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10.1245/ASO.2004.08.016
Annals of Surgical Oncology 11:362-374 (2004)
© 2004 Society of Surgical Oncology
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Right arrow Sentinel lymph node

EDUCATIONAL REVIEW

Lymphatic Mapping in the Molecular Era

Timothy M. Pawlik, MD, Merrick I. Ross, MD and Jeffrey E. Gershenwald, MD

From the Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas.

Correspondence: Address correspondence and reprint requests to: Jeffrey E. Gershenwald, MD, Department of Surgical Oncology, Unit 444, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030; Fax: 713-745-4926; E-mail: jgershen{at}mdanderson.org

Key Words: Sentinel lymph node • Melanoma • Serial sectioning • Immunohistochemistry • PCR

Historically, surgical treatment of clinically negative regional lymph nodes in patients with melanoma has been controversial. Some surgeons have chosen to dissect these nodes in patients who are at increased risk for nodal basin metastasis even when the nodes appear clinically normal (elective lymph node dissection [ELND]), whereas other surgeons perform lymphadenectomy only in cases of clinically evident nodal metastases (therapeutic lymph node dissection [TLND]). More recently, intraoperative lymphatic mapping and sentinel lymph node (SLN) biopsy, also termed sentinel lymphadenectomy, has been used to assess nodal basins at risk for regional metastasis, and then formal lymph node dissections are performed on a selective basis. This technique represents a targeted approach to the regional nodal basin based on the concept that finite regions of the skin drain via an organized system of afferent lymphatic channels specifically to the SLNs within the regional nodal basin. Numerous studies have confirmed that sentinel lymphadenectomy accurately identifies the node or nodes most likely to contain disease, if any are involved.1–5 Moreover, the pathologic status of the SLNs has been shown to be the most important predictor of disease recurrence and survival in patients with clinically negative lymph nodes.6,7

Because of the tremendous interest in the SLN biopsy technique and, in particular, because this technique produces a specimen that contains fewer lymph nodes than that from a formal lymph node dissection, there has been an evolution in the approach to assessing SLN pathologic status. Historically, histological assessment of lymph nodes has involved conventional hematoxylin and eosin (H&E) examination. The SLN biopsy technique, however, facilitates the application of a more intense investigative process that uses serial sectioning, immunostaining, or both, and it has become the standard approach in most melanoma centers.8,9 More recently, molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) have been used in assessing SLNs to detect submicroscopic disease that might not be detectable by conventional histological methods.

The purpose of this review is to discuss the evolution of the lymphatic mapping/SLN biopsy technique, evaluate new developments in the pathologic analysis and molecular methods used to assess SLNs, and present an overview of ongoing clinical trials involving SLN assessment.

THE EVOLUTION FROM ELND TO SENTINEL LYMPHADENECTOMY

The management of clinically negative regional lymph nodes has been the focus of a long and sometimes contentious debate. Two alternative surgical strategies have evolved since the initial recommendation for lymph node dissection for melanoma was presented more than 100 years ago10: (1) prophylactic lymph node dissection or ELND of the regional nodal basin at risk or (2) TLND performed only after regional lymph node recurrence occurs. Advocates of ELND argue that patients with clinically negative, histologically positive lymph nodes at ELND have a better chance for survival (50%–60%) than do patients in whom clinically apparent metastases develop in the regional lymph nodes during follow-up (15%–35%).11–15 However, none of four randomized, prospective studies assessing ELND have demonstrated an overall survival advantage for this technique.16–20 The first two trials, one from the World Health Organization (WHO) and another from the Mayo Clinic, performed in the 1970s, were ultimately criticized because the study populations were at low risk for occult nodal disease and were unlikely to benefit from the proposed surgical treatment. In contrast, more recent analyses of two contemporary randomized prospective trials that targeted higher-risk, clinically node-negative patients (WHO ELND Trial and the Intergroup Melanoma Trial) suggest that ELND may have some survival benefit.15,19–21 Updated results from the WHO ELND trial of patients with truncal melanoma >1.5 mm thick have demonstrated that patients with microscopic nodal disease in the ELND treatment arm had improved overall survival compared with patients in whom palpable adenopathy developed after wide excision of the primary melanoma alone.20 In similar fashion, long-term results from the Intergroup ELND trial of patients with melanomas 1 to 4 mm thick demonstrated that prospectively stratified subgroups (1–2 mm and all nonulcerated primary tumors) had a survival benefit with ELND.21

Regardless of whether a consensus has been reached on the benefit of ELND, these data call into question recommendations to delay lymphadenectomy until palpable nodal disease develops; the data also support the use of alternative approaches to earlier identification of occult nodal disease. However, if all high-risk melanoma patients underwent ELND, most would be exposed to the morbid consequences of an operation with little, if any, potential for clinical benefit, because most of these patients do not harbor microscopic nodal metastasis. Hence, several investigators have proposed the sentinel node approach as a minimally invasive procedure for identifying the approximately 20% of patients who harbor occult microscopic disease.22,23 Over the past 10 to 15 years, the SLN biopsy technique has gained increasing acceptance and has been substantially refined.

