Annals of Surgical Oncology

, 16:177 | Cite as

Molecular Staging of Pathologically Negative Sentinel Lymph Nodes from Melanoma Patients Using Multimarker, Quantitative Real-Time RT-PCR

  • Josep M. Hilari
  • Cristina Mangas
  • Liqiang Xi
  • Cristina Paradelo
  • Carlos Ferrándiz
  • Steven J. Hughes
  • Cindy Yueh
  • Ivy Altomare
  • William E. Gooding
  • Tony E. Godfrey
Melanomas

Abstract

The aim of this study was to evaluate the prognostic potential of quantitative reverse-transcription, polymerase chain reaction (qRT-PCR) in melanoma patients with pathologically negative sentinel lymph nodes (SLN). Our study included 195 node-negative melanoma patients with a Breslow thickness greater than 0.76 mm (n = 158), or less than 0.76 mm but who had Clark level IV–V, microscopic ulceration, or pathological signs of regression (n = 32), and five patients with melanoma of unknown thickness. SLNs were examined by serial-section histopathology. A portion of each SLN was frozen for qRT-PCR analysis using markers Tyrosinase, MART1, SSX2, MAGEA3, PAX3, and GalNAc-T. In addition, two other markers (PLAB and L1CAM) were evaluated for melanoma specificity but not for SLN analysis. Median follow-up was 64 months, during which time there were 15 (7.7%) recurrences. A total of 370 lymph nodes were analyzed by qRT-PCR. No association was found between quantitative expression level of any marker and disease recurrence. Previously published primer designs were tested for PAX3 and GalNAc-T and revealed that alternative PAX3 transcripts are differentially expressed in melanoma and benign lymph nodes. No associations with recurrence were found regardless of the transcripts amplified by different primer sets. PLAB and L1CAM did not appear to differentiate between malignant melanoma and benign melanocytes or lymph nodes in our analysis. We conclude that, in this large cohort of patients, multimarker qRT-PCR analysis of SLNs did not correlate with disease recurrence. Our data support specific PAX3 splice variants but not GalNAc-T, PLAB or L1CAM as possible markers for melanoma metastasis to SLNs.

Supplementary material

10434_2008_183_MOESM1_ESM.pdf (240 kb)
MOESM1 [Evaluation of Potential Markers for Detection of Lymph Node Metastasis inMelanoma Using Quantitative RT-PCR] (PDF 241 kb)
10434_2008_183_MOESM2_ESM.pdf (17 kb)
Supplemental Figure 2MOESM1 [Exon structure of PAX3 alternative transcriptsindicating the location of the two PCR primer designs used in this study.Our primer design amplifies all 7 known transcripts whereas the design ofTakeuchi et al. only amplifies 3 transcripts.] (PDF 18 kb)
10434_2008_183_MOESM3_ESM.pdf (62 kb)
Supplemental Table 1MOESM1 [Oligonucleotide primer and probe sequences used] (PDF 63 kb)

