Sentinel lymph node melanoma metastases: Assessment of tumor burden for clinical prediction of outcome in the first Multicenter Selective Lymphadenectomy Trial (MSLT-I).

PURPOSE: As clinical management decisions in patients with Stage III melanoma have become more complex, precise pathologic characterization of sentinel lymph node (SLN) metastases has become critical to guide management. The extent of SLN involvement correlates with risk of adverse outcomes, but reported methods of disease quantification vary. We examined SLN metastases from patients participating in an international clinical trial and compared several methods of tumor burden quantification. METHODS: SLNs from 146 node-positive patients in the first Multicenter Selective Lymphadenectomy Trial (MSLT-I) were centrally-reviewed and characterized by number of tumor-positive nodes, percent nodal area tumor replacement, maximum dimension of largest metastasis, tumor penetrative depth, number of tumor foci, metastasis microanatomic location, and extracapsular extension. These data were analyzed for correlation with non-SLN metastasis and melanoma-specific survival (MSS). RESULTS: The median number of tumor-involved SLNs was 1. The median maximum metastasis dimension was 1.11 mm. Median SLN area involvement was 1.5%. Tumor burden measures were highly correlated with each other. Factors associated with non-SLN metastasis by univariable analysis were primary tumor ulceration and extent of metastases. Tumor thickness, ulceration, non-SLN metastasis and multiple measures of SLN tumor burden were significantly related to MSS on univariable analysis. After multivariable adjustment, number of involved SLNs (p = 0.05) and percent nodal area tumor replacement (p = 0.02) were independent predictors of MSS. CONCLUSION: Central review of MSLT-I pathology indicates that primary tumor and SLN tumor characteristics predict non-SLN metastasis and MSS. Percent nodal involvement was more powerfully prognostic than the more commonly used maximum dimension of largest metastasis.

Is sentinel node susceptibility to metastases related to nodal immune modulation?

Sentinel lymph nodes (SLNs), the initial site of regional metastases, directly receive lymph containing immune-modulatory cytokines and tumor cells from primary melanomas. Immune-suppressed SLNs are ideal for studies of tissue susceptibility to metastases. They show reduced antigen-presenting dendritic cells, activated T cells, high endothelial venules, and transvenular immigration of T cells. Tumor-induced immune suppression contributes to establishment of nodal metastases. SLNs may serve as an effective model to study reversal of tumor-induced immune suppression. We reviewed this topic in Nature Reviews of Immunology in 2006. We here summarize the Nature paper and provide additional results from ongoing studies and the recent literature.

"Stealth" melanoma cells in histology-negative sentinel lymph nodes.

A proportion of patients who develop regional and distant recurrences of melanoma after a pathologically negative sentinel lymph node (SN) biopsy are reported to have enhanced signals for melanoma-associated messenger ribonucleic acid (mRNA) when sensitive molecular approaches such as reverse transcriptase polymerase chain reaction (RT-PCR) are used to evaluate their SN tissue. The significance of these findings remains controversial, because the cellular source of the augmented signals cannot be known as the nodal tissue is destroyed during preparation for RT-PCR. Nevertheless, it is claimed that the source of the augmented signal is covert metastatic melanoma cells. To determine whether there are histologically occult metastases in SN and whether there are sources of augmentable melanoma-associated mRNA other than melanoma cells, we applied reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) to formalin-fixed paraffin-embedded nodal tissue. This approach amplifies small amounts of melanoma-associated mRNA and permits identification of cells that express that mRNA. Cells containing MART-1 mRNA were detected in 6 of 21 SNs (29%) and 2 of 16 nonsentinel lymph node (NSNs) (13%) that were tumor negative on hematoxylin and eosin and on immunohistochemical assessment for S-100, MART-1, and HMB-45. In patients with microscopic evidence of melanoma in their SN, MART-1 mRNA-positive cells were identified in 2 of 7 NSNs (29%) that were histologically tumor free. MART-1 mRNA-positive cells were also detected in tumor-negative SN sections from 6 of 7 (86%) nodes that had tumor present in areas of the node not represented in the studied sections. Some cells that expressed MART-1 mRNA that was diffusely distributed in the cytoplasm appeared to be melanoma cells, whereas others resembled macrophages. The latter cells expressed augmented mRNA on granules that were intermixed with melanin granules. In other cases, MART-1 mRNA-positive macrophage-like cells contained nuclei and nucleoli more typical of melanoma cells and may represent the macrophage-melanoma hybrids that have been previously reported. Combination of RT in situ PCR for MART-1 mRNA and immunohistochemistry for CD68 revealed that CD68 was colocalized in some cells that expressed MART-1 mRNA. Some lymph nodes that are tumor negative by histology and immunohistochemistry contain cells that express mRNA for MART-1. Some of these cells may be interpreted as "stealth" melanoma cells in which, despite the presence of MART-1 mRNA, there is an absence of immunohistochemically detectable MART-1 protein. Other cells that contain MART-1 mRNA are clearly not melanoma cells or may represent melanoma hybrids. These findings should be taken into account when interpreting and applying the results of RT-PCR analysis of nodal (and other) tissues.

