mail: [email protected];[email protected]Search for more papers by this authorC Farisoglio, C Farisoglio Department of Dermatology,Search for more papers by this authorG Tucci, G Tucci Department of Dermatology,Search for more papers by this authorF Facchetti, F Facchetti Department of Pathology I,Search for more papers by this authorR Farfaglia, R Farfaglia Department of General Surgery II,Search for more papers by this authorC Pizzocaro, C Pizzocaro Department of Nuclear Medicine, University Hospital Spedali Civili, Brescia, ItalySearch for more papers by this authorM Ungari, M Ungari Department of Pathology I,Search for more papers by this authorPG Calzavara-Pinton, PG Calzavara-Pinton Department of Dermatology,Search for more papers by this author AM Manganoni, Corresponding Author AM Manganoni Department of Dermatology, *Corresponding author, Department of Dermatology, University Hospital Spedali Civili, P.le Spedali Civili 1, 25123 Brescia, Italy, tel. +390303995300; fax +390303995016; E-mail: [email protected];[email protected]Search for more papers by this authorC Farisoglio, C Farisoglio Department of Dermatology,Search for more papers by this authorG Tucci, G Tucci Department of Dermatology,Search for more papers by this authorF Facchetti, F Facchetti Department of Pathology I,Search for more papers by this authorR Farfaglia, R Farfaglia Department of General Surgery II,Search for more papers by this authorC Pizzocaro, C Pizzocaro Department of Nuclear Medicine, University Hospital Spedali Civili, Brescia, ItalySearch for more papers by this authorM Ungari, M Ungari Department of Pathology I,Search for more papers by this authorPG Calzavara-Pinton, PG Calzavara-Pinton Department of Dermatology,Search for more papers by this author First published: 14 July 2008 https://doi.org/10.1111/j.1468-3083.2007.02538.x DOI: 10.1111/j.1468-3083.2007.02538.x Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume22, Issue8August 2008Pages 1008-1009 RelatedInformation

Human High Molecular Weight–Melanoma-Associated Antigen: Utility for Detection of Metastatic Melanoma in Sentinel Lymph Nodes

Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection. Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection. Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases. HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Why Does Sentinel Lymph Node Biopsy Not Increase Survival in Patients With Melanoma?

demonstrate. The findings of this study—published recently—indicate that this is not the case, since survival in the group of patients who initially underwent SLNB was similar to that of patients who were initially allocated to observation alone. 5 Here, we should emphasize a second point: long-term survival in patients with stage III melanoma is approximately 30%, 2 suggesting that delayed or therapeutic lymphadenectomy can be curative in a significant percentage Before trying to answer the question posed by the title of this article, we should perhaps ask another question: why might we expect sentinel lymph node biopsy (SLNB) to increase survival in patients with melanoma at all? If we think of SLNB as an essentially diagnostic technique, we would not expect the technique to provide therapeutic benefit or to directly (as opposed to indirectly) prolong survival in patients with melanoma. SLNB offers prognostic information of undoubted value, as reflected in the extensive review of the subject by Mangas et al 1 published in this same issue of Actas Dermo-Sifiliograficas. The prognostic value of detecting the presence of melanoma micrometastases in sentinel lymph nodes has led to SLNB being incorporated into the most recent tumor staging system published by the American Joint Committee on Cancer. 2 In my personal opinion, this inclusion is fully supported by the available data. It is at this point, however, that I shall place special emphasis on a first observation essential to following the line of argument in this article: positive SLNB is indicative of poor prognosis even though lymph node micrometastases are detected, in theory, at a very early stage in the process of melanoma spread. This poor prognosis associated with positive SLNB is a strong indication in itself that the technique detects a problem at a very early stage but that, in most cases, we are unable to resolve it (or at least, not to a greater extent than when the problem is detected later). However, a recent study by Essner 3 affirmed that debate about whether SLNB is a diagnostic or therapeutic intervention persists and Ferrandiz and Mangas, 4 in another recent article, concluded that we might indeed expect some direct therapeutic benefit from SLNB in patients with melanoma. Moreover, the design of the Multicenter Selective Lymphadenectomy Trial-I (MSLT-I) 5 was based on the belief that SLNB would improve overall survival in patients with melanoma, and that was also what the trial aimed to