SENTINEL LYMPHADENECTOMY

Several studies have demonstrated that the SLNs are the first nodes to contain metastasis, if any, and in this way, the pathologic status of the SLNs reflects that of the entire regional basin.6,22,24,25 Therefore, if the SLN lacks metastasis, the remainder of the regional lymph nodes are unlikely to contain disease, and a completion lymphadenectomy need not be performed. Conversely, although less than one fifth of patients with a positive SLN will have additional melanoma in the nonsentinel nodes, a completion lymphadenectomy remains the standard of care for all patients with a positive SLN. Multiple studies have demonstrated that the false-negative rate for sentinel lymphadenectomy is low (<4%), with a predictive value of a negative sentinel node approaching 99%.3,26,27 Other studies have confirmed the validity of the SLN concept and the accuracy of sentinel lymphadenectomy as a staging procedure.6,23–25,28

Advances in the SLN Technique
When wide excision of the primary tumor is performed, a vital blue dye (isosulfan blue 1%) is injected into the patient intradermally around the intact melanoma or biopsy site. The blue dye is taken up by the lymphatic system and carried via afferent lymphatics to the SLN.1,22–24 The draining nodal basin is explored, and the afferent lymphatic channels and first-draining lymph nodes (the SLNs) are identified by the uptake of the blue dye. With this single-modality technique, the SLN is identified approximately 85% of the time.1,22–24,29 Although this initial approach was promising, 15% of the patients could not benefit from a selective approach because no SLN was identified.

Subsequently, two techniques were incorporated and have significantly improved SLN localization: (1) preoperative lymphoscintigraphy and (2) 99Tc-labeled sulfur colloid accompanied by intraoperative use of the handheld gamma probe. Preoperative lymphoscintigraphy with 99Tc-labeled sulfur colloid permits the identification of patients with multiple draining nodal basins and those with lymphatic drainage to SLNs located outside the standard nodal basins, including epitrochlear, popliteal, or ectopic sites.30–34 In patients with melanoma that drains to multiple regional nodal basins, the histological status of one draining basin does not predict the pathology of the other basins. Therefore, it is particularly important to identify and assess all at-risk regional nodal basins to properly stage the patient.35

Probably the most important development in the SLN biopsy technique, however, has been the introduction of intraoperative lymphatic mapping by using a handheld gamma probe.24,36,37 In this approach, .5 to 1.0 mCi of 99Tc-labeled sulfur colloid is injected intradermally 1 to 4 hours before surgery; during surgery, a handheld gamma probe is used to transcutaneously identify the SLNs that will be removed. Using both blue dye and radiocolloid increases the surgeon’s ability to identify the SLN (>96%–99% accuracy) compared with the use of blue dye alone (84% accuracy).3,22,24,29,38 Although most clinicians use this combined-modality approach, some favor the single-agent strategy of 99Tc-labeled sulfur colloid alone, and they have reported similarly excellent results.25,36,39

The Goals and Benefits of Regional Lymph Node Treatment
To better understand the issues related to sentinel lymphadenectomy, an overview of the goals of regional lymph node treatment in patients with melanoma would be helpful. The goals are (1) pathologic regional lymph node staging, (2) regional disease control, (3) potential cure, and (4) reduction of surgery-induced morbidity compared with formal lymphadenectomy.

SLN Staging
The prognostic significance of SLN pathologic status has recently been convincingly demonstrated. Gershenwald et al.6 showed that SLN status was the most significant clinicopathologic prognostic factor with respect to survival. In their study, the 3-year survival rate for patients with SLN positive disease was 55.8%, compared with 88.5% for patients with SLN-negative disease. Several other multivariate regression analyses have shown that regional lymph node status is the most powerful predictor of recurrence and survival,6,7,21,40–42 even among patients with thick melanomas.7,27,43,44 According to a recent analysis of the American Joint Commission on Cancer database, 5-year survival rates for stage III disease range from 69% for patients with a nonulcerated melanoma and only one microscopically positive lymph node to 13% for patients with ulcerated primary tumors and clinically evident nodal disease with more than three pathologically involved nodes.40 The prognostic importance of distinguishing between microscopically and macroscopically positive lymph nodes has been emphasized by its incorporation into the newly revised melanoma staging system.21,40,45 The SLN microscopic tumor burden has been shown to be an important predictor of survival in patients with stage III melanoma as determined by SLN biopsy.46,47 As a result of these and future studies, refinements in American Joint Commission on Cancer staging criteria may ultimately occur, potentially providing improved stratification for patients in this expanding population of microscopic stage III disease. Furthermore, knowledge of SLN pathologic status will also allow patients to be identified for adjuvant treatment and to be accurately stratified in adjuvant therapy clinical trials.

