Originally published as JCO Early Release 10.1200/JCO.2002.08.009 on July 1 2002

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Tyrosinase mRNA in Blood of Patients With Melanoma Treated With Adjuvant Interferon

From the Medical Oncology Department, Institut de Malalties Hemato-Oncològiques, and Dermatology Department, Hematopathology Laboratory, and Epidemiology Department, Institut de Investigacions Biomèdiques August Pi i Sunyer Hospital Clínic, University of Barcelona, Barcelona, Spain.

Address reprint requests to Begoña Mellado, MD, Medical Oncology Department, Hospital Clínic, University of Barcelona, C/Villarroel, 170, 08036 Barcelona, Spain; email: bmellado{at}clinic.ub.es

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate the clinical significance of the detection of circulating melanoma cells in patients treated with adjuvant interferon and to determine their potential value as a marker of interferon response.

PATIENTS AND METHODS: We prospectively analyzed 616 peripheral-blood samples from 120 melanoma patients with stage IIA (n = 33), IIB (n = 22), III (n = 50), or IV (surgically resected) (n = 15) disease receiving adjuvant interferon alfa-2b therapy. Tyrosinase mRNA was assayed by reverse transcriptase polymerase chain reaction (RT-PCR) as a marker of circulating melanoma cells before the start of interferon and every 2 to 3 months thereafter.

RESULTS: With a median follow-up time of 32.3 months (range, 7.1 to 77.5 months), 47 patients (39.8%) relapsed and 31 (26%) died. During adjuvant interferon treatment, 76 patients (64%) had undetected circulating melanoma cells and 44 patients (36%) had a positive RT-PCR result in at least one sample. Actuarial 5-year disease-free survival was 62% in patients with persistently negative RT-PCR during interferon treatment and 38% for patients with positive RT-PCR during interferon (P = .02). Actuarial 5-year overall survival was 75% and 50%, respectively (P = .03).

CONCLUSION: Patients with melanoma and tyrosinase mRNA detected in the blood during adjuvant interferon therapy had a worse prognosis compared with patients with undetected tyrosinase mRNA during treatment. Further investigation into the detection of circulating melanoma cells as a surrogate marker of response to adjuvant interferon therapy is warranted.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
INTERFERON HAS recently became a widely used adjuvant therapy for high-risk melanoma patients since several studies have reported that it might reduce the risk of relapse and death of patients with surgically resected high-risk melanoma.^1-4 Unfortunately, many patients still relapse despite of the administration of adjuvant interferon. In this scenario, it would be important to find markers of interferon response, with the ultimate goal of identifying those patients who are more likely to benefit from interferon therapy and those who would need newer therapeutic strategies.

A series of studies have addressed the prognostic implications of the detection of circulating tumor cells in the blood from patients with melanoma treated surgically. Since the seminal study by Smith et al,⁵ many studies have assessed the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the blood from melanoma patients as a marker of circulating melanoma cells. Tyrosinase mRNA is specifically expressed in melanocytes, Schwann cells, and melanoma cells. Since melanocytes do not circulate, the detection of tyrosinase mRNA in blood translates to the presence of circulating melanoma cells. Subsequent studies confirmed the ability to detect mRNA tyrosinase by RT-PCR in the blood of melanoma patients and investigated the potential clinical implications of this assay.^6-28 We and others have reported that the presence of circulating malignant cells detected by the amplification of tyrosinase mRNA and/or other melanoma markers by RT-PCR has an adverse prognostic value in patients with melanoma.^{10-13,16-18,23-28} Additionally, we detected tyrosinase mRNA in the blood of patients months or years after the initial treatment. The finding of circulating melanoma cells during the late follow-up of clinically disease-free melanoma patients was also associated with a poor outcome.¹⁷ These data led us to look at melanoma as a systemic disease and to believe that a potential end point of adjuvant systemic immunotherapy could be to maintain patients with undetectable malignant cells in peripheral blood.

