Pathologic examination of sentinel lymph nodes from melanoma patients

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In melanoma patients, the sentinel node biopsy (SNB) procedure is a highly accurate staging method, and the tumor-harboring status of the sentinel node (SN) is the most important prognostic factor for patients with early stage disease. For the SN to provide accurate prognostic information, however, it is essential that all “true” SNs are removed and examined diligently. Pathologists should examine multiple hematoxylin–eosin and immunohistochemically stained sections from each SN, but it is unclear from the currently available evidence what is the most appropriate sectioning and staining protocol. Relevant factors to consider include the accuracy of the procedure, the time, labor, and costs involved, and clinical follow-up data which are likely to vary between institutions; hence, individual protocols should be developed locally by pathologists in consultation with their surgical colleagues. At the Sydney Melanoma Unit, four sequential sections of both halves of each SN are examined. The first and fourth sections are stained with hematoxylin–eosin, the second section is stained for S-100 protein, and the third section is stained for HMB-45. Pathologists should not only identify the presence of melanoma metastases within the SN, but also record the size of the largest metastatic focus, tumor penetrative depth (measured from the inner margin of the node capsule to the deepest tumor cell within the SN), and the percentage nodal cross-sectional area involved (as measured on the slides). Potential diagnostic pitfalls in SN evaluation include the misinterpretation of nevus cells, macrophages, or antigen-presenting interdigitating dendritic cells as melanoma. Careful assessment of the morphologic characteristics of the cells and their immunohistochemical profile should prevent misdiagnosis. Routine frozen section examination of SNs from melanoma patients is not recommended. The utility of ultrasound to detect SN metastases (confirmed by fine needle biopsy) is currently being investigated. Whereas potentially this may avoid the need for formal sentinel lymphadenectomy and histopathologic evaluation in some patients, the lack of sensitivity of currently available ultrasound technologies to detect the small micrometastases (<2 mm in diameter), that are typically present in most melanoma patients with a positive SN, limits its current role. In the future, other techniques, such as the use of carbon particles or antimony analysis, may better localize the site of metastases within SNs and permit more focused and efficient pathologic examination of SNs. At present, the role of nonhistopathologic methods of SN evaluation, such as reverse transcription polymerase chain reaction (RT-PCR) and magnetic resonance spectroscopy, remains unclear, and these techniques require further evaluation.

Section snippets

Preparing sentinel nodes for pathologic examination: what the pathologist should request of the surgeon

SNs should be removed intact, preferably with a thin rim of surrounding adipose tissue and be devoid of crush or diathermy artifacts that may complicate pathologic assessment. Surgeons may apply marking sutures to the SN to indicate the site of entry of afferent lymphatic channels (because this region of the SN is the most likely site to harbor a metastasis)15 or to highlight particular areas of concern. The pathology request form should indicate the number of removed SNs and their anatomic

Macroscopic examination of sentinel nodes

Following 12 to 24 hours of fixation, each SN specimen is dissected. The size of the specimen, the number of lymph nodes present, and the location and orientation of any marking sutures should be recorded. Usually there is only one lymph node in each specimen; however, if more than more one putative SN is present, each should be handled separately as a SN. Excessive adipose tissue surrounding the SN that may hinder optimal tissue processing should be removed, although preservation of a small

Pathologic sampling of sentinel nodes for microscopic examination

Pathologists should examine multiple sections from each half of the SN, including sections stained with hematoxylin–eosin (HE) and sections stained immunohistochemically for melanoma-associated antigens. It is unclear from the currently available evidence what is the most appropriate number of sections to examine, at what levels these should be cut from the tissue blocks, and the most appropriate number and combinations of immunostains that should be assessed.

