| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
SUPPLEMENT |
From the Departments of Pathology and Laboratory Medicine and Surgery, University of California, Los Angeles School of Medicine, Los Angeles, California.
Correspondence: Address correspondence and reprint requests to: Alistair J. Cochran, MD, Departments of Pathology and Laboratory Medicine and Surgery, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095–1732; Fax: 310-267-2058; E-mail: acochran{at}mednet.ucla.edu
ABSTRACT
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.
Key Words: Clinical staging • Cutaneous melanoma • Melanoma-specific survival • Nonsentinel node metastases • Sentinel mode • Tumor burden
Accurate staging of clinically localized primary melanoma by sentinel node (SN) biopsy assumes correct identification of the SN(s). However, the clinical protocol that identifies SNs is not completely accurate; thus, there is at least a possibility that a node identified as sentinel by blue dye and/or a radiopharmaceutical may not be the true SN. There is a clear need for objective indices that the pathologist can use to confirm the identity of the SN.
There is also a need for indices to determine the risk of metastasis beyond the SN. If this node contains tumor, the patient usually undergoes complete dissection of the nodal basin. However, only about one-third of completion lymphadenectomy specimens from these patients contain tumor.1 Accurate identification of patients whose nodal metastasis is confined to the SN would avoid unnecessary completion lymphadenectomy; instead, this more extensive surgery could be reserved for patients with a substantial likelihood of metastases in nonsentinel nodes.
LABORATORY CONFIRMATION OF A SENTINEL NODE
Although the blue dye has usually dissipated through the excised tissues by the time the specimen arrives in pathology, blue coloration may be visible to the naked eye in fresh tissues, in a frozen tissue block, and even in a paraffin-embedded tissue block. However, it usually cannot be seen on microscopic inspection of stained tissue sections. Similarly, the isotope decays rapidly from peak values measured in the operating room, and pathology laboratories are not usually equipped to measure tissue radioactivity.
We have successfully used particulate carbon as an inert marker to confirm the identity of the SN.2–4 Carbon particles injected with the blue dye accumulate preferentially and usually exclusively in the SN, where they settle in the subcapsular sinus and in the lymphoid tissues adjacent to the area where the afferent lymphatic enters the node (Fig. 1). Because the area of the SN that receives lymph from the primary tumor is often relatively limited in extent, the use of an inert marker such as carbon can also indicate which portion of this node is the most likely site of metastatic tumor cells. Intensive evaluation of nodal tissues can focus on this area of the node. However, carbon cannot be applied if the patient has a permanent tattoo in the relevant lymphatic drainage areas, because carbon-based particles from the tattoo often migrate to the regional nodes.
|
EVALUATION OF THE SENTINEL NODE FOR TUMOR
Intraoperative Frozen Sections
We do not recommend frozen section assessment of SNs. Although intraoperative evaluation of frozen sections allows immediate completion lymph node dissection if the SN contains melanoma cells,9 it is less reliable than analysis of formalin-fixed and paraffin-embedded preparations. This is because the preparation of a full-face frozen section, i.e., whole cross-sections of the SN, often sacrifices relatively substantial amounts of tissue; because SN metastases are often small and usually narrowly located on either side of the nodal meridian, diagnostic tissue may be completely destroyed. Additionally, it is often difficult to prepare a truly full-face section from frozen material; however, a less-than-full-face section may exclude the subcapsular sinus, the preferential site of early metastases. Further tissue may be lost during subsequent formalin fixation of thawed tissue. Finally, the tissue and cell morphology in frozen sections is generally inferior to that in formalin-fixed and paraffin-embedded permanent sections. If intraoperative assessment is necessary, cut faces of the node can be examined with a hand lens or dissecting microscope; alternatively, cell smears may be obtained by scraping the nodal cut surfaces, or tumor imprints may be prepared by pressing the cut surfaces of the nodes onto glass slides.
EXAMINATION OF A SENTINEL NODE
The optimal SN specimen has no crush or cautery artifacts and has been placed in 10% formalin (at least 10 times the SN volume) after surgical removal, before it is sent to pathology. In pathology, the color of the node is immediately evaluated and compared with the operative note of its color. Surgeon-reported radioactivity is recorded. Ideally, the surgeon has marked the blue-stained sector of the node with clips or stitches of varying length and color and has clearly indicated the significance of these markers in the requisition form.