Regional Disease Control
Sentinel lymphadenectomy has an important therapeutic role in achieving regional nodal control and possible cure. Although previous experience with ELND is controversial with regard to survival, lymphadenectomy has clearly enhanced regional disease control and has helped provide effective local palliation. Gershenwald et al.47 showed that regional nodal control is not compromised by previous SLN biopsy in patients with melanoma. Recurrence in the previously dissected nodal basin was observed in only 10% of patients who had undergone successful lymphatic mapping and SLN biopsy, and none had recurrence in the mapped regional nodal basin as the sole site of recurrence. Overall, these low rates of in-basin recurrence compare favorably with those observed after formal lymphadenectomy in patients who have clinically evident nodal disease (9%–36%) and are also similar to the in-basin failure rates of patients who have undergone ELND with proven microscopic disease.47–52 It is important to note that the completion lymph node dissection specimen is not routinely histologically assessed in the same fashion as the SLN. Therefore, there may be additional disease in the completion nodal specimen that goes undetected with standard histological techniques. This disease could represent a potential source of subsequent recurrence if it is not removed. Because such recurrences are difficult to treat surgically and potentially contribute to significant morbidity, completion dissections performed for microscopic disease provide the potential for improved regional control, thus lending additional support to the SLN approach.

Potential Cure
SLN biopsy can accurately identify clinically node-negative patients who harbor microscopic disease and are therefore most likely to benefit from completion lymphadenectomy. Historically, the 5-year survival rate after lymphadenectomy has ranged from 19% to 60%.11,53,54 Some of this variation is due to patient selection: patients who present with clinically detectable disease have worse 5-year survival rates than those with subclinical disease.11–15,55 On the basis of recent data from the WHO ELND Trial and the Intergroup Melanoma Trial (described previously), which suggest that some patients may benefit from early intervention of microscopically involved regional nodes, identification of patients with minimal disease burden by using the SLN approach may help determine that group of patients who may derive an improved survival benefit from early therapeutic lymphadenectomy while sparing most patients with node-negative disease from requiring further surgical intervention.

Limited Morbidity
Compared with ELND, which is characterized by complete regional nodal extirpation, a regional lymph node strategy using sentinel lymphadenectomy is associated with less surgery in most patients and, therefore, a lower morbidity rate, because most patients (i.e., those with a negative SLN) are not subjected to the potential morbidity that can result from formal lymphadenectomy.28,56 Moreover, the SLN biopsy technique is itself associated with a low morbidity.57 There are substantially fewer postoperative complications after SLN biopsy compared with ELND58; rates of lymphedema, pain, and numbness and loss of active motion range are lower when compared with a full anatomical dissection.59–61

EVOLUTION OF PATHOLOGIC ANALYSIS

Because staging criteria and many therapeutic decisions are based on pathologic lymph node status, accurate pathologic assessment of the SLNs is of paramount importance. Pathologists have traditionally examined the multitude of lymph nodes from lymphadenectomy specimens by submitting the lymph node and examining one H&E-stained section from each paraffin block. This approach represents the standard of care for pathologic evaluation of formal lymphadenectomy specimens regardless of the type of neoplasia. With the advent of the SLN biopsy technique, however, pathologists can limit their examination to an average of 2 lymph nodes per nodal basin rather than the 10 to 35 nodes per basin typically submitted in a formal lymph node dissection specimen. With fewer lymph nodes to analyze, the pathologist can focus on those nodes at highest risk—the SLNs. This makes a much more detailed examination of this nodal tissue more feasible.

Intraoperative Assessment of the SLN
During the development phase of the SLN technique, frozen-section analysis was often used for histological analysis.22 Intraoperative pathologic assessment of the SLN could identify individuals with microscopic regional nodal (i.e., stage III) disease, who would then undergo concomitant lymphadenectomy. Unfortunately, multiple studies have shown that intraoperative pathologic assessment of the SLNs in patients with melanoma has a low sensitivity due, among other reasons, to the inferior quality of frozen sections when compared with permanent sections.62,63 Most metastatic deposits in patients with microscopic stage III disease are <2 mm in diameter. It is interesting to note that frozen-section analysis failed to identify micrometastases <2 mm in diameter in up to 30% of patients.46,47,64 Other studies have similarly reported a low sensitivity for frozen-section analysis (range, 43%–56%).63,65,66 Given that the a priori chance of detecting a lymph node metastasis of melanoma is approximately 20% (ultimately based on the actual primary tumor prognostic factor mix in the cohort being studied)4,6,22 and that the sensitivity of the test is approximately 50%, occult metastases would be identified by frozen-section analysis in only 10% of patients with melanoma.66 Even among patients with thick melanomas (>4.0 mm), the low sensitivity of frozen-section analysis combined with the pretest probability of metastatic disease does not justify its routine use.

