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Journal of Clinical Oncology, Vol 23, No 7 (March 1), 2005: pp. 1473-1482
© 2005 American Society of Clinical Oncology.
DOI: 10.1200/JCO.2005.07.168
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Prognostic Significance of Activated Akt Expression in Melanoma: A Clinicopathologic Study of 292 Cases

Derek L. Dai, Magdalena Martinka, Gang Li

From the Department of Medicine, Division of Dermatology; Department of Pathology, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Address reprint requests to Gang Li, MD, Jack Bell Research Centre, 2660 Oak St, Vancouver, BC V6H 3Z6, Canada; e-mail: gangli{at}interchange.ubc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
PURPOSE: Akt is a serine/threonine kinase that leads to stimulation of cell cycle progression, cell proliferation, and inhibition of apoptosis. To investigate the role of Akt in melanoma pathogenesis, we examined the expression of phospho-Akt (p-Akt; Ser-473) in melanocytic lesions at different stages and analyzed the correlations between the p-Akt expression level and clinicopathologic factors and patient survival.

PATIENTS AND METHODS: We evaluated the p-Akt expression in 12 cases of normal nevi, 58 cases of dysplastic nevi, 170 cases of primary melanomas, and 52 cases of melanoma metastases using tissue microarray and immunohistochemistry.

RESULTS: Strong p-Akt expression was observed in 17%, 43%, 49%, and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma, and melanoma metastases, respectively. Significant differences for p-Akt staining pattern were observed between normal nevi and primary melanomas (P < .05), and between primary melanomas and melanoma metastases (P < .001). Furthermore, our Kaplan-Meier survival curves showed that strong p-Akt expression is inversely correlated with both overall and disease-specific 5-year survival of patients with primary melanoma (P < .05 for both). Strikingly, our multivariate Cox regression analysis revealed that p-Akt is an independent prognostic factor in low-risk melanomas (thickness ≤ 1.5 mm; relative risk, 6.44; 95% CI, 1.28 to 32.55; P = .018).

CONCLUSION: The expression of p-Akt increases dramatically with melanoma invasion and progression and is inversely correlated with patient survival. In addition, p-Akt may serve as an independent prognostic marker and help to identify those patients with low-risk melanomas who are at increased risk of death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
The incidence of cutaneous malignant melanoma is increasing more rapidly than that of any other tumors.1 It is estimated that the lifetime risk for melanoma has increased by 15-fold in the past 60 years, with 54,200 new cases expected to be diagnosed in 2003 in the United States.2–4 Malignant melanoma is associated with one of the highest mortality rates, particularly for advanced disease. Although early melanomas are curable with surgical excision,5 up to 20% of patients will develop metastatic tumors owing to its high capability of invasion and rapid metastasis to other organs.6 Patients with metastatic melanoma have a poor prognosis, with a median survival of only 6 to 10 months.7

The main obstacle in treating melanoma is its resistance to conventional chemotherapy, with an overall response rate of less than 20%.8,9 Although the molecular mechanism for drug resistance in melanoma is still poorly understood, it seems that the low therapeutic efficacy in this disease is likely due to a relative inability to induce apoptosis.10 However, unlike other tumor types, melanoma displays a very low mutation rate of the p53 gene,11–13 suggesting that dysregulation of other pathways rather than p53 apoptosis pathway may contribute to distorted apoptosis of melanoma cells, and thus, disease progression.

Recently, growing attention has been focused on the oncogene Akt (also known as protein kinase B [PKB]), the cellular homolog of the retroviral oncogene v-Akt.14–16 To date, three isoforms of Akt (Akt 1, 2, and 3) have been identified that are closely related to each other, sharing up to 80% of amino acid homology.17–20 Akt is a serine-threonine kinase that is partially activated through phosphorylation of Thr-308 and that reaches its maximum activity after phosphorylation of Ser-473 in tandem with that of Thr-308.21–24 As a key effector of phosphatidylinositol 3-kinase (PI3K) signaling pathway, Akt has been shown to play a critical role in controlling the balance between cell survival and apoptosis.25 Upon activation, Akt delivers antiapoptotic signals by phosphorylating Bad and procaspase-9,26 as well as the Forkhead family of transcription factors such as AFX, FKHR, and FKHRL1, which in turn induce the expression of proapoptotic factors.27–31 In addition, Akt can promote cell survival by indirectly activating the prosurvival transcription factor NF-{kappa}B through the phosphorylation of I-{kappa}B kinase.32,33 Besides its antiapoptotic function, Akt also acts to promote cell proliferation and growth34–38 and angiogenesis.39,40

To date, Akt overexpression or activation has been shown to be correlated with poor prognosis in several tumor types, including gastric carcinomas, hepatocellular carcinoma, leukemia, breast cancer, and pancreatic cancer.41–45 To investigate the role of Akt activity in melanoma progression, we used tissue microarray (TMA) technology and immunohistochemistry to evaluate the Akt activity in different stages of human melanocytic lesions. Our data demonstrated that increased phospho-Akt (p-Akt) expression is significantly associated with melanoma progression and a worse patient survival. Furthermore, we found that p-Akt is an independent prognostic marker in low-risk melanomas (thickness ≤ 1.5 mm).


