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10.1245/ASO.2006.12.010
Annals of Surgical Oncology 13:910-918 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Prognostic Significance of Molecular Staging Study of Sentinel Lymph Nodes by Reverse Transcriptase-Polymerase Chain Reaction for Tyrosinase in Melanoma Patients

Cristina Mangas, MD1, Josep M. Hilari, BS1, Cristina Paradelo, MD1, Jordi Rex, MD1, Maria Teresa Fernández-Figueras, MD, PhD2, Manel Fraile, MD, PhD3, Antoni Alastrue, MD4 and Carlos Ferrándiz, MD, PhD1

1 Department of Dermatology, Hospital Universitario Germans Trias i Pujol, Carretera Canyet s/n. 08916-Badalona, Spain
2 Department of Pathology, Hospital Universitario Germans Trias i Pujol, Universidad Autónoma de Barcelona, Carretera Canyet s/n. 08916-Badalona, Spain
3 Department of Nuclear Medicine, Hospital Universitario Germans Trias i Pujol, Universidad Autónoma de Barcelona, Carretera Canyet s/n. 08916-Badalona, Spain
4 Department of Surgery, Hospital Universitario Germans Trias i Pujol, Universidad Autónoma de Barcelona, Carretera Canyet s/n. 08916-Badalona, Spain

Correspondence: Address correspondence and reprint requests to: Carlos Ferrándiz, MD, PhD; E-mail: cferrandiz.germanstrias{at}gencat.net.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We performed this study to evaluate the clinical effect of microscopic and submicroscopic metastases in sentinel lymph nodes (SLNs) from patients with early-stage melanoma.

Methods: Patients with confirmed cutaneous melanoma (American Joint Committee on Cancer stages I and II) underwent standard lymphoscintigraphy and SLN biopsy. Serial sections were divided between routine histopathology with hematoxylin and eosin plus immunohistochemistry for HMB-45 and molecular analysis by nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay for tyrosinase (using ß-actin as a control).

Results: Of 180 patients analyzed (318 SLNs), 38 (21%) patients had positive SLN(s) by routine hematoxylin and eosin and immunohistochemistry (microscopic disease; group 1), and 142 (79%) had negative histological results. Analysis by RT-PCR detected tyrosinase in at least 1 SLN from 124 (69%) patients. Among patients with histologically negative SLN(s), tyrosinase was detected in 86 (48%) patients (submicroscopic disease; group 2), whereas 40 (22%) patients had negative results by both histology and RT-PCR (group 3). Sixteen (9%) patients had histologically negative SLNs and ambiguous RT-PCR results (group 4). Among 138 patients in the analysis of recurrence (mean follow-up, 45 months), only 18 patients had a recurrence: 11 (31%) of 35 in group 1, 5 (10%) of 51 in group 2, and 2 (5%) of 37 in group 3. No recurrences were seen in group 4. Only group 1 had a significantly shorter disease-free survival and overall survival compared with the other groups.

Conclusions: After a long follow-up period, molecular upstaging by tyrosinase RT-PCR failed to detect a subgroup of patients with an increased probability of recurrence.

Key Words: Melanoma • Molecular staging • Reverse transcriptase-polymer chain reaction • Tyrosinase • Sentinel lymph node


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most powerful predictor of recurrence and overall survival in patients with primary cutaneous melanoma is the pathologic status of the regional lymph nodes at the time of diagnosis.1 However, the pathologic study of lymph nodes from a complete elective lymphadenectomy is time consuming and expensive. With the introduction of the sentinel lymph node (SLN) biopsy procedure, which allows pathologists to focus on a more detailed pathologic examination of just one to four lymph nodes, this problem was partially solved.2 This minimally invasive procedure is currently performed as the standard of care in many major melanoma centers as an alternative to elective lymphadenectomy.3

