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Molecular Biology and Genetics |
Department of Molecular Oncology [H. T., C. K., D. S. B. H.], Division of Biostatistics [H-J. W.], and John Wayne Cancer Institute [D. L. M.], Saint John’s Health Center, Santa Monica, California 90404
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have used several MAAs in multimarker RT-PCR assays that were developed for detecting occult metastatic melanoma cells in blood or tumor-draining lymph nodes (21, 22, 23, 24) . The heterogeneity of expression of differentiated MAAs in melanomas contributes to the inconsistency of using a single MAA as a marker for detecting metastatic melanoma. Also, it is known that these MAAs may not be expressed, or are expressed in very low levels, by some melanomas, such as hypomelanotic melanomas (22) . It is not uncommon for metastatic melanomas to be less pigmented than primary tumors. Advanced stages of tumors have been known to revert to become less differentiated and resort to a more fetal phenotype similar to the origin of the tumor cells. The association between clinical significance with reduction or loss of differentiated MAAs during tumor progression has not been well studied.
Host-tumor immunity has been shown to control melanoma progression and maintain stability (5 , 6 , 12) . The efficacy of this tumor immunity may be dependent on multiple factors such as individual or multiple MAA expression levels, host immune system status, tumor growth rate, MAA presentation and recognition, and so forth (5 , 19 , 20 , 25 , 26) . Host immunity can also play a significant role in immunodeletion of a particular antigen phenotype(s) of a tumor. Although there are studies suggesting that specific MAA down-regulation correlates with reduced T-cell recognition, association with disease outcome has not been well demonstrated (20) . To date, there has been problems in assessment of MAA expression levels in melanomas and correlation with disease outcome. Studies with IHC have been hampered by the lack of availability of MAA-specific antibodies for paraffin-embedded tissue sections, and also by their sensitivity. The utilization of RT-PCR has been documented in the past for determining the presence of MAA; however, quantitative analysis has been problematic and tedious primarily because of the lack of availability of reproducible methodologies.
Majority of patients with distant metastasis are treated with chemotherapy and/or radiotherapy, which have minimal effect on overall survival. There is a major need of improvement in characterization of advanced-stage melanomas to improve management and develop targeted therapeutics. Although patients with distant metastasis have generally a highly unfavorable prognosis, there is a subset of patients who do have prolonged survival with their disease. It is intriguing and not well understood why some AJCC stage IV patients have a better survival rate than other patients with similar or less disease burden. We have previously shown that cytoreductive surgery of visceral metastases in AJCC stage IV patients can improve survival (25) . In these studies significant improved survival was afforded by the complete resection of multiple organ-site metastasis (25 , 27) . However, complete removal of all clinically evident disease is not always possible. It is inevitable that subclinical disease remains, even in patients who are made clinical disease free. The correlation between mRNA expression levels of MAAs and disease outcome in advanced-stage melanoma patients has yet to be investigated. The validity of quantifying MAAs using either molecular analysis or IHC, to date, has been difficult to establish. However, the recent development of the qRT assay allows the rapid and reproducible quantitative analysis with high sensitivity and specificity if correctly designed. The previous approaches of quantitative RT-PCR have been very tedious and not reproducible as routine assays, particularly in the assessment of multiple markers on a large number of patients.
TYR, TRP-2, and MART-1 are major melanocyte-differentiation antigens that are immunogenic in patients and well expressed in melanomas (10 , 11 , 15) . In addition to being immunogenic, TYR and TRP-2 are involved in key processes of the melanin synthesis pathway (28 , 29) . These MAAs have been used individually, in combination, or as components in various formulations in vaccine clinical trials (6 , 12 , 15 , 30 , 31) . Active-specific immunotherapy containing these MAAs have shown augmentation of MAA-specific immunity (20) . These MAAs induce both T-cell responses and antibody responses (15, 16, 17) to several epitopes; however, it is not known which type of immune response or which MAA epitope-specific response is dominant in effective antimelanoma immunity. Melanomas from different stages of disease are often heterogeneous in expression of these MAAs (22) . All three of these MAA are intracellularly expressed and considered predominantly as T-cell recognition antigens in coordination with different HLA class I and II antigens. It is logistically difficult to make an overall immune assessment of melanoma patients and determine whether any individual MAA immune response played a significant role in tumor control or regression. Therefore, our approach was to directly measure the actual mRNA copies of MAA in the melanomas to provide a more reliable quantitative measure of MAA expression, which may be a predictor of disease outcome.
