Serum S100B and LDH Are Not Useful in Predicting the Sentinel Node Status in Melanoma Patients

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1 Serum and Are Not Useful in Predicting the Sentinel Node Status in Melanoma Patients FRIEDERIKE EGBERTS 1, ANNIKA MOMKVIST 1, JAN-HENDRIK EGBERTS 2, KATHARINA C. KAEHLER 1 and AXEL HAUSCHILD 1 1 Department of Dermatology, Schleswig-Holstein University Hospital, Campus Kiel, Germany; 2 Department of General and Thoracic Surgery, Schleswig-Holstein University Hospital, Campus Kiel, Germany Abstract. Background: Serum and, as well as the status of the sentinel node, have been reported as prognostic markers in melanoma patients. The purpose of this study was to determine the value of serum S-100B and in melanoma patients prior to sentinel lymph node dissection (SLND) with respect to the clinical outcome. Patients and Methods: Serum and were measured prior to SLND in 259 melanoma patients between 2000 and Upper institutional limits were 0.12 μg/l for and 240U/l for. Results: The median follow-up time was 27.1 months. The median S-100B value for SN-negative and SNpositive patients was 0.06 μg/l and 0.05 μg/l, respectively (p=0.291). Similarly for, the values were U/l and U/l, respectively (p=0.763). Neither of the proposed markers were a statistically significant prognostic parameter for disease-free survival (DFS), distant metastasis-free survival (DMFS) and overall survival (OS). Conclusion: In the present study neither serum nor prior to SLND were useful in predicting the histopathological status of the sentinel node. None of them correlated with DFS, DMFS or OS. In metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) the prognosis is still poor and the median overall survival time is 6 to 12 months only (1). There is an ongoing debate about the appropriate medical treatment for patients with advanced metastatic melanoma. Randomized trials have failed to demonstrate the benefits of one regimen (2). In addition to an improvement in the available therapeutic regimens for better response and overall Correspondence to: Axel Hauschild, MD, Professor of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Department of Dermatology, Schittenhelmstr. 7, Kiel, Germany. Tel.: , Fax: , ahauschild@ dermatology.uni-kiel.de Key Words: Melanoma, Sentinel node, serum S100,, prognosis, metastasis. survival rates, it is essential to identify patients who will more likely respond to systemic treatment with better longterm outcomes. Identifying prognostic factors for predicting the clinical course of the disease is therefore essential. Routine imaging methods and laboratory tests are part of the regular follow-up protocol for detecting and treating metastases as early as possible. Monitoring examinations in melanoma patients with distant metastasis revealed that Lactate dehydrogenase () serum concentrations correlate with prognosis, and changes reflect progression and regression of metastatic disease accordingly (3-6). is not specific to melanoma metastasis, but has been used in several studies as a prognostic marker in melanoma patients and subsequently been implemented in the current AJCC melanoma staging system (7). Melanoma-associated molecules are also potential candidates for measurement in the blood of patients with melanoma. Among such markers, protein S-100B has shown to be of prognostic value since a clear correlation of S-100B serum concentrations, clinical stage of disease and survival has been found in various clinical investigations (8-12). A luminoimmunometric assay (LIA) to measure S-100B serum concentrations is commercially available for the monitoring of advanced metastatic melanoma (13). Clinical studies have demonstrated that changes of S- 100B concentrations reflect the course of disease in the majority of melanoma patients (14-16). Sentinel lymph node dissection (SLND) was implemented as a staging procedure in primary melanoma patients without clinically detectable lymph node metastases more than 15 years ago (17). Since SLND does not lead to a clear improvement in survival, its value has been the subject of controversy. Some authors do not recommend SLND for routine use until there is further evidence of benefits for the patients (18-22). On the other hand, the histopathology status of the sentinel node (SN) has been demonstrated to be an important prognostic factor for disease-free (DFS) and overall survival (OS) of patients with primary melanoma (23). The three-year DFS rates for patients with tumor-free and tumorcontaining SNs are 88.5% and 55.8%, respectively (24) /2010 $