In the past, nodal staging was based on routine histopathologic analysis with hematoxylin and eosin (H&E) stains and immunohistochemical (IHC) examinations of a central cross section of the node, which studies < 1% of the submitted nodal tissue. As a consequence, the proportion of patients with occult nodal involvement was often underestimated.4 Serial sectioning and IHC examination have been shown to improve the identification of melanoma cells in SLN(s) compared with routine cross-sectioning and H&E staining.5 Studies that reevaluated (with serial sectioning, IHC, or both) histologically negative SLN(s) from patients who experienced a recurrence have identified occult nodal metastases in up to two thirds of these patients.6 Moreover, it has become evident that 5% to 10% of patients with primary melanomas thicker than 1 mm who have histologically negative SLN(s) will have a recurrence. This suggests that melanoma patients who have submicroscopic nodal disease, which is not readily detected by routine histopathologic or IHC examination, are at risk for recurrence.6,7

The amplification of tyrosinase messenger RNA (mRNA) by nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been adapted for analysis of SLN(s) from patients with primary cutaneous melanoma to increase the sensitivity of tumor cell detection.8,9 This method can identify patients who have submicroscopic metastatic disease that may be missed with routine histopathologic and IHC examination of serial SLN sections. A study using tyrosinase RT-PCR to reevaluate the histologically negative SLN(s) of patients who experienced a recurrence demonstrated that 92% were positive.10 However, little is known about the biological and clinical relevance of finding evidence of submicroscopic metastatic disease in the SLN(s) of patients with primary cutaneous melanoma. Preliminary data suggested that there may be a large percentage of false-positive results or that patients with submicroscopic disease rarely have a recurrence, because most of the patients who were RT-PCR positive never developed a recurrence. This implies that RT-PCR might be too sensitive for clinical use. Recently, however, other markers, in addition to tyrosinase, have been used to enhance the sensitivity and specificity of RT-PCR analysis,11–13 and these newer methods can even be applied to paraffin-embedded archival tissue.14

This study evaluated the rate of recurrence and the prognostic significance of microscopic and submicroscopic metastases in SLN(s) from patients with American Joint Committee on Cancer (AJCC) clinical stage I or II melanoma. SLNs were examined by both routine histopathology and tyrosinase RT-PCR.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Patients with a biopsy-confirmed diagnosis of cutaneous melanoma were required to meet the following eligibility criteria: diagnosis of melanoma within 1 month of study entry; Breslow thickness >.76 or ≤.76 mm but with a Clark level of IV or V, microscopic ulceration, or pathologic signs of regression; no clinical evidence of regional lymph node or distant metastases by physical examination and staging evaluations (chest radiograph, abdominal ultrasonography, computed tomography, or bone scintigraphy); no evidence of multiple synchronous primary melanoma; no pregnancy; no changes in lymphatic flow caused by disruption of afferent lymphatic channels secondary to surgery (i.e., excision of primary tumor with wide margins); no radiotherapy; and no inflammation or infection around the biopsy site or surgical wound. These criteria ensured that all patients enrolled in the study were AJCC clinical stage I or II.15 All patients provided informed written consent.

Study Design
All patients underwent preoperative lymphoscintigraphy to define the regional lymphatic basins at risk for metastatic disease and to identify the number and location of SLN(s) as described previously.16,17 Wide local excision of the primary lesion and SLN biopsy were performed. Immediately after surgery, SLNs were cut with a scalpel into slices approximately 1 mm thick parallel to the longitudinal axis, except for the first 70 patients, in whom SLNs were bivalved. SLNs were evaluated by both histopathologic and molecular methods. The even slices were submitted for routine H&E staining and IHC study, and the odd slices were used for molecular analysis. The slices were alternately processed for RT-PCR studies and for paraffin inclusion. Patients were followed up for melanoma recurrence and survival.