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
TMA Construction
Formalin-fixed, paraffin-embedded tissues from 16 human normal nevi, 66 dysplastic nevi, 204 primary melanomas, and 58 metastatic melanomas were used for our present study. All specimens were obtained from the 1990 to 1998 archives of the Department of Pathology, Vancouver General Hospital. The use of human skin tissues in this study was approved by the medical ethical committee of the University of British Columbia and was performed in accordance with the Declaration of Helsinki guidelines. The most representative tumor area was carefully selected and marked on the hematoxylin and eosin-stained slide. The TMAs were assembled using a tissue-array instrument (Beecher Instruments, Silver Spring, MD). Duplicate 0.6-mm-thick tissue cores were taken from each biopsy specimen. Three composite high-density TMA blocks containing 107, 126, and 111 cases from a total of 344 patients were designed. Multiple 4-µm sections were cut with a Leica microtome (Leica Microsystems Inc, Bannockburn, IL) and transferred to adhesive-coated slides. One section from each TMA was routinely stained with hematoxylin and eosin. The remaining sections were stored at room temperature for immunohistochemical staining.

Immunohistochemistry of TMA
The TMA slides were dewaxed by heating at 55°C for 30 minutes and by three washes, 5 minutes each, with xylene. Tissues were rehydrated by a series of 5-minute washes in 100%, 95%, and 80% ethanol, and distilled water. Antigen retrieval was performed by heating the samples at 95°C for 30 minutes in 10 mmol/L sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 minutes. After blocking with universal blocking serum (DAKO Diagnostics, Mississauga, Ontario, Canada) for 30 minutes, the samples were incubated with a polyclonal rabbit antiphospho-Ser-473 of Akt antibody (1:100 dilution; Cell Signaling Technology, Beverly, MA) at 4°C overnight. The sections were then incubated with biotin-labeled secondary antibody and streptavidin-peroxidase for 30 minutes each (DAKO Diagnostics). The samples were developed with 3,3'-diaminobenzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) and counterstained with hematoxylin. Then, the slides were dehydrated following a standard procedure, and sealed with coverslips. Negative controls were included by omitting p-Akt antibody during the primary antibody incubation.

Evaluation of Immunostaining
The p-Akt staining in TMAs was examined blinded by two, independent observers (including one dermatopathologist) simultaneously, and a consensus score was reached for each core. The positive reaction of p-Akt was scored into four grades according to the intensity of the staining: 0, 1+, 2+, and 3+. The percentages of p-Akt-positive cells were also scored into four categories: 0 (0%), 1 (1% to 33%), 2 (34% to 66%), and 3 (67% to 100%). In the cases with a discrepancy between duplicated cores, the higher score from the two tissue cores was taken as the final score. The sum of the intensity and percentage scores is used as the final staining score. The staining pattern of the biopsies was defined as follows: 0, negative; 1 to 2, weak; 3 to 4, moderate; 5 to 6, strong.

Statistical Analysis of TMA
Statistical analysis was performed with the SPSS 11.5 software (SPSS, Chicago, IL). The {chi}2 test was used to compare the quantitative differences of p-Akt staining in different stages of melanoma progression. The association between p-Akt staining and the clinicopathologic parameters of the primary melanoma patients, including age, sex, tumor thickness, ulceration, histological subtype, location, and American Joint Committee on Cancer (AJCC) stage, was also evaluated by {chi}2 test. The Kaplan-Meier method and log-rank test were used to evaluate the correlations between p-Akt expression and patient survival. Cox regression model was used for multivariate analysis. A P value of less than .05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Clinicopathologic Features of TMAs
Due to loss of biopsy cores or insufficient tumor cells present in the cores, 12 cases of normal nevi, 58 cases of dysplastic nevi, 170 cases of primary melanomas, and 52 cases of melanoma metastases could be evaluated for p-Akt staining. For the 170 primary melanoma cases, there were 95 men and 75 women, with age ranging from 21 to 93 years (median, 57 years). For melanoma staging, we used Breslow thickness and AJCC stages as our criteria for evaluating p-Akt expression: 107 tumors were ≤ 1.5 mm thick (low-risk melanoma), and 63 were larger than 1.5 mm (high-risk melanoma); 112 tumors were at AJCC stage I, 49 were at stage II, and nine were at stage III. Thirty-three melanomas were located in sun-exposed sites (head and neck), and 137 were located in sun-protected sites (other locations). Ulceration was observed in 28 cases (Table 1).


