Originally published as JCO Early Release 10.1200/JCO.2007.13.7653 on November 3 2008

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Impact of Molecular Staging Methods in Primary Melanoma: Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) of Ultrasound-Guided Aspirate of the Sentinel Node Does Not Improve Diagnostic Accuracy, But RT-PCR of Peripheral Blood Does Predict Survival

Christiane A. Voit, Gregor Schäfer-Hesterberg, Martina Kron, Alexander C.J. van Akkooi, Juergen Rademaker, Ansgar Lukowsky, Alfred Schoengen, Markus Schwürzer-Voit, Wolfram Sterry, Markus Krause, Joachim Röwert-Huber, Alexander M.M. Eggermont

From the Department of Dermatology of the Charité, Humboldt University, Berlin; Departments of Biometry and Medical Documentation and Medical Oncology, University of Ulm, Armed Forces Hospital, Ulm, Germany; Department of Surgical Oncology, Erasmus University Medical Center, Daniel den Hoed Cancer Center, Rotterdam, the Netherlands; and Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY

Corresponding author: Christiane Voit, MD, Klinik für Dermatologie, Venerologie und Allergologie, Charité–Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; e-mail: christiane.voit{at}t-online.de

	ABSTRACT

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Purpose This study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients.

Patients and Methods Between 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter.

Results Thirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001).

Conclusion US-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

	INTRODUCTION

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The technique of sentinel lymph node biopsy (SLNB) was originally defined as selective removal of the first draining node (or nodes) from a tumor, called the sentinel node (SN). After the surgical removal of the SN, the SN is thoroughly examined by means of an extensive pathology protocol. This technique identifies early metastatic involvement in the regional lymph node(s).¹

Ultrasound (US), in combination with fine-needle aspiration cytology (FNAC) of regional lymph nodes, is able to detect lymph node metastasis both at the time of presentation of a primary melanoma and for surveillance at follow-up.^2-5 US-guided FNAC of sentinel nodes is not the standard of care yet, but it is enjoying rapid implementation in many clinics, because it was shown to eliminate the need for unnecessary surgical SN procedures in approximately 30% of all patients with breast cancer.^6,7 We have previously shown that US-guided FNAC is highly accurate and eliminates the need for SLNB in 16% of all examined cases.⁸ SLNB has been reported to have a false-negative rate that ranges from 6% to 13%.⁹ Approximately one third of these initially false negative SNs can be proven to be positive by increasing the number of step sections of the sentinel nodes.¹⁰ There is some evidence that undetected SN metastases will not necessarily become clinically apparent and that these small, most frequently subcapsular metastases are not necessarily relevant to overall survival.^11,12

The aims of the present study are to evaluate the accuracy of (1) additional reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates retrieved through preoperative US-guided FNAC, (2) RT-PCR of the excised SN, and (3) RT-PCR of peripheral blood.

	PATIENTS AND METHODS

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Patients
In this prospective study, 127 consecutive patients scheduled for SLNB after the excision of a melanoma were enrolled after providing written informed consent. All patients had a melanoma of at least 1.00 mm in thickness or, in patients with melanomas less than 1.00 mm, they were Clark grade IV or V, ulcerated, or showed regression. The study was approved by the local ethical committee and conducted in accordance with the Declaration of Helsinki.

Methods
Evaluation of patients was performed according to the following schedule. First, a lymphoscintigraphy was performed, followed by US-guided FNAC of the sentinel node. Subsequently, patients underwent SLNB within 24 hours of the lymphoscintigraphy. The SN was retrieved through the use of the triple technique of lymphatic mapping, which includes the preoperative lymphoscintigraphy (already made), intraoperative use of patent blue dye, and the intraoperative use of a handheld gamma probe. Finally, a second FNAC was performed of the SN after it was excised operatively. All FNAC material was used in part for cytology and another part was shock-frozen for molecular biology with tyrosinase RT-PCR. Furthermore, small pieces of the hilum region and of the afferent draining lymphatic vessels were cut out of the excised SN, which were also examined for the presence of melanoma cells by tyrosinase RT-PCR.

The SNs then proceeded to the pathologist for regular work-up. All excised SNs and the corresponding afferent lymphatic drainage are examined in step sections in hematoxylin and eosin staining and staining against HMB-45 and Melan-A according to the European Organisation for Research and Treatment of Cancer Melanoma Group protocol for the work-up of SNs.¹³

A blood sample was taken at time of SLNB for all patients. Further blood samples were collected when patients presented for clinical examination during their regular scheduled follow-up at 3- or 6-month intervals depending on the American Joint Committee on Cancer stage, which is in accordance with the guidelines of the German Dermatology Society.