Pathologic Examination
Each even slice was embedded in a separate paraffin block, and consecutive sections were cut from each block. At least three sections were stained with conventional H&E stain, and the two adjacent sections were studied by IHC staining by using polyclonal rabbit anti-cow S100 (DAKO Corporation, Carpinteria, CA) diluted 1/200 and monoclonal mouse anti-human melanoma antigen HMB-45 (DAKO) diluted 1/200. When the primary tumor was known to be negative for HMB-45 or when a group of suspicious cells was positive for S100 but negative for HMB-45, a consecutive slice was stained for monoclonal mouse anti-human Melan A clone 103 (DAKO) diluted 1/25. All sections were systematically examined for micrometastases starting from the marginal sinus. When negative cases on IHC staining were positive by RT-PCR, two additional sections (.2 mm deep) were cut and stained for H&E and HMB-45. In all cases, the same pathologist performed the examination. A lymph node was considered positive by histopathology if tumor cells were identified either by H&E or by IHC staining.

RT-PCR Assay
Tissue blocks were snap-frozen in liquid nitrogen and kept at –80°C until RNA isolation. The tissue was mechanically homogenized, and total RNA was extracted by using a standard kit (Ultraspec; Biotecx Laboratories, Houston, TX). The RNA concentration and purity were measured by spectrophotometry, and the integrity was visualized by 1.5% agarose gel. To assess the RNA integrity and the reverse transcription procedure, the ß-actin gene complementary DNA was simultaneously amplified with tyrosinase. Two different regions of the tyrosinase complementary DNA were amplified by using two different sets of primers, HTYR1-HTYR2/HTYR3-HTYR4,18 and a new set of primers, TyrF1-TyrR1/TyrF2-TyrR2 (Table 1Go). The result was considered definitive when it was reproduced by using both sets of primers. Both tests were repeated when the results were discordant, and in those cases, the test was labeled as positive or negative only when three of four tests were concordant. Cases in which only two of four tests were positive for tyrosinase were considered ambiguous results.


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  		TABLE 1. Oligonucleotide primers used for tyrosinase and ß-actin amplification
 
To reduce sample manipulation, we performed one-step RT-PCR reactions by using .5 µg of total RNA, which was added to the mix containing 4 ng/µL of random primer, 2.5 mM of MgCl2, .2 µM of tyrosinase primers (either HTYR1-HTYR2 or TyrF1-TyrR1; TIB MolBiol), and .03 µM of the ß-actin primers (ActF and ActR; TIB MolBiol, Berlin, Germany). The RT-PCR reactions were performed as follows: 30 minutes at 42°C followed by 35 cycles at 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 20 seconds (HTYR1-HTYR2 primers) or 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 20 seconds (TyrF1-TyrR1 primers).

Nested PCR was performed with 1 µL of the RT-PCR product in 25 µL of final volume with the following conditions: .1 mM of each deoxyribonucleoside triphosphate (deoxyribonucleoside triphosphate solutions; Ecogen, Barcelona, Spain), .4 µM of tyrosinase primers, .4 µM of ß-actin primers, and 1.25 U of Taq polymerase (Eco Taq; Ecogen). Twenty-five PCR cycles were performed as described previously for the HTYR3-HTYR4 primers and TyrF2-TyrR2 primers. The sensitivity of the assay was assessed by serial dilutions of the melanoma cell line COLO 853 in the lymph cell line MOLT-4. The sensitivity was optimized to detect 3 melanoma cells in 107 lymphocytes.

After the patients provided informed consent, lymph nodes obtained from 16 patients undergoing noncancer surgery (vascular surgery) were used as negative controls for tyrosinase RT-PCR. Only 1 (6.2%) of the 16 lymph nodes was positive for tyrosinase (false-positive rate), but 2 lymph nodes had ambiguous results (i.e., 2 positive and 2 negative reactions). We included water as a nontemplate control in every PCR, and the result was excluded if the control was contaminated.

Adjuvant Therapy and Follow-Up
Patients whose SLN(s) were positive by histopathology underwent a complete lymph node dissection. Those patients and patients with a primary tumor thickness > 4 mm—regardless of the pathologic status of their SLN(s)—were offered adjuvant high doses of interferon alfa-2b immunotherapy.19,20 No clinical decisions for further surgical and/or adjuvant medical treatment were based on the results of the RT-PCR assay except for 34 patients who had histologically negative SLNs that were positive by RT-PCR. These patients agreed to be enrolled in a pilot therapeutic trial. These 34 patients and patients with <3 months of follow-up were not considered for recurrence and survival analysis. Follow-up was considered since the date of the SLN biopsy. Follow-up evaluations were performed at 6-month intervals and consisted of a careful physical examination and routine blood investigations, including lactate dehydrogenase levels. At least once a year, a chest radiograph and ultrasound abdominal examination were performed. Computed tomography, magnetic resonance imaging, and bone scintigraphy were performed only in patients with clinical findings suggestive of meta-static disease.