2 Therefore, the status of the SNs was included as a prognostic parameter in the current AJCC staging system for melanoma (7). To contribute to the discussion about prognostic serological markers in patients with malignant melanoma 1mm, this single-center study was performed. The prognostic significance of serum and serum concentrations prior to SLND with regards to the histopathological status of the SN and to DFS, distant metastasis-free survival (DMFS) and OS, were assessed. Patients and Methods Patient population and treatment. The study population consisted of 259 consecutive patients with primary cutaneous melanoma and no preoperatively detected metastases, in whom SLND was performed between October 2000 and September 2006 and at least 1 SN was evaluated histopathologically. Indications for SLND were a Breslow tumor thickness of 1 mm or <1 mm if regression or ulceration of the primary melanoma was histologically documented. Exclusion criteria were palpable lymph node metastases or signs of distant metastases. Patients provided written informed consent, and this study was approved by an Institutional Review Board. Baseline clinical data on the patients and tumor-related data, such as location of the primary tumor, tumor thickness, and Clark s level, were available from their medical records. Information on the type of adjuvant treatment as well as on morbidity and mortality was collected (median follow-up time: 27.1 months). Staging of the patients was performed according to the AJCC classification of 2002 (7). All patients included in the study had undergone a standard technique of preoperative lymphoscintigraphy with radioactive Tc99-nanocolloid and double-staining of the SNs with the intraoperative use of blue dye and a hand-held gamma probe. In addition, a local excision of the primary tumor with standard safety margins according to the German Melanoma guidelines was performed (25). The SNs were analyzed histopathologically by sectioning them into 200 μm slices and proceeding with conventional staining with hematoxylin and eosin (H&E) and immunohistochemical staining with antibodies to S-100, Melan-A and HMB-45. When no tumor cells were detected by either H&E or immunohistochemistry, the SN was considered negative. Patients with a malignant melanoma in the head and neck area were operated on in the ENT department of our university hospital. All patients with positive SNs underwent complete lymph node dissection using established surgical techniques. In the case of axillary metastases, levels I to III of the axillary lymph nodes were removed. When groin metastases were detected, patients underwent complete ilioinguinal dissection, whereas in patients with neck metastases, a modified neck dissection was performed. Tumor marker assays. Serum samples were investigated immediately for concentrations. For protein measurement, the blood samples were collected, separated and frozen immediately after blood extraction, as recommended by Djukanovic et al. (26). Samples were stored at 20 C for a maximum of 1 week. Total activity was measured with an automated controlled system (reagent and analyser: Roche/Hitachi 747; Roche Diagnostics, Mannheim, Germany). The assay was performed according to the manufacturer s instructions. For protein measurement, we used the Sangtec S-100 LIA, a two-site sandwich assay based on three different monoclonal antibodies (SMST 12, SMSK 25+28) that specifically bind to the B-unit of the S-100B protein. The assay detects the BB- and ABdimers of the S-100B protein in serum. The functional lower detection limit of the assay was 0.02 μg/l, defined as the concentration where the coefficient of variation is exceeding 20%, as indicated in the manufacturer s instructions (Sangtec Medical, Bromma, Sweden). A serum concentration of 0.12 μg/l for S-100B was used as the upper institutional limit according to the manufacturer s instructions. For interpretation of levels in serum the upper institutional limits of 240 units per liter were chosen. 219 serum probes and 214 probes were used for evaluation. Follow-up and further treatment. All patients were routinely followed up according to the German Melanoma guidelines (25) at 3-month intervals for the first 5 years. Thereafter, follow-up information was obtained from patient interviews or questionnaires sent to family practitioners. In addition to the medical history and clinical examinations, imaging techniques including chest X-rays, lymph node and abdominal ultrasound (for SN-negative patients) and CT scans of the abdomen and chest as well as an MRI of the brain (for SN-positive patients) were routinely performed. If progressive disease (PD) was detected, surgical excisions were performed whenever possible. Otherwise, chemotherapy or combined systemic treatment was administered. Statistical methods. The data were analyzed using SPSS for Windows (Version 13.0, Chicago, IL, USA). Baseline descriptive statistics included proportions and mean or median values, as appropriate by data distribution. Normality distribution was checked by Kolmogorov-Smirnov test. To assess the statistical significance of the difference in the mean Serum and concentrations for each group, the Mann-Whitney U-test and the unpaired t-test were used, respectively. To investigate the capability of both and S-100B in detecting the presence of micrometastatic disease, sensitivity and specificity as well as the positive predictive values and likelihood ratios were calculated. Estimated survival curves were constructed by the Kaplan-Meier method. Melanoma-related deaths were considered as events in overall survival. DFS, DMFS