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Quantification of Melanoma Micrometastases in Sentinel Lymph Nodes Using Real-Time RT-PCR

2005; Elsevier BV; Volume: 124; Issue: 3 Linguagem: Inglês

10.1111/j.0022-202x.2005.23633.x

1523-1747

Thomas Giese, Monika Engstner, Ulrich Mansmann, Wolfgang Hartschuh, Bernhard Arden,

Molecular Biology Techniques and Applications

Detection of micrometastases in the regional tumor-draining lymph nodes is critical for staging and prognosis in melanoma patients. We applied a quantitative multiple-marker RT-PCR assay to improve the detection of occult melanoma cells in the sentinel lymph node (SLN). From 139 patients with primary cutaneous melanoma who underwent sentinel lymphadenectomy, a total of 235 SLN were assessed for Melan-A and tyrosinase expression by real-time quantitative RT-PCR. Twenty-three patients (17%) were positive by histopathology and expressed messenger RNA of one or two markers. Of the patients with histopathologically negative SLN 39 (28%) were reclassified by positive RT-PCR. Patients were examined for tumor recurrences during a median follow-up period of 29 mo. Tumor recurrences were demonstrated in eight patients (35%) with histopathologically positive SLN, in four patients (10%) with submicroscopic tumor cells detected exclusively by real-time RT-PCR, and in none of the patients negative by both methods. The differences in recurrence rates were statistically significant (p=0.01). These data indicate that real-time quantitative RT-PCR for the detection of minimal residual melanoma in SLN improves the prediction of disease-free survival. Detection of micrometastases in the regional tumor-draining lymph nodes is critical for staging and prognosis in melanoma patients. We applied a quantitative multiple-marker RT-PCR assay to improve the detection of occult melanoma cells in the sentinel lymph node (SLN). From 139 patients with primary cutaneous melanoma who underwent sentinel lymphadenectomy, a total of 235 SLN were assessed for Melan-A and tyrosinase expression by real-time quantitative RT-PCR. Twenty-three patients (17%) were positive by histopathology and expressed messenger RNA of one or two markers. Of the patients with histopathologically negative SLN 39 (28%) were reclassified by positive RT-PCR. Patients were examined for tumor recurrences during a median follow-up period of 29 mo. Tumor recurrences were demonstrated in eight patients (35%) with histopathologically positive SLN, in four patients (10%) with submicroscopic tumor cells detected exclusively by real-time RT-PCR, and in none of the patients negative by both methods. The differences in recurrence rates were statistically significant (p=0.01). These data indicate that real-time quantitative RT-PCR for the detection of minimal residual melanoma in SLN improves the prediction of disease-free survival. confidence interval hematoxylin and eosin immunohistology messenger RNA sentinel lymph node In early stages of primary cutaneous melanoma (American Joint Committee on Cancer stages I and II), Breslow's tumor thickness was demonstrated to be the single most important prognostic factor (Morton et al., 1993Morton D.L. Davtyan D.G. Wanek L.A. Foshag L.J. Cochran A.J. Multivariate analysis of the relationship between survival and the microstage of primary melanoma by Clark level and Breslow thickness.Cancer. 1993; 71: 3737-3743Crossref PubMed Scopus (124) Google Scholar). With the development of regional lymph node metastasis (American Joint Committee on Cancer stage III), the 5-y survival rate decreases 40%. At this tumor stage prognostic factors of primary melanoma no longer contribute significantly to survival prediction. The extent of nodal involvement is strongly associated with survival rates (Balch et al., 1996Balch C.M. Soong S.J. Bartolucci A.A. et al.Efficacy of an elective regional lymph node dissection of 1 to 4 mm thick melanomas for patients 60 years of age and younger.Ann Surg. 1996; 224: 255-263Crossref PubMed Scopus (550) Google Scholar). Accurate nodal staging is useful for guiding adjuvant therapy (Kirkwood, 1998Kirkwood J.M. Adjuvant IFNα2 therapy of melanoma.Lancet. 