Statistical Analysis
Standard statistical techniques were used. Categorical variables were compared between groups by {chi}2 test. Patients’ survival functions were generated for overall and disease-free survival by using the Kaplan-Meier method.21 Survival was calculated from the date of SLN biopsy to the date of first recurrence. Statistical inference on survival functions between subgroups was based on the long-rank test for equality of the survival functions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A prospective cohort of 195 consecutive patients with AJCC stage I or II primary cutaneous melanoma were enrolled from September 1998 to June 2003 at a single institution (Hospital Universitario Germans Trias i Pujol, Badalona, Spain), where lymphatic mapping and SLN biopsy were successfully performed in all but 3 patients. These three patients had multiple SLNs, one of which was not found. After processing of the SLN(s), patients who tested negative by RT-PCR for the ß-actin control (n = 10) and five patients with aggregates of nevus cells were removed from the study. From the remaining 180 patients, a total of 318 SLNs, ranging from 1 to 4 (mean, 2.73) per patient, were obtained and analyzed. The demographic, clinical, and histopathologic characteristics of these 180 patients are listed in Table 2Go.


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  		TABLE 2. Demographic, clinical, and histopathologic characteristics of patients included in the analysis (n = 180)
 
Pathologic and RT-PCR Examination
After routine examination by H&E and IHC staining with HMB-45, 38 (21%) patients were found to have metastatic melanoma cells in 1 or more SLN(s), and 142 (79%) patients had negative results. Among patients with histologically positive SLNs, tyrosinase mRNA was detected in all but three nodes (considered RT-PCR false-negative results). Expression of tyrosinase mRNA by using RT-PCR was detected in at least 1 SLN from 124 (69%) patients. Among patients with histologically negative nodes, tyrosinase mRNA was detected in 86 (48%) patients, whereas 40 (22%) patients tested negative by RT-PCR, and 16 (9%) had ambiguous RT-PCR results (i.e., 2 positive and 2 negative reactions). As expected, the number of patients with histologically positive SLN(s) increased with both the tumor thickness and clinical stage (P < .001), whereas the RT-PCR results showed a statistically significant correlation only with Breslow thickness (Table 3Go).


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  		TABLE 3. Pathologic and molecular status of SLNs depending on T value, clinical stage, and Breslow thickness (n = 180)
 
A total of 138 patients were included in the analysis of recurrence and survival. Thirty-four patients with histologically negative but RT-PCR–positive SLN(s) were excluded from this analysis because they were enrolled in another clinical trial. Patients were classified into four groups according to their SLN status (Table 4Go), and the incidence of recurrence was evaluated for each group after a mean follow-up of 45 months (median, 45 months; range, 4–78 months). Group 1 included 35 patients with histologically positive SLN(s) (i.e., microscopic metastases). All patients in this group underwent a complete lymphadenectomy, and five (14%) had micrometastases in additional nodes. Twenty-six (65%) patients in this group received adjuvant therapy with high-dose interferon alfa, but only 17 (70%) were able to complete at least 80% of their scheduled dose. Group 2 included 51 patients with histologically negative but RT-PCR–positive SLN(s) (i.e., submicroscopic metastases). Group 3 included 37 patients with negative SLN(s) by both diagnostic techniques. Group 4 included 15 patients with histologically negative SLN(s) and ambiguous RT-PCR results.