Tumor cells are usually few in number and dispersed singly or as microcolonies (less than or equal to 20 cells) in the subcapsular sinuses.10,11 Less often tumor cells are identified in afferent lymphatics, nodal lymphoid tissues, or deeper sinuses. Because melanomas metastasize first to the subcapsular sinus, the SN should be bisected through its longest meridian; full-face sections are essential. Both cut surfaces should be evaluated by hand lens or dissecting microscope for blue coloration, tumor deposits, and foci of carbon particles.4 Nodal halves are placed (cut face down) into cassettes, formalin-fixed for 24 to 48 hours, paraffin-embedded, and serially sectioned at 4-µm intervals.
We prepare at least 10 serial sections from each lymph node half; hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) with antibodies to S100, HMB-45, and Melan-A/MART-1 are used alternately (Fig. 2). The entire slide is scanned at low power and the different nodal compartments are assessed with particular attention to the subcapsular sinus. High-power (x400) fields are examined for single or clustered tumor cells. Melanoma metastases may extend into the lymphoid parenchyma or central nodal sinus. Extracapsular invasion is noted,12 as are the number of metastatic foci and the greatest dimension of the largest focus. These foci are assessed by a micrometer13 or by measuring the percentage area of the node occupied by tumor.14
|
False-positive assessment of SN tissue may result from misidentification of nonmelanoma cells in immunostained sections. Confusion between melanin-containing macrophages and immunopositive melanoma cells may be reduced by the use of aminoethylcarbazole (red) rather than diaminobenzidine (brown) for antibody visualization. Other potential sources of false-positive interpretation are S-100-reactive dendritic cells, capsular and trabecular nevi, histiocytes, intranodal and perinodal nerves, and ganglion cells. HMB-45 reactivity in the absence of melanoma cells may be seen in the focally calcified trabeculae of inguinal or pelvic lymph nodes, most markedly in older patients (capsular/trabecular nevi).
Discrimination of benign melanocytic and other cells from metastatic melanoma requires consideration of nodal architecture and cytologic features of the cells. Melanoma cells are usually larger than nevocytes and occur individually and in small aggregates within the subcapsular sinus and lymphoid tissues. Histiocytes and macrophages have small, kidney bean–shaped nuclei with small nucleoli. Features that facilitate identification of melanoma cells include their large size, high nuclear-to-cytoplasmic ratio, prominent nucleoli, mitotic figures (especially atypical forms), and the fine punctate melanin granules that indicate a cell that synthesizes melanin.
Nevocytic melanocytes (nevocytes) stain positive for S-100 and MART-1 but have weak to absent reactivity for HMB-45 and are negative for Ki67. Paracortical dendritic cells stain positive for S-100 but negative for MART-1, HMB-45, and tyrosinase. Similarly, intranodal nerves stain positive for S-100 but are negative for the other melanoma-associated markers.
PREDICTION OF NONSENTINEL NODE METASTASIS
We examined the ability of SN tumor area, primary tumor thickness, and the density and distribution of SN interdigitating dendritic cells (IDC) to predict nonsentinel metastasis and survival among 90 patients who underwent complete lymph node dissection after identification of metastatic melanoma in SN sections stained by H&E or IHC.16 For each patient we evaluated the micrometer-measured thickness of the primary tumor. We then used a computer-assisted image analysis program (Planar Morphometry, version 2.1; Southern Micro Instruments, Atlanta, GA) to determine the area of the node occupied by tumor and expressed this as a percentage of the total SN area.17 In sections stained for S-100 protein, we assessed the area of the node occupied by IDC in the paracortex and expressed this as a percentage of the total area of the node.6–8 We also determined the density of IDC/mm2 in the nodal paracortex by the technique described by Huang et al.7 We assessed and correlated primary tumor thickness, the amount of tumor in the SN, and the area and density of IDC in the SN with the presence of melanoma in the nonsentinel nodes. We also related these parameters to disease-free and overall survival.