Clinically relevant information from sentinel lymph node biopsies of melanoma patients.

Careful laboratory evaluation of sentinel nodes (SNs) is critical to determine the presence of tumor. This requires evaluation of multiple full-face sections from the SN, using H&E staining and immunohistochemistry. In the future, molecular and genetic approaches may be adjunctive to microscopy. Number of tumor-positive SNs and the amount and location of tumor in SNs correlate with tumor spread to non-sentinel nodes, subsequent recurrences and melanoma death, permitting patient assignment to a risk category.

IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence.

Downregulation of the immune system facilitates tumor progression at different stages of cutaneous melanoma. Sentinel nodes, the first lymph nodes on lymphatics draining directly from a primary melanoma, are immune downregulated by tumor-generated immunosuppressive cytokines, including interleukin-10 (IL-10). To better understand the kinetics of sentinel node suppression, we investigated IL-10 expression by melanoma cells and tumor-associated macrophages and lymphocytes at different stages of primary melanoma evolution. We used reverse-transcriptase in situ PCR to identify the cellular sources of IL-10 mRNA in 39 melanomas. IL-10 mRNA was identified in tumor cells of 2 of 6 melanomas in situ (33%), of 17 of 21 invasive melanomas (81%) and of 11 of 12 metastatic melanomas (92%). Higher IL-10 expression correlates with tumor progression, with differences between melanoma in situ, invasive melanoma and metastatic melanoma. In primary melanomas, the IL-10 mRNA content of tumor cells correlates with Clark's level. There was significantly more IL-10 mRNA in vertical growth-phase melanoma cells than in radial growth-phase cells. In a logistic regression model, moderate-to-high IL-10 mRNA expression by tumor cells was significantly associated with vertical growth-phase melanoma. IL-10 mRNA was detected in melanoma-associated macrophages and lymphocytes. In invasive melanomas, IL-10 mRNA reactivity of macrophages decreased as Clark's level increased. Alterations of immunity by IL-10 derived from melanoma cells and melanoma-associated macrophages and lymphocytes potentially facilitate evolution of the primary melanoma and render regional lymph nodes susceptible to metastases.

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi.

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.

Update on lymphatic mapping and sentinel node biopsy in the management of patients with melanocytic tumours.