Classification by US. Our classification by US and FNAC were previously reported.^4,5,14 Preoperatively, we performed a high-resolution US examination of the lymphatic basin and the lymphatic drainage of the tumor by using the high-end device Technos (ESAOTE, Genoa, Italy) equipped with three transducers between 3.5 and 13 MHz.

FNAC. Methods and results of FNAC were previously reported.^3,15 The fine needle only has a diameter of 0.4 mm (26G). Multiple aspirates were obtained for cytology and for tyrosinase RT-PCR. The smears were dehumidified before staining. To get a representative result, a number of 100 cells per smear are expected.

Tyrosinase RT-PCR of Fine Needle Aspirates (FNA-PCR)
RNA isolation: FNAC material. Approximately 0.3 µL of aspirate was shock-frozen in liquid nitrogen and stored at –80°C for subsequent molecular biologic evaluation. Total RNA was extracted from the mini-cell pellet by means of RNeasy total RNA kit (Qiagen, Hilden, Germany). RNA was quantified by ultraviolet spectrophotometry at 260 nm and 280 nm and stored at –80°C. A total of 1.5 µg of total RNA was transcribed.

cDNA synthesis. Reverse transcription was performed with the SuperScript First-Strand System for RT-PCR (Invitrogen, Carlsbad, CA) according to instructor's protocol. The cDNA synthesis was carried out with 1.5 µg of RNA, 500 ng of Oligo (dT)_12-18 primer, 10 nmol/L of dNTP-Mix, 200 U of SuperScript II (Invitrogen, Carlsbad, CA), followed by an Escherichia coli RNase H treatment (2 U).

Nested tyrosinase PCR. Primers for a nested tyrosinase PCR were used as described.^14,16 Thirty cycles were run using a schedule described elsewhere.^14,17 To amplify the β2m housekeeping gene, PCR was performed under the same condition and concentration as for tyrosinase. β2m PCR product size is 165 base pairs (for sequences of primers for β2m and porphobilinogen deaminase, refer to Max et al^18,19).

Quality control experiments. After an inter-laboratory trial of the European Organisation for Research and Treatment of Cancer for inter-laboratory quality assurance,²⁰ several steps were taken to detect any cross-over contamination and were performed after the protocol as published.²¹ If results of patient samples were discordant, the procedure starting from RNA preparation was rerun twice until two concordant results could be seen. The specificity of PCR products was examined by sequencing all positive samples.

Quantitative real-time PCR. Real-time PCR was conducted on a LightCycler instrument (Roche, Mannheim, Germany) using primer specific for PBGD and tyrosinase (HTYR3, HTYR4) in a single-round PCR. Probes for LightCycler PCR (PBDD-3FL, PBGD-5LC, Tyr-3FL, Tyr-5LC) were purchased from Metabion, Martinsried, Germany. The sequences as used are described by Keilholz et al.²²

Statistics
To evaluate the diagnostic value of RT-PCR of the US-guided FNAC before patients underwent SLNB and of the second FNAC of the surgically excised SN, sensitivity, specificity, and positive and negative predictive values were calculated separately for each of these methods. These results were compared with the sensitivity and specificity of the cytology of US-guided FNAC alone (no RT-PCR) and the final histologic evaluation by the pathologist.

Disease-free survival (DFS) time and overall survival (OS) time were calculated from the date of the SLNB (first blood sampling) until first recurrence or death, respectively. DFS and OS were analyzed with the method of Kaplan and Meier. Furthermore, because serial tyrosinase blood RT-PCR measurements per patient were performed, a proportional hazards model with RT-PCR as a time-dependent covariate was fitted and a hazard ratio with 95% CIs and P values was calculated. RT-PCR result was considered as a time-dependent prognostic factor because several RT-PCR results per patient were available, and RT-PCR results changed over time.²³ Statistical analyses were performed with SAS version 8.2 (SAS Institute, Cary, NC).

	RESULTS

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In total, we evaluated 141 SNs in 127 patients. No US- or FNAC-related morbidity or complications occurred during this study.