Since adjuvant interferon treatment is now widely used in high-risk melanoma patients, we decided to investigate, in a prospective fashion, whether the prognostic value of circulating melanoma cells was maintained in patients treated with adjuvant interferon. We hypothesized that the presence of circulating melanoma cells during adjuvant interferon treatment could be a surrogate marker of tumor resistance, which may indicate the need to change the therapeutic strategy. To study this hypothesis, we prospectively tested tyrosinase mRNA, before and, on a serial basis, during treatment, in 120 melanoma patients who where rendered clinically free of disease by surgery and were receiving adjuvant interferon alfa-2b therapy at our institution. To our knowledge, this is the first time that such a study has been conducted, since in previously published studies patients were not treated with adjuvant interferon.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
The inclusion criterion was a histologically documented diagnosis of melanoma, stage IIA, IIB, III, or IV (metastasis surgically resected), treated by surgery followed by adjuvant interferon alfa-2b therapy. History of another malignancy was an exclusion criterion. The study was approved by the ethics committee of the hospital, and informed consent was obtained from each patient for participation in the study. Blood for RT-PCR was obtained at the same time as the blood extraction performed during previously established follow-up: the first collected 15 mL of blood was used for standard biochemistry and cell blood count; the next 10 mL were used for RT-PCR studies. Clinical stage and therapy were documented at the time of entry onto the study. The clinical outcome of the patients was prospectively followed. Clinical staging consisted of medical history, physical examination, cell blood count, blood biochemistry, and chest x-ray. Other complementary examinations were performed if clinically indicated. Clinical stage was defined based on American Joint Committee on Cancer guidelines.

All patients received adjuvant interferon alfa-2b (Schering-Plough, Kenilworth, NJ). Patients with stage IIA or higher disease who refused high-dose interferon or in whom it was contraindicated received low-dose interferon (3 million units [MU] 3 days per week subcutaneously for 2 years). Patients with stage IIB, III, or IV disease received high-dose interferon (20 MU/m²/d intravenously 5 days per week for 4 weeks, followed by 10 MU/m²/d 3 days per week for 1 year). Intermediate-dose interferon (10 MU/d subcutaneously 5 days per week for 4 weeks, followed by 5 MU/d 3 days per week for 2 years) was administered to a subgroup of stage II and III patients included in an internal adjuvant study performed at our institution.

A blood sample for RT-PCR was obtained 3 to 6 weeks after surgical treatment, before the start of interferon therapy (baseline PCR). During interferon therapy, patients were tested for tyrosinase mRNA in blood every 2 to 3 months until the end of the treatment (serial PCR). No clinical decisions were made based on the results of the RT-PCR assay.

Samples
Ten milliliters of blood were collected in EDTA anticoagulant from each patient. The mononuclear cell fraction of peripheral blood was isolated by Ficoll gradient as described by Boyum.²⁹ Total RNA was isolated from the mononuclear cell fraction by guanidinium thiocyanate extraction using the method described by Chomczynski and Sacchi.³⁰ Positive controls were processed separately to avoid contamination.

RT-PCR Method
RT-PCR was performed according to the manufacturers’ instructions (Gibco BRL, Paisley, United Kingdom) using 1 µg of total cellular RNA. First-strand cDNA was generated with 50 ng of specific primer (HTR2), deoxynucleotide triphosphate 0.5 mmol/L, 1 unit of RNAsin (Promega, Madison, WI), and 200 units of murine Moloney leukemia virus reverse transcriptase (Gibco) in 20 µL of final volume. A 10-µL aliquot of this reaction was used in the first round of PCR using 50 ng of each primer (HTR1 and HTR2), MgCl2 1.6 mmol/L, deoxynucleotide triphosphate 0.2 mmol/L, and 1.5 units of Taq polymerase (Gibco) under the following conditions: one cycle of 5 minutes at 95°C for template denaturation, followed by 30 cycles of 65 seconds of denaturation at 95°C, 65 seconds at 55°C for primer annealing, and 50 seconds for polymerase extension at 72°C. All PCR reactions were terminated with a 10-minute extension at 70°C. For the second round of PCR, 5 µL of a 1/100 dilution of the first-round PCR product was used in combination with 50 ng of HTR3 and HTR4 primers in a 25-µL final volume. Cycling conditions were the same as the first round of PCR. Final products were electrophoresed on 2% agarose gel and analyzed by direct visualization after ethidium bromide staining. Every RT-PCR reaction was repeated at least twice to confirm results.

For synthetic oligonucleotides, primer sequences were devised from published sequences of tyrosinase gene.⁹ Outer primers were as follows: HTYR1 (sense), TTGGCAGATTGTCTGTAGCC; and HTYR2 (antisense), AGGCATTGTGCATGCTGCT. Nested primers were as follows: HTRY3 (sense), GTCTTTATGCAATGGAACGC; and HTYR4 (antisense), GCTATCCCAGTAAGTGGACT. The outer primers amplified a PCR product of 284 base pairs (bp) and the nested primers amplified a fragment of 207 bp.

Integrity of RNA for RT-PCR assay was determined by performing parallel RT-PCR reactions using primers specific for beta-globin¹¹ and/or by running RNA in a 1% agarose gel. Samples that failed to show the 28s and 18s ribosomal bands were considered degraded and ineligible for the study.

