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Melastatin Expression and Prognosis in Cutaneous Malignant Melanoma

From the Dermatopathology Unit and Departments of Pathology and Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston; and Millennium Predictive Medicine, Inc, Cambridge, MA.

Address reprint requests to Lyn M. Duncan, MD, Dermatopathology Unit, WRN827, Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114; email duncan{at}helix.mgh.harvard.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Melastatin (MLSN-1), a novel melanocyte-specific gene recently identified using a genomic approach, is expressed in murine and human melanoma cells at levels inversely proportional to metastatic rates in vivo. We studied the relationship between expression of melastatin mRNA in the primary cutaneous tumor and prognosis in patients with localized malignant melanoma.

PATIENTS AND METHODS: Melastatin mRNA was evaluated by in situ hybridization in primary cutaneous melanoma from 150 patients with localized disease (American Joint Committee on Cancer [AJCC] stage I and II). Multivariate Cox proportional hazards regression analysis was performed to assess the prognostic utility of melastatin mRNA expression while adjusting for other prognostic indicators.

RESULTS: Uniform melastatin mRNA expression in the primary tumor was correlated with prolonged disease-free survival (P < .0001). Multivariate analysis revealed that melastatin status, mitotic rate, and tumor thickness influence disease-free survival independently. The 8-year disease-free survival rate in AJCC stage I patients whose tumors diffusely expressed melastatin mRNA was 100%, whereas in stage I patients with melastatin loss, the disease-free survival rate was 77% ± 15% (median ± SE). In patients with stage II disease whose tumors diffusely expressed melastatin mRNA, the 8-year disease-free survival rate was 90% ± 7%, whereas in patients with melastatin loss, the disease-free survival rate was 51% ± 8%.

CONCLUSION: Downregulation of melastatin mRNA in the primary cutaneous tumor is a prognostic marker for metastasis in patients with localized malignant melanoma and is independent of tumor thickness and other variables. Used in combination, melastatin status and tumor thickness allow for the identification of subgroups of patients at high and low risk of developing metastatic disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE INCIDENCE OF cutaneous melanoma is increasing more rapidly than any other cancer in the United States. Among white Americans, the lifetime risk of malignant melanoma is more than one in 75. Approximately 44,000 new patients will be diagnosed in 2000 in the United States.^1,2 Surgical excision of localized primary cutaneous melanoma (American Joint Committee on Cancer [AJCC] stages I and II; approximately 75% of diagnoses) may lead to cure in many patients. The overall 5-year survival rate for these patients is approximately 80%,^3,4 suggesting that approximately 20% of stage I and II patients may have micrometastatic disease at the time of diagnosis. Melanoma is one of the most lethal of the cancers: in 1996 melanoma accounted for approximately 2% of all cancer-related deaths in the United States.⁵ Currently, there is no cure for patients who present with melanoma metastatic to distant sites, and the median survival of these patients is only approximately 6 months.⁶ Identifying patients at high risk of developing metastases is one of the most critical issues in the management of this cancer.

We have taken a genomic approach toward characterizing the progression of melanoma from localized to metastatic disease. Using a modified differential mRNA analysis of murine B16 melanoma sublines with divergent metastatic potential in vivo, a novel gene whose expression correlated with in vivo aggressiveness was discovered. The gene, named melastatin, was uniformly expressed in the indolent B16 F1 cell line, featured reduced expression in cell lines with intermediate metastatic potential, and was essentially absent in the highly aggressive B16 F10 subline.⁷ Subsequent to cloning of the human melastatin gene (MLSN-1), Northern blot analysis demonstrated melastatin expression only in melanocytic cells and the choroid; no expression of melastatin in other tissues was detected. In situ hybridization analysis of human cutaneous melanocytic tissues revealed that melastatin mRNA was uniformly expressed in benign melanocytic nevi. On the other hand, primary cutaneous melanomas showed variable expression of melastatin mRNA. Notably, melastatin status was correlated with the thickness of the primary tumor. Preliminary data also suggested that melastatin expression was inversely associated with human metastatic disease. These findings suggested that melastatin expression might be a prognostic marker in patients with melanoma.