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  		Table 1. Clinicopathologic Characteristics of 170 Primary Melanomas

 
Increased p-Akt Expression Correlates With Melanoma Progression
Various levels of p-Akt staining were observed in nevi and melanoma biopsies (Fig 1). Strong p-Akt staining was recorded in 17%, 43%, 49%, and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma, and melanoma metastases, respectively (Fig 2). Significant differences for p-Akt staining pattern were observed between normal nevi and primary melanomas (P = .028, {chi}2 test) and between primary melanomas and melanoma metastases (P < .001, {chi}2 test; Fig 2). However, there is no significant difference in p-Akt staining between normal nevi and dysplastic nevi (P = .087, {chi}2 test) or between dysplastic nevi and primary melanoma (P = .406, {chi}2 test; Fig 2).



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  		Fig 1. Representative images of phospho-Akt (p-Akt) immunohistochemical staining in human melanocytic lesions. (A and E) Normal nevus with negative p-Akt expression. (B and F) Dysplastic nevus with moderate p-Akt expression. (C and G) Primary melanoma with moderate p-Akt expression. (D and H) Melanoma metastasis with strong p-Akt expression. Magnification x150 for A, B, C, and D; x400 for E, F, G, and H.

 


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  		Fig 2. Phospho-Akt (p-Akt) expression in normal nevi (NN), dysplastic nevi (DN), primary melanomas (PM), and melanoma metastases (MM). There is significantly more p-Akt expression in melanoma metastases compared with primary melanomas (P < .001, {chi}2 test) and more p-Akt expression in primary melanomas compared with normal nevi (P < .05, {chi}2 test). Neg., negative.

 
p-Akt Expression in Melanoma and Clinicopathologic Parameters
To assess whether Akt phosphorylation correlates with clinicopathologic parameters of the patients, we examined the expression of p-Akt (Ser-473) in 170 primary melanomas at various stages of invasion. First, we analyzed the expression level of p-Akt in tumors with different thickness and AJCC stages, as tumor thickness and AJCC stages are well-known prognostic markers for patients with primary melanoma. We found that strong p-Akt expression significantly correlates with advanced stages of primary melanoma. As shown in Figure 3A, while strong p-Akt expression was detected in 60% of high-risk melanomas (Breslow thickness > 1.5 mm), only 43% of low-risk melanomas (Breslow thickness ≤ 1.5 mm) showed strong p-Akt expression (P = .029, {chi}2 test). For AJCC stages, strong p-Akt expression was found in 57% and 59% of AJCC stage II and III melanomas, respectively, compared with only 36% in stage I melanomas (Fig 3B; P = .040, {chi}2 test). There is no significant difference between p-Akt expression in stage II or III melanomas (P = .901, {chi}2 test). Interestingly, we also found that strong p-Akt staining was detected in 62% of male patients compared with only 33% of female patients who had strong p-Akt expression (Fig 4; P < .001, {chi}2 test). No correlation was found between p-Akt expression and patient age, tumor subtype, location, or ulceration status of tumors (data not shown).



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  		Fig 3. Correlation between phospho-Akt (p-Akt) expression and tumor invasion in primary melanomas. (A) Tumors thicker than 1.5 mm have a significantly higher percentage of strong p-Akt expression compared with tumors ≤ 1.5 mm thick (P < .05, {chi}2 test). (B) Tumors in American Joint Committee on Cancer stage II or III have a significantly higher percentage of strong p-Akt expression compared with tumors in stage I (P < .05, {chi}2 test). Neg., negative.

 


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  		Fig 4. Correlation between phospho-Akt (p-Akt) expression and sex of the patients in primary melanomas. Male patients showed a significantly higher percentage of strong p-Akt expression than female patients (P < .001, {chi}2 test). Neg., negative.

 
Survival Analysis
To evaluate whether strong p-Akt expression in human primary melanomas correlates with a worse prognosis, Kaplan-Meier survival curves were constructed using overall or disease-specific 5-year survival to evaluate the biopsies stained negative to moderate versus those stained for strong p-Akt expression. Our data revealed that strong p-Akt expression in primary melanoma tissue is inversely correlated with both overall and disease-specific 5-year survival (P = .049 and P = 0.014, respectively, log-rank test; Fig 5). When the primary melanoma lesions were further divided into two subgroups according to tumor thickness, we found that strong p-Akt expression is significantly correlated with poor patient overall survival in low-risk melanoma patients (≤ 1.5 mm; P = .037, log-rank test; Fig 6A). Strong p-Akt staining did not significantly correlate with disease-specific patient survival in this subgroup (P = .181, log-rank test). This may be due to the fact that a very small number of disease-specific deaths occurred in the low-risk melanoma patients within 5 years: only one death in the negative to moderate p-Akt group and three deaths in strong p-Akt group. Nevertheless, there is still a trend toward a strong p-Akt correlation with poor, disease-specific patient survival (Fig 6B). However, in the high-risk melanoma group (> 1.5 mm), p-Akt expression did not significantly correlate with overall and disease-specific patient survival (P = .929 and P = .239, respectively, log-rank test; Fig 6C and D).



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  		Fig 5. Correlation between phospho-Akt (p-Akt