Negative controls of the RT-PCR reaction included a sample without RNA. The human melanoma-derived cell line SK-mel 28 (American Type Culture Collection, Rockville, MD) was used as a positive control. The sensitivity of our RT-PCR was determined by performing serial dilutions of SK-mel 28 cells in 10^-7 normal mononucleated cells. With the described conditions, we were able to detect one SK-mel 28 cell in 10⁶ mononucleated cells by RT-PCR and one SK-mel 28 cell in 10⁷ mononucleated cells by Southern blot analysis.^11,17 Southern blot analysis was performed to confirm that our RT-PCR–amplified products were indeed from tyrosinase. After electrophoresis gels were transferred overnight to a nitrocellulose membrane with 20 times standard saline citrate buffer. Membranes were hybridized with an oligonucleotide complementary to a region in the tyrosinase c-DNA, using a nonisotopic fluorescein 3'-oligolabeling system (Amersham, Buckinghamshire, United Kingdom).

Every sample was tested in two different RT-PCR assays. If both assays showed a 207-bp band, the sample was classified as positive. If no band was detected in either of the two assays, the sample was considered negative. If a discordance in a result was observed between two reactions (a positive and a negative result of the same sample), the sample was subjected to two additional independent RT-PCR assays. If one or both additional assays were positive, the sample was classified as positive. If the two additional assays were negative, the sample was classified as negative. We only had 24 (3.8%) of 616 samples with a single positive assay that could not be confirmed in any additional RT-PCR reaction. Southern blot analysis after nested RT- PCR was performed in these samples, and none of them became positive with RT-PCR plus Southern blot analysis. We considered these samples to be negative for further analysis.

Statistic Analysis
Arithmetic mean, SD, and percentage of cases observed constituted the descriptive analysis. Disease-free survival (DFS) and overall survival (OS) were calculated from the time of inclusion in the study until relapse or death, respectively. DFS and OS were analyzed by the Kaplan-Meier method. Curves were compared by the log-rank test. Cox regression models were used to predict the chance of relapse and death. Multivariate models were estimated using P < .05 as enter criterion and P > .10 as remove criterion. 95% Confidence intervals were calculated for each of the estimates. Significance level was 5%. Software for analysis was STAT (STATA Corp, College Station, TX, 1997).

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
A total of 120 patients participated in this study. Patients’ characteristics and treatment are described in Table 1. Clinical stage was IIA for 33 patients, IIB for 22 patients, III for 50 patients, and IV for 15 patients. Sites of metastasis in stage IV patients were skin in five patients, lymph nodes in five patients, skin and lymph nodes in two patients, single lung node in two patients, and the intestines in one patient. The median follow-up time was 32.3 months (range, 7.1 to 77.5 months). At the time of this analysis, two patients had been lost to follow-up and were not included in the prognosis studies. Forty patients received low-dose interferon (25, stage IIA; five, IIB; four, III; six, stage IV), 16 received an intermediate dose (eight, stage IIA; seven, IIB; one, III), and 64 received high-dose interferon (10, stage IIB; 45, III; nine, IV). In eight patients treated with high-dose interferon, treatment was discontinued because of severe toxicity (neurotoxicity in one patient, psychiatric toxicity in two patients, hepatic toxicity in two patients, retinal thrombosis in one patient, endocarditis in one patient, and Reynaud syndrome in one patient). In the low-dose group, therapy was discontinued in two patients: one patient developed myasthenia gravis and another patient spontaneously decided to stop therapy. In the intermediate-dose group, no discontinuations of treatment occurred. Six hundred sixteen blood samples were tested for tyrosinase mRNA. The median number of RT-PCR tests per patient was six (range, two to 11).

View larger version (33K): [in this window] [in a new window]   Fig 1. (A) RNA samples. (B) RT-PCR products visualized on a 2% agarose gel with ethidium bromide staining. Lane 1, negative control; lanes 2-11, samples from melanoma patients; lane 12, positive control (SK mel-28). (C) Southern blot showing hybridization of fluorescein-labeled tyrosinase probe of the RT-PCR–positive samples.

Clinical stage and dose of interferon were factors associated with DFS and OS in the univariate analysis. Patients receiving high-dose interferon had a significantly lower DFS and OS than patients receiving an intermediate or low dose. Median DFS was 27.6 months for the low-dose group, 36.6 months for the intermediate-dose group, and 19.3 months for the patients receiving a high dose of interferon (P = .04). Median OS was 30.7 months for the patients treated with low-dose interferon, 42.6 months for the intermediate-dose group, and 31.4 months for the high-dose group (P = .04). These results are consistent with the fact that patients in the high-dose group had a more advanced stage of disease. In the multivariate analysis, the dose of interferon was not an independent prognostic factor for DFS and OS, while clinical stage maintained its prognostic value for DFS (P = .000073) and OS (P = .00072).