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  		TABLE 4. Number and sites of recurrence by sentinel lymph node (SLN) status (n = 138)
 
There were 18 recurrences in this cohort of 138 patients, and the sites of first tumor recurrence are listed in Table 4Go. There were 11 recurrences in group 1 (31%), 5 in group 2 (10%), 2 in group 3 (8%), and none in group 4 at the last follow-up. The mean time to first recurrence was 14 months (range, 2.58–40.2 months) for all recurrences, 14.5 months in group 1, 11.3 months in group 2, and 38.3 months in group 3. Ten of 138 patients died: 7 in group 1 and 3 in group 2.

Kaplan-Meier estimates of disease-free survival by SLN status are shown in Fig. 1aGo. Patients in group 1 had significantly shorter disease-free survival than patients in group 2 (P = .007), group 3 (P = .0021), or group 4 (P = .0074). Among patients with histologically negative SLN(s), those who had positive SLN(s) by RT-PCR (group 2) had no difference in the probability of recurrence compared with those who had negative results by RT-PCR (group 3; P = .5) or those who had ambiguous RT-PCR results (group 4; P = .2). Moreover, there was no difference in disease-free survival between groups 2 and 3 whether patients in group 4 were considered to be RT-PCR negative (P = .7) or RT-PCR positive (P = .2).


Figure 1
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  		FIG. 1. Kaplan-Meier estimate of disease-free survival (a) and overall survival (b) by sentinel lymph node (SLN) status. Group 1 includes patients with histologically positive SLN(s), group 2 includes patients with histologically negative but reverse transcriptase-polymerase chain reaction (RT-PCR)–positive SLN(s), group 3 includes patients with histologically negative SLN(s) that were also negative by RT-PCR, and group 4 includes patients with ambiguous RT-PCR results.

 
Regarding overall survival, patients in group 1 had worse survival than patients in group 2 (P = .006), group 3 (P = .002), or group 4 (P = .009). No other difference between groups was significant (Fig. 1bGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The reported rates of histologically detectable micrometastasis in the SLN(s) of patients with AJCC stage I or II primary cutaneous melanoma are quite uniform—approximately 20% (21% in our series)—despite different criteria used to select patients for SLN biopsy and different sampling methods and pathologic approaches to analyzing SLNs.22,23 However, the frequency of submicroscopic metastases in SLNs (histologically negative and RT-PCR positive) varies widely, ranging from 30% to 52%24 (48% in our series; Table 5Go).7,10,24–29 These data may result from different approaches to molecular studies, such as using nested RT-PCR or single RT-PCR, different primers and conditions for RT-PCR, and different criteria for labeling a sample as positive. In our approach, a subset of ambiguous PCR results (i.e., two negative and two positive RT-PCR reactions) in 10% of the patients could be positive or negative depending on both the criteria used to label a sample as positive and the efficiency of RT, PCR, or both. The fact that patients with ambiguous RT-PCR results had had no recurrences at last follow-up suggests that these patients may have such a small tumor burden, probably at the limit of detection by RT-PCR, that the tyrosinase amplification is not always reproducible. As far as we know, this is the first time that this has been reported, but probably this is also one of the reasons to explain the wide variability of the results among series.


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  		TABLE 5. Summary of recent molecular detection studies of melanoma metastases in sentinel lymph nodes
 
The really important issue and the primary goal of all these molecular reports is the prognostic significance of submicroscopic metastasis in SLN(s). Most of the previous studies seem to confirm significant differences in the rate of recurrence for patients with microscopic versus submicroscopic nodal metastases and compared with patients with no molecular evidence of nodal metastases.7,10,14,24–28 However, we did not find any difference in disease-free survival between patients with submicroscopic metastasis (group 2) and those without any evidence of nodal metastasis (group 3). In accordance with our results, Kammula et al.29 recently reported that with a median follow-up of 67 months, there was no longer a statistical difference in disease-free survival among the histologically negative patients who were stratified by tyrosinase RT-PCR. Also similar to the results of Kammula et al. is the observation that RT-PCR–negative patients experience disease recurrence significantly later than RT-PCR–positive patients, whereas no significant differences between pathologically positive results and pathologically negative/PCR-positive results were found. The later recurrences in the PCR-negative group can explain why other shorter follow-up studies found prognostic significance with the molecular upstaging method. This observation points out, as Kammula et al. previously suggested, that these tumors with negative SLNs by both molecular and pathologic methods have a different biological behavior and remain dormant for longer periods. However, the main problem with the nested RT-PCR tyrosinase approach is the high percentage of positive results (approximately 50% in most series; Table 5Go). Certainly, the smaller probability of recurrence of the pathologically negative/PCR-positive group compared with the pathologically positive group suggests that there may be a large percentage of RT-PCR false-positive results. False-positive results for tyrosinase detection can result from several causes: illegitimate transcription, contamination, or capsular nevi.30 Alternatively, the RT-PCR method may simply be too sensitive and may identify subclinical metastatic disease that is unlikely to lead to a clinical recurrence.

