Division of 1 Medical Oncology and 4 Plastic Surgery, University Hospital, Pisa; 2 Regional Center of Nuclear Medicine, 3 Pathology Unit, Department of Surgery and 5 Division of Dermatology, University of Pisa Medical School, Pisa, Italy
* Correspondence to: Dr A. Romanini, Division of Medical Oncology, Santa Chiara University Hospital, Via Roma, 67, 56126 Pisa, Italy. Tel: +39-050-992645; Fax: +39-050-992070; E-mail: a.romanini{at}ao-pisa.toscana.it
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Abstract |
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Patients and methods: Patients with primary stage III cutaneous melanoma underwent radioguided sentinel lymphadenectomy. Their SLNs were assessed for tyrosinase (Tyr) and melanoma antigens recognized by T-cells (MART-1) mRNA expression using RTPCR, in parallel with hematoxylin and eosin staining and immunohistochemistry. Tyr and MART-1 expression in the SLNs were correlated with PATH assay results, standard prognostic factors, time to progression and overall survival.
Results: Twenty-three of the 124 patients (18.5%) had positive SLNs by both PATH and RTPCR (PATH+/PCR+). Sixteen patients (13%) were negative by PATH and positive by RTPCR (PATH/PCR+). Eighty-five patients (68.5%) had SLNs that were negative by both PATH and RTPCR (PATH/PCR). At a median follow-up of 30 months, recurrence rates among the three cohorts were statistically different (PATH+/PCR+, 60%; PATH/PCR+, 31%; PATH/PCR, 9.4%). Seven of 23 (30%) and two of 16 (12.5%) patients died in the PATH+/PCR+ and PATH/PCR+ SLN groups, respectively, whereas no patient died in the PATH/PCR SLN group.
Conclusions: RTPCR is more sensitive than PATH to detect SLN metastases and it is a reliable predictor of disease relapse in stage III melanoma patients.
Key words: lymph node metastasis, melanoma, prognosis, RTPCR, sentinel lymph node
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Introduction |
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The regional tumor-draining lymph node basin is the most frequent site of early melanoma metastases [3]. As originally proposed [4
] the sentinel lymph node (SLN) is defined as the first lymph node in the regional basin that receives a cutaneous afferent lymphatic vessel from the primary melanoma. If the SLN is negative for metastases, then the probability for the remainder of the nodes in the basin to harbor melanoma cells is less than 1% [4
]. Therefore, full nodal classification can be obtained with a conservative SLN biopsy that is less invasive, less morbid and entails lower costs than elective lymph node dissection. Lymphadenectomy can thus be reserved for only those patients in whom a positive SLN has been detected (selective lymphadenectomy) [5
]. Another major advantage of lymphatic mapping is that only one or two SLNs per patient (instead of 1040 lymph nodes deriving from complete lymphadenectomy) are processed for pathology. Since SLNs are the nodes most likely to contain metastatic disease, it is possible to analyze them more accurately, for instance with serial sectioning (5 µm in thickness) and immunohistochemical (IHC) analysis with antibodies to S-100 [6
] or HMB-45 [7
]. This procedure has 10% to 30% higher sensitivity for identifying micrometastases compared with conventional hematoxylin and eosin (H&E) staining [8
]. Moreover, when SLNs of patients who had developed recurrence within 1.53 years were re-evaluated with serial sectioning and/or IHC, occult metastases (that had previously gone undetected with conventional H&E staining) were found in 6% to 11.5% of the cases [9
11
].
More recently, application of the reverse transcriptase polymerase chain reaction (RTPCR) technique enabled the upstaging of an additional 13% to 30% of patients whose SLNs were negative when analyzed by H&E and IHC staining (conventional pathology, PATH) [1215
]. Messenger-RNA (mRNA) codifying for tyrosinase (Tyr, a key enzyme in the synthesis of melanin) and for the melanoma antigen recognized by T-cells (MART-1, expressed in most melanoma cell lines) were chosen as markers for the presence of melanoma cells in SLNs [16
].
The purposes of this study were (i) to validate the usefulness of RTPCR for Tyr and MART-1 mRNA for improving the identification of minimal residual disease in SLNs of melanoma patients versus PATH, and (ii) to determine whether PATH/PCR+ SLN patients had increased risk of relapse compared with PATH/PCR SLN patients.