Our hypothesis is that the mRNA level of MAA expression in metastatic melanoma can be used as a predictor of overall disease outcome in advanced metastatic melanomas. In corollary, we hypothesize that lower MAA mRNA copy levels in metastatic melanoma would correlate with tumor progression and more aggressive disease. We analyzed TYR, MART-1, and TRP-2 mRNA copy levels using a qRT assay on melanoma cell lines and surgically resected metastatic melanoma specimens. A significant correlation between individual MAA mRNA copy levels and number of MAAs positive in tumors and disease outcome was determined.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Melanoma Cell Lines.
Fifteen melanoma cell lines established and characterized at the JWCI were assessed: MA, MB, MC, MD, ME, MF, MG, MH, MI, MJ, MK, ML, MM, MN, and MO. All established cell lines were grown in RPMI 1640 supplemented with 100 ml/liter heat-inactivated FCS, penicillin, and streptomycin (Life Technologies, Inc., Grand Island, NY) in a T75-cm2 flask as described previously (24)
. Total RNA was extracted from cells when cell cultures reached 70–80% confluence.
RNA Isolation.
Total cellular RNA from cell lines and tissue specimens was extracted, isolated, and purified using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as described previously (23
, 24) . All of the RNA extractions were performed in a designated sterile laminar flow hood using RNase-free lab ware. RNA was quantified and assessed for purity by UV spectrophotometry and RIBOGreen detection assay (Molecular Probes, Eugene, OR). Tissue processing, RNA extraction, RT-PCR assay set-up, and post-RT-PCR product analysis were performed in separate designated rooms and facilities to prevent cross-contamination, as reported previously (23)
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Primers and Probes.
Primer and probe sequences were designed for the qRT assay using Oligo Primer Analysis Software, version 5.0 (National Biomedical Systems, Plymouth, MN). To avoid possible amplification of contaminating genomic DNA, primers were designed so that each PCR product covered at least one intron. Fluorescence resonance energy transfer probe (32)
sequences were as follows: TYR, 5'-FAM-TTCACCATGCATTTGTTGACAGTATT-BHQ-1–3'; MART-1, 5'-CAL RED-CAGAACAGTCACCACCACCTTATT-BHQ-2–3'; TRP-2, 5'-FAM-TCACATCAAGGACCTGCATTTGTTA-BHQ-1–3'; GAPDH, 5'-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1–3'; and ß2M, 5'-CAL RED-TCCATGATGCTGCTTACATGTCT CGA-BHQ-2–3'. Control melanomas and nonmelanoma tissues and cell lines were used to optimize the assay. GAPDH and ß2M were used as internal reference housekeeping genes for status of sample mRNA assessed.
RT-RealTime PCR Assay.
All of the reverse-transcriptase reactions were performed using Moloney murine leukemia virus reverse-transcriptase (Promega, Madison, WI) with oligo-dT priming as previously described (23)
. The qRT assay was performed using iCycler iQ RealTime thermocycler (four-color) detection system (Bio-Rad Laboratories, Hercules, CA). The PCR reaction mixture consisted of cDNA template from 250 ng of total RNA, 1 µM of each primer, 0.3 µM fluorescence resonance energy transfer probe, 1 unit AmpliTaq gold polymerase (Applied Biosystems, Branchburg, NJ), 200 µM each dNTP, 4.5 mM MgCl2, 10 µg of BSA, and 10x AmpliTaq buffer to a final volume of 25 µl. Samples were amplified with a precycling hold at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min for GAPDH and TYR (annealing at 59°C for ß2M and MART-1, at 60°C for TRP-2), and extension at 72°C for 1 min.