AIMS: To communicate best practices for sentinel lymph node evaluation and assessment of prognosis for patients with melanoma. METHODS: Description and justification of approaches derive from experience with management of more than 2000 melanoma patients evaluated by lymphatic mapping and sentinel node biopsy (LMSNB). RESULTS: Pathologists, by detecting blue dye or carbon particles or alterations in nodal cell populations should attempt to confirm that a node submitted as sentinel is truly sentinel. Pathologists must adequately sample the node by examining multiple tissue sections and determine the presence or absence of metastatic melanoma using sections stained by H&E and immunocytochemistry. Approximately 20% of patients have melanoma in the sentinel node (SN) and accurate evaluation of SN tumour status is the most precise technique for staging clinically localised cutaneous melanoma. The remaining non-sentinel nodes (NSN) in the basin are tumour-free in 67% of patients with melanoma in the SN. Breslow thickness of the primary, the area of tumour in the SN (relative to total nodal area) and density of dendritic leukocytes in the SN paracortex (factors that are combinable in prognostic algorithms) predict metastases in the NSN and the likelihood of recurrence and melanoma-specific death. CONCLUSIONS: Careful pathological analysis is essential to determine the presence or absence of metastatic melanoma in sentinel nodes, findings that indicate whether completion lymphadenectomy is required. Quantitative analysis of the primary melanoma and the amount of tumour in the sentinel node, with evaluation of the dendritic cells in that node, provide invaluable information that predicts non-sentinel node tumour status with increased accuracy and the likelihood of future recurrence and death from melanoma. While these activities require considerable effort from pathologists, their clinical impact justifies the increased workload.

Prediction of metastatic melanoma in nonsentinel nodes and clinical outcome based on the primary melanoma and the sentinel node.

Lymphatic mapping and sentinel node biopsy are well-established techniques for staging and managing patients with melanoma, breast cancer and other malignancies that spread initially to the regional lymph nodes. Identification of tumor in the sentinel node is the most precise staging technique currently available. The sentinel node is the site of metastatic melanoma in approximately 20% of melanoma patients and if tumor is present in the sentinel node it is customary to perform a complete dissection of the lymph nodes of the affected nodal basin. This may be overtreatment for some patients as tumor is identified in the nonsentinel nodes of only one-third of sentinel node-positive melanoma patients treated by completion lymphadenectomy. If it were possible accurately to identify the minority of patients with tumor in the nonsentinel nodes, the patients most likely to benefit from lymphadenectomy, the remaining patients could be spared a potentially morbid operation that is unlikely to confer clinical advantage. In 90 patients with a melanoma-positive sentinel node, who subsequently had a completion lymphadenectomy, we evaluated and compared the capacity of characteristics of the primary melanoma and of the sentinel node to predict individuals likely to have tumor in nonsentinel nodes. We assessed the Breslow thickness of the primary, the amount of tumor in the sentinel node (relative tumor area) and, as an index of immune modulation of the sentinel node, the density of dendritic leukocytes in the nodal paracortex. The relative area of tumor in the sentinel node and Breslow thickness of the primary melanoma most accurately predicted the presence of tumor in the nonsentinel nodes (P=0.0001 in both cases-Wilcoxon rank sums). The presence of melanoma in the nonsentinel nodes was also predicted by the density of dendritic leukocytes in the paracortex (P=0.008-Wilcoxon rank sums). These three observations assessed alone and in combination predict the presence of tumor in the nonsentinel nodes with high accuracy. The same characteristics also significantly correlated with tumor recurrence (tumor burden, P=0.0001, Breslow, P=0.0001 and dendritic cell density, P=0.0007) and death from melanoma (tumor burden, P=0.0001, Breslow, P=0.0001 and dendritic cell density, P=0.0026).

Optimized assessment of sentinel lymph nodes for metastatic melanoma: implications for regional surgery and overall treatment planning.

Correct identification of the sentinel node (SN) and accurate evaluation of this node's tumor status constitute the most precise technique for staging clinically localized cutaneous melanoma. However, even if tumor is present in the SN (as in approximately 20% of patients), the remaining nodes in the basin are often tumor-free. We have found that the Breslow thickness of the primary, the relative area of tumor in the SN (with respect to the area of the SN), and the density of dendritic leukocytes in the SN paracortex not only can predict the likelihood of nonsentinel node metastases but also are correlated with likelihood of tumor recurrence and melanoma-specific survival. The most robust of these predictors is relative tumor area, and this may be used as the basis of practical predictive algorithms.