There is also the concern that frozen-section analysis may result in the loss of diagnostic tissue, because some potentially informative tissue may be lost as the frozen-section block is cut. It is interesting to note that when a frozen-section specimen prepared from a lymph node is less than a complete section, the portion most often omitted is the subcapsular sinus, which is the portion of the node most likely to contain occult tumor cells.67,68 In addition, when the frozen tissue is processed for paraffin-embedded, permanent sections, there is also a loss of tissue.

To avoid the problem of losing diagnostic nodal tissue, some pathologists have suggested the use of intraoperative imprint cytology, or "touch preps." Although the literature pertaining to the use of such cytological techniques to evaluate SLNs in patients with melanoma is limited, the data suggest that the sensitivity of imprint cytology compared with that of paraffin-embedded permanent sections is also low.69 This approach, however, may represent a useful initial diagnostic strategy for intraoperative assessment of patients with a grossly suspicious SLN when concomitant lymphadenectomy is being considered.

In summary, routine intraoperative SLN frozen-section or imprint cytological assessment has a low sensitivity. Frozen-section analysis is also potentially harmful because it can destroy diagnostic tissue. Frozen-section analysis of SLNs in patients with melanoma should not be performed routinely; however, if intraoperative assessment of the SLN suggests gross involvement, imprint cytology or a limited frozen-section analysis may be appropriate to confirm the diagnosis so that concomitant completion lymphadenectomy can be performed, provided that this approach has been discussed before surgery with the patient.

Beyond Conventional Histological Analysis: Step Sectioning and Immunohistochemistry
As previously noted, traditional histological evaluation was based on conventional H&E staining of one or two sections from the cross section of the node. In 1988, Cochran et al.67 first suggested that the SLN be bivalved along its longest axis and that both sides be examined microscopically. Although this method of pathologic analysis examines <1% of the volume of the node, initial studies by Cochran et al.67 suggested that melanoma nodal metastases are found predominantly around the central plane of the lymph node and that deeper examination of the remainder of the lymph node was not necessary because further studies failed to reveal additional metastases. Simple bivalving of the SLN was considered sufficient because it detected occult tumor cells in approximately 20% of SLNs, a figure similar to the frequency of subsequent regional metastases that occurred in patients treated by wide excision alone.68 However, more recent studies suggest that the incidence of clinical nodal failure after wide local excision alone is underestimated by routine pathologic evaluation of the lymph nodes. It is therefore not surprising that recent pathologic studies have reported finding more metastases with deeper sectioning and immunohistochemistry, thus increasing the sensitivity of the histological examination.9,70–73 Gershenwald et al.72 examined the problem of pathologic failure, defined as occult metastatic disease present in an accurately identified SLN but not detected on histological evaluation. Although the overall recurrence rate among 243 patients initially characterized as having a negative SLN on conventional histological analysis (i.e., single H&E stain, which was the standard approach in the early 2000s) was low (11% at a median of 35 months of follow-up), the mapped nodal basin was the most common first site of recurrence. In 8 of the 10 patients who developed regional nodal failure in a mapped nodal basin as a component of their first site of recurrence, reassessment of the SLNs by serial sectioning or immunohistochemistry (with S-100 and HMB-45) revealed microscopic disease that was initially undetected on conventional histological examination. Data from this study and other studies suggest that the failure to use specialized pathologic techniques, rather than the failure to identify the correct SLN, represents the most important explanation for understaging the nodal basin and subsequent nodal basin recurrence after otherwise successful lymphatic mapping and SLN biopsy.1,67,72,74,75 As a result, intense conventional histological evaluation of SLNs, including bread-loafing or step-sectioning, and immunohistochemical analysis—or both—now represent a standard approach for SLN assessment.9

Evidence for the improved pathologic accuracy of serial sectioning was first noted in 1948, when it was shown that by performing serial sections on axillary lymph node specimens from patients with breast carcinoma, an additional 33% of cases initially diagnosed as negative for metastases were found to be positive.76 In theory, the only way to maximize pathologic detection of micrometastases would be to serially section the SLN to extinction,77 but such an approach would be prohibitively expensive and impractical. Therefore, a balance between maximum detection rate and technical feasibility is required for SLN pathologic analysis. The limiting step—determining the number of sections cut, stained, and examined—remains expensive, time consuming, and controversial.78 Cochran79 initially recommended examining 10 sections from each half of the lymph node. This method yielded a 17% positivity rate, which was similar to that achieved in other studies with the conventional technique. More recently, others have recommended extending pathologic analysis to include 50-, 100-, and 150-µm serial sections.77 This more extensive approach produced up to a 27% rate of detection of metastases, much as Bieligk et al.70 had previously reported.

The implementations of serial sectioning and immunohistochemistry for SLN assessment have occurred simultaneously. Many groups now consider the combination of immunohistochemistry and serial sectioning with H&E to be an essential diagnostic tool.9,80–82 One estimate of the general sensitivity of immunohistochemical staining is that this approach can detect 1 abnormal cell in a background of 105 normal cells.83 It has been estimated that 5% to 15% of metastatic nodes may be missed with H&E alone3,6,8,9,67,84,85 and that serial sectioning and immunohistochemical staining might double the yield of positive dissections.86,87

Several antibodies directed against melanoma-associated antigens are routinely used for evaluation of paraffin-embedded specimens.88 An overview of the relevant biology of common targets and the sensitivity and specificity profile of associated antibodies as they relate to melanoma detection follows.