A number of clinical and histologic prognostic indicators of metastatic potential and mortality in melanoma patients have been examined, including tumor thickness, patient age at diagnosis, sex, site of the primary tumor, anatomic level of invasion (Clark level), mitotic rate, presence of ulceration, regression, and distribution of tumor-infiltrating lymphocytes.^8-12 In addition, a number of novel molecular markers have been evaluated for prognostic utility in melanoma patients. Although immunohistochemical evaluation of markers such as HLA, intercellular adhesion molecule–1 (ICAM-1), cathepsins, integrins alpha-2 beta-1 and alpha-v beta-3, metallothionein, and others show some prognostic value, they add little to tumor thickness and other morphologic markers now in routine use.^9,10,13-15

Despite the identification of novel markers, prognosis based on tumor thickness (first described 30 years ago) remains the standard for predicting outcome and determining treatment in patients with primary cutaneous melanoma.¹¹ For patients determined to be at low risk of developing metastasis by virtue of having a relatively thin tumor (1.5 mm), there is no therapeutic or further surgical intervention recommended beyond complete excision of the primary tumor. Patients with thicker tumors undergo sentinel lymph node biopsy and, if nodal disease is detected, patients are offered adjuvant therapy. Although tumor thickness remains the primary prognostic factor used in making clinical management decisions, there is room for improvement: some patients with very thin tumors go on to die of melanoma, whereas others with deeply invasive thick melanomas may experience long-term disease-free survival.

To determine the clinical significance of melastatin mRNA expression, we analyzed primary cutaneous melanomas from a cohort of patients who presented with AJCC stage I and II (localized) disease. Because our initial study focused on the correlation between melastatin expression and melanoma-free survival, we chose a cohort of patients with the potential for long-term clinical follow-up. This initial cohort predated the routine use of sentinel lymph node mapping, a surrogate marker for poor outcome in melanoma patients. In this study, melastatin distribution was compared with other known prognostic factors to determine the utility of melastatin in predicting metastasis-free survival in patients with at least 4 years of follow-up.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Population
Patients were identified through review of the surgical pathology files of the James Homer Wright Pathology Laboratories at the Massachusetts General Hospital and the records of the Massachusetts General Hospital Cancer Registry for the years 1988 to 1994. Tissue blocks of formalin-fixed paraffin-embedded primary cutaneous tumors were obtained from patients with AJCC stage I or stage II melanoma. Thus all patients were diagnosed with localized cutaneous melanoma at the time of diagnosis. Six patients received elective lymph node dissection at the time of dissection, and one patient underwent sentinel lymph node sampling. The results of these procedures revealed no evidence of lymph node metastasis. Clinical follow-up was acquired from the patients’ physicians and through review of medical records. Four hundred ten AJCC stage I or II patients were identified. Primary tumor blocks were not available for 203 patients (most diagnosed at outside institutions). Tumor blocks from 19 additional patients were found to be depleted of invasive tumor. Three patients were excluded because they had more than one primary melanoma. Five patients’ tumors had lack of RNA integrity in the specimen. Two cases were excluded because of technical inadequacy of the in situ hybridization assay. The final analyses were limited to the 150 remaining patients who had at least 4 years of follow-up. The diagnosis of melanoma for all 150 cases was confirmed at the Massachusetts General Hospital at the time of original diagnosis and reconfirmed by one of the authors (L.M.D.) at the time of this study. This protocol was approved by the Institutional Review Board Subcommittee on Human Studies at the Massachusetts General Hospital (no. 98-7024).