Detailed patients and tumor characteristics are listed in Table 1. Of the 120 SNs recognized by US-guided FNAC before the SLNB, 34 were histologically malignant. The combined technique of the preoperative US-guided FNAC of the SN correctly classified 28 of 34 histologically malignant cases as positive, thus achieving a sensitivity of 82%. This combination of US and FNAC also correctly identified 62 of 86 histologically proven benign SNs as not involved, reflecting a specificity of 72% (Table 2).⁸

Table 3 shows an overview of the results with regard to histology (hematoxylin and eosin and/or immunostains), FNAC, and RT-PCR. For patients with discordant results by these techniques (ie, cases where all three results were not all positive or all negative), discrepancies and final survival outcome are listed in Table 4.

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. (A) Disease-free survival from the time of first blood sampling stratified for reverse-transcriptase polymerase chain reaction (RT-PCR) that is always negative and RT-PCR that is positive at least once. (B) Overall survival from the time of first blood sampling stratified for RT-PCR that is always negative and RT-PCR that is positive at least once.

In an additional real-time RT-PCR assay for quantization of tyrosinase according to Keilholz et al²² and for porphobilinogen deaminase, housekeeping gene was used. Melanoma aspirates from SNs and their paired peripheral blood samples were analyzed only if they tested positive in qualitative RT-PCR. Tyrosinase-mRNA was not detected in healthy donor blood samples. Patients with stage III disease expressed this marker more frequently and at higher levels in peripheral blood as compared with those with earlier stage disease. The diagnostic sensitivity was optimal in blood samples containing more than 0.1 pg/µL of porphobilinogen deaminase.

	DISCUSSION

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In this study, we show that US-guided FNAC is reliable in detecting metastatic involvement of the SN in patients with melanoma, but that the results are not improved by RT-PCR of the aspirate. The 16% pre-SNLB identification of a positive SN by US-guided FNAC alone and, more importantly, the 82% identification rate of an involved SN by US-guided FNAC is virtually identical to that which has been reported for US-guided FNAC in the Rotterdam breast cancer study.²⁴

However, this is not improved by additional RT-PCR of the aspirate nor of the excised SN. In fact, RT-PCR of aspirated pre-SLNB material shows virtually identical sensitivity and specificity compared with the FNAC results of the same node. Whereas RT-PCR of post-SLNB material does not improve the sensitivity compared with the final histology results, the use of both FNAC and RT-PCR of US-guided material of one and the same SN does increase the sensitivity of this pre-SLNB technique to 82%. So in approximately 16% of all patients, an SLNB procedure can be avoided by performing US-guided FNAC of the lymph nodes up front.⁸

Regarding the prognostic value of micrometastases in the SN, their predictive role for the involvement of the non-SNs and the clinical consequences of a false-negative staging as a result of undetected micrometastases are still under debate.²⁵ The Multicenter Selective Lymphadenectomy Trial-1 trial has demonstrated that implementation of SNLB in patients with primary melanomas does not seem to improve survival.⁹ In this trial, SLNB did not seem to improve survival in the overall population of 2001 patients, nor in the 1,327 patients with intermediate-thickness melanomas (1.2 to 3.5 mm). At the same time, the trial report suggests that SNLB is beneficial for SN-positive patients with 1.2- to 3.5-mm melanomas. This conclusion concerns a subgroup analysis, which shows a much smaller difference in survival when corrected for false negatives.

Recently, the largest experience by far of RT-PCR evaluation of SNs indicated that this procedure did not further enhance prognostic value of SN staging.²⁶ This Sunbelt trial report, which represents the largest experience (more than 1,400 patients) could not prove any benefit of this examination for the additional work-up of the SN.²⁶ A total of 1,446 patients with histologically negative SNs underwent RT-PCR analysis and showed no difference in DFS, distant metastasis–free survival (DMFS), or OS between the RT-PCR–positive and RT-PCR–negative patients.

PCR-based detection of melanoma cells in SNs of patients was reviewed in a recent meta-analysis covering 22 studies with 4,019 patients who underwent SLNB for clinical stage I to II cutaneous melanoma. PCR status of SN was shown to have a clinically valuable prognostic power in patients with melanoma, but caution is warranted to avoid overestimating of results.²⁷

This is accordance with the conclusions of the present study and of another observation regarding the lack of prognostic value of the presence of submicrometastasis (< 0.1 mm) in the SN, because these cases had the same DFS, DMFS, and OS prognosis as SN-negative patients.¹² These observations would indicate that clinically relevant disease and disease volume are related and that the detection of tumor cells below a certain threshold (< 0.1 mm and/or by RT-PCR alone in the absence of hematoxylin and eosin/immunohistochemical histopathologic evidence) does not represent clinically relevant metastatic disease.