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Patients and methods |
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Lymphatic mapping
Lymphoscintigraphy was performed 46 h prior to SLN biopsy to define the draining lymphatic basin at risk for metastatic disease and to identify the corresponding SLN. Aliquots of 0.10.2 ml containing 48 MBq of 99mTc-HSA nanocolloid (Nanocoll®; Amersham Health, Amersham, UK) were injected intradermally around the primary melanoma, or on skin margins of the surgical scar if the primary lesion had previously been excised. A large field-of-view gamma camera (Camstar XRT; GE Medical System, Milwaukee, WI, USA) equipped with a parallel-hole, high-resolution collimator was used for imaging, with a 10% window centered on the 140 keV energy peak of 99mTc. Early dynamic scintigraphic images helped to visualize the lymphatic draining from the primary lesion. Subsequently, delayed static orthogonal and oblique images were acquired to better localize the nodes also depthwise. A hand-held -probe (Scintiprobe MR 100 Pol.hi.tech®; Carsoli, Italy) was used to confirm by external counting the approximate location of the SLN, whose skin projection was marked using indelible ink [18
].
Radioguided SLN biopsy
SLN biopsy was performed under intraoperative hand-held -probe guidance [18
]. The criteria for
-probe identification of a SLN were based on detecting a focal zone of radioactivity with a count ratio (hot spot to background) >10. We define background as the average count rates of the surrounding non-sentinel nodes and lymph node basin. Any additional lymph node whose counting rate exceeded 20% of the hottest node was considered an additional SLN. Complete removal of the SLNs was confirmed by reduction of the counting rate in the surgical area to background level. In patients who had melanomas in areas that drained into more than one nodal basin, a SLN was removed from each nodal basin. Wide local excision of the primary tumor was usually performed after the removal of all SLNs, except in cases where the nodal basin was close to the primary melanoma than initial excision of the tumor was necessary to reduce interfering radioactivity [18
].
Specimen collection
The harvested SLNs were immediately bisected, one half was sent for H&E and IHC, the other half was placed in Trizol (Life Technology) to preserve RNA and sent for RTPCR molecular analysis.
Histopathology
After fixation in 10% formalin, the dimensions of the SLN specimen and its consistency were evaluated. The hilar region was identified as a landmark for dividing the specimen into two halves along the longitudinal axis, thus obtaining a wider area for sectioning. Several 5-µm thick sections were cut, the first was stained with H&E and the three adjacent ones were immunohistochemically tested for S-100 and HMB-45 proteins, and MART-1 in cases that were negative to the previous evaluations [19]. Histopathologic examination included scanning of the entire slide with magnifications of x40 and x100 in order to detect discrete foci of tumor cells. The sinusoids were examined at a magnification of x250 in order to detect single tumor cells and/or small clumps.
RNA isolation and RTPCR assay
Following surgical resection, SLN specimens for molecular biology analysis were immediately placed in a RNAse-free guanidinium isothiocyanate solution (Trizol; Life Technologies) and stored at 80°C until use. Total RNA was extracted from homogenized tissues according to the manufacturer's protocol, and integrity and purity of the sample were checked by agarose-formaldehyde gel electrophoresis under denaturing conditions and spectrophotometrical reading. Nucleic acids were then stored in aliquots under ethanol at 80°C until analysis.
Total RNA (2 µg) were reverse-transcribed into cDNA in 20 µl reaction mixture containing 100 pmol random hexamers (Boehringer Mannheim), 6 U reverse transcriptase (RT; Promega), 1.5 mm MgCl2, 0.2 mm each dNTP, 8 U RNAse inhibitor (Life Technologies). Samples were incubated at 25°C for 10 min, 42°C for 1 h and RT denatured at 95°C for 5 min in a 2400 Perkin Elmer thermal cycler. cDNA samples were subsequently amplified for the target sequences by using a single-step PCR and the following reaction mixture: 5 µl cDNA, 1.5 mm MgCl2, 0.2 mm each dNTP, 1 U of AmpliTaq Gold DNA polymerase (Perkin Elmer), 20 pmol each primer and PCR buffer 1x in 50 µl final volume. To prevent contamination and false positive results, RNA extraction, preparation of reaction mixtures and amplifications were carried out in separated rooms. Each RTPCR experiment included a sample with no RNA as negative controls and RNA extracted from SK-mel 23 melanoma cell line as positive control that were added to each run. Each sample was amplified for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA to assess RNA integrity and absence of DNA polymerase inhibitors, and the melanoma-associated markers, Tyr and MART-1 mRNAs.