S-100 Protein
The S-100 protein is expressed by essentially all benign and malignant melanocytic lesions.89–91 Because anti–S-100 antibodies stain virtually 100% of melanomas, use of this antibody can significantly increase tumor identification.80,92–94 Cochran et al.92 noted S-100 positivity in 56 of 56 cutaneous melanomas, regardless of their primary or metastatic status. In a subsequent study performed by the same group before the SLN era, compared with the H&E technique, immunohistochemical staining of lymph nodes with anti–S-100 antibodies identified significantly more (29% vs. 10%) tumor-positive nodes containing single tumor cells that were adjacent to nodes that were partly or wholly replaced by melanoma.95

Despite its high sensitivity, a significant limitation of using S-100 as a marker of nodal disease is that other cells commonly found in nodal tissue contain S-100 protein, including neural-derived cells (e.g., Schwann cells) and antigen-presenting cells (e.g., interdigitating reticular cells and Langerhan cells).92,96 Moreover, benign nevus rest cells are also a potential source of false-positive S-100 results in the examination of SLNs for metastatic disease. Nevus cells are usually seen within the fibrous substance of the capsule, as opposed to melanoma cells, which are frequently subcapsular or parenchymal in distribution.97 Carson et al.98 found that nodal nevi are located most commonly in the peripheral capsule (93%) compared with the internal trabecula (7%) and are significantly more common in SLNs (3.9%) compared with non-SLNs (.01%). Although the detection of benign nevi can usually be accomplished by using H&E alone, immunohistochemistry is sometimes needed.98 A review of the histological appearance and morphology of nodal cells that stain positive for S-100 is therefore important to avoid misinterpretation of benign cells as melanoma metastases. Another immunohistochemical approach to help distinguish benign cells from metastatic melanoma involves staining with a high-sensitivity marker, such as S-100, along with a more specific marker, such as HMB-45, and the proliferation marker Ki67.99

HMB-45
The monoclonal antibody HMB-45 is specific against the protein gp100, which is ultrastructurally localized to premelanosomes.100–102 As such, HMB-45 represents a more specific marker of melanocytes than S-100.103–105 Unlike S-100, use of HMB-45 results in the staining of melanocytes without staining of nonmelanoma cells such as antigen-presenting cells. Nevus cells are usually negative with HMB-45.106 Although HBM-45 is characterized by improved specificity, HMB-45 staining may be patchy and may fail to stain 10% to 30% of metastatic melanomas.99,107–109

Melan-A/MART-1
A product of the MART-1 gene, Melan-A is a melanocytic differentiation antigen. Anti–MART-1 antibody recognizes a 20- to 22-kDa doublet mostly present in melanocytes. Jungbluth et al.110 noted that among 75 metastatic melanomas, 81% were Melan-A positive, whereas 75% were HMB-45 positive. In this study, Melan-A showed homogenous staining in significantly more cases than HMB-45. On the basis of similar work, Blessing et al.111 recommended Melan-A as a useful addition to antibody panels, because it is apparently specific for melanocytic lesions and is more sensitive than HMB-45. Others have suggested that Melan-A specificity is roughly equivalent to that of HMB-45, and they therefore prefer to use HMB-45.108

MAGE3
The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types but is silent in normal adult tissues other than testis and placenta. Whereas this antibody is reported to be very specific for metastatic melanoma, MAGE3 seems to have a low sensitivity (approximately 50%).112

Tyrosinase
Although not widely used in the clinical setting, tyrosinase has been explored as a target for immunohistochemical analysis in SLN specimens. Tyrosinase is a rate-limiting enzyme involved in the initial stages of melanin biosynthesis in melanocytes and melanoma cells.113 Although initial studies with an antityrosinase antibody revealed an overall sensitivity of 94%, the sensitivity correlated inversely with clinical stage.114 In a more recent study, Clarkson et al.107 compared anti-tyrosinase with S-100, HMB-45, and anti–Melan-A. They found that anti-tyrosinase had an intermediate sensitivity somewhere between that of S-100 (high) and HMB-45 (low) and comparable to that of anti-Melan-A. Although S-100 still seemed to be more sensitive, anti-tyrosinase seemed to display greater specificity.115

Overall, the use of step sectioning and immunohistochemical analysis identifies microscopic SLN metastasis in significantly more cases than routine pathologic analysis alone. Unfortunately, no single antibody satisfies the criteria for an ideal marker that is capable of detecting all relevant microscopic nodal disease. Because certain markers lack specificity (S-100), whereas others lack sensitivity (HMB-45, MAGE3, and anti-tyrosinase), a panel of antibodies may be used. At M. D. Anderson Cancer Center, an antibody cocktail that contains HMB-45 and anti–MART-1 is currently used for SLN assessment.9