Positive controls (melanoma cell lines MC, MF, and MN), negative controls from peripheral blood lymphocytes of healthy donors and tumor-free lymph nodes from nonmelanoma patients, and reagent controls (reagent alone without RNA or cDNA) for the qRT assays were included in each assay run. Lymphocytes and lymph nodes were processed and RNA prepared as described previously (23 , 33) . Each assay was performed at least twice to verify the results, and the mean copy number was used for analysis. SD between assays was not significant for all MAAs studied.
The standard curve for quantifying mRNA copy number was established amplifying nine aliquots of templates with known copy numbers (100 to 108 copies). Specific MAA cDNA was synthesized as follows; RT-PCR and sample RNA was performed, run on 2% agarose gel electrophoresis, and the cDNA was extracted using the QIAquick gel extraction method (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The MAA cDNA was ligated into pCR II-TOPO cloning vector (Invitrogen, San Diego, CA), the cDNA clones were transformed into Escherichia coli DH5-
cells, and cultures were expanded as described previously (24)
. Plasmids containing the target gene were purified and quantified for use in the qRT setup. To confirm that the inserted PCR product size is correct, plasmids were digested with specific restriction enzymes, and the cDNA clone PCR products were then run on gel electrophoresis.
Statistical Analysis.
Patient groups based on individual MAA levels were compared using Mann-Whitney U test, Kappa analysis and Spearman correlation coefficient analysis. The outcome measurement was overall survival based from the time of surgery of stage IV disease in which tumor was removed, to death or last follow-up. The cumulative survival rates for patient groups were calculated using the Kaplan-Meier methods and compared by using the log-rank test (21)
. Because there are limitations of known melanoma prognostic factors for this stage of patients, multivariate analysis was not performed. No marker outcome correlated with known relevant prognostic factors such as gender, age, or Breslow thickness. All of the patients had some form of visceral metastasis, and a majority had more than one organ-site metastasis removed. All of the Ps that were two-sided at a value of 
0.05 were considered to be statistically significant.
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RESULTS |
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. The PCR amplification of the serially diluted cDNA standard templates of each marker showed a logarithmic signal increase (Fig. 1)
. The standard curve was generated by using the threshold cycle (Ct) of templates in known numbers of copies. The Ct of each sample was plotted on the standard curve, and the mRNA copy number was calculated by the iCycler iQ RealTime Detection System Software (Bio-Rad Laboratories). All three of the MAAs were expressed in every melanoma cell line. The TYR mRNA copy number ranged from 439 to 3.22 x 107 copies (median, 4.77 x 106 copies) per 250 ng of total RNA from 15 individual melanoma cell lines. The MART-1 mRNA copy number ranged from 429 to 6.41 x 107 copies (median, 1.71 x 107 copies), and the TRP-2 mRNA copy number ranged from 1.77 x 104 to 2.14 x 108 copies (median, 6.06 x 107 copies). None of the three mRNA markers were expressed under optimal qRT conditions in the blood (n = 7) of healthy donors and pathology-defined cancer-negative lymph nodes from carcinoma patients (n = 7).
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and 3
). The patients ranged in age from 25 to 81 years (mean ± SD, 53 ± 14 years), consisting of 21 males and 14 females. The mean Breslow’s thickness was 4.33 mm ±3.91 SD. Thirty patients (86%) received adjuvant immunotherapy in the form of a cancer vaccine (CANVAXIN, CancerVax Corp., Carlsbad, CA; Refs. 7
, 16
). Five patients did not receive the vaccine, one patient received no therapy, and four received biochemotherapy and/or radiotherapy. TYR mRNA was detected in 29 (83%) of 35 melanoma specimens with copies ranging from 0 to 3.41 x 106 mRNA copies (median, 3.85 x 104 copies) per 250 ng of total RNA from melanoma specimens. MART-1 mRNA was detectable in 25 (71%) metastatic melanoma specimens. The MART-1 mRNA copy number ranged from 0 to 2.32 x 106 copies (median, 3.84 x 104 copies) per 250 ng of total RNA from melanoma specimens. TRP-2 mRNA expression was detected in 31 (89%) specimens with mRNA copy number ranging from 0 to 3.99 x 107 copies (median, 8.49 x 105 copies).