In Situ Hybridization
Four micron–thick sections of formalin-fixed paraffin-embedded tissues were deparaffinized, rehydrated, and postfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes. After washing with PBS, the tissue sections were digested with 2 µg/mL proteinase K at 37°C for 15 minutes and again incubated with 4% paraformaldehyde/PBS for 10 minutes. The tissue sections were then washed with PBS and subsequently incubated with 0.2 N HCl for 10 minutes, 0.25% acetic anhydride/1 mol/L triethanolamine for 10 minutes, and dehydrated with graded ethanols. Hybridizations were performed with single-stranded 35S-radiolabeled (5 x 10⁷ cpm/mL) cRNA probes encoding a 1.9-kB segment of the coding region of the human cDNA (accession no. AF071787)⁷ or a 1-kB segment of the coding region of the human H4 histone gene, in the presence of 50% formamide, 10% dextran sulfate, 1x Denhardt’s solution, 600 mmol/L NaCl, 10 mmol/L DTT, 0.25% sodium dodecyl sulfate and 100 µg/mL tRNA for 18 hours at 55°C. After hybridization, slides were washed with 5x standard saline citrate (SSC) at 55°C for 30 minutes, 50% formamide/2x SSC at 55°C for 30 minutes, 10 mmol/L TrisHCl (pH 7.6)/500 mmol/L NaCl/1 mmol/L EDTA (TNE) at 37°C for 10 minutes, incubated in 10 µg/mL RNase A in TNE at 37°C for 30 minutes, washed in TNE at 37°C for 10 minutes, incubated once in 2x SSC at 50°C for 30 minutes, twice in 0.2x SSC at 50°C for 30 minutes, and dehydrated with graded ethanols. Localization of mRNA transcripts was detected by dipping slides in Kodak NTB2 photoemulsion (Eastman Kodak, Rochester, NY) and exposure for 10 to 14 days at 4°C.^16,17 In addition to the positive control probe for H4 histone, sense probes were used as a negative control in representative tissue samples and showed no signal above background levels. Before dehydration and coverslipping of slides, sections were counterstained using Myers hematoxylin and alcoholic eosin Y.

Scoring of Melastatin Assay
Slides were scored as having loss of melastatin if a nest of at least five melanoma cells in a dermal tumor focus showed no expression, determined by presence of only background levels of photoemulsion grains over the cells.¹⁸ The majority of cases showing loss of melastatin exhibited a striking absence of expression in a significantly large and presumably clonal tumor nest. In cases where the entire primary tumor exhibited absence of gene expression, benign melanocytes elsewhere in the section were used as internal positive controls for sample RNA integrity. All cases displayed melastatin mRNA expression in benign melanocytes. Samples with potential compromising assay artifacts were repeated, and those lacking positive benign melanocytes or displaying diffuse dermal loss of melastatin were examined for H4 histone expression to determine RNA integrity. Melastatin mRNA expression for each tumor was scored independently by two readers in the absence of patient outcome information. Discordant scoring results between readers were resolved by joint review.

Statistical Analysis
All analyses were performed using the SAS statistical package (Version 6.12, SAS Institute Inc, Cary, NC). The association of melastatin mRNA status with other covariates was tested with the ² test for categorical data and the Wilcoxon test for continuous data. Logistic regression analysis was used for multivariate analysis of covariates associated with melastatin. The Kaplan-Meier method was performed to examine the influence of melastatin and other covariates on disease-free survival using the SAS LIFETEST procedure. Patients alive with metastatic disease or dead of melanoma-related causes were considered to have experienced metastasis; disease-free interval for such patients was measured as the time from initial diagnosis to first metastasis, or date of death where no date of metastasis was noted before death. Those alive with no evidence of disease were censored. Comparison of survival functions for different strata was assessed with the log-rank statistic. Multivariate Cox proportional hazards analysis was carried out using the SAS PHREG procedure with backwards elimination of covariates.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Situ Hybridization
Normal intraepidermal melanocytes expressed MLSN-1 mRNA, detected in bright-field microscopy as black grains overlying the cells and in dark field as bright white spots (Fig 1). In cases of melanoma with contiguous benign nevi, the benign nevomelanocytic component showed uniform MLSN-1 mRNA expression. MLSN-1 mRNA was detected in the intraepidermal melanoma in all of the tumors tested. In contrast, variable expression of MLSN-1 mRNA was observed in the dermal component of the melanomas. Primary cutaneous tumors in 36% of patients showed uniform MLSN-1 mRNA expression throughout the dermal melanoma (Fig 2), whereas 64% of tumors showed complete absence of signal for MLSN-1 mRNA in a focus of the dermal melanoma (Fig 3).