Molecular Detection of Submicroscopic Disease
With histopathologic and immunohistochemical examination of the SLN, identification of isolated melanoma cells or oligocellular deposits remains difficult and depends on the section of the SLN being examined. A possible alternative to this tedious examination is the use of a molecular-based diagnostic approach such as RT-PCR, which, in this setting, is used to detect tumor-specific messenger RNA (mRNA) in SLN tissue.116–119 This method uses expression of melanocyte-specific mRNA as a target for tumor cell characterization and is based on the premise that expression of tissue-specific mRNA represents submicroscopic metastatic disease that may be undetectable by conventional histological techniques. Using RT-PCR techniques to detect mRNA coding for melanocyte-specific genes, investigators can theoretically detect 1 melanoma cell in a 106 to 107 background of normal cells.119,120 The technique involves (1) isolating SLN mRNA; (2) synthesizing complementary DNA (cDNA) by reverse transcription; (3) generating tumor-specific oligonucleotide primers to amplify, by PCR, a portion of the specific gene of interest; and (4) identifying the PCR product. The integrity of each mRNA sample and the cDNA synthesized can be further enhanced by using a second set of nested RT-PCR primers. It is important to construct the correct primer sets for the PCR to avoid amplification of pseudogenes, which could lead to false-positive results.121,122 Others have argued that to achieve a high specificity, the PCR cycle number should be optimized for each marker, thereby avoiding the production of unwanted illegitimate transcripts or specific transcripts from other low-abundance nodal cell types (e.g., benign nevus).123 Ultimately, the specificity of RT-PCR as a diagnostic tool depends in large part on target gene selection; ideally, the genes should be expressed exclusively in the tumor cells and not in other cells, such as lymphoid or neural tissue. Several melanocyte-associated genes have been identified as potential targets for molecular-based assessment of SLNs.

Tyrosinase
Because tyrosinase is the enzyme in the rate-limiting step of melanin biosynthesis, its expression may be used to identify cells of melanocytic lineage in the SLNs and thus potentially identify submicroscopic metastatic disease.124 Smith et al.117 were the first to describe the use of RT-PCR for tyrosinase mRNA to detect melanoma cells in peripheral blood. Others subsequently modified this technique to detect micrometastases in lymph nodes.85,119,125 Shivers et al.118 compared RT-PCR with standard step sectioning and H&E staining. Perhaps not surprisingly, more than half of the patients with pathologically negative nodes were found to be positive by RT-PCR. In another study, RT-PCR analysis of the SLNs was compared with tyrosinase and MART-1 immunohistochemical staining.126 In this study, using the more sensitive immunohistochemistry as a comparison, significantly fewer patients were found to have SLNs that were negative by immunohistochemical examination but positive by RT-PCR.126 Corroborating these findings, Blaheta et al.127 found that 18% of patients had at least one SLN positive by immunostaining, but 49% had a positive SLN by RT-PCR for tyrosinase. More recently, Li et al.128 reported on the use of the tyrosinase RT-PCR assay in a patient population also examined by serial sectioning and S-100 immunohistochemistry. S-100 immunohistochemistry identified 31% of the patients as having metastatic disease. In contrast, when RT-PCR for tyrosinase mRNA was used to detect metastatic melanoma cells in the SLNs, 70% of patients were classified as positive. Logistic regression analysis found that PCR positivity correlated with tumor thickness, Clark level, and tumor ulceration.128

Use of tyrosinase RT-PCR as a sole molecular criterion has been criticized; suboptimal specificity has been cited as one of the reasons for the unexpectedly high SLN "positivity" rates observed with this technique. Some have argued that the use of tyrosinase alone may not dependably differentiate between benign nevi and melanoma.80 Because nonmelanoma cells that may be present in the SLNs (including capsular and trabecular nevocytes, macrophages, and Schwann cells) express tyrosinase, the specificity of this technique may be limited.98 Moreover, because the SLN tissue used for mRNA preparation is de facto destroyed as part of the preparative phase, histological examination is not feasible.68 The in situ RT-PCR approach may eventually overcome this problem by allowing PCR analysis on slide-mounted tissue sections, thus theoretically preserving histological integrity.129,130 Unfortunately, this approach is technically demanding and is associated with some of the same problems as histological assessment; only the tissue contained on the slide (approximately 5–20 µm) is analyzed, thus creating a potential for sampling error. Finally, tyrosinase expression in SLNs can also be the result of illegitimate transcription caused by imperfect transcription.131,132 For these reasons, tyrosinase expression in lymph nodes may cause false-positive results; additional markers are necessary to increase the specificity of the RT-PCR process.