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Spearman correlation coefficient analysis and Kappa analysis (Table 4)
revealed significant correlation among TYR, MART-1, and TRP-2 mRNA copy levels. There was a significant correlation (P < 0.0001) in comparison of TYR versus MART-1, TYR versus TRP-2, and TRP-2 versus MART-1 with the correlation coefficient 0.818, 0.814, and 0.814, respectively. These MAAs have no significant nucleic acid homologies with each other.
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). There was no significant correlation between MART-1 or TRP-2 mRNA copy number and known clinicopathological prognostic factors. Although not significant, analysis of the postoperative survival rate showed a trend of higher MART-1 mRNA copy level associated with better overall survival (Fig. 2B)
. TRP-2 mRNA was frequently detected and, when present, had a significantly overall higher median level than the other MAAs. Therefore, TRP-2 analysis was assessed differently using a specific copy number cutoff based on overall profile of all of the specimens. For TRP-2 mRNA, the postoperative survival rate was significantly lower for eight patients with low expression (<105 copy number) than for 27 patients with high expression (
105 copy number; P = 0.009; Fig. 2C
). Moreover, when the TRP-2 mRNA copy level is normalized with GAPDH mRNA expression level, the survival rate for 11 patients with low TRP-2 mRNA expression (TRP-2:GAPDH ratio, <0.1) was significantly lower than for 24 patients with high TRP-2 mRNA expression (TRP-2:GAPDH ratio, 0.1; P = 0.006; Fig. 2D
). This cutoff value of TRP-2:GAPDH ratio approach was also used for TYR:GAPDH ratio and MART-1:GAPDH ratio. The survival rate for 30 patients with low TYR mRNA expression (TYR:GAPDH ratio, <0.1) was significantly lower than for five patients with high TYR mRNA expression (TYR:GAPDH ratio, 0.1; P = 0.037). For MART-1 mRNA expression survival was significantly lower when MART-1:GAPDH ratio was <0.1 (P = 0.040).
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. Patients with melanomas expressing low or no mRNA of at least two of the three MAAs were associated with a significantly (P = 0.024) worse prognosis (Figs. 3A–C)
. Patients expressing no MAA (median, 2.9 months) versus one to three MAAs (median 16 months) had a significantly (P = 0.013) poorer overall survival.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used an absolute value of the target mRNA copy number per specific amount of total RNA for analysis without correcting for the internal reference gene (34 , 35) . The results indicated that the analysis using the ratio (TRP-2:GAPDH) was very similar to that of TRP-2 mRNA copies (absolute value). In addition, the results showed that the two commonly used internal reference housekeeping genes, GAPDH and ß2M, in metastatic melanoma specimens were not always concordant in mRNA expression. Therefore, using housekeeping genes as internal reference or standards in qRT for the calculation of specific marker levels may be misleading. A standard curve with specific copy numbers of the target cDNA allows a more accurate estimate of mRNA copies in the amount of sample being tested. This approach also allows comparison between markers and specimens to assess inter- and intra-assay variations. Various approaches of analyzing qRT have been reported, most report on the assessment of relative amounts of mRNA based on housekeeping gene reference standards. These have been problematic in that they can mislead in the interpretation of the true mRNA copy levels in a specimen. For example, tumors including melanomas have been documented to have down-regulation or loss of ß2M expression (20 , 36) . This suggests that the use of ß2M as an internal reference marker for quantitative mRNA analysis would be inaccurate and inconsistent particularly for melanomas. Individual gene transcripts are regulated by different mechanisms and have different half-life. In cancer cells, often gene expressions are skewed; thus, using reference housekeeping genes as standards of baseline mRNA levels of the cells can be misleading. Also the fact that housekeeping genes are commonly expressed in normal cells (i.e., endothelial and immune cells), which usually are found in most melanoma tumors, may also skew results.
