In a recently reported collaborative blinded study with the Sydney Cancer Centre, we confirmed that qRT assay could identify occult metastasis in SLNs that were negative by initial histopathology: qRT assessment of 74 archived PE SLNs from 33 patients upstaged melanoma in 4 patients (12%).10
That study also found that although both conventional histopathologic assessment and qRT assay depend on accurate surgical and nuclear medicine techniques, qRT assessment of SLN sections has greater sensitivity. But do qRT-detected tumor cells have clinical relevance?
In this long-term follow-up study, we demonstrate that patients with qRT-positive SLNs had significantly worse OS, MSS, DFS, and DDFS than patients with histopathology-negative/qRT-negative SLNs. Since our 2004 report of this cohort, the OS and DDFS curves for qRT-positive and histopathology-positive patients have separated: by about 7 years, qRT-positive patients had a worse OS; by about 8 years, they also had a worse DDFS. Interestingly, the pattern of recurrence associated with histopathology-negative/qRT-positive SLNs was comparable with the pattern of recurrence that we previously reported for patients with a falsely negative SLN.17
Although our findings suggest that molecular detection of melanoma metastasis in the SLN predicts an increased risk of distant as well as locoregional recurrence, they must be qualified by the reminder that all patients with histopathology-positive SLNs underwent immediate CLND, whereas patients with histopathology-negative/qRT-positive SLNs received treatment only for recurrence. Prospective analysis of the prognostic significance of qRT-positive SLNs is underway in the second Multicenter Selective Lymphadenectomy Trial. This randomized international phase III trial is the first study to stratify patients for treatment (CLND or nodal ultrasono-graphic observation) on the basis of the molecular status of the SLN specimen. Results not only will determine whether CLND is necessary in all patients with SLN involvement, but also will establish the clinical utility of nodal upstaging on the basis of detection of molecular biomarkers.
The qRT biomarker panel that was used in this study is currently being applied in second Multicenter Selective Lymphadenectomy Trial. The panel comprises comprised biomarkers with a high level of clinical utility for disease outcome in melanoma patients. This selection criterion eliminated tyrosinase and gp100, because these 2 well-known proteins of the melanogenesis pathway14,18–20
have unacceptably high false-positive rates in the regional lymph nodes.21–23
Of the 4 biomarkers used in this study, MART1
is a melanoma-associated antigen commonly expressed by melanomas but absent in nonmelanoma malignancies and normal non-cancer lymph nodes13,20,24
is a melanoma-associated antigen not produced in normal tissues except male germline cells and placenta.25 GalNAc-T, PAX3,
are not detected in nevi or differentiated melanocytes. Importantly, these biomarkers are from nonoverlapping malignant pathways.13,18,23,26–28
For example, GalNAc-T,
a key enzyme for biosynthesis of tumor-associated gangliosides GM2 and GD2,29,30
is a biomarker for aggressive melanoma progression, as shown previously by our group.31 PAX3
a transcription factor involved in melanoma proliferation and is resistance to apoptosis, migration, melanogenesis regulation, and differentiation.32,33 PAX3
is activated during fetal-tissue development; it is suggested to block terminal differentiation and thereby maintain the stem cell function of melanoblasts.34,35 PAX3
expression was a significant hazard for disease recurrence in this study ().
Several large studies have found no link between molecular biomarkers and prognosis in patients with clinically localized melanoma.24,25,36
The Sunbelt Melanoma Trial, a multicenter prospective randomized study of melanoma staging and adjuvant therapy, reported no difference in DFS, DDFS, or OS between reverse transcriptase-PCR (RT-PCR)–positive and -negative patients followed up for a median of 30 months.37
However, this trial included TYR
as a weighted molecular biomarker for positivity; also, it used a nonquantitative gel-based assay, and it did not standardize SLN pathology sampling. Both histopathology- and RT-PCR–positive patients were randomized to different adjuvant treatment arms with α
-interferon, which would have diluted differences in outcomes. In an unusual result, Hilari et al21
recently reported low specificity for any melanoma biomarker (TYR
, and L1CAM
) and no association with disease recurrence. However, significant low recurrence rates and a high percentage of thin melanomas in that study would have biased its interpretation. Several of the biomarkers used have been shown to be of clinical utility by our group and other groups.21
A recent meta-analysis review by Mocellin et al23
examined 22 large studies of PCR upstaging of the SLN. Three of the studies used quantitative RT-PCR assays; most of the remaining studies used standard or nested gel-based RT-PCR. These traditional RT-PCR assays have limited sensitivity. Only 12 studies included at least 2 biomarkers in the RT-PCR analysis, and 11 of the 12 studies used tyrosinase. The heterogeneity of technique complicated the analysis but did not prevent some useful conclusions about RT-PCR as a prognostic factor. Overall, the RT-PCR positivity rate of 51.1% was significantly higher than the histopathology positivity rate of 20.3%, a finding that is in agreement with the data presented in our current report. The RT-PCR was positive in 42.3% of histopathology-negative SLNs, and the false-negative rate for RT-PCR was only 4.9%. The disease-recurrence rate was significantly higher when RT-PCR results were positive versus negative (16.8% vs 8.7%). A negative predictive value of 91.3% indicates a useful prognostic factor. These data suggest that molecular staging of the SLN yields valuable, informative prognostic findings despite variability in molecular assays used.
The data presented in this report are limited by the era in which the PE specimens were generated (1992–1999), as would be the case for any study of long-term outcomes based on retrospective analysis of specimens. Since the introduction of SLND for melanoma in 1992,3
histologic evaluation of the SLN has become more efficient and more detailed in some centers.4
Use of the current standard of SLN ultra-staging may have increased the yield of histopathology-positive SLNs in our study, decreasing the number of histopathology-negative/qRT-positive SLNs and influencing the survival curves. However, it would not have decreased the number of qRT-positive SLNs and likely would not alter the results of the multivariate analysis. As recently reported by our group, the multiple disciplines involved in SLN detection and staging increase the potential for variations and errors but do not diminish the ability of molecular staging to detect occult micrometastases missed by conventional techniques.10
Another consideration is the evolution of melanoma staging, which has become more accurate and sophisticated in recent years. The presence of ulceration may predict a higher likelihood of recurrence even when the SLN is histologically negative. Thus, our study’s inclusion of ulcerated lesions may account for its high rate of recurrence; the 30% recurrence rate is higher than expected at 10 years of follow-up. However, the 19% ulceration rate is in line with previously reported rates of ulceration for primary-tumor depths of 1 to 2 mm.38
Again, changes in state-of-the-art diagnosis and treatment are inevitable limitations faced by any longitudinal prognostic biomarker study.
Our study of molecular upstaging for histopathology-negative SLNs from American Joint Committee on Cancer stage I/II patients has the longest follow-up for molecular staging of regional lymph nodes in any solid cancer. The study is also the first to demonstrate the significant prognostic utility of molecular upstaging on the basis of PE tissues. The data confirm a prognostic role for multimarker qRT assessment of SLN PE tissue from patients with clinically localized melanoma and support molecular analysis as part of the comprehensive prognostic assessment.