We found that a relatively simple assessment of tumor area relative to nodal area14,17,18 provided a strong indication of the likelihood of metastases in nonsentinel nodes. Seventeen of 26 patients (65.4%) with metastatic melanoma that occupied more than 4% of the SN (high tumor burden) had tumor in at least one nonsentinel node. Nonsentinel node tumor was significantly less frequent when tumor occupied less than 4% of the SN (low tumor burden; 2 of 64 patients, or 3.1%; P = .001). Patients with low tumor burden in the SN could be further subdivided: nonsentinel metastasis was identified in none of the 52 patients whose SN tumor burden was less than 1% but in 2 of 12 patients (16.7%) whose SN tumor burden was 1% to 4% (P = .03). Among patients with a low tumor burden in the SN, those with a reduced number and density of IDC (a finding that likely indicates relative immune suppression of the SN8,19) were more likely to have nonsentinel metastasis.
The thickness of a primary melanoma, the amount of tumor in the SN, and to a lesser extent the density of IDC in the paracortex were very closely correlated with clinical outcome. As shown in Fig. 3, 20 of 26 patients (77%) whose SN tumor burden exceeded 4% died of melanoma. In contrast, 14 of 64 (22%) with a smaller SN tumor burden died of melanoma (P = .0001, log rank test). The 64 individuals who had a relatively small SN tumor burden could be further categorized by the density of paracortical dendritic cells: 11 of 33 (33%) who had <65 IDC/mm2 died of melanoma, compared with 3 of 31 (10%) who had at least 65 IDC/mm2 (P = .0185, log rank test).
|
In patients with primary melanomas greater than or equal to 2 mm, careful nodal examination by H&E combined with IHC detects occult metastatic melanoma at a frequency identical to the rate of regional nodal recurrence in thickness-matched patients treated by wide excision alone.4 However, conventional microscopy may fail to detect occult melanoma in patients with a thinner primary; such cases may require more extensive sampling and possibly evaluation by newer approaches such as reverse-transcriptase polymerase chain reaction (RT-PCR).4,15 The ability of RT-PCR techniques to identify melanoma markers in SNs that contain no immunohistopathologic evidence of tumor when sampled at a level that is practical may lead to an extremely accurate approach that combines the strengths of molecular and microscopic assessment.20 It is not feasible to abandon microscopy and analyze the nodes only by RT-PCR, because the preparative techniques for RT-PCR destroy the tissues; the cell of origin for an enhanced signal therefore cannot be identified. Also, if the signal is caused by capsular and trabecular nevocytes, intranodal nerves, macrophages, or melanoma-macrophage hybrids, interpretation based strictly on RT-PCR could cause a false-positive result and lead to the risk of overtreatment.21 The efficacy and clinical impact of molecular analysis of the SN will be examined in the second Multicenter Selective Lymphadenectomy Trial, sponsored by the National Cancer Institute (Principal Investigator, Donald L. Morton, John Wayne Cancer Institute, Santa Monica, CA).
Even if the SN is truly positive for tumor, there is still a risk of overtreatment. In most patients with SN metastasis, tumor cells are confined to a single node. As a predictor of nonsentinel involvement, measurement of tumor area is admittedly cumbersome. However, we have shown that that maximum tumor diameter measured by an ocular micrometer is an acceptable surrogate for tumor area in evaluating tumor burden in lymph nodes.17 The use of an ocular micrometer to evaluate the diameter of metastases in the SN has been reported by Starz et al.,13 Wagner et al.,22 and Ranieri et al.23 The relative efficiency of micrometer measurements versus tumor area assessment will be tested in cooperative studies between UCLA and the Augsburg group and in the upcoming Multicenter Selective Lymphadenectomy Trial.
Some pathologists are concerned about the increased cost and time required for these detailed evaluations of the SN. This concern must be related to the critical and practical nature of the data obtained. The role of the pathologist in the sentinel node technique is pivotal and unlikely to diminish.
FOOTNOTES
The Breslow thickness of the primary, the relative area of tumor in the sentinel node (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.
Received for publication December 7, 2003. Accepted for publication December 16, 2003.
REFERENCES
This article has been cited by other articles:
 |
|
 |
|
  A. Govindarajan, D. M. Ghazarian, D. R. McCready, and W. L. Leong Histological Features of Melanoma Sentinel Lymph Node Metastases Associated with Status of the Completion Lymphadenectomy and Rate of Subsequent Relapse Ann. Surg. Oncol., February 1, 2007; 14(2): 906 - 912. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