The MAAs for the study were selected based on their differentiation properties, expression frequency in primary melanomas, and high specificity in the RT-PCR assay. They are frequently found in melanomas and in all melanocytes (22) . The MAAs studied all play a role in the melanogenesis pathway and are not expressed in normal tissues, except some neuroectoderm-derived tissues (22 , 37) . All three of the MAAs were expressed in 100% of melanoma cell lines; however, the mRNA copy number for individual MAAs varied in individual cell lines. Copy numbers were higher in melanoma cell lines than in melanoma tissues, as expected. The observation of cell lines expressing a higher frequency of tumor-associated antigens than tumor-tissue specimens is not uncommon (38 , 39) . Culture conditions often accentuate tumor marker expression through selective growth advantages and clonal selection. Moreover, tumor cell lines in in vitro culture often represent a more clonal population than tumor cells of tissue specimens (22 , 33 , 40) . Established cell lines are not always representative of the in vivo conditions and can give false interpretations of gene expression. Absence of individual MAA was apparent in some melanoma lesions.
Often, as tumors progressively evolve, the tumor cells become more aggressive and less differentiated toward a phenotype similar to their fetal cell origin. This is particularly observed in malignant tumors that are allowed sufficient time to evolve before the host expires. The frequency of TYR, MART-1, and TRP-2 mRNA detection in metastatic melanoma specimens was found to be 83, 71, and 89%, respectively. These results coincided with our previous qualitative studies using the RT-PCR plus Southern blot assay (22) . There was a significant correlation between the expression of individual MAA in the melanoma specimens. The mechanism or pathway underlying the correlation has not been identified. Previous studies on melanoma have indicated that RT-PCR analysis and IHC do not always coincide (20) . This has been attributable to the technical problems, sensitivity, and specificity of both of the assays in the past. The availability as well as the specificity of antibodies to TYR, TRP-2, and other MAAs for IHC analysis on paraffin-embedded sections has been a problem to date. MAA expression levels and corresponding IHC analyses vary from laboratory to laboratory because of the different techniques and the subjective nature of interpretation of IHC data. Only HMB-45 (anti-GP100) is considered a reliable MAA and verified antibody among laboratories for melanoma diagnosis (23 , 41) . The quantitative approach of using qRT for assessing MAA is far more practical and specific than IHC methodologies. Studies have shown that mRNA levels of MAAs can correlate with T-cell recognition; however, this is dependent on the MAA in question (42) . For this study, we did not assess the MAA GP100 for several reasons. The MAA GP100 is well expressed in melanomas and immunogenic; however, there are problems in specificity in that it can be found in cells other than melanoma (43) . In addition, GP100 is known to have alternative exon splicing and exon-intron transcript splicing variants, thus making it a problem in RT-PCR assays.