Computational model predicts effective delivery of 188-Re-labeled melanin-binding antibody to metastatic melanoma tumors with wide range of melanin concentrations

Metastatic melanoma is almost always deadly and new methods of treatment are urgently needed. Recently, we established the feasibility of radioimmunotherapy (RIT) for experimental melanoma in mice using a 188-rhenium (188Re)-labeled monoclonal antibody (mAb) 6D2 (IgM) to melanin. Our objective was to determine the effects of varying tumor melanin concentration and of different diffusivities and lymphatic clearance rates of the normal tissue, on the absorbed dose to the tumor in simulated therapy, in preparation for a clinical trial of RIT for melanoma. Using finite element analysis (FEA), we created a pharmacokinetic model that describes melanin-targeting RIT of a melanoma micrometastasis (1.3-mm radius) imbedded in normal tissue (14.3-mm radius). Our method incorporates antibody plasma kinetics, transcapillary transport, interstitial diffusion, and lymphatic clearance. Michaelis-Menten kinetics was used to model mAb binding to tumor melanin for melanin concentrations of 76, 7.6, 0.76, 0.076, and 0.0076 micromol/l. An absorbed dose was calculated, after accounting for direct and crossfire irradiation, on the basis of a 7.4-GBq intravenous dose of 188Re-6D2. The results showed that penetration of mAb into the tumor was inversely proportional to tumor melanin concentration. Decreased diffusivity and increased lymphatic clearance of the surrounding normal tissue decreased the dose to the tumor. The formation of mAb-melanin complex was remarkably similar within a 1000-fold range of melanin concentration, resulting in total doses of 2840, 2820, 2710, and 1990 cGy being delivered to tumors with melanin concentrations of 76, 7.6, 0.76, and 0.076 micromol/l, respectively. In conclusion, RIT of metastatic melanoma can be effective over a wide range of tumor melanin concentrations. The results can be useful in the design of a clinical trial of melanin-targeting RIT in patients with metastatic melanoma.

Issues affecting molecular staging in the management of patients with melanoma

Prediction of metastatic potential remains one of the main goals to be pursued in order to better assess the risk subgroups of patients with melanoma. Detection of occult melanoma cells in peripheral blood (circulating metastatic cells [CMC]) or in sentinel lymph nodes (sentinel node metastatic cells [SNMC]), could significantly contribute to better predict survival in melanoma patients. An overview of the numerous published studies indicate the existence of several drawbacks about either the reliability of the approaches for identification of occult melanoma cells or the clinical value of CMC and SNMC as prognostic factors among melanoma patients. In this sense, characterization of the molecular mechanisms involved in development and progression of melanoma (referred to as melanomagenesis) could contribute to better classify the different subsets of melanoma patients. Increasing evidence suggest that melanoma develops as a result of accumulated abnormalities in genetic pathways within the melanocytic lineage. The different molecular mechanisms may have separate roles or cooperate during all evolutionary phases of melanocytic tumourigenesis, generating different subsets of melanoma patients with distinct aggressiveness, clinical behaviour, and response to therapy. All these features associated with either the dissemination of occult metastatic cells or the melanomagenesis might be useful to adequately manage the melanoma patients with different prognosis as well as to better address the different melanoma subsets toward more appropriate therapeutic approaches.

Melanoma micrometastases (&lt;0.1 mm) in sentinel nodes can be regarded as negative

Melanoma micrometastases (&lt;0.1 mm) in sentinel nodes can be regarded as negative

Clinical relevance of melanoma micrometastases (&lt;0.1 mm) in sentinel nodes: are these nodes to be considered negative?