The gp100 Gene
As with immunohistochemistry (with HMB-45), expression of the gp100 gene has also been proposed as a more specific marker for RT-PCR detection within SLNs. Gutzmer et al.133 examined the specificity of using gp100 versus tyrosinase PCR for detection of submicroscopic disease in SLNs. Whereas tyrosinase PCR had a specificity of 82.5%, gp100 PCR had a specificity of 100%. Unfortunately, the sensitivity of gp100 PCR in the study was only 7.7%; this low sensitivity makes gp100 untenable as an isolated marker for PCR-based screening. At least one study has also questioned the specificity of gp100 gene expression. Brouwenstijn et al.134 reported on the "illegal" expression of gp100, finding gp100 transcripts in cell lines of nonmelanocytic origin, including renal cell carcinoma, ovarian carcinoma, and breast carcinoma. Collectively, these data suggest that the screening of patients for gp100 expression should be carefully used in a setting where other molecular markers can corroborate the diagnosis.

Multimarker RT-PCR
Because melanoma tumor-associated gene expression can be heterogeneous, melanoma cells may go undetected when only a single-marker RT-PCR assay is used.135 Using primers against several different melanoma markers may help to avoid this pitfall. Theoretically, by using a multimarker assay for the detection of occult melanoma in SLNs, overall accuracy by RT-PCR should increase. Goydos et al.126 examined this concept by looking at the RT-PCR detection rate by using the combination of the melanocyte-specific proteins MART-1 and tyrosinase. In this study, all 10 patients with positive SLNs by histology had positive PCR products for both MART-1 and tyrosinase. Only one patient with a negative histopathologic examination was positive for MART-1 but negative for tyrosinase. Thus, it seemed that the addition of MART-1 to tyrosinase RT-PCR seemed to have only a marginal benefit. In a separate study, Bostick et al.71 examined the mRNA expression of three melanocyte-associated antigens (MART-1, MAGE3, and tyrosinase) and concluded that the combined use of these markers was more accurate in predicting the true incidence of nodal metastases. At least 2 mRNA markers were expressed in 16 (94%) of 17 patients with histopathologically proven metastases and in 20 (36%) of 55 patients with histopathologically proven melanoma-free SLNs. On the basis of these data, Bostick et al.71 concluded that H&E and immunohistochemical staining may underestimate the true incidence of metastatic melanoma in the SLNs, whereas the use of multiple-marker RT-PCR may be more accurate in detecting micrometastatic disease in the SLNs.

Clinical Relevance of Submicroscopic Disease
Positivity rates in studies using a variety of RT-PCR approaches to evaluate SLNs for micrometastatic melanoma range from 55% to 73%.119,136–138 On the basis of known clinicopathologic risk factors and patterns of recurrence reported in studies before the application of molecular approaches to the SLN, a recurrence rate of approximately 30% may be expected.139 The reported molecular-based positivity rates for submicroscopic metastatic disease in lymph nodes exceeds the relapse rates of patients seen in historical controls.71,128 This discrepancy between the high rate of PCR positivity and the lower rates of historical clinical recurrence highlights the uncertain clinical significance of PCR-positive-only SLNs. It also suggests that these molecular approaches at present may overestimate clinically relevant disease.

The data from RT-PCR–based SLN analysis suggest that the prognosis of patients with an SLN that is RT-PCR positive but negative by histology or immunohistochemistry is worse than that of patients who have negative results by both techniques. Bostick et al.71 reported that patients with SLNs that were histopathologically melanoma free but were positive by multiple–mRNA marker RT-PCR were at an increased risk of recurrence compared with patients with SLNs that were positive for fewer mRNA markers. Li et al.128 also reported an increase in recurrence and death rates in a cohort of patients with an SLN that was positive for disease by RT-PCR detection alone. The recurrence rate for histologically negative and RT-PCR–negative patients was only 1.6%, compared with 10.1% for histologically negative but RT-PCR–positive patients. Patients with an SLN that was positive by both histology and RT-PCR had the highest rate of recurrence (36.6%). Survival rates followed a similar pattern: patients with an SLN positive by both immunohistochemistry and RT-PCR had the highest rate of death (18.4%) compared with patients who had a negative SLN by both immunohistochemistry and RT-PCR (1.6%). The prognostic significance of a negative SLN by RT-PCR has been recently corroborated. Ribuffo et al.140 reported that no patient with RT-PCR–negative nodes developed a recurrence and that the prognostic significance of RT-PCR–negative nodal status was independent of Breslow tumor thickness.