1998; 351: 1901-1903Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The technique of sentinel lymph node (SLN) biopsy is based on the concept that it identifies the first node to receive a cutaneous afferent lymphatic from the primary tumor. If the SLN does not contain melanoma cells, then the probability that a non-sentinel node in the draining lymphatic basin contains melanoma cells is less than 1% (Morton et al., 1992Morton D.L. Wen D.R. Wong J.H. et al.Technical details of intraoperative lymphatic mapping for early stage melanoma.Arch Surg. 1992; 127: 392-399Crossref PubMed Scopus (3628) Google Scholar). Serial sectioning and immunohistology (IH) using HMB-45 and S-100 antibodies improves the detection of occult melanoma cells, compared with conventional hematoxylin and eosin (HE) staining alone (Cochran et al., 1984Cochran A.J. Wen D.R. Herschman H.R. Occult melanoma in lymph nodes detected by antiserum to S-100 protein.Int J Cancer. 1984; 34: 159-163Crossref PubMed Scopus (65) Google Scholar; Morton et al., 1992Morton D.L. Wen D.R. Wong J.H. et al.Technical details of intraoperative lymphatic mapping for early stage melanoma.Arch Surg. 1992; 127: 392-399Crossref PubMed Scopus (3628) Google Scholar). More recently, the technique of RT-PCR has been demonstrated to increase substantially the sensitivity of tumor cell detection (Blaheta et al., 1999Blaheta H.-J. 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: 822-828Crossref PubMed Scopus (75) Google Scholar). Using tyrosinase as messenger RNA (mRNA) marker SLN were analyzed by nested RT-PCR in comparison with histopathology including IH. Tumor recurrences after a median follow-up period of 19 mo were observed in 67% of patients with histopathologically positive SLN, in 25% of patients with submicroscopic tumor cells detected by RT-PCR alone, and 6% of patients negative by both methods (Blaheta et al., 2000Blaheta H.-J. Ellwanger U. Schittek B. et al.Examination of regional lymph nodes by sentinel node biopsy and molecular analysis provides new staging facilities in primary cutaneous melanoma.J Invest Dermatol. 2000; 114: 637-642Crossref PubMed Scopus (150) Google Scholar). Histological and RT-PCR status of the SLN were demonstrated to be the only significant prognostic factors for disease-free survival. The results of two other studies equally demonstrated significant differences of tumor relapse for the three groups of patients determined by their SLN status (Shivers et al., 1998Shivers S.C. Wang X. Li W. et al.Molecular staging of malignant melanoma: Correlation with clinical outcome.JAMA. 1998; 280: 1410-1415Crossref PubMed Scopus (297) Google Scholar; Bostick et al., 1999Bostick P.J. Morton D.L. Turner R.R. 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: 3238-3244Crossref PubMed Scopus (236) Google Scholar). The molecular assay based on nested PCR is designed to detect approximately three melanoma cells in 107 lymph node cells (Wang et al., 1994Wang X. Heller R. VanVoorhis N. et al.Detection of submicroscopic lymph node metastases with polymerase chain reaction in patients with malignant melanoma.Ann Surg. 1994; 220: 768-774Crossref PubMed Scopus (233) Google Scholar), but the question of whether such minimal amounts of cancer cells are clinically important remains controversial. We reasoned that in particular for the patient group exclusively positive by RT-PCR, it would be instructive to perform a quantitative RT-PCR analysis of tumor-associated gene expression. It is conceivable that values of mRNA marker gene expression above a certain cut-off level may be a more powerful predictor of disease relapse than values below this cut-off. A single-step real-time RT-PCR assay has recently been established for accurate and reproducible quantitation of the melanoma-associated tyrosinase and Melan-A mRNA (Abrahamsen et al., 2004Abrahamsen H.N. Nexo E. Steiniche T. Hamilton-Dutoit S.J. Sorensen B.S. Quantification of melanoma mRNA markers in sentinel nodes. Pre-clinical evaluation of a single-step real-time reverse transcriptase-polymerase chain reaction assay.J Mol Diagn. 