References

  1. 1.
    Morton DL, Wen DR, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg. 1992;127:392–9.CrossRefPubMedGoogle Scholar
  2. 2.
    Reintgen D, Cruse CW, Wells K, et al. The orderly progression of melanoma nodal metastases. Ann Surg. 1994;220:759–67.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Thompson JF, Uren RF. Lymphatic mapping in management of patients with primary cutaneous melanoma. Lancet Oncol. 2005;6:877–85.CrossRefPubMedGoogle Scholar
  4. 4.
    Roberts AA, Cochran AJ. Pathologic analysis of sentinel lymph nodes in melanoma patients: current and future trends. J Surg Oncol. 2004;85:152–61.CrossRefPubMedGoogle Scholar
  5. 5.
    Spanknebel K, Coit DG, Bieligk SC, et al. Characterization of micrometastatic disease in melanoma sentinel lymph nodes by enhanced pathology: recommendations for standardizing pathologic analysis. Am J Surg Pathol. 2005;29:305–17.CrossRefPubMedGoogle Scholar
  6. 6.
    Yee VS, Thompson JF, McKinnon JG, et al. Outcome in 846 cutaneous melanoma patients from a single center after a negative sentinel node biopsy. Ann Surg Oncol. 2005;12:429–39.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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.CrossRefPubMedGoogle Scholar
  9. 9.
    Shivers SC, Wang X, Li W, et al. Molecular staging of malignant melanoma: correlation with clinical outcome. JAMA. 1998;280:1410–5.CrossRefPubMedGoogle Scholar
  10. 10.
    Blaheta HJ, 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;114:637–42.CrossRefPubMedGoogle Scholar
  11. 11.
    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.CrossRefPubMedGoogle Scholar
  12. 12.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Scoggins CR, Ross MI, Reintgen DS, et al. Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma. J Clin Oncol. 2006;24:2849–57.CrossRefPubMedGoogle Scholar
  14. 14.
    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.CrossRefPubMedGoogle Scholar
  15. 15.
    Ribuffo D, Gradilone 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.CrossRefPubMedGoogle Scholar
  16. 16.
    Ulrich J, Bonnekoh B, Bockelmann R, et al. Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients. Eur J Cancer. 2004;40:2812–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Mangas C, Hilari JM, Paradelo C, et al. Prognostic significance of molecular staging study of sentinel lymph nodes by reverse transcriptase-polymerase chain reaction for tyrosinase in melanoma patients. Ann Surg Oncol. 2006;13:910–8.CrossRefPubMedGoogle Scholar
  18. 18.
    Gradilone A, Ribuffo D, Silvestri I, et al. Detection of melanoma cells in sentinel lymph nodes by reverse transcriptase-polymerase chain reaction: prognostic significance. Ann Surg Oncol. 2004;11:983–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Mocellin S, Hoon DS, Pilati P, Rossi CR, Nitti D. Sentinel lymph node molecular ultrastaging in patients with melanoma: a systematic review and meta-analysis of prognosis. J Clin Oncol. 2007;25:1588–95.CrossRefPubMedGoogle Scholar
  20. 20.
    Scoggins CR, Ross MI, Reintgen DS, et al. Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma. J Clin Oncol. 2006;24:2849–57.CrossRefPubMedGoogle Scholar
  21. 21.
    Abrahamsen HN, Sorensen BS, Nexo E, et al. Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART–1 and tyrosinase messenger RNA. Clin Cancer Res. 2005;11:1425–33.CrossRefPubMedGoogle Scholar
  22. 22.
    Tucker MA, Fraser MC, Goldstein AM, et al. A natural history of melanomas and dysplastic nevi: an atlas of lesions in melanoma-prone families. Cancer. 2002;94:3192–209.CrossRefPubMedGoogle Scholar
  23. 23.
    Holt JB, Sangueza OP, Levine EA, et al. Nodal melanocytic nevi in sentinel lymph nodes. Correlation with melanoma-associated cutaneous nevi. Am J Clin Pathol. 2004;121:58–63.CrossRefPubMedGoogle Scholar
  24. 24.
    Starz H, Haas CJ, Schulz GM, Balda BR. Tyrosinase RT-PCR as a supplement to histology for detecting melanoma and nevus cells in paraffin sections of sentinel lymph nodes. Mod Pathol. 2003;16:920–9.CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Talantov D, Mazumder A, Yu JX, et al. Novel genes associated with malignant melanoma but not benign melanocytic lesions. Clin Cancer Res. 2005;11:7234–42.CrossRefPubMedGoogle Scholar
  27. 27.
    Rex J, Paradelo C, Mangas C, et al. Single-institution experience in the management of patients with clinical stage I and II cutaneous melanoma: results of sentinel lymph node biopsy in 240 cases. Dermatol Surg. 2005;31:1385–93.PubMedGoogle Scholar
  28. 28.
    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.PubMedGoogle Scholar
  29. 29.
    Tassone F, Hagerman RJ, Taylor AK, et al. Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet. 2000;66:6–15.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Talantov D, Mazumder A, Yu JX, et al. Novel genes associated with malignant melanoma but not benign melanocytic lesions. Clin Cancer Res. 2005;11:7234–42.CrossRefPubMedGoogle Scholar
  31. 31.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Yee VS, Thompson JF, McKinnon JG, et al. Outcome in 846 cutaneous melanoma patients from a single center after a negative sentinel node biopsy. Ann Surg Oncol. 2005;12:429–39.CrossRefPubMedGoogle Scholar
