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© 2003 American Society for Clinical Oncology Molecular Tumor Markers in the Blood: Early Prediction of Disease Outcome in Melanoma Patients Treated With a Melanoma Vaccine
From the Department of Molecular Oncology, John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica; and Department of Biomathematics, University of California Los Angeles School of Medicine, Los Angeles, CA. Address reprint requests to Dave S.B. Hoon, PhD, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; email: hoond{at}jwci.org.
Purpose: Patients with American Joint Committee on Cancer (AJCC) stage III melanoma are at high risk of recurrence and death. We hypothesized that a multiple-marker reverse transcriptase polymerase chain reaction (MM-RT-PCR) blood assay could predict, early in the course of therapy, those patients destined to experience treatment failure with a melanoma vaccine (MV) previously shown to improve survival in a phase II clinical trial. Patients and Methods: After complete surgical resection, prospectively collected cryopreserved peripheral-blood lymphocyte specimens (n = 90) from the serial bleeds of 30 patients with AJCC stage III melanoma were studied by MM-RT-PCR, using the markers tyrosinase, melanoma antigen recognized by T cells-1 (MART-1), and universal melanoma antigen gene-A (uMAG-A). All patients were enrolled in a phase II MV trial during the period of blood draws, and were selected for this study in a blinded fashion. Median duration of clinical follow-up was 74 months for the 13 survivors and 11 months for the 17 nonsurvivors. Results: The presence of at least one melanoma-specific RT-PCR marker was associated with an increased risk of disease recurrence (risk rate, 3.12; P = .02) and decreased risk of survival (relative rate, 2.62; P = .0496) by multivariate analysis. Conclusion: MM-RT-PCR of the blood provided early prediction of subsequent disease recurrence and death in clinically disease-free AJCC stage III melanoma patients enrolled in a MV phase II trial. On the basis of the results of this pilot study, the MM-RT-PCR blood assay should be considered as a clinically important monitoring tool for assessing patient response to adjuvant therapy, and in the surveillance of clinically disease-free patients for the earliest signs of recurrence.
THICKNESS OF the primary tumor and regional lymph node status are the most significant clinicopathologic factors associated with the development of distant metastasis in patients with melanoma.1–3 When melanoma is localized to the primary tumor site at the time of diagnosis (American Joint Committee on Cancer [AJCC] stages I and II), 5-year survival is more than 90%, whereas only 5% of patients with stage IV disease survive for 5 or more years.1,4,5 However, patients with AJCC stage III disease represent an enigmatic group in which approximately half of the patients will succumb to their disease in 5 years. Thus, these intermediate-stage patients are a major focus of adjuvant treatment trials. At present, there are no validated clinical assays capable of accurately predicting response to adjuvant therapy. Thus, an assay capable of providing accurate and early prediction of those patients destined to experience disease recurrence or die of their disease would be of considerable clinical importance, particularly for intermediate-stage patients receiving adjuvant therapy. Patients identified as being at high risk for ultimately experiencing adjuvant treatment failure, if identified early enough, might then be considered for aggressive alternative treatment protocols before the onset of clinically detectable metastasis, and at a point when such treatment plan modifications would be likely to have the greatest potential impact. The majority of AJCC stage III melanoma patients are candidates for adjuvant therapy after surgical resection. In this group of patients, adjuvant therapy with interferon alfa-2b or melanoma vaccines may improve both disease-free survival (DFS) and overall survival (OS).6–10 The John Wayne Cancer Institute (JWCI) has developed and assessed a melanoma vaccine (MV) in a phase II clinical trial, with further evaluation currently underway in a randomized United States Food and Drug Administration–approved phase III multicenter trial (CANVAXIN, CancerVax Corp, Carlsbad, CA). The phase II clinical trial showed evidence of improved 5-year survival among AJCC stage III patients treated with a 5-year course of MV compared with patients receiving either bacillus Calmette-Guérin vaccine alone, an earlier generation MV, or chemotherapy.7,9–11 Because there are at present no validated blood markers for melanoma that can definitively predict patient response