2004; 6: 253-259Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In a prospective diagnostic study, we have developed a similar quantitative multiple marker RT-PCR that uses tyrosinase and Melan-A mRNA expression to detect occult melanoma cells in comparison with conventional IH in SLN. In this study, the prognostic impact of the presence of nodal melanoma micrometastases detected by real-time quantitative RT-PCR was evaluated. The question was addressed as to whether a patient group with a high risk for developing melanoma recurrence and a favorable prognostic group of patients with a low risk of relapse may be distinguished by quantitative RT-PCR. A major disadvantage of nested PCR is the high risk of false-positive results because of PCR artifacts and contamination, making this technique unsuitable for clinical diagnostics. We therefore investigated whether a closed-tube real-time PCR would be a practical approach to overcome these difficulties. Best suited for our demands of fast and reliable real-time PCR, we chose the LightCycler system. Absolute mRNA levels of tyrosinase and Melan-A were determined in 235 SLN from a total of 139 patients with primary cutaneous melanoma using real-time quantitative PCR. The sample collection and preparation was modified for future automated settings. To exclude samples with low mRNA yield, a cut-off level of 12,000 transcripts of β-actin per μL cDNA was implemented. Detection of single transcripts is possible; we used, however, a concentration of 10 transcripts per 10 μL of cDNA as cut-off for the detection of the melanoma-specific parameters. Two housekeeping genes, β-actin and cyclophilin B, were quantified as internal standards (see supplement). Reproducibility and sensitivity of tumor cell detection were tested as described in the supplement. We performed a quantitative comparison between Melan-A and tyrosinase expression using identical lymph node samples. There was a strict correlation between Melan-A and tyrosinase transcript numbers (Spearman's correlation coefficient (rs)=0.94; 95% confidence interval (CI): 0.89–0.98). The regression equation can be interpreted as having a slope of 1.0 within the 95% CI (see supplement), thus yielding a 2.2-fold (95% CI: 1.58–3.13) higher transcript number for Melan-A than for tyrosinase. For comparison, lymph nodes with occurrence of benign melanocytic nevus cells contained between 1.9 and 552 transcripts per μL of Melan-A and displayed ratios of Melan-A to tyrosinase expression between 2.3 and 5.2. A sample from a clinically palpable lymph node metastasis showed 25,892 Melan-A transcripts per μL and a ratio of 1.9. Nevus cells as well as the lymph node metastasis are not included. We then compared mRNA levels with the classification by histopathology. Of 25 SLN positive by HE staining, 21 were real-time PCR positive with a range of 1.2 to 73,714 Melan-A transcripts per μL (median 1466), whereas six of nine SLN positive by IH but negative by HE staining were real-time PCR positive ranging from 1.6 to 37 Melan-A transcripts per μL (median 7.6). Thirty-two SLN negative by histopathology (including IH) but real-time PCR positive ranged from 1.0 to 1336 Melan-A transcripts per μL (median 4.0, Figure 1). Melan-A transcript numbers were significantly different between the SLN groups defined by positive and negative histopathology (p 0.75 mm) from April 1999 to September 2002 at the Department of Dermatology of the University Hospital Heidelberg. None of the patients had clinical evidence of regional or metastatic disease (American Joint Committee on Cancer stages I and II). The protocol for the study was approved by the Ethics Committee of the University of Heidelberg. Patients gave their informed consent for all aspects of the investigation according to the Declaration of Helsinki principles. SLN biopsy was performed as described previously (Kahle et al., 2000Kahle B. Hoffend J. Hartschuh W. Petzoldt D. Sentinel lymph node sonography in malignant melanoma.Hautarzt. 