In an effort to increase the sensitivity and specificity of SLN molecular staging, different approaches have been proposed. Some investigators have used a multimarker method11–14,25,26 consisting of a single round of RT-PCR for different melanoma-associated antigens, such as melanocytic differentiation antigens (i.e., tyrosinase, melanoma antigen recognized by T cells [MART-1/Melan A], melanocity lineage-specific antigen gp-100 [gp-100], melanocity protein Pmel-17 precursor [Pmel-17], tyrosine-related protein-1 [TRP-1], and tyrosine-related protein-2 [TRP-2]) or those associated with tumor transformation (i.e., melanoma antigen family A [MAGE], B melanoma antigen [BAGE], and G antigen [GAGE]). The optimal combination of these different markers remains unknown, although a recent study showed a combination of markers with independent prognostic significance.26 Simultaneously, real-time PCR is being developed with the aim of quantifying the metastatic disease burden and establishing the optimal threshold to identify patients at high risk for recurrence.31,32 Takeuchi et al.26 with a follow-up similar to that of Kammula et al. and longer than that in our series, found significance with a multimarker quantitative method. To clarify whether a different molecular approach in our case would show a statistically significant difference with molecular upstaging, quantification of several markers through real-time PCR is now in progress.

Another interesting observation is that the recurrence rate in both groups 1 and 2 was slightly lower than that reported in other series with similar or less follow-up (30% vs. 29%–67% and 10% vs. 10%–25%, respectively), as shown in Table 5Go. Overall, our different criteria for selecting patients for SLN biopsy, including those with a Breslow thickness < 1 mm but with pathologically evident signs of regression, might increase the percentage of good-prognostic patients. Another explanation could be the relatively better prognosis for melanoma in women,15,33 because our cohort of patients has a marked female predominance compared with other series. In this sense, a leveling off tendency in melanoma mortality rates in South European countries such as Spain has been recently published.34 In patients with microscopic metastases, the extraordinary heterogeneity of metastatic risk for stage III melanoma with a 5-year survival rate ranging from 67% to 26%15 can also explain our less frequent rate of recurrence in this group of patients because, in our series, > 50% of the clinical stage III melanoma patients had clinical stage IIIA disease (data not shown). Moreover, treatment with complete lymphadenectomy plus adjuvant high-dose interferon, which was used in this study, differs from other series and could be an explanation for the apparent disease-free survival advantage in this group of patients.

Although the number of recurrences was small, thus limiting the interpretation, the different patterns of recurrence in patients with microscopic and submicroscopic disease are interesting. In our study, only one patient in the group with histologically negative and RT-PCR–positive SLN(s) had a distant metastasis. The first site of recurrence in this group was most often regional. In contrast, in the group with histologically positive SLN(s), most of the first recurrences were at distant sites. These results raise the question of whether the regional nodal recurrences in patients with histologically negative but RT-PCR–positive SLN(s) really had false-negative results in the SLN biopsy procedure or in the pathologic analysis. However, in contrast to the results of this study, the New Jersey Cancer Institute series showed a high rate of visceral metastasis in patients with both microscopic and submicroscopic nodal metastases,28 thus suggesting that completion lymphadenectomy in patients with only RT-PCR–detectable nodal disease may be ineffective. In our opinion, considering the discordant published results, questions regarding the best therapeutic strategy for patients with RT-PCR–positive SLN(s) remain unsolved. The Sunbelt Melanoma Trial and Multicenter Selective Lymphadenectomy Trial II may elucidate this polemical issue.33

In conclusion, the results reported in this study corroborate the previous observation that a longer follow-up period is necessary to understand the biological significance of metastatic molecular detection by RT-PCR alone. Further investigation with a different molecular approach using multiple markers and with a longer follow-up may help us to understand the meaning of the presence of occult disease in SLNs from melanoma patients.