As only about 20% of sentinel node (SN) positive melanoma patients have additional non-SN lymph node involvement in the Completion Lymph Node Dissection (CLND) specimen, we tried to identify a SN positive patient group, which can be spared CLND. Micro anatomic analyses of metastatic SNs were performed to identify patient/tumor and/or SN factors predicting additional non-SN positivity as well as disease-free and overall survival. SN positivity was found in 77 of 262 stage I/II patients, included into a prospective database (10/97-5/04). Of 74 patients pathology material was available for re-evaluation. Micro anatomic analyses categorized topography of SN-metastases, Starz classification and amount of SN tumor burden. Additional non-SN positivity, DFS, OS and was calculated for all analyses. Mean Breslow thickness was 3.5 mm (0.8-12.0); mean FU was 35 (6-81) months. There was no additional non-SN positivity for SN-micrometastases <0.1 mm. Topography of SN involvement had no impact on OS. Estimated 5-year OS rates for the different groups of <0.1 mm, 0.1-1.0 mm and >1.0 mm SN tumor burden were 100%, 63% and 35% respectively. Distant metastases were exceedingly rare (1/16 = 6.3%) in <0.1 mm SN-positive patients. On multivariate analysis the SN tumor burden was the most important prognostic factor for DFS (P = 0.005) and OS (P = 0.03). Distant metastasis-free survival was identical (91%) to the 5-yr OS of SN negative patients, the estimated 5-yr OS was 100% for these patients and additional non-SN positivity was not observed. Therefore, our data suggest that patients with sub-micrometastases (<0.1 mm) in the SN may be judged as SN negative, as non-stage III, and are highly unlikely to benefit from CLND, which we no longer recommend.

Intraoperative Frozen-Section Evaluation Can Reduce Accuracy of Pathologic Assessment of Sentinel Nodes in Melanoma Patients

Ariyan and colleagues recently reported their experience with sentinel lymph node (SLN) biopsies from 263 patients with primary cutaneous melanoma. The SLNs were evaluated by intraoperative frozen section (F/S). Paraffin-embedded sections of the SLNs, stained with hematoxylin and eosin and immunohistochemically for S100 and HMB45, were assessed subsequently. Twentyeight patients (10.6%) had melanoma in a SLN, 23 (82%) of whom had tumor identified intraoperatively on F/S. The authors concluded that their series “attests to the reliability of frozen sections in identifying SLN harboring metastases.” Although we do not dispute that F/S examination of SLNs, when performed by a practiced and committed group such as that of Dr Aryian and his colleagues, can have an occasional role in some specialist melanoma treatment centers, we are concerned that use of this technique in other professional contexts can reduce the accuracy and reliability of the SLN biopsy procedure. Although the authors went to some length to suggest that their low yield of positive nodes (10.6%) compared with most large series (15% to 34%) reflected the high percentage of patients with thin primary tumors and fewer thick melanomas in their series, we cannot draw the same conclusion from the limited data presented. Indeed, the rate of positive SLNs for most groups of tumor thicknesses is lower than in the other series referenced in their article, and considerably lower than in a published (but uncited) report from the M D Anderson Cancer Center (Table 1). Unfortunately, the very limited clinicopathologic details provided prevent statistical comparison with other series. The inexplicably low rate of SLN positivity in the study raises the disturbing possibility that use of F/S might have led to a substantial number of false-negative SLNs resulting from loss of diagnostic tissue during the technical manipulations required for the F/S procedure, and/or that subsequent permanent section analysis may have been rendered more difficult by earlier freezing of some or all of the tissue. Our extensive combined experience with intraoperative F/S evaluation of SLNs, obtained during development of the modern SLN approach, was associated with a failure to identify melanoma micrometastases in 4% to 8% of ultimately positive patients. In view of this, and the practical considerations detailed below, we have long discontinued performing routine F/S examination on SLNs from melanoma patients. There is a major possibility that limited amounts of relatively localized diagnostic material in an SLN may be lost as a consequence of examining the node by F/S. First, substantial tissue is inevitably lost when “facing up” the frozen tissue block. Facing up is removal of tissue to obtain a section that contains all areas of the tissue. Melanoma micrometastases are most likely to lodge in the subcapsular region of an SLN and in adjacent and abutting lymphoid tissues around the central plane through the hilum and longest dimension of the node. It is absolutely critical that this portion of the SLN is included in the F/S and examined carefully. To obtain a full-face F/S that includes the subcapsular sinus region of the entire SLN, it is unavoidable that substantial nodal tissue is lost during facing up. Because it is difficult to make the cut surface of the SLN sit flat during freezing of the tissue, some discarded tissue inevitably derives from the critical subcapsular region. When a fresh SLN is bisected, the nodal capsule retracts and central tissue bulges from the cut surface. Metastases that are localized only in the central part of the node, as is occasionally observed, may also be discarded as a result of the facing-up procedure. Second, because tissue and cellular preservation is generally inferior in frozen