With even more sophisticated histopathologic protocols,²⁸ the detection rate of submicroscopic disease will increase and lead to the reporting of higher SN-positive rates, which may not be clinically relevant. The struggle to find earlier, prognostically better, and more reliable tumor markers is not new.²⁹ However, to date, the conventional staging methods by SN histology and US-guided FNAC are the best we have.

RT-PCR testing of more than one marker in peripheral blood was associated with shorter DFS and DMFS but no change in OS in the Sunbelt trial.²⁶ Here, a single-marker RT-PCR for the detection of tyrosinase was used, although in the literature, the use of multimarker assays in peripheral blood was reported to increase sensitivity (sometimes, however, at the expense of specificity).^30,31 Apart from the often reported inter-laboratory differences between use of quantitative versus qualitative RT-PCR schedules, peripheral blood testing was positive in two smaller studies,^21,32 but more importantly, it was negative in this large study (Sunbelt trial; n = 1,446).²⁶

However, our own recent study had an extremely long median follow-up duration (from the first blood sample to the last follow-up examination or death) of 6.3 years (range, 0.9 to 8.6 years).²¹ Second, here the study population comprised exclusively patients with stage II and III melanoma (ie, patients who have a higher probability to have recurrence) as compared with the study population in the Sunbelt trial. Third, the tyrosinase result was modeled as a time-dependent variable, because results changed over time. This study had shown a strong association between PCR detected in peripheral blood and OS. In a proportional hazards regression analysis, PCR positivity was an important predictor (hazard ratio = 12.6; 95% CI, 3.4 to 46.3; P < .001).²¹ A recent letter by Qualgino et al³³ stressing the role of the time-dependent calculations of the serial tyrosinase measurements was directed to the authors of the Sunbelt trial, and the answer was that after recalculations in the proposed manner, the blood tyrosinase turned out to be predictive of outcome. Even a single positive test result in tyrosinase RT-PCR from peripheral blood seemed to be a warning for metastases, and several positive test results might be taken as reliable hint for disease progression.³²

In conclusion, US-guided FNAC had previously been proven to be an accurate staging method for patients before they undergo an SN procedure. Additional RT-PCR of fine-needle aspirates or of the SN did not further improve results. We recommend US-guided FNAC pre-SNLB as a simple method to reduce the number of operative SN procedures. RT-PCR could be valuable when taken from peripheral blood of patients with high-risk melanoma to predict recurrence and/or survival.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Christiane A. Voit, Juergen Rademaker, Markus Schwürzer-Voit, Alexander M.M. Eggermont

Financial support: Wolfram Sterry

Administrative support: Juergen Rademaker, Alfred Schoengen, Markus Schwürzer-Voit, Wolfram Sterry, Alexander M.M. Eggermont

Provision of study materials or patients: Christiane A. Voit, Gregor Schäfer-Hesterberg, Ansgar Lukowsky, Alfred Schoengen, Markus Krause, Joachim Röwert-Huber

Collection and assembly of data: Christiane A. Voit, Gregor Schäfer-Hesterberg

Data analysis and interpretation: Martina Kron, Alexander C.J. van Akkooi, Alexander M.M. Eggermont

Manuscript writing: Christiane A. Voit, Martina Kron, Alexander C.J. van Akkooi, Alexander M.M. Eggermont

Final approval of manuscript: Christiane A. Voit, Gregor Schäfer-Hesterberg, Martina Kron, Alexander C.J. van Akkooi, Juergen Rademaker, Ansgar Lukowsky, Alfred Schoengen, Markus Schwürzer-Voit, Wolfram Sterry, Markus Krause, Joachim Röwert-Huber, Alexander M.M. Eggermont

	NOTES

 
published online ahead of print at www.jco.org on November 3, 2008.

Supported by Deutsche Krebshilfe (Grant No. 70-2791-Vo I).

C.A.V. and G.S.-H. contributed equally to this work.

Presented in part at the Melanoma Session of the 42nd Annual Meeting of the American Society of Clinical Oncology, June 2-6, 2006, Atlanta, GA.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

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Submitted July 29, 2007; accepted July 24, 2008.

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