to adjuvant therapy, we hypothesized that the multiple-marker reverse transcriptase polymerase chain reaction (MM-RT-PCR) assay could be used to detect the presence of occult circulating tumor cells early in the course of adjuvant treatment. We also hypothesized that if the presence of occult tumor cells in the peripheral-blood lymphocytes (PBLs) of melanoma patients with extensive clinical follow-up correlated with eventual melanoma recurrence or death caused by disease, such findings would be of considerable potential clinical importance. Alternative treatment strategies might then be implemented while patient tumor burden remained at microscopic levels, and when adjuvant treatments have the greatest chance of being clinically effective. Therefore, we applied the MM-RT-PCR assay to the cryopreserved PBLs of AJCC stage III melanoma patients who were previously enrolled onto the phase II MV trial, and for whom we had extensive clinical follow-up data. In this study, the presence of melanoma-associated mRNA markers for tyrosinase, melanoma antigen recognized by T cells-1 (MART-1), and universal melanoma antigen gene-A (uMAGE-A) in the PBLs of stage III melanoma patients was assessed using an MM-RT-PCR assay established in our laboratory.4,12 Tyrosinase is primarily a component of the melanogenesis-associated pathway, whereas MART-1 is frequently expressed (> 85%) in melanoma tumor cells and melanoma cell lines, but is not expressed in nonmelanoma malignancies or in the blood of humans without cancer. Genes in the MAGE-A family of melanoma-associated antigens are selectively expressed by a variety of human tumors, including melanoma. Among noncancer tissues, only the placenta and testes have been shown to express MAGE-A genes. The uMAGE-A primer set of the MM-RT-PCR assay is capable of detecting the mRNA transcripts of at least six genes within the MAGE-A gene family. Therefore, the MM-RT-PCR blood assay is able to detect the mRNA of eight different melanoma-associated genes under optimal assay conditions.
Patient Factors Thirty patients (17 males and 13 females) with AJCC stage III melanoma were selected to participate in this preliminary MM-RT-PCR blood assay study (Table 1  ). All study patients were selected by JWCI biostatisticians from AJCC stage III patients participating in a phase II MV trial that accrued stage III and IV patients between 1993 and 1997. Selection of eligible patients was based on the availability of cryopreserved blood samples from the three specific serial time points chosen for evaluation and on the availability of complete clinical follow-up data. Before onset of vaccine therapy, all study patients were rendered clinically free of disease after complete surgical resection of all detectable disease. Patients were accrued into the MM-RT-PCR study on the basis of the following survival criteria: death caused by disease within 5 years of entering the MV trial (n = 19) and survival more than 5 years after MV trial entry (n = 11). All staff participating in MM-RT-PCR assays were blinded to patient identity and clinical history, including survival outcomes, until all data had been collected and statistically analyzed.
The MV contains three well-characterized allogeneic melanoma cell lines (M10, M24, and M101); each patient dose consists of 24 x 106 irradiated cells (8 x 106 cells per MV cell line).7 During the phase II MV trial, all patients were observed monthly with serial clinical and laboratory examinations, and every 3 months with radiographic examinations, until completion of the 5-year course of vaccine treatment or the patient’s death.9,10 Cryopreserved PBL specimens were selected for all MM-RT-PCR study patients from three serial bleed time points: the first day of vaccination (day 0), and the vaccinations at 2 months (week 8) and 4 months (week 16). All blood samples were drawn before vaccine injection. A total of 90 PBL specimens were analyzed (three serial bleeds per patient) for the presence of tyrosinase, MART-1, and uMAGE-A mRNA. These time points were selected on the basis of our previous observations that humoral and cellular immune responses to MV peak at 8 to 12 weeks after initial vaccine administration, whereas objective clinical tumor response typically follows initial vaccination within 3 months.9,11
The median duration of clinical follow-up from time of entry into the MV trial to initiation of PBL processing for this MM-RT-PCR study was 30 months for all 30 patients, 74 months for survivors (n = 13), and 11 months for nonsurvivors (n = 17; Table 1 Institutional review board approval was obtained in advance, and all study patients provided informed consent for the collection of their blood specimens for investigational testing.