Molecular classification of melanoma using real‐time quantitative reverse transcriptase‐polymerase chain reaction

The early detection and characterization of metastatic melanoma are important for prognosis and management of the disease. Molecular methods are more sensitive in detecting occult lymph node metastases compared with standard histopathology and are reported to have utility in clinical diagnostics. Using real-time quantitative reverse transcriptase-polymerase chain reaction ([q]RT-PCR), the authors examined 36 samples (30 melanomas, 4 benign nevi, and 2 reactive lymph nodes) for the expression of 20 melanoma-related genes that function in cell growth and differentiation (epidermal growth factor receptor [EGFR], WNT5A, BRAF, FOS, JUN, MATP, and TMP1), cell proliferation (KI-67, TOP2A, BUB1, BIRC5, and STK6), melanoma progression (CD63, MAGEA3, and GALGT), and melanin synthesis (TYR, MLANA, SILV, PAX3, and MITF). In addition, samples were tested for mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3). Hierarchical clustering analysis of the expression data was able to distinguish between the melanoma and nonmelanoma samples and further stratified the melanoma samples into two groups differentiated by high expression of the genes involved in beta-catenin activation (EGFR and WNT5A) and the MAPK/ERK pathway (BRAF, FOS, and JUN). Eighteen of the 28 patients (64%) were found to have mutations in either exon 15 of BRAF (V599 substitution) or codon 61 of NRAS. The mutations were mutually exclusive and did not appear to be associated with the different expression subtypes. The results of the current study demonstrate that real-time qRT-PCR can be analyzed using hierarchical clustering to identify expression patterns that differentiate between melanomas and other tissue types. Using a supervised analysis of the data, the authors found that the best discriminators for molecularly distinguishing between melanoma, benign nevi, and lymph nodes were MLANA, CD63, and BUB1. These markers could have diagnostic utility for the detection of melanoma micrometastasis in sentinel lymph nodes.

Predictors of Nonsentinel Lymph Node Positivity in Patients with a Positive Sentinel Node for Melanoma

Patients found to harbor melanoma micrometastases in the sentinel lymph node (SLN) are recommended to proceed to complete lymph node dissection (CLND), although the majority of patients will have no additional disease identified in the nonsentinel lymph nodes (NSLNs). We sought to assess predictive factors associated with finding positive NSLNs, and identify a subset of patients with low likelihood of finding additional disease on CLND. We queried our prospective melanoma database for patients from January 1996 to August 2003 with a positive SLN. Univariable logistic regression models were fit for multiple factors and a positive NSLN. To derive a probabilistic model for occurrence of one or more positive NSLN(s), a multivariable logistic model was fit using a stepwise variable selection method. Of 980 patients who underwent SLN biopsy for cutaneous melanoma, 232 (24%) had a positive SLN; 221 (23%) followed by CLND. Of these patients, 34 (15%) had one or more positive NSLN(s). In multivariable analysis, male gender (odds ratio [OR] 3.6 [95% CI 1.33, 9.71]; p = 0.01), Breslow thickness (OR 4.58 [95% CI 1.28, 16.36]; p = 0.019), extranodal extension (OR 3.2 [95% CI 1.0, 10.5]; p = 0.05), and three or more positive sentinel nodes (OR 65.81 [95% CI 5.2, 825.7]; p = 0.001) were all associated with the likelihood of finding additional positive nodes on CLND. Of 47 patients with minimal tumor burden in the SLN, only 1 (2%) had additional disease in the NSLN. These results provide additional data to plan clinical trials to answer the question of who can safely avoid CLND after a positive SLN. Patients with minimal tumor burden in the SLN might be the most likely group, although defining "minimal tumor burden" must be standardized. Serial sectioning and immunohistochemistry on the NSLN in any "low-risk" group must be performed in a clinical trial to confirm that residual disease is unlikely before avoiding CLND can be recommended.