   ACKNOWLEDGMENTS
 
Supported by grants from Fondo de Investigaciones Sanitarias (98/0449 and 02/933) and the Red Española de Centros de Genómica del Cáncer (C03/10).


   FOOTNOTES
 
C. Mangas and J. M. Hilari contributed equally to this work.

Received for publication December 10, 2004. Accepted for publication February 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Balch CM. Cutaneous melanoma: prognosis and treatment results worldwide. Semin Surg Oncol 1992; 8:400–14.[Medline]
  2. Morton DL, Wen DR, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127:392–9.[Abstract]
  3. McMasters KM, Reintgen DS, Ross MI, et al. Sentinel lymph node biopsy for melanoma: controversy despite widespread agreement. J Clin Oncol 2001; 19:2851–5.[Abstract/Free Full Text]
  4. Cochran AJ, Wen DR, Morton DL. Occult tumor cells in the lymph nodes of patients with pathological stage I malignant melanoma. An immunohistological study. Am J Surg Pathol 1988; 12:612–8.[Medline]
  5. Yu LL, Flotte TJ, Tanabe KK, et al. Detection of microscopic melanoma metastases in sentinel lymph nodes. Cancer 1999; 86:617–27.[CrossRef][Medline]
  6. Gershenwald JE, Colome MI, Lee JE, et al. Patterns of recurrence following a negative sentinel lymph node biopsy in 243 patients with stage I or II melanoma. J Clin Oncol 1998; 16:2253–60.[Abstract]
  7. Shivers SC, Wang X, Li W, et al. Molecular staging of malignant melanoma: correlation with clinical outcome. JAMA 1998; 280:1410–5.[Abstract/Free Full Text]
  8. Wang X, Heller R, Van Voorhis N, et al. Detection of submicroscopic lymph node metastases with polymerase chain reaction in patients with malignant melanoma. Ann Surg 1994; 220:768–74.[Medline]
  9. Blaheta HJ, Schittek B, Breuninger H, et al. Lymph node micrometastases of cutaneous melanoma: increased sensitivity of molecular diagnosis in comparison to immunohistochemistry. Int J Cancer 1998; 79:318–23.[CrossRef][Medline]
  10. Li W, Stall A, Shivers SC, et al. Clinical relevance of molecular staging for melanoma: comparison of RT-PCR and immunohistochemistry staining in sentinel lymph nodes of patients with melanoma. Ann Surg 2000; 231:795–803.[CrossRef][Medline]
  11. Hoon DS, Wang Y, Dale PS, et al. Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay. J Clin Oncol 1995; 13:2109–16.[Abstract/Free Full Text]
  12. Sarantou T, Chi DD, Garrison DA, et al. Melanoma-associated antigens as messenger RNA detection markers for melanoma. Cancer Res 1997; 57:1371–6.[Abstract/Free Full Text]
  13. Hatta N, Takata M, Takehara K, Ohara K. Polymerase chain reaction and immunohistochemistry frequently detect occult melanoma cells in regional lymph nodes of melanoma patients. J Clin Pathol 1998; 51:597–601.[Abstract]
  14. Kuo CT, Hoon DS, Takeuchi H, et al. Prediction of disease outcome in melanoma patients by molecular analysis of paraffin-embedded sentinel lymph nodes. J Clin Oncol 2003; 21:3566–72.[Abstract/Free Full Text]
  15. Balch CM, Buzaid AC, Soong S-J, et al. Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 2001; 19:3635–48.[Abstract/Free Full Text]
  16. Paradelo C, Fraile M, Ferrandiz C, Alastrue A, Bigata X. Lymphoscintigraphy in the study of lymphatic drainage patterns in patients with melanoma. Med Clin (Barc) 1999; 113:281–4.
  17. Rex J, Paradelo C, Mangas C, et al. Single-institutional experience in the management of patients with clinical stage I and II cutaneous melanoma. Results of the sentinel lymph node biopsy in 240 cases. Dermatol Surg 2005; 31:1385–93..[Medline]