View larger version (95K): [in this window] [in a new window]   Fig 1. Melastatin-positive normal melanocytes. Solitary intraepidermal melanocytes show a positive signal for MLSN-1 mRNA. (A) bright field, (B) dark field. In situ hybridization using S35-labeled MLSN-1 riboprobe in formalin-fixed paraffin-embedded archival tissue sections, hematoxylin and eosin counterstain.

View larger version (121K): [in this window] [in a new window]   Fig 2. Melastatin-positive invasive cutaneous melanoma. A positive signal for MLSN-1 mRNA is observed in the intraepidermal and dermal melanoma cells. (A,C) Bright field; (B,D) dark field (same fields as A and C, respectively).

View larger version (119K): [in this window] [in a new window]   Fig 3. Invasive cutaneous melanoma with loss of melastatin mRNA expression. (A,B) Melastatin-positive melanoma (B, upper left) with an adjacent focus of melanoma without melastatin signal (B, lower right). (C,D) complete loss of MLSN-1 mRNA expression in invasive melanoma. (A,C) Bright field; (B,D) dark field.

View larger version (12K): [in this window] [in a new window]   Fig 4. Kaplan-Meier analysis of disease-free survival in patients with primary cutaneous malignant melanoma stratified by melastatin status.

View larger version (15K): [in this window] [in a new window]   Fig 5. Kaplan-Meier analysis of disease-free survival in patients with primary cutaneous malignant melanoma stratified by AJCC stage and melastatin status.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

In our results described herein, we find that in situ hybridization analysis of MLSN-1 mRNA in primary cutaneous tumors provides independent prognostic information in patients with stage I and II malignant melanoma. Patients with stage I melanoma have primary tumors less than 1.5 mm thick and Clark levels 3; stage II patients have tumors less than 1.5 mm thick and Clark levels more than 3 or tumors 1.5 mm thick. Neither stage I or II patients have evidence of metastatic melanoma at presentation. Clinicians vary in terms of whether they incorporate other prognostic factors besides AJCC stage into their decision-making analysis. Ulceration, mitotic activity, age, site, and sex are factors that have been used as an adjunct to tumor thickness in the analysis of melanoma prognosis.^27-29 In our study, many of these factors showed prognostic utility for metastatic disease when analyzed individually. When analyzed alone, ulceration is a significant predictor of outcome in our study (P = .03) and is of borderline significance when analyzed in conjunction with tumor thickness as a continuous variable (P = .08). When ulceration is evaluated along with Breslow thickness and mitotic count, however, ulceration falls out as not significant (P = .85). Similarly, when ulceration is studied along with tumor thickness and melastatin status, ulceration is not a significant predictor (P = .24). Of all of the variables studied, melastatin, tumor thickness, and mitotic count were the only factors that remained as independent predictors of disease-free survival in multivariate analyses.

The mitotic count is a continuous variable indicating the mitotic activity of the dermal vertical growth phase component of primary cutaneous melanoma.^8,30,31 Increased mitotic activity in the primary tumor has been demonstrated by others to be associated with a poor prognosis.^8,32 Clark et al⁸ studied the utility of mitotic count in predicting 8-year survival rates of 264 patients with vertical growth phase melanoma. Patients with tumors that contained no mitoses, one to six mitoses per mm², and more than six mitoses per mm² had survival rates of 95%, 79.4%, and 38.2%, respectively, similar to the survival rates found in our population. Physicians vary in their use of mitotic count in pathologic examination of tumors, and the measure is currently not part of the AJCC/UICC staging system for melanoma.