GAPDH primers were: sense 5' GGT CGG AGT CAA CGG ATT TG 3', antisense 5'ATG AGC CCC AGC CTT CTC CAT 3', yielding a 320 bp amplicon [20]. Tyr expression was determined by using HTR3 sense 5' GTC TTT ATG CAA TGG AAC GC 3' and HTR4 antisense 5' GCT ATC CCA GTA AGT GGA CT 3' primers, generating a 207 bp PCR product and used as inner primer set of a previously described nested-PCR reaction [21
]. MART-1 was amplified with M1 sense 5' AGA TGC CAA GAG AAG ATG CTC 3' and M2 antisense 5' GCT CTT AAG GTGAAT AAG GTG G 3', yielding a 364 bp PCR product [22
]. The amplification cycling profile was as follows: initial denaturation 95°C for 5 min; cycling: 95°C for 30 s, 55°C for 30 s and 72°C for 30 s (five cycles); 95°C for 20 s, 55°C for 20 s and 72°C for 20 s for 55 cycles (35 cycles for GAPDH); final extension 72°C for 10 min. Amplifications were carried out in a 2400 Perkin Elmer thermal cycler and, after reaction completion, 20 µl of PCR samples were run on 2% agarose gel electrophoresis and samples scored positive when amplicons of the expected size were readily visible under UV light [23
].
Adjuvant therapy
Patients with nodal metastases identified by PATH underwent complete node dissection and were offered adjuvant high-dose interferon--2b (HD-IFN, Intron-A; Schering-Plough, Kenilworth, NJ, USA) therapy [24
]. No lymphadenectomy or adjuvant IFN therapy were offered to patients with SLNs negative by PATH even though they were positive by RTPCR (PATH/PCR+).
Statistical analysis
To assess the correlation of clinicopathologic risk factors with PATH and RTPCR Tyr and MART-1 mRNA expression in the SLN, univariate (using 2-test or Student's t-test for continuous variables to compare the difference in mean Breslow thickness) and multivariate (using logistic regression model) analyses were used.
The KaplanMeier method was used to plot disease-free survival (DFS) and overall survival (OS), while the log-rank test was used to compare curves [25, 26
]. DFS was calculated from the day of SLN biopsy until the ascertainment of disease progression or the last follow-up visit. OS was calculated from the day of SLN biopsy until death or the last follow-up visit.
Cox proportional hazard regression models were performed to examine the association of pathologic and molecular risk factors with DFS. Data were analyzed using SPSS/PC+11.5 statistical software. In all statistical analyses, P < 0.05 was considered significant.
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Results |
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Histology, IHC and RTPCR analysis
Thirty (15%) of the 197 SLNs were positive for metastatic involvement by conventional pathology (H&E, IHC with S-100 and HMB-45). In one SLN, IHC for MART-1 was performed to confirm an uncertain pathological diagnosis. Overall, 144 of 197 SLNs (73%) were negative for metastatic involvement by both PATH and RTPCR analysis, while 30 of 197 SLNs (15%) were positive by both PATH and RTPCR analysis (RTPCR being positive in all SLNs that were positive by PATH). On the other hand, 23 of 197 SLNs (12%) were negative by PATH, yet they turned out positive for metastatic involvement by RTPCR analysis.
SLN status and clinical outcome
Local recurrence or stage at first relapse and number of deaths, on a patient by patient basis, are reported in Table 2. Eighty-five patients (68.5%) had SLNs that were negative by both PATH and RTPCR (PATH/PCR). Twenty-three of the 124 patients (18.5%) had positive SLNs by PATH, and all of them were also positive by RTPCR (PATH+/PCR+). Sixteen patients (13%) were negative by PATH and positive by RTPCR (PATH/PCR+). Out of the 85 PATH/PCR patients, only one recurred locally (after 9 months) and seven relapsed in distant organs, yet none have died, thus identifying a group with favorable prognosis (9.4% recurrences/relapses, 0% deaths). Out of the 23 PATH+/PCR+ patients, 14 relapsed and seven died, thus identifying a group with poor prognosis (60% recurrences/relapses, 30% deaths). Finally, out of the 16 PATH/PCR+, three recurred (after 9, 16 and 24 months), two relapsed and two died, thus identifying a group with intermediate prognosis (31% recurrences/relapses, 12.5% deaths). The patient with two melanoma lesions with SLNs negative at both PATH and RTPCR analysis has not recurred after 44 months of follow-up.