Although these preliminary findings demonstrating that PCR-based SLN diagnostics identify subsets of patients at higher risk of recurrence within the histologically negative SLN group are intriguing, the true clinical significance of positive PCR results in an otherwise histologically tumor-negative SLN is still unknown. In some studies, a large portion of the nodal tissue is used for PCR analysis and is therefore unavailable for comparative histological examination. This raises concern about sampling bias, because different amounts of tissue are sometimes analyzed with each technique. Also, in some of the earlier studies (for which follow-up is more mature), molecular analysis of the SLN was not compared with current standard histological assessment, such as step sectioning and immunohistochemistry, thereby possibly emphasizing an apparent advantage of the PCR detection method.118 Most PCR studies reported to date have also had very short follow-up times. It therefore remains difficult to draw conclusions about the prognostic importance of SLNs that are positive by PCR but negative by immunohistochemistry and step sectioning. Whether submicroscopic molecular disease can truly be considered micrometastatic disease remains unclear. Some authors have argued that current RT-PCR techniques overestimate the number of patients who have clinically significant melanoma metastases and that alterations in technique (i.e., PCR cycle number or use of cancer testis antigen) need to be incorporated before RT-PCR is adopted as a routine clinical practice.123,141 Ongoing clinical trials that use RT-PCR as a diagnostic tool will undoubtedly provide additional insight as these studies mature.

ONGOING CLINICAL TRIALS

Prospective, randomized clinical trials, such as the Sunbelt Melanoma Trial and the Multicenter Selective Lymphadenectomy Trial (MSLT), are currently under way. In the Sunbelt Melanoma Trial, SLNs from melanoma patients that are negative by histology and immunohistochemistry are also examined by RT-PCR by using the tyrosinase, MART1, MAGE3, and gp100 markers. If tyrosinase and at least one other marker are detected by RT-PCR, the node is considered positive in this study. After stratification by ulceration and tumor thickness, patients are randomized to observation, completion lymphadenectomy, or completion lymphadenectomy plus adjuvant interferon alfa-2b. End points include both recurrence and overall survival. The results of this trial should help to elucidate the prognostic importance of molecular detection of melanoma cells in the SLNs.

The recently completed MSLT was designed to assess whether a selective approach to regional lymphadenectomy, limiting complete nodal dissection to patients with occult disease in the SLN, confers a survival benefit compared with wide excision of the primary tumor with observation alone of the regional nodal basin. Patients whose primary cutaneous melanoma was >=1 mm or was a Clark level IV or V tumor with any Breslow thickness were eligible for the trial. Patients were randomized to either wide excision alone plus observation or wide excision plus lymphatic mapping with subsequent completion lymphadenectomy if the SLN is positive. Although early results from this trial are still not available, the MSLT findings should help to define whether a survival benefit is associated with selective lymphadenectomy.

CONCLUSIONS

The surgical management of the regional lymph node basins in patients with melanoma has been revolutionized after the development and introduction of sentinel lymphadenectomy. Debates regarding ELND and TLND have been supplanted by a widespread acceptance of a selective approach to the regional nodal basin guided by SLN biopsy. Advances in both surgical technique and pathologic analysis have combined to make sentinel lymphadenectomy a powerful tool. The data strongly support the notion that histological examination of the first nodes to receive primary lymphatic drainage, the SLNs, accurately reflects the overall status of the regional nodal basin. The SLNs are therefore able to identify those patients who are most likely to benefit from completion lymphadenectomy while sparing patients with negative nodes the morbidity of an additional surgical procedure.

The advent of the SLN technique has helped usher in a new era of molecular pathologic analysis of nodal tissue. Rather than traditional routine histological examination, the SLN is now subjected to more extensive pathologic examination with step sectioning, immunohistochemistry, and RT-PCR. In the future, cDNA microarray technology will make possible a genomic approach to melanoma classification, potentially allowing the identification of genetic markers or expression profiles that might be important for diagnosis, prognosis, and even therapy.142,143 Which combination of examinations should be used to maximize both pathologic sensitivity and specificity (i.e., overall accuracy) remains debatable. Furthermore, the clinical significance of detecting a single cluster of cells by immunohistochemistry or submicroscopic disease found by PCR remains unclear. Treatment of such minimal disease may represent overtreatment for many patients, because less than one fifth of patients with a positive SLN have additional melanoma in the nonsentinel nodes removed during the completion lymphadenectomy. Recent reports suggest that the extent of microscopic tumor burden in the SLNs not only is a strong predictor of clinical outcome, but also may be a key determinant of likely involvement of additional nodes in the same basin.46,47,144 Indeed, one may conjecture that in a setting of truly submicroscopic disease, the SLN biopsy procedure itself may be both therapeutic and diagnostic; however, this requires further study. Long-term results of studies such as the Sunbelt Melanoma Trial and the MSLT will, it is hoped, help to answer many of these questions.

ACKNOWLEDGMENTS

Supported in part by an award from The University of Texas M. D. Anderson Cancer Center Physician-Scientists Program (JEG). The authors thank M. D. Anderson Cancer Center’s Department of Scientific Publications for their assistance with this manuscript.

FOOTNOTES

In this review, we outline the evolution of lymphatic mapping and sentinel lymphadenectomy in melanoma. In particular, we discuss developments in surgical technique, pathologic analysis, and molecular methods used to assess SLNs and present an overview of ongoing clinical trials in the field.

Received for publication August 22, 2003. Accepted for publication November 21, 2003.

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