and OS were calculated from time of sentinel lymph node excision until recurrence of the disease (locoregional/distant) or death, respectively. Probabilities of less than 0.05 were considered to be statistically significant. Results Patient characteristics. A total of 259 patients (136 females, 123 males) with a median age of 57.5 (range 14-80) years were classified as having AJCC stage IA-IIC disease. In 44 out of 259 patients (17.0%), micrometastatic disease of the SNs was detected. In 31 patients (12.0%), only one SN was involved, and in 13 patients (5.0%) two or more SNs contained melanoma cells. In 18.2% of the patients with a positive SN further tumor deposits in non-sns were found upon complete lymph node dissection (CLND) (n=8). Table I summarizes the patient characteristics according to the histopathological status of the SN. 1800

3 Egberts et al: Serum and in Melanoma Table I. Patient characteristics. Negative SN Positive SN Total (n=215) (n=44) (n=259) Age, years, median (range) 57.5 (14-80) 57.5 (20-75) 57.5 (14-80) Gender Male 99 (46.0%) 24 (54.5%) 123 (47.5%) Female 116 (54.0%) 20 (45.5%) 136 (52.5%) Breslow s thickness Mean, mm ±SD 2.13± ± ±1.56 Median, mm (range) 1.7 ( ) 2.5 ( ) 1.9 ( ) Ulceration Present 74 (34.4%) 22 (50.0%) 96 (37.1%) Absent 141 (65.6%) 22 (50.0%) 163 (62.9%) AJCC stage before SLND (T classification) IA 18 (94.7%) 1 (5.3%) 19 (100%) IB 88 (92.6%) 7 (7.4%) 95 (100%) IIA 61 (77.2%) 18 (22.8%) 79 (100%) IIB 37 (75.5%) 12 (24.5%) 49 (100%) IIC 11 (64.7%) 6 (35.3%) 17 (100%) SN: Sentinel node, AJCC: American Joint Committee on Cancer. Table II. Estimated 5-year survival rates according to the status of the sentinel node. DFS DMFS OS SN 80.0% 83.9% 87.6% SN % 50.1% 77.2% SN: Sentinel node, DFS: disease-free survival, DMFS: distant metastases-free survival, OS: overall survival. Survival analysis. The median follow-up time in our study was 27.1 months (range 0-83). Since more than 50% of the patients were still alive, the mean overall survival time of the entire cohort was estimated as 75.8 months (SD: 1.7; 95% CI: ). The mean disease-free and distant metastasis-free survival time was 68.1 months (SD 2.2; 95% CI: ) and 70.9 months (SD 2.0, 95% CI: ), respectively. The estimated overall survival rates of our patient population were as following: 1-year OS rate: 100%, DFS rate: 94.9%, DMFS rate: 98.1%; 5-year survival rate: OS: 85.2%, DFS: 73.0%, DMFS: 76.4%. Significant differences were observed between SN-negative and SN-positive patients for DFS (p<0.001), DMFS (p<0.001) and OS (p=0.039). The estimated 5-year survival rates are summarized in Table II. Diagnostic and prognostic value of and. There was no statistically significant correlation between the histopathologic SN status and the preoperatively obtained serum markers and. The median S-100B value Table III. Probability of detecting metastatic disease in the sentinel node(s) for serum and. (95%-CI) (95%-CI) Sensitivity 16.13% 5.88% ( ) ( ) Specificity 87.23% 98.33% ( ) ( ) Positive predictive value 17.24% 40.00% ( ) ( ) Positive likelihood ratio ( ) ( ) Table IV. Log-rank test regarding OS, DFS and DMFS (serum and ). Mean Survival Factor survival Events Censored (months) (n (%)) (n (%)) p-value OS DFS DMFS < 0.12 μg/l (6.3%) 178 (93.7%) μg/l (6.9%) 27 (93.1%) < 240 U/l (5.7%) 197 (94.3%) U/l (20.0%) 4 (80.0%) < 0.12 μg/l (12.1%) 167 (87.9%) μg/l (20.7%) 23 (79.3%) < 240 U/l (12.9%) 182 (87.1%) U/l (20.0%) 4 (80.0%) < 0.12 μg/l (20.5%) 170 (89.5%) μg/l (20.3%) 26 (89.7%) < 240 U/l (10.0%) 188 (90.0%) U/l (20.0%) 4 (80.0%) OS: Overall survival, DFS: disease-free survival, DMFS: distant metastasis-free survival. for SN-negative and SN-positive patients was 0.06 μg/l (range: μg) and 0.05 μg/l (range: μg/l), respectively (p=0.291). The median value for SNnegative and SN-positive patients was U/l (range: U/l) and U/l (range: U/l), respectively (p=0.763). Table III illustrates the calculated sensitivities, specificities, predictive values and positive likelihood ratios for and. Overall, the sensitivities of both markers at predicting the tumor status of the SN were rather low. Furthermore, a statistically significant correlation between preoperatively obtained and levels and non-sn positivity upon CLND could not be established (: p=0.521; : p=0.250). 1801

4 Figure 1. Kaplan-Meier survival for patients stratified by serum levels (cut-off: 0.12 μg/l). There was no statistically significant difference between groups regarding OS, DFS and DMFS (OS: Overall survival; DFS: disease-free survival; DMFS: distant metastasis-free survival). Figure 2. Kaplan-Meier survival for patients stratified by serum levels (cut-off: 240 U/l). There was no statistically significant difference between groups regarding OS, DFS and DMFS. (OS: Overall survival; DFS: disease-free survival; DMFS: distant metastasis-free survival). 1802