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Univariate and multivariate analyses
After pathologic staging of the primary tumor and SLN, 69 of 125 lesions (55.2%) were stage I, 33 lesions (26.4%) were stage II and 23 (18.4%) were stage III. Age, sex, primary tumor site, Breslow thickness, Clark level, AJCC stage, ulceration and histotype were the variables independently considered as risk factors in univariate analysis (Table 3). Nodular histotype (P = 0.002), Breslow thickness (P = 0.0001), mean Breslow thickness, compared using Student's t-test for continuous variables (P = 0.0001), and AJCC stage of the primary lesion (P = 0.0001) were significant predictors of metastatic involvement of SLN as identified by PATH.
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DFS and OS curves
KaplanMeier DFS and OS curves (Figures 1 and 2, respectively) show the profound impact of the PATH and PCR status on both parameters. In particular, patients whose SLNs were metastatic at both PATH and PCR analysis had the shortest median DFS (29 months; 95% CI 1345). Patients whose SLNs were PATH/PCR+ had a slightly, but not significantly, longer median DFS (33 months; 95% CI not determined). Finally, in patients with PATH/PCR SLNs the median DFS was not reached and was anyway significantly longer (P < 0.05) than in patients whose SLNs were PATH+/PCR+ (P < 0.0001) or PATH/PCR+ (P = 0.0057).
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Cox hazard regression analysis showed that the risk of disease progression was of the same order of magnitude in patients whose micrometastases in SLNs were detected with molecular markers (PCR+) only or with both PATH and RTPCR assays (P = 0.003) (Table 4). However, most of the relapses and deaths observed in the patients with metastatic SLNs identified only by RTPCR (PATH/PCR+) occurred relatively later in follow-up versus patients with metastatic involvement detected by both PATH and RTPCR (PATH+/PCR+).
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Discussion |
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To increase sensitivity in the detection of nodal metastases, the molecular biology technique RTPCR has been introduced to ascertain the presence of mRNA linked to genes highly expressed in malignant melanoma [1214
, 30
32
]. Tyr, the key enzyme in melanin biosynthesis, is expressed both in normal melanocytes and in melanoma cells [33
]. Nerve cells have also been reported as a source of Tyr expression [34
]. On the other hand, MART-1, a melanoma-associated antigen recognized by T-cells, is a 118 amino acid transmembrane protein (13 kDa) with tissue specificity for melanoma, melanocytes and retina. In order to increase sensitivity, we considered both markers, Tyr and MART-1, to assess the prognostic value of the SLN status [35
]. By applying this approach, we identified an additional 12% metastatic SLNs over those already detected by conventional pathology.
In our patient population, local recurrence occurred in three of 16 and distant metastases in two of 16 patients whose SLN was negative by PATH but positive by PCR analysis. On the other hand, only one of 85 patients with SLN negative by PCR (and obviously also by PATH, since no false-negative results were observed for PCR) had local recurrences and seven other patients had distant metastases, indicating that PCR is both sensitive and accurate in identifying patients more likely to recur because of their melanoma. Moreover, the patients with SLNs negative by PATH but positive by PCR analysis who would have been classified in a low-risk category if based solely on PATH analysis, had an approximate four-fold risk of developing metastases compared with patients whose SLNs were negative both by PATH and PCR analysis (Table 4). The fact that 10 out of these 16 patients had AJCC stage I disease (data not shown) indicates that the molecular assay is more accurate in detecting micrometastases even in early-stage melanomas. Out of the nine patients who have died in our entire group, seven had SLN metastases detected by both techniques (PATH and PCR), while in two patients the SLN metastasis was detected only by PCR (Table 2). These considerations further support the statement that, among those patients whose SLNs are negative by PATH, molecular analysis identifies a subset of patients who could benefit from closer follow-up or from an adjuvant treatment.