RT-PCR Results During Interferon Therapy and Prognosis
The RT-PCR results in blood samples during interferon therapy were significantly associated with patient outcome. Among the 118 patients with follow-up, 44 (37%) had at least a positive sample during treatment and 74 (63%) were persistently negative. The detection of a positive RT-PCR result for tyrosinase mRNA in blood during the interferon treatment significantly correlated with a lower DFS and OS. The median DFS was not reached for the group with negative RT-PCR during interferon therapy, while it was 20 months for the positive. Actuarial 5-year DFS was 62% for the negative group versus 38% for the patients with positive RT-PCR results during interferon therapy (P = .02) (Fig 2). Median OS was not reached for the group with negative results during interferon therapy, while it was 52 months for the group with positive results. Actuarial 5-year OS was 75% and 50% for the groups with negative and positive RT-PCR results during interferon therapy, respectively (P = .03) (Fig 3).

View larger version (13K): [in this window] [in a new window]   Fig 2. RT-PCR results during interferon therapy and DFS. DFS was calculated by the Kaplan-Meier method. The statistical significance of the difference in DFS in RT-PCR–negative versus RT-PCR–positive patients was calculated by the log-rank test.

View larger version (13K): [in this window] [in a new window]   Fig 3. RT-PCR results during interferon adjuvant therapy and OS. OS was calculated by the Kaplan-Meier method. The statistical significance of the difference in OS in RT-PCR–negative versus RT-PCR–positive patients was calculated by the log-rank test.

RT-PCR Positivity Before and/or During Interferon Therapy and Prognosis
Among the 118 patients with follow-up, 52 (44%) had at least a positive result for tyrosinase RT-PCR, before treatment, during treatment, or both. Among the patients with a positive result before and/or during therapy, 27 patients (51.9%) relapsed and 19 (36.5%) died. For the group of patients with persistently negative results (n = 66), 20 (30%) relapsed and 12 (18.8%) died. Median DFS was not reached for the negative patients, and it was 30.1 months for the positive group (P = .04). Median OS was not reached in either of the two groups (P = .03).

RT-PCR Results and Dose of Interferon
Since the dose of adjuvant interferon differed among patients, we separately analyzed the value of the RT-PCR results under interferon in the three different treatment groups. Table 3 shows the RT-PCR results, before and during interferon therapy, in the three therapeutic groups. It also shows the 5-year actuarial DFS and OS per each group of interferon dose, according to the RT-PCR results. The RT-PCR results before the start of interferon therapy did not correlate with DFS or OS in any dose group (Table 3). Similarly, in patients receiving low-dose (n = 39) or intermediate-dose (n = 15) interferon, there were no statistically significant differences in OS or DFS according RT-PCR results during therapy. However, in patients receiving high-dose therapy (n = 64) with positive RT-PCR during interferon, median DFS was significantly lower (20 months) than in patients with negative results (median DFS not reached; P = .02) (Fig 4). Similarly, patients in the high-dose group with positive RT-PCR results during therapy had a lower OS than negative patients, with a median OS of 35 months in the positive patients versus not reached in the negative (P = .02) (Fig 5). Patients in the high-dose group who had at least a positive RT-PCR result before and/or during therapy also had a significantly lower OS and DFS than the group of always-negative patients (Table 3).

View larger version (13K): [in this window] [in a new window]   Fig 4. RT-PCR results during interferon therapy and DFS in patients treated with high-dose interferon. DFS was calculated by the Kaplan-Meier method. The statistical significance of the difference in DFS in RT-PCR–negative versus RT-PCR–positive patients was calculated by the log-rank test.

View larger version (12K): [in this window] [in a new window]   Fig 5. RT-PCR results during interferon adjuvant therapy and OS in patients treated with high-dose interferon. OS was calculated by the Kaplan-Meier method. The statistical significance of the difference in OS in RT-PCR–negative versus RT-PCR–positive patients was calculated by the log-rank test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

In the present work, we specifically studied patients receiving adjuvant interferon therapy. Our hypothesis was that interferon may be able to eliminate microscopic disease present at the time of diagnosis of high-risk melanoma patients, and that the persistence of circulating cells in patients receiving interferon may be indicative of treatment failure. Two studies support that a persistently negative RT-PCR result during interferon treatment would be associated with the lack of circulating melanoma cells rather than downregulation of tyrosinase mRNA caused by interferon. In a study published by Brossart et al,⁹ tyrosinase mRNA was detected in blood and bone marrow in patients with stage IV melanoma treated with interferon and interleukin-2, before and during therapy. The same group showed, with a semiquantitative RT-PCR assay for tyrosinase, that the amount of transcript correlated with response to immunotherapy.⁸ Here, we observed that positivity for tyrosinase mRNA in blood during the interferon treatment significantly correlated with a lower DFS and OS. Overall, these data provide novel evidence for the detection of circulating melanoma cells as an adverse prognostic marker during adjuvant interferon.