Detection of multiple melanoma-associated markers in melanoma cell lines by RT in situ PCR.

New surgical oncology techniques, such as lymphatic mapping and sentinel node biopsy, require precise identification of the presence of even very small numbers of tumor cells. The gold standard for such analysis remains microscopic assessment of tissue sections, stained conventionally or by immunohistochemistry for appropriate tumor markers. This approach is limited by sampling constraints and requires a high degree of expertise from the microscopist. Recent studies have demonstrated a subgroup of patients whose sentinel nodes are negative on microscopy, but whose nodes yield an enhanced signal for melanoma markers when evaluated by RT-PCR. These enhanced signals reflect a mixture of signal sources, including small numbers of melanoma cells and cells other than melanoma cells that express the relevant markers(s). Because the preparative techniques for RT-PCR destroy the structural integrity of the tissues and disrupt individual cells, the exact cellular source of enhanced signal from a tissue cannot be demonstrated by conventional RT-PCR. RT in situ PCR, in which the RT-PCR technique is applied on a tissue section, does identify the cells that are the source of signal. We have attempted to optimize this interesting approach and have applied it to the detection of relevant melanoma markers in tissue culture lines.

Selective Modulation of Paracortical Dendritic Cells and T-Lymphocytes in Breast Cancer Sentinel Lymph Nodes.

Sentinel nodes (SNs), the nodes nearest a primary tumor on the direct lymphatic drainage path, are the site of earliest metastases, and in melanoma show striking immune modulation. We evaluated SNs from breast cancer patients for evidence of similar immune perturbation. The purpose of this study was to evaluate whether SNs from patients with breast cancer show the alterations in the histology and cytology of the paracortical areas seen in SNs from patients with melanoma. Formalin-fixed and paraffin-embedded sections from 32 SNs and 32 nonsentinel nodes (NSNs) from patients with breast cancer were evaluated. Sections were stained with hematoxylin and eosin and with antibodies to S-100 protein and HLA-DR, DQ, and DP to identify interdigitating dendritic cells (IDCs), and by an antibody to CD43RA to delineate T lymphocytes. By computerized image analysis we evaluated the distribution, frequency, immunophenotype, and activation status of IDCs and associated T lymphocytes in SNs and NSNs. Average areas occupied by S-100-positive dendritic cells (DCs) in SNs and NSNs were 0.13% and 19.98%, respectively, of total nodal area (p < 0.0001). The average density of S-100-positive IDCs in SNs was 11.00/mm2 and in NSNs was 257.88/mm2 (p < 0.0001). In SNs 43.55% of DCs (4.93/mm2) were nondendritic, 51.92% (5.69/mm2) had short dendrites, and 5.2% were mature with long dendrites (0.62/mm2). In SNs the ratio of immature to mature IDCs was 7.95:1. In NSNs, 8.09% of DCs (8.5/mm2) were nondendritic, 28.22% (67.46/mm2) had short dendrites, and 63.07% (145.96/mm2) were mature DCs with long dendrites. The ratio of immature to mature DCs in NSNs was 1:6.66. The average areas occupied by HLA class II-positive DCs in SNs and NSNs were 4.21% and 31.82%, respectively, of total nodal area. The frequency of coexpression of S-100 and HLA class II by immature IDCs without dendrites was 11.27% in SNs and 15.00% in NSNs. In both SNs and NSNs (p < 0.001) all mature S-100-positive IDCs with long dendrites expressed HLA class II. CD43RA-positive T lymphocytes occupied 20.06% of total nodal area in SNs and 63.57% in NSNs (p < 0.0001). The SNs from breast cancer patients are profoundly immune modulated with, by comparison to NSNs, markedly reduced paracortical areas, densities of paracortical DCs, frequency of S-100-positive IDCs coexpressing HLA class II, and a predominance of immature nondendritic and poorly dendritic DCs.