2000; 51: 915-919Crossref PubMed Scopus (1) Google Scholar). SLN were harvested and cut in half through their longitudinal axis. One half of each SLN was fixed in 5% formaldehyde and sent for standard pathologic evaluation. It was embedded in paraffin and the sections were examined with HE staining. In all instances further sections were evaluated by IH by use of a monoclonal antibody to HMB-45 (Hatta et al., 1998Hatta 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: 597-601Crossref PubMed Scopus (33) Google Scholar), and antiserum to S-100 protein (Cochran et al., 1988Cochran A.J. Wen D.R. Morton D.L. Occult tumor cells in the lymph nodes of patients with pathological stage I malignant melanoma. An immunohistological study.Am J Surg Pathol. 1988; 12: 612-618Crossref PubMed Scopus (198) Google Scholar). An SLN was defined as positive by histopathology if tumor cells were identified by HE staining and/or by immunostaining. From the other half of each SLN aliquots were collected in 1 mL of RNAlater (Ambion, Huntingdon, UK). After disruption of the tissue using a RiboLyser (Thermo Hybaid, Heidelberg, Germany), mRNA was isolated with the MagNA Pure LC robotic work station (Roche Applied Science, Mannheim, Germany). cDNA was synthesized using AMV-RT and oligo(dT) as primer (first strand cDNA synthesis kit, Roche Applied Science). After termination of the cDNA synthesis, the reaction mix was diluted to a final volume of 500 μL and stored at -20°C until PCR analysis. Parameter-specific primer sets optimized for the LightCycler (Roche Applied Science) were developed and kindly provided by SEARCH-LC GmbH (Heidelberg, Germany). The PCR was performed with the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Applied Science) according to the protocol provided in the parameter-specific kits. cDNA input was normalized by the average expression of the two housekeeping genes β-actin and cyclophilin B. The data of two independent analyses for each sample and parameter were averaged and presented as adjusted transcripts per μL cDNA. Multiplying this number by 3000 gives an estimate of the transcripts in an average biopsy. Capsular nevocytes might be a source for tyrosinase or Melan-A mRNA detection by RT-PCR. All SLN sections were, therefore, carefully examined for the presence of benign melanocytes. Lymph node specimens with nevus cell aggregates were excluded from our analysis. Examination findings were judged true positive when metastasis was confirmed by additional examination techniques (e.g., histopathology or other imaging techniques) or by the further course of the disease (documented growth of metastasis during the following 6 mo). Conversely, if confirmation by these criteria was lacking, findings were judged false positive. The analysis related to tumor thickness is based on a non-parametric approach using median and quartiles to describe location and scale parameters. Differences between groups were assessed with the Mann–Whitney test. Strip-plots were used to present the data graphically. Linear regression methodology was used to gauge two PCR measurements that were transformed on a log 10 base. The 95% CI for intercept and regression coefficient β were also calculated. In case of inclusion of the value 1 in the CI for β, the retransformed intercept was interpreted as the factor of change between both measurements. The approach chosen also allowed to calculate 95% CI for the factor of change. The correlation between two measurements was quantified by rs and its 95% CI. The calculations were performed using SPLUS 2000 (MathSoft, Seattle, Washington) and Statxact (V.3, Cytel Software Corporation, Boston, Massachusetts). We thank U. Schulze-Halberg, G. Glensch, and S. Meisel for their excellent technical assistance. We thank Dr D. Petzoldt and Dr H. Näher for their kind support of this work. Download .jpg (.03 MB) Help with files Figure S1Serial dilution of melanoma cells in control SLN lysates. Download .jpg (.04 MB) Help with files Figure S2Correlation of Melan-A and tyrosinase transcript numbers. Download .doc (.03 MB) Help with doc files Table S1Patient characteristics. Download .doc (.02 MB) Help with doc files Table S2Sites of first melanoma recurrence.