5 Egberts et al: Serum and in Melanoma Upon univariate analysis, neither nor serum levels prior to SLND showed a statistically significant prognostic impact regarding the clinical outcome (DFS, DMFS, OS) (Table IV, Figures 1and 2). Discussion Contradictory results have been published regarding the prognostic impact of several serum markers in malignant melanoma. The prognostic value of in patients with advanced metastatic melanoma has been underscored by the implementation of this marker into the current AJCC melanoma classification system in 2001 (7). Moreover, Deichmann and co-workers found levels to be the most relevant parameter compared to and melanoma inhibitory activity (MIA) (27). In contrast, several other authors reported that is an appropriate marker in advanced metastatic melanoma and superior to serum concentration (14, 28-31). Serum S100 may be useful for the evaluation of response to treatment and prediction of early distant relapse and survival in both stage III and IV melanoma (32, 33). It has also been shown that serum levels may increase before disease progression becomes clinically apparent (10, 34-35). Recently, Tarhini et al. evaluated the serum levels from 670 high-risk surgically resected melanoma patients. A high baseline or increasing serum was an independent prognostic marker of risk for mortality, and lower values at baseline and during follow-up were associated with longer survival (36). The histopathology status of the SN has been reported by many authors to be a meaningful and independent prognostic marker for the clinical outcome of melanoma patients (24, 35). It provides information that might be helpful for both the patient and the attending physician in the decision about further treatment options such as adjuvant therapy with interferons or, alternatively, participation in a clinical trial. To date it is unclear, whether a serum marker exists which is able to predict micrometastatic disease in the SN. In the present study, we were unable to find a positive correlation between serum markers and prior to SLND and the histopathological status of the SN. Similarly, the sensitivities of and in predicting tumor status of the SN were rather low, and there was no statistically significant prognostic impact of either marker regarding the clinical outcome. One explanation may be that in these patients, neither marker is produced in sufficient quantities to obtain a measurable increase in serum levels. The findings presented here are consistent with other reports in the literature. Smit et al. showed, in a smaller study of only 89 melanoma patients, that levels are not useful in predicting the histopathological status of the SN. However, this study did not compare the levels to other serum markers such as (38). In the study of Acland et al. on 31 melanoma patients with a primary tumor thickness >1 mm, the authors also concluded that could not predict the presence of micrometastatic disease in the SNs. In this relatively small patient cohort, the authors did not compare and levels. Moreover, they did not provide any follow-up information, including the impact of the serum markers on the clinical outcome (39). In a comparative study on serum and FDG-positron-emission tomography on 332 melanoma patients, neither method was able to provide sufficient information on the SN status but were useful for detecting recurrence or metastases (40). Our present study demonstrates that neither serum nor are a marker of limited metastatic disease, nor are they able to predict the presence of micrometastatic disease in regional lymph nodes. and therefore can neither replace SLND, nor provide predictive information in the staging of melanoma patients with more than 1 mm tumor thickness. Furthermore, the detection of circulating tumor cells in melanoma patients using the tyrosinase mrna RT-PCR method failed to be sensitive enough to be used as a progression marker in the early stages of the disease. In a large-sized prospective study on 1,446 patients, RT-PCR analysis on SN and peripheral blood mononuclear cells did not provide additional prognostic information when compared to the conventional histopathological examination of the SNs (41). Ultrasound-guided fine-needle aspiration cytology of SNs is reliable in detecting metastatic involvement and may eliminate the need for surgical SN procedures in 16% of cases (42). However, as this procedure is not yet standardized, it is only suitable for highly specialized centers. To date, no serum analysis or standardized non-invasive investigation is able to provide reliable information about the histopathological status of the SN. Therefore, SLND remains an essential staging procedure for patients with a clinically localized melanoma. In this setting, the tumor load within the SN will be of highest prognostic importance regarding the clinical outcome and further therapeutic strategies. Acknowledgements We thank Ulrike Schulz, Medistat, Kiel, Germany, for her substantial input to the statistical analyses presented in this study. References 1 Brochez L and Naeyaert JM: Understanding the trends in melanoma incidence and mortality: where do we stand? Eur J Dermatol 10: 71-75; quiz 76, Bulliard JL and Cox B: Cutaneous malignant melanoma in New Zealand: trends by anatomical site, Int J Epidemiol 29: ,

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