Loss of TYR expression and low TRP-2 expression significantly correlated with poor survival. Moreover, the patients who demonstrated loss or low expression of at least two markers had a significantly worse prognosis. IHC studies have reported that the TYR expression level was significantly lower in metastatic melanoma than in its primary tumor (44) . This suggests that MAA loss or down-regulation may be a relatively late event corresponding to tumor progression and dedifferentiation. Moreover, the decrease in MAA expression may represent a progression to a more undifferentiated melanoma. Thirty (86%) of the 35 patients tested had received CANVAXIN therapy. The vaccine consists of three melanoma cell lines, which express TYR, TRP-2, and MART-1. The association between the efficiency of immunotherapy and expression of immunogenic MAA remains unclear. It is likely that melanoma cells with decreased expression or loss of MAA are of a selected phenotype with greater advantage to progress and escape host immunity, thus adversely affecting patients’ overall survival (26 , 45) . These studies suggest that advanced-staged patients with melanomas expressing the three MAAs would benefit from cytoreductive therapy followed by adjuvant active-specific immunotherapy targeting these MAAs. Host-tumor immunity may exert pressures on melanomas, thus leading to deletions and selections of specific tumor cell phenotypes. If only patients who received immunotherapy were assessed (n = 30), there were significantly correlations of all MAAs level of expression including MART-1 with disease outcome. Even after complete resection of disease in these patients, there is a high likelihood of subclinical metastases remaining. Cytoreductive surgery in itself may be an indirect form of immunotherapy intervention, in that removing tumor burden allows the host immune system to be more effective against any minimal residual disease remaining, and it reduces suppressive factors released or induced by large tumor burden (4 , 25) . Although MAA immunity may be present, it is a race between tumor growth and the regulatory effect of the host immune system on the tumor.
Genetic instability may also influence in the shutting down of specific enzyme gene sites associated with the melanogenesis pathway, thus resulting in a more undifferentiated tumor phenotype. Although MAAs may be synthesized, the processing and presentation by the tumor cell may be disrupted by multiple cellular physiological events. Several factors, such as loss of HLA class I/II antigens, disruption of peptide transporter protein pathways, truncation of proteins, rapid degeneration of mRNA transcripts, alternative exon splicing, and defective translation, can all influence MAA expression (20 , 26 , 36 , 45) . All of these events occur at different levels in melanoma cells; thus, it is difficult to assess them collectively and correlate to disease outcomes.
In summary, we used a qRT to demonstrate expression of TYR, MART-1, and TRP-2 mRNA in melanoma cell lines and individual metastatic melanomas. The reduction of specific MAA mRNA copy levels was associated with worse prognosis. The approach we have shown is rapid, requires a small amount of sample, and is repeatable and quantitative. This investigation has provided a potential mechanism of why a subset of AJCC stage IV melanomas patients survive longer than others. This may be an important stratification factor in treatment design and management. Interestingly, many of the stage IV patients lived more than 2 years after being diagnosed. This is contrary to the belief that AJCC stage IV melanoma patients survive less than 2 years after being diagnosed. This study also indicates that a subset of stage IV patients with a particular MAA gene expression may benefit from individualized targeted immunotherapy to maintain disease stability. Additional studies on a larger cohort of patients in a defined trial group will be needed to validate these findings. Studies on biological behavior of the melanoma cells expressing these specific MAAs may allow the development of new strategies in immunotherapy for patients with advanced metastatic melanoma.
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FOOTNOTES |
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1 Supported in part by: NIH National Cancer Institute PO1 Grants CA 29605 Project II, and CA 12528 Project II. 
2 To whom requests for reprints should be addressed, at Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404. E-mail: Hoon{at}jwci.org
3 The abbreviations used are: MAA, melanoma-associated antigen; ß2M, ß2-microglobulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MART-1, melanoma antigen recognized by T cells 1; RT-PCR, reverse transcription-PCR; qRT, quantitative RT-RealTime PCR; TRP-2, TYR-related protein 2; TYR, tyrosinase; IHC, immunohistochemistry; AJCC, American Joint Committee on Cancer; JWCI, John Wayne Cancer Institute.
Received 7/11/02. Accepted 11/12/02.