  18. Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991; 338:1227–9.[CrossRef][Medline]
  19. Reintgen D, Balch CM, Kirkwood J, Ross M. Recent advances in the care of the patient with malignant melanoma. Ann Surg 1997; 225:1–14.[CrossRef][Medline]
  20. Kirkwood JM, Ibrahim JG, Sondak VK, et al. High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol 2000; 18:2444–58.[Abstract/Free Full Text]
  21. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53:457–81.[CrossRef]
  22. Wagner JD, Corbett L, Park HM, et al. Sentinel lymph node biopsy for melanoma: experience with 234 consecutive procedures. Plast Reconstr Surg 2000; 105:1956–66.[Medline]
  23. Albertini JJ, Cruse CW, Rapaport D, et al. Intraoperative radio-lympho-scintigraphy improves sentinel lymph node identification for patients with melanoma. Ann Surg 1996; 223:217–24.[CrossRef][Medline]
  24. Ribuffo D, Gardilone A, Vonella M, et al. Prognostic significance of reverse transcriptase-polymerase chain reaction-negative sentinel nodes in malignant melanoma. Ann Surg Oncol 2003; 10:396–402.[Abstract/Free Full Text]
  25. Bostick PJ, Morton DL, Turner RR, et al. Prognostic significance of occult metastases detected by sentinel lymphadenectomy and reverse transcriptase-polymerase chain reaction in early-stage melanoma patients. J Clin Oncol 1999; 17:3238–44.[Abstract/Free Full Text]
  26. Takeuchi H, Morton DL, Kuo C, et al. Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients. J Clin Oncol 2004; 22:2671–80.[Abstract/Free Full Text]
  27. Blaheta HJ, Schittek B, Breuninger H, et al. Detection of melanoma micrometastasis in sentinel nodes by reverse transcription-polymerase chain reaction correlates with tumor thickness and is predictive of micrometastatic disease in the lymph node basin. Am J Surg Pathol 1999; 23:822–8.[CrossRef][Medline]
  28. Goydos JS, Patel KN, Shih WJ, et al. Patterns of recurrence in patients with melanoma and histologically negative but RT-PCR-positive sentinel lymph nodes. J Am Coll Surg 2003; 196:196–204.[CrossRef][Medline]
  29. Kammula US, Ghossein R, Bhattacharya S, Coit DG. Serial follow-up and the prognostic significance of reverse transcriptase-polymerase chain reaction-staged sentinel lymph nodes from melanoma patients. J Clin Oncol 2004; 22:3989–96.[Abstract/Free Full Text]
  30. Bautista NC, Cohen S, Anders KH. Benign melanocytic nevus cells in axillary lymph nodes. A prospective incidence and immunohistochemical study with literature review. Am J Clin Pathol 1994; 102:102–8.[Medline]
  31. Johansson M, Arstrand K, Hakansson A, Lindholm C, Kagedal B. Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction. Melanoma Res 2000; 10:213–22.[Medline]
  32. Davids V, Kidson SH, Hanekom GS. Melanoma patient staging: histopathological versus molecular evaluation of the sentinel node. Melanoma Res 2003; 13:313–24.[CrossRef][Medline]
  33. McMasters KM, Noyes RD, Reintgen DS. Lessons learned from the Sunbelt Melanoma Trial. J Surg Oncol 2004; 86:212–23.[CrossRef][Medline]
  34. Cayuela A, Rodriguez-Dominguez S, Lapetra-Peralta J, Conejo-Mir JS. Has mortality from malignant melanoma stopped rising in Spain? Analysis of trends between 1975 and 2001. Br J Dermatol 2005; 152:997–1000.[CrossRef][Medline]



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