The fact that most of the relapses and deaths observed in patients with PATH/PCR+ SLNs occurred relatively later in follow-up compared with PATH+/PCR+ SLN patients, is possibly correlated to the lower tumor burden at diagnosis in the former group. In fact, the majority of such patients had melanomas with <2 mm Breslow thickness and AJCC stage IA or IB, while the group with both PATH and PCR positive SLNs had a significantly higher proportion of patients with >2 mm Breslow thickness (P = 0.009) and AJCC stage II (P = 0.004) melanomas (data not shown). On the other hand, patients with melanoma Breslow thickness >2 mm and AJCC stage IIAIIB had worse DFS and OS compared with patients with lower Breslow thickness lesions and lower AJCC stage disease (P < 0.05; data not shown).
We identified Breslow thickness, AJCC stage and nodular histotype as significant predictors of disease progression (Table 3). Of the three factors only nodular histotype was never reported as significant predictor of disease progression in other studies evaluating SLN status.
Owing to the low number of events observed, correlation of these variables with survival was not performed.
The present results are consistent with, and extend data from, recent studies stratifying patients according to combined PATH and PCR analysis (Table 5) [1214
, 30
32
]. In this regard, it should be emphasized that, at variance with some, but not all reports, our study includes two separate RTPCR markers in addition to conventional PATH, no false-negative case was found at PCR analysis, and the correlation between RTPCR results and DFS is based on 30 month follow-up. In fact, consistent with data in the literature [36
], in this period 85% of our SLN-positive patients recurred, with median time to recurrence of 17 months (range 645). Out of the seven studies including over 50 melanoma patients and evaluating fresh SLNs, only three included a larger patient population than ours (but PCR analysis was based on one marker only), only two utilized multiple molecular markers (based, however, on much smaller groups of patients, or on a much shorter follow-up), and finally three followed patients for >30 months (but with one molecular marker only, or with a smaller group of patients). All eight studies, including our own, reported statistically significant differences in the proportion of patients with recurring disease between groups with PATH and PCR both negative and groups with both parameters positive. Only in three studies (including our own) was there a statistically significant difference in OS between the groups with PATH and PCR both negative and, respectively, negative PATH but positive PCR (Table 5). Other recently published papers on this topic are not included in Table 5 because of different patient selection [37
] or PCR techniques [38
, 39
] and/or study design [15
]. The recent paper by Kammula et al. [40
] reports a statistically significant difference in DFS between PATH/PCR+ and PATH/PCR patients at the 42-month follow-up, while the difference in DFS was no longer statistically significant at extended follow-up (67 months). Based on these findings, the authors conclude that future studies evaluating molecular staging will require approximately 5 years of median follow-up to accurately define outcome for patients with occult melanoma metastases. However, such conclusions are hampered by a number of considerations. In fact, in a consistent proportion of their PATH/PCR patients (28%) the ulceration status was unknown. Given the small sample size, the ensuing misclassification based on SLN status alone might heavily affect survival analysis, as ulceration is an independent adverse prognostic factor in patients with 24 mm Breslow thickness lesions [41
]. Furthermore, Kammula et al. did not report their false-positive rate at RTPCR of SLNs, a possible occurrence when employing Tyr alone, which is expressed also in melanocytes and nerve cells, normal cells that can be found in non-metastatic lymph nodes. Thus, the study by Kammula et al. should not jeopardize the importance of molecular staging in distinguishing a subgroup of patients with intermediate prognosis between the PATH/PCR and PATH+/PCR+ groups. In fact, even if this advantage is short-lived, this information could guide clinicians in performing a more intensive follow-up after the third year of diagnosis or offering patients adjuvant high-dose IFN or a more aggressive chemoimmunotherapy treatments in the attempt to achieve a longer survival. The study by Morton et al. [15
] pioneered the design of the MSLT-1 trial, the third interim analysis of which was recently reported at the ASCO meeting: SLN status definitely has a statistically prognostic significance, and lymphadenectomy after detection of clinically occult SLN metastases significantly prolongs survival compared with therapeutic lymphadenectomy when the draining lymph nodes grow to a palpable size [42
]. Uncertainty still remains as to how to treat PATH/PCR+ SLN patients, a still open issue that is being addressed by the Sunbelt melanoma trial that compares the effect of regional lymphadenectomy (with or without HD-IFN) on DFS and OS in patients with early nodal metastases detected by PATH or by PCR analysis [43
].
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Acknowledgements |
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Received for publication April 11, 2005. Revision received June 10, 2005. Accepted for publication July 15, 2005.
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