Most patients diagnosed with cutaneous melanoma present with stage I or II disease. These patients have 8-year disease-free survival rates of approximately 91% and 60%, respectively (this study). Although the diagnosis of melanoma in our 150 patients preceded the routine use of sentinel node sampling at our institution, today patients predicted to have a high risk of metastasis may undergo sentinel lymph node analysis, leading to the identification of microscopic melanoma metastases in regional lymph node basins.^33-35 In the United States, many patients with primary cutaneous melanoma more than 1.0 mm in thickness are offered sentinel lymph node mapping, but fewer than 20% of these patients have melanoma cells detected in their sentinel lymph nodes.^36,37 Adjuvant therapy is usually reserved for this subset of patients with histologically confirmed lymph node metastasis.

The poor outcome of patients with metastatic melanoma is a reflection of the existing treatment regimens; none offer an increased survival for patients with advanced stage melanoma. Although chemotherapy and immunotherapy may prolong the interval to recurrence, the high toxicity of these therapies raises questions regarding their utility in the absence of a demonstrated effect on survival.³⁸ Tools that identify a subgroup of stage II patients who are likely to develop metastases may lead to the use of these adjuvant therapies in these patients before the development of documented metastatic disease. Along these lines, there has been a recent trial of interferon alfa-2a adjuvant therapy in patients with stage II melanoma that demonstrates some efficacy.³⁹ However, because of the toxicity of even low-dose interferon therapy, most oncologists reserve interferon for patients with documented melanoma metastases to regional lymph nodes.³⁸ As novel therapies such as melanoma vaccines are developed, markers that identify early-stage patients with a high risk of developing metastases will permit targeting of therapy to these patients. If the data presented herein are replicated in ongoing studies, it may be appropriate for patients with early-stage melanoma and loss of melastatin to be considered for participation in studies of these melanoma vaccines.

Used in conjunction with AJCC stage, melastatin allows for the identification of a large subset of patients (33 of 150, or 22%) who have a 100% chance of surviving at least 8 years. Melastatin may also be used to select patients who may benefit from additional diagnostic modalities. Specifically, for the 43% (25 of 58) of stage I patients in this study with loss of melastatin expression in their tumors, the 8-year survival rate was 77%. These patients may benefit from sentinel lymph node analyses, independent of the thickness of their melanoma. On the other hand, 23% (21 of 92) of the patients with stage II disease in this study showed no loss of melastatin. Similar to stage I patients, these patients had a high likelihood of an 8-year survival (approximately 90%). We are currently evaluating the correlation of melastatin expression with sentinel lymph node analysis. We anticipate that melastatin expression will be positive in patients with negative sentinel lymph node analyses; these patients are expected to have long-term disease-free survival. Unfortunately, long-term clinical follow-up data are not available in retrospective cohorts, and therefore we will be following these patients in a prospective fashion. The remaining 77% of stage II patients who showed loss of melastatin had an 8-year disease-free survival rate of 51%. This subset of stage II patients may benefit from more aggressive therapy. The results of our multivariate analyses suggest that evaluation of melastatin expression adds significant, independent prognostic information. By incorporating an analysis of melastatin expression into clinical practice, physicians and patients may be able to more accurately and cost-effectively target the use of sentinel lymph node mapping studies and other invasive diagnostic modalities.

Although antimelastatin antibodies are in development, none has been demonstrated to be suitable for immunohistochemical analysis. A colorimetric technique for the detection of melastatin mRNA is also being developed. This study did not evaluate the role of other molecular markers for assessing metastatic potential in malignant melanoma. Future studies should explore melastatin together with other immunohistochemical and molecular markers to determine whether there is added prognostic utility in the combined marker set for predicting metastatic potential. Similarly, studies that examine melastatin status as a predictor of sentinel lymph node positivity may provide valuable information to help direct lymph node biopsy. If found to be a useful predictor, melastatin could be used to identify the small proportion of patients found on sentinel lymph node biopsy to have micrometastatic disease,^36,37 avoiding unnecessary procedures in the remaining patients.

	ACKNOWLEDGMENTS

 
We thank Laura Carleu, Jennifer Morgan, Patricia Della Pelle, and Maria Forcellati for data and specimen collection and preparation. We also thank the many individuals who provided critical feedback on the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted June 19, 2000; accepted August 22, 2000.

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