  33. 33.
    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.CrossRefPubMedGoogle Scholar
  34. 34.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Shivers SC, Wang X, Li W, et al. Molecular staging of malignant melanoma: correlation with clinical outcome. JAMA. 1998;280:1410–5.CrossRefPubMedGoogle Scholar
  37. 37.
    Blaheta HJ, 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;114:637–42.CrossRefPubMedGoogle Scholar
  38. 38.
    Ulrich J, Bonnekoh B, Bockelmann R, et al. Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients. Eur J Cancer. 2004;40:2812–9.CrossRefPubMedGoogle Scholar
  39. 39.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    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.CrossRefPubMedGoogle Scholar
  41. 41.
    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.CrossRefPubMedGoogle Scholar
  42. 42.
    Ribuffo D, Gradilone 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.CrossRefPubMedGoogle Scholar
  43. 43.
    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.PubMedGoogle Scholar
  44. 44.
    Rimoldi D, Lemoine R, Kurt AM, et al. Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods. Melanoma Res. 2003;13:511–20.CrossRefPubMedGoogle Scholar
  45. 45.
    Giese T, Engstner M, Mansmann U, Hartschuh W, Arden B. Quantification of melanoma micrometastases in sentinel lymph nodes using real-time RT-PCR. J Invest Dermatol. 2005;124:633–7.CrossRefPubMedGoogle Scholar
  46. 46.
    Mangas C, Hilari JM, Paradelo C, et al. Prognostic significance of molecular staging study of sentinel lymph nodes by reverse transcriptase-polymerase chain reaction for tyrosinase in melanoma patients. Ann Surg Oncol. 2006;13:910–8.CrossRefPubMedGoogle Scholar
  47. 47.
    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.CrossRefPubMedGoogle Scholar
  48. 48.
    Scoggins CR, Ross MI, Reintgen DS, et al. Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma. J Clin Oncol. 2006;24:2849–57.CrossRefPubMedGoogle Scholar
  49. 49.
    Mocellin S, Hoon DS, Pilati P, Rossi CR, Nitti D. Sentinel lymph node molecular ultrastaging in patients with melanoma: a systematic review and meta-analysis of prognosis. J Clin Oncol. 2007;25:1588–95.CrossRefPubMedGoogle Scholar
  50. 50.
    Scoggins CR, Ross MI, Reintgen DS, et al. Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma. J Clin Oncol. 2006;24:2849–57.CrossRefPubMedGoogle Scholar
  51. 51.
    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.CrossRefPubMedGoogle Scholar
  52. 52.
    Mangas C, Hilari JM, Paradelo C, et al. Prognostic significance of molecular staging study of sentinel lymph nodes by reverse transcriptase-polymerase chain reaction for tyrosinase in melanoma patients. Ann Surg Oncol. 2006;13:910–8.CrossRefPubMedGoogle Scholar
  53. 53.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Blaheta HJ, 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;114:637–42.CrossRefPubMedGoogle Scholar
  55. 55.
    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.CrossRefPubMedGoogle Scholar
  56. 56.
    Ribuffo D, Gradilone 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.CrossRefPubMedGoogle Scholar
  57. 57.
    Ulrich J, Bonnekoh B, Bockelmann R, et al. Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients. Eur J Cancer. 2004;40:2812–9.CrossRefPubMedGoogle Scholar
  58. 58.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Giese T, Engstner M, Mansmann U, Hartschuh W, Arden B. Quantification of melanoma micrometastases in sentinel lymph nodes using real-time RT-PCR. J Invest Dermatol. 2005;124:633–7.CrossRefPubMedGoogle Scholar
  60. 60.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Giese T, Engstner M, Mansmann U, Hartschuh W, Arden B. Quantification of melanoma micrometastases in sentinel lymph nodes using real-time RT-PCR. J Invest Dermatol. 2005;124:633–7.CrossRefPubMedGoogle Scholar
  62. 62.
    Balch CM, Soong SJ, Atkins MB, et al. An evidence-based staging system for cutaneous melanoma. CA Cancer J Clin. 2004;54:131–49.CrossRefPubMedGoogle Scholar
  63. 63.
    Talantov D, Mazumder A, Yu JX, et al. Novel genes associated with malignant melanoma but not benign melanocytic lesions. Clin Cancer Res. 2005;11:7234–42.CrossRefPubMedGoogle Scholar

Copyright information

© Society of Surgical Oncology 2008

Authors and Affiliations

  • Josep M. Hilari
    • 1
  • Cristina Mangas
    • 1
  • Liqiang Xi
    • 2
  • Cristina Paradelo
    • 1
  • Carlos Ferrándiz
    • 1
  • Steven J. Hughes
    • 3
  • Cindy Yueh
    • 2
  • Ivy Altomare
    • 2
  • William E. Gooding
    • 4
  • Tony E. Godfrey
    • 2
    • 5
  1. 1.Department of Dermatology, Hospital Universitario Germans Trias i Pujol, BadalonaUniversidad Autónoma de BarcelonaBarcelonaSpain
  2. 2.Department of PathologyMount Sinai School of MedicineNew YorkUSA
  3. 3.Department of SurgeryUniversity of Pittsburgh Medical CenterPittsburghUSA
  4. 4.Biostatistics Facility, Cancer InstituteUniversity of PittsburghPittsburghUSA
  5. 5.Department of SurgeryUniversity of Rochester Medical CenterNew yorkUSA

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