Patient PBL Specimen Preparation
MM-RT-PCR Assay Total cellular RNA was extracted from all specimens using TRI-Reagent according to the manufacturer’s instructions (Molecular Research Center, Cincinnati, OH).12,14 The concentration, purity, and amount of total RNA were determined by ultraviolet spectrophotometry. The integrity of all RNA specimens was verified by performing RT-PCR with the housekeeping gene porphobilinogen deaminase. All PCR products were confirmed by a hybridization probe–based quantitative electrochemiluminescence (ECL) detection assay (ORIGEN Analyzer, IGEN International, Gaithersburg, MD) as previously described.4,15 PBLs from healthy donors without cancer were used as negative controls, and were incorporated throughout all MM-RT-PCR assay steps.16 Molecular biology–grade water was used in place of RNA and cDNA samples as negative controls for all RT, PCR, and ECL reactions to detect potential assay contamination. All assays were repeated at least once to verify results. All negative-control PCR samples confirmed the absence of assay contamination (data not shown). RNA extraction, RT-PCR assay setup, and PCR product ECL detection steps were carried out in separate designated rooms to prevent cross-contamination, as previously described.12,15 Biotin-labeled oligonucleotide primers for the PCR assays and Tris(2,2'-bipyridine)-ruthenium(II) chelate-labeled nucleic acid hybridization probes for the ECL assays were designed in our laboratory and synthesized by GenoMechanix (Alachua, FL) and the Midland Certified Reagent Company (Midland, TX), respectively, as previously described.12,13 Before this study was initiated, optimization of the MM-RT-PCR assay was first performed on the cryopreserved PBLs of AJCC stage III and IV melanoma patients, including patients who both received and did not receive MV. All RT reactions were carried out with oligo-deoxythymidine priming as described previously.4,14 PCR primer sequences for each marker were designed to span at least one intron region to avoid amplification of genomic DNA.14,15 RT products were amplified using a PCR panel consisting of the markers tyrosinase, MART-1, and uMAGE-A. The specificity and sensitivity of these three markers have been demonstrated previously on blood and lymphoid tissues.12,14,16 All three MM-RT-PCR tumor markers were previously validated by gel electrophoresis analysis and marker-specific probe-based Southern blotting.13 Primer and ECL probe design for MM-RT-PCR tumor markers, as well as PCR conditions, have been previously described.4,12 Optimal PCR conditions were maintained using a Hybaid thermocycler (Hybaid, Middlesex, United Kingdom). The MM-RT-PCR assay was considered positive when the ECL assay results for each marker were greater than three SDs above the mean ECL values of PBL from healthy volunteer donors. Assays were repeated at least twice to verify results.
Statistical Analysis
MM-RT-PCR Assay Sensitivity Assay sensitivity was one to five melanoma cells diluted in 107 PBLs from healthy donors using the MM-RT-PCR assay.4,12 Tyrosinase, MART-1, and uMAGE-A were expressed by all three melanoma cell lines, but were not expressed in any of the healthy donor PBL specimens. Porphobilinogen deaminase mRNA was detected in all melanoma cell lines and in all 90 PBL specimens.
Tumor Marker Detection in Patient PBL Specimens and Clinical Correlation
Among the 30 patients assessed in this study, 19 (63%) experienced disease recurrence during follow-up (Table 2
Seventeen (57%) of 30 patients died as a result of melanoma during follow-up, and nine (53%) of these 17 patients had at least one positive RT-PCR tumor marker present in at least one PBL specimen (Table 2  ). In contrast, 13 (43%) of 30 patients were alive at the time of this study, but only two of these 13 patients had positive melanoma markers in their PBL (Table 2). The presence of at least one RT-PCR tumor marker in any PBL specimen correlated significantly (P = .04, log-rank test) with decreased patient survival by univariate analysis (Fig 2). Multivariate modeling and stepwise regression analysis showed a significant correlation between RT-PCR marker status and death caused by disease. The presence of at least one RT-PCR tumor marker in any PBL specimen was associated with a nearly three-fold risk (RR, 2.62; 95% CI, 1.00 to 6.79; P = .0496, Wald test) of death caused by disease when compared with patients with MM-RT-PCR marker–negative PBL (Table 2).