Since the doses of interferon differed among patients, we performed a subset analysis of the study to explore a possible dose effect. In this analysis, the significant prognostic value of RT-PCR was maintained in patients treated with high-dose interferon. In the low- and intermediate-dose groups, the prognosis of RT-PCR–positive patients was also worse than that for RT-PCR–negative patients, but the differences were not statistically significant. Although we cannot formally rule out a dose effect, this result may also be due to the fact that the majority of patients received high doses of interferon and, in addition, the fact that the high-dose group included the patients with poorest prognosis who had a higher number of relapses/deaths. These two points result in a higher statistical power to detect significant differences in the high-dose group in comparison with the intermediate- or low-dose group. Additional patients would be required to further characterize this observation.

In the present study, positivity for baseline tyrosinase mRNA in peripheral blood before the start of interferon therapy did not correlate with clinical stage or prognosis. This result differs from the correlations seen with stage and prognosis observed in our first series.¹¹ This difference among the current and prior study¹¹ might be due at least to the following considerations. First, there was a tighter grouping of intermediate/high-risk patients in this study compared with previous ones. In our previous publication,¹¹ we included patients who had from stage I to bulky visceral stage IV disease, whereas in the present study, we included only patients with stage II and III disease, as well as a very limited group of patients with resected stage IV disease. Second, the timing of blood extraction must be considered. In our previous study, samples were drawn during the perioperative period, whereas in the present study, the first blood extraction was 3 to 6 weeks after surgery. We cannot rule out that this difference in the timing of blood extraction might have affected the results. Third, in the first study, patients did not receive adjuvant interferon, whereas in the present study, all patients were treated with interferon, which might possibly affect the prognostic implication of a baseline RT-PCR sample. We have to note, however, that a correlation between the effects (or the lack thereof) of interferon on individual patient outcomes and the presence or absence of tyrosinase mRNA in blood cannot be established because we did not have a parallel, matched control population not receiving interferon during the period of the study. To characterize this issue, it would be necessary to conduct a study to assay sequentially the mRNA tyrosinase in blood from patients randomized to receive interferon versus control.

An as yet unresolved issue of the detection of circulating cells by RT-PCR is the low reproducibility of the results among different laboratories^19,20 and in the same laboratory, when samples from the same patient are taken on different day time-points²⁰ or even at the same time.¹² The low reproducibility has been attributed to technical differences in sample preparation, RNA and cDNA synthesis, RT-PCR protocols, the small number of patients per study, the intermittent shedding of tumor cells into the blood, and to the small amount of circulating tumor cells. In our study, we observed isolated RT-PCR–positive samples that could not be confirmed in three additional separate RT-PCR reactions, and we considered them to be negative for further analysis. These unconfirmed "positive" results represent 3.8% of the analyzed samples. They may be false-positive results of the technique or indicate the presence of a very small amount of tyrosinase transcript in the blood. The last possibility is supported by results of a real-time PCR semiquantitative study²¹ showing that lower amounts of transcript of tyrosinase or MART-1 were found in samples with low reproducibility. In contrast, the amount of transcript of the target mRNA was significantly higher in samples with a positive result confirmed in four separate reactions. We cannot exclude that this minority of patients represents a different group with a lower volume of disease and a different clinical outcome.

In conclusion, the detection of tyrosinase mRNA in the blood of patients with melanoma during adjuvant interferon therapy is associated with an increased risk of relapse and death from the disease. The use of a multiple marker assay and/or a semiquantitative RT-PCR assay may improve the usefulness of the serial detection of circulating melanoma cells as a surrogate marker of adjuvant treatment efficacy in melanoma.

	ACKNOWLEDGMENTS

 
This article was published ahead of print at www.jco.org.

Supported by Plan Nacional I+D (SAS97-0093) (to T.C., B.M.). L.G. was supported by Amgen (1996) and a research grant from Asociación Española de la Lucha Contra el Cáncer (1997). M.C.V. was supported by Beca Instituto de Salud Carlos III.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted July 31, 2001; accepted May 15, 2002.

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