Melanoma Controversies: Clinical Significance of Nodal Micrometastases and the Future of Melanoma Vaccines

There is no greater impediment to progress in the sciences than the desire to see it take place too quickly.—G.C. Lichtenberg (1742–1799), German physicist, philosopher Failure after long perseverance is much grander than never to have a striving good enough to be called a failure.—George Eliot [Mary Ann Evans] (1819 –1880), British novelist Progress is not an illusion, it happens, but it is slow and invariably disappointing.—George Orwell (1903–1950), British novelist In this issue of World Journal of Surgery, Leong et al. [1] evaluate the clinical significance of lymph node metastases in melanoma detected by sentinel lymph node (SLN) biopsy, and Spagnoli et al. [2] review the progress in development of active antigen-specific immunotherapy. These articles provide a convenient excuse to reflect upon the progress in melanoma staging and treatment. Leong and colleagues studied 363 patients with cutaneous melanoma for whom they have a median follow-up of 4.8 years. Sentinel nodes were evaluated by routine histopathology with hematoxylin and eosin (H&E) staining and immunohistochemical stains. However, the first 294 patients did not undergo immunohistochemistry (IHC) testing for evaluation of the sentinel nodes. Therefore the nodal metastases detected by H&E staining alone in these 294 patients likely represent larger and more obvious foci of metastatic disease in the lymph node. Overall, 18% of their patients had positive sentinel nodes. Mortality was significantly associated with sentinel lymph node status as well as lymphovascular invasion, ulceration, mitotic index, and Breslow thickness. This is yet another study indicating that SLN biopsy can accurately stage the regional lymph nodes and that SLN status is an independent prognostic factor predicting recurrence and survival. Most (80–85%) patients with a ‘‘positive’’ SLN have only a single SLN that contains metastatic disease. With serial section histopathology and IHC analysis, it is possible to identify extremely small foci of microscopic disease within the SLN—sometimes even single cells. Do patients with a few melanoma cells in the SLN have the same prognosis as those whose disease is measured in millimeters? Until recently, there had been little controversy over the clinical significance of nodal micrometastases in melanoma. It was thought by many that adequate data were available to substantiate the fact that patients with a positive sentinel node detected by histology or IHC had a worse prognosis. On the other hand, the clinical significance of nodal micrometastases in breast cancer has generated enormous controversy. Do nodal micrometastases in melanoma have a different significance than they do in breast cancer? To shed light on this issue, several investigators have now evaluated various means of measuring the microscopic tumor burden in the sentinel nodes of melanoma patients and have found a strong correlation with disease-free and overall survival [3–6]. It is becoming increasingly clear that it is no longer sufficient to simply call sentinel nodes ‘‘positive’’ or ‘‘negative’’—it is likely that the amount and pattern of microscopic disease in the lymph node has important prognostic significance. Whether micromorphometry-based classification systems [3, 4] or other means of classifying the tumor burden in the sentinel node [5, 6] ultimately are the best way to evaluate SLN pathology remains to be determined. Nonetheless, there may be some minimum volume and distribution of melanoma cells in the SLN that have little impact on prognosis. We may also find ageand gender-related differences in the clinical significance of minimal nodal disease. It is therefore imperative that future studies incorporate careful analysis of the tumor burden in the sentinel nodes. Why is this so important? Because it affects treatment decisions, including the decision to perform a completion lymph node dissection and the decision to give adjuvant therapy. Completion lymph node dissection is currently recommended for patients with a positive sentinel node, regardless of the amount of the tumor burden in the lymph node [7, 8]. The rate of finding additional positive lymph nodes upon completion lymph node dissection ranges from 12% to 14% for the lowest-risk group of patients—those who have had positive sentinel nodes detected only by IHC [8, 9]. Whether patients can safely avoid the potential morbidity of completion lymph node dissection is a topic being Correspondence to: Kelly M. McMasters, M.D., Ph.D., e-mail: mcmasters@louisville.edu World J. Surg. 29, 681–682 (2005) DOI: 10.1007/s00268-005-1113-7