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H. N. Abrahamsen, B. Sandahl Sorensen, E. Nexo, S. J. Hamilton-Dutoit, J. Larsen, and T. Steiniche 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., February 15, 2005; 11(4): 1425 - 1433. [Abstract] [Full Text] [PDF]
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 K. K. Sra, M. Babb-Tarbox, S. Aboutalebi, P. Rady, G. L. Shipley, D. D. Dao, and S. K. Tyring Molecular Diagnosis of Cutaneous Diseases Arch Dermatol, February 1, 2005; 141(2): 225 - 241. [Abstract] [Full Text] [PDF]
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N. Umetani, H. Takeuchi, A. Fujimoto, M. Shinozaki, A. J. Bilchik, and D. S. B. Hoon Epigenetic Inactivation of ID4 in Colorectal Carcinomas Correlates with Poor Differentiation and Unfavorable Prognosis Clin. Cancer Res., November 15, 2004; 10(22): 7475 - 7483. [Abstract] [Full Text] [PDF]
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 H. N. Abrahamsen, E. Nexo, T. Steiniche, S. J. Hamilton-Dutoit, and B. S. Sorensen 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., August 1, 2004; 6(3): 253 - 259. [Abstract] [Full Text]
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 K.-i. Yasumoto, H. Watabe, J. C. Valencia, T. Kushimoto, T. Kobayashi, E. Appella, and V. J. Hearing Epitope Mapping of the Melanosomal Matrix Protein gp100 (PMEL17): RAPID PROCESSING IN THE ENDOPLASMIC RETICULUM AND GLYCOSYLATION IN THE EARLY GOLGI COMPARTMENT J. Biol. Chem., July 2, 2004; 279(27): 28330 - 28338. [Abstract] [Full Text] [PDF]
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H. Takeuchi, D. L. Morton, C. Kuo, R. R. Turner, D. Elashoff, R. Elashoff, B. Taback, A. Fujimoto, and D. S.B. Hoon Prognostic Significance of Molecular Upstaging of Paraffin-Embedded Sentinel Lymph Nodes in Melanoma Patients J. Clin. Oncol., July 1, 2004; 22(13): 2671 - 2680. [Abstract] [Full Text] [PDF]
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H. Takeuchi, A. Fujimoto, M. Tanaka, T. Yamano, E. Hsueh, and D. S. B. Hoon CCL21 Chemokine Regulates Chemokine Receptor CCR7 Bearing Malignant Melanoma Cells Clin. Cancer Res., April 1, 2004; 10(7): 2351 - 2358. [Abstract] [Full Text] [PDF]
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A. Fujimoto, H. Takeuchi, B. Taback, E. C. Hsueh, D. Elashoff, D. L. Morton, and D. S. B. Hoon Allelic Imbalance of 12q22-23 Associated with APAF-1 Locus Correlates with Poor Disease Outcome in Cutaneous Melanoma Cancer Res., March 15, 2004; 64(6): 2245 - 2250. [Abstract] [Full Text] [PDF]
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H. Watabe, J. C. Valencia, K.-i. Yasumoto, T. Kushimoto, H. Ando, J. Muller, W. D. Vieira, M. Mizoguchi, E. Appella, and V. J. Hearing Regulation of Tyrosinase Processing and Trafficking by Organellar pH and by Proteasome Activity J. Biol. Chem., February 27, 2004; 279(9): 7971 - 7981. [Abstract] [Full Text] [PDF]
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A. J. Miller, J. Du, S. Rowan, C. L. Hershey, H. R. Widlund, and D. E. Fisher Transcriptional Regulation of the Melanoma Prognostic Marker Melastatin (TRPM1) by MITF in Melanocytes and Melanoma Cancer Res., January 15, 2004; 64(2): 509 - 516. [Abstract] [Full Text] [PDF]
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J. Szenajch, B. Jasinski, A. Synowiec, J. Kulik, M. Chomicka, J. Struzyna, Z. Nowecki, P. Rutkowski, W. Ruka, W. Kupsc, et al. Prognostic Value of Multiple Reverse Transcription-PCR Tyrosinase Testing for Circulating Neoplastic Cells in Malignant Melanoma Clin. Chem., September 1, 2003; 49(9): 1450 - 1457. [Abstract] [Full Text] [PDF]
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, standards. RFU, relative fluorescence unit.