Various histopathologic sectioning

Sentinel node sampling

The most commonly used method for pathologic examination of SNs is that described by Cochran and colleagues.29 This involves bisecting SNs through their longest dimension in the central meridian through the hilar plane and cutting 10 full face sections from each face of the bivalved SN following “facing up” (ie, trimming of the paraffin block until a section that includes all areas of tissue is obtained). At the Sydney Melanoma Unit (SMU), the pathologic protocol used for the examination of SNs

Increased histopathologic sampling of sentinel nodes detects more melanoma metastases

A number of studies have assessed the detection rate of metastatic melanoma in SNs using different pathologic sampling protocols. Most of these studies have shown that increased histopathologic sampling of SNs from melanoma patients detects more melanoma, although the extent of the increase in detection rates reported has differed in each of the studies (Table 2).24

In 2003, Cook and coworkers25 reported the detection rate of melanoma metastases within SNs using 3 different histopathologic

Practical issues of relevance to increased sampling protocols

In addition to detection rates, time and economic considerations are of great relevance when considering the adoption of a more extensive sampling and histopathologic examination protocol for SNs. What are the practical implications of examining 20 sections per block of each SN? At our institution, we perform an average of 4 SN procedures per day on melanoma patients. If 20 sections were examined per block (rather than the 4 sections that are currently examined), using the assumptions referred

Microscopic examination of sentinel nodes

It is a pathologist's responsibility to examine all sections cut and stained on each SN. We recommend that each slide be scanned at 100× magnification (10× objective and 10× eyepiece). Particular attention should be paid to the subcapsular sinus region of the SN because this is the most likely site to harbor tiny micrometastases.32 Other regions of the SN and perinodal tissue should also be assessed, including the paracortical region, deeper parenchyma, and perinodal lymphatics. High-power

Melanoma tumor burden and location in sentinel nodes

A number of recent studies have shown that the location and extent of tumor deposits within SNs (Figure 5) correlates with the presence of positive non-SNs in CLND specimens and provides important prognostic information. If there are only a few metastatic cells in the subcapsular sinus of the SN, the prognosis is good and the chance of finding additional metastatic disease in a CLND specimen is extremely small. If, on the other hand, there are multiple large foci of tumor which extend deeply

Are all melanoma metastases within sentinel nodes clinically relevant?

Some have recently suggested that very small tumor deposits within the subcapsular sinuses of SNs may not be clinically relevant.48, 49 Careful, prolonged follow-up of such cases in prospective clinical trials will be necessary to answer this question. Although some studies have been interpreted as suggesting that very small melanoma metastases within SNs may not be clinically significant, these studies have had short follow-up periods, are likely to be confounded by lead time bias, and

Nonhistopathologic causes of false-negative sentinel nodes

Regional node field recurrence of melanoma following a reportedly negative SNB sometimes occurs (failure rates of up to 7.9% have been reported in some series; see Table 1) as a consequence of errors in lymphatic mapping, the surgical procedure of sentinel lymphadenectomy, and histopathologic assessment. At the SMU, measurement of antimony levels in SNs has been used to identify surgical causes of false-negative SNB. In Australia, technetium-99m-labeled antimony sulfide colloid is used for

Techniques to better localize sites of melanoma metastases within sentinel nodes

If it was possible to better localize the anatomic site of micrometastases within SNs, more concentrated examination and sectioning could be focused on the area most likely to harbor metastases, thereby improving the efficiency of pathologic examination of SNs. Investigators from the John Wayne Cancer Institute have studied the use of carbon particles injected with the blue dye into the primary melanoma site immediately before the SNB procedure. They found that the carbon particles entered the

Nonhistopathologic methods of sentinel node evaluation in melanoma patients

Other methods that are potentially more cost efficient and at least as accurate as histopathologic examination for the evaluation of SNs have been studied. Molecular methods (including RT-PCR to detect mRNA for melanoma-associated markers) suggest that submicroscopic disease may be present in 28% to 41% of patients with pathologically negative SNs, but there is continuing debate about which markers should be used. Early studies used tyrosinase, but it now appears that this is not a good marker,

Conclusions

The tumor-harboring status of SNs is the most important prognostic factor for early stage melanoma patients, and pathologists are therefore obliged to make a diligent assessment of those nodes. Determination of the most appropriate histopathologic sampling, sectioning, and staining protocol for SNs must take into account the accuracy of the procedure, the time, labor, and costs involved, and clinical follow-up data where it is available. The balance between these competing factors will vary

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