Immunohistochemical detection of incipient melanoma micrometastases. Relationship with sentinel lymph node involvement

It is acknowledged that tumour thickness, ulceration and lymph node invasion are the most important prognostic factors for cutaneous melanomas. Other histopathological features may also be informative. The aim of this study was to ascertain whether immunohistochemical methods can improve the detection of satellite micrometastases in primary melanoma patients. In addition, the predictive value of cutaneous satellite micrometastases for sentinel lymph node involvement was evaluated. A total of 265 primary cutaneous melanomas and 68 of the respective sentinel nodes were studied using a panel of seven antibodies directed against melanocyte-related antigens. In 12.4% of the 265 cases, small satellite micrometastases were detected by immunohistochemistry. Sentinel lymph node metastases were found in 14% of the 68 cases. Invasion of the sentinel lymph node correlated with the presence of cutaneous satellite micrometastases. It is concluded that the presence of cutaneous satellite micrometastases may be an indication for the performance of sentinel lymph node biopsy, and this finding calls for a closer follow-up of these patients.

Pathology of melanocytic lesions: new, controversial, and clinically important issues.

Patients with primary cutaneous melanocytic lesions rely not only on the knowledge, skills, and experience of their treating clinician but also on the fundamentally important input of their pathologist for accurate diagnosis and appropriate management. Free and precise communication between pathologists and surgeons is important and undoubtedly improves patient care, particularly when managing difficult or complicated cases. To provide both patient and surgeon with the necessary information they require to make the most appropriate decisions, the pathology report should include all pathologic factors that are important in determining the patient's prognosis and management. Use of a synoptic format for pathology reporting of melanomas can facilitate this. Recent studies have established that the dermal mitotic rate of a primary cutaneous melanoma is a major prognostic determinant, and have shown that its assessment and that of other important histopathologic prognostic variables are reproducible between pathologists. Sentinel node (SN) biopsy has provided a minimally invasive procedure that can accurately predict the regional node status of melanoma patients. It is well demonstrated that the use of immunohistochemical stains assists in the detection of melanoma micrometastases in SNs, although it remains unclear which is the optimal pathologic protocol for SN evaluation and whether there is a role for reverse transcriptase polymerase chain reaction (RT-PCR) in SN assessment. False negative SN biopsies may occur as a result of errors in lymphatic mapping or sentinel lymphadenectomy, or because of a deficiency in the process of histopathologic evaluation. Recent studies have shown that the likelihood of non-SN involvement when the SN is positive correlates mostly with the extent of SN involvement, in particular the tumor penetrative depth (defined as the maximum distance of melanoma cells from the inner margin of the SN capsule). It appears that assessment of the micromorphometric features of positive SNs may be useful in predicting which patients have a low probability of having metastatic tumor in non-SNs, and therefore in selecting patients who potentially may be spared a completion lymph node dissection. It is likely that future advances in our understanding of the molecular biology of melanoma will provide new insights into tumor classification, improve diagnostic accuracy and prognostic ability, and lead to the development of more precisely targeted therapies.