Despite ongoing debate, there is accumulating evidence that occult melanoma cells circulating in the blood may serve as surrogates for eventual disease outcome.20–24 The long-term follow-up for the patients is both unique and important for this assay utility assessment. This extended period of follow-up provides a more accurate basis for assessing the true clinical importance of occult circulating tumor cells detected by MM-RT-PCR, and particularly in intermediate-stage patients who are clinically free of residual disease. Although most melanoma recurrences in patients with stage III disease occur within 3 to 5 years after complete resection, melanoma has a known predilection for recurring much later. This consideration adds to the value of more prolonged periods of clinical follow-up in evaluating the predictive value of molecular detection assays.25 However, a major limitation of melanoma RT-PCR studies, to date, has been the relatively brief duration of patient follow-up, typically encompassing only 2 to 3 years. In addition, in view of the intermittent nature of tumor cell shedding into the blood and the nominal volume of blood in any single venipuncture specimen, the lack of serial bleed analysis in most RT-PCR studies may also be an important limiting factor. Therefore, our successful use of cryopreserved PBLs from the serial bleeds of melanoma patients with long-term clinical follow-up provides a highly optimized setting in which to demonstrate accurately the potential clinical utility of the MM-RT-PCR blood assay, in addition to providing a novel approach to molecular analysis of the blood. The use of multiple tumor markers is also important in optimizing RT-PCR assay sensitivity. Previously, we have shown that an MM-RT-PCR melanoma assay is highly sensitive in detecting occult tumor cells in the blood and sentinel lymph nodes, and that this information is pathologically and clinically useful.4,20 In view of the heterogeneous expression of tumor-associated mRNA markers by individual cancer cells within a tumor, as well as between clonal populations of cells within both primary and metastatic tumors, single-marker RT-PCR assays are less efficient at detecting occult disease than is an MM-RT-PCR assay.4,15,20 Because of the poor clinical predictive power of single RT-PCR marker assays performed on blood, this study has emphasized the application of an MM-RT-PCR assay.
Importantly, the ability of the MM-RT-PCR blood assay to predict disease outcome in this study was based entirely on the analysis of PBL specimens that were prospectively collected within the first 16 weeks of a 5-year course of vaccine therapy, and after surgical resection of all clinically detectable disease. This interval is particularly important with respect to patient response to vaccine therapy because it is during this period that immune system response to MV is maximally stimulated, but is before the earliest observable objective clinical responses in many patients.9,11 The ability of the MM-RT-PCR assay to predict eventual patient response to lengthy courses of adjuvant treatment, and at such an early time point after initiation of therapy, may have important clinical implications. Indeed, in this preliminary study, the MM-RT-PCR assay was especially powerful in predicting a low risk of future disease recurrence and disease-related mortality when no melanoma markers were detected. This predictive power for disease relapse, and for death caused by disease, was sustained over many years after collection of the blood samples tested by MM-RT-PCR (Figs 1 Our findings indicate that the MM-RT-PCR may be useful as a powerful clinical tool with which to assist in the management of patients with AJCC stage III melanoma, as well as other solid tumors that are assessable by RT-PCR. Such a sensitive and specific early-warning assay would be a particularly useful surrogate for determining which patients should be selected for more aggressive clinical monitoring or alternative treatment strategies before the onset of clinically detectable metastatic disease. The MM-RT-PCR assay also seems to select patients who, by virtue of the absence of detectable circulating melanoma markers, are likely to have a favorable long-term clinical outcome. The MM-RT-PCR assay might also play an important role in the stratification of patients within phase II and III adjuvant treatment protocols. Using such data should improve the accuracy of designing and monitoring study protocols in addition to the use of AJCC stage and other traditional clinicopathologic parameters. Indeed, on the basis of the results of this and previous MM-RT-PCR studies at our institution, the MM-RT-PCR blood assay is currently being used at JWCI to monitor prospectively patients enrolled onto the ongoing international phase III melanoma MV multicenter trial and onto other clinical research protocols. Additional evaluation of the MM-RT-PCR blood assay with larger numbers of patients will then be used to validate further the findings of this preliminary study. Another important potential application of the MM-RT-PCR blood assay would be to supplement currently used clinicopathologic staging information in counseling cancer patients and their families, both in terms of prognostic predictions and treatment options. The availability of such highly specific and individualized prognostic information may considerably reduce the subjectivity inherent in the counseling of patients with newly diagnosed cancer, and can assist both patients and physicians in making critical treatment and follow-up decisions. In summary, on the basis of this preliminary study of 30 patients at three different early time points in their treatment, MM-RT-PCR of the blood can predict the risk of future recurrence and death caused by disease in intermediate-stage melanoma patients undergoing adjuvant therapy. In this group of AJCC stage III melanoma patients with a more than 5-year clinical follow-up, our results significantly demonstrate the potential clinical utility of molecular analysis in the detection of subclinical disease, and the powerful prognostic value of such assays. We propose that the MM-RT-PCR blood assay is a potentially useful adjunct with which to monitor patients receiving adjuvant therapy for AJCC stage III malignancies. Additional evaluation and validation of the MM-RT-PCR blood assay seems warranted for melanoma and other solid tumors, on the basis of the intriguing results of our study.
We thank the John Wayne Cancer Institute clinical and technical staff and IGEN International for their contributions to this study.
Supported in part by National Institutes of Health, National Cancer Institute P01 Grants CA 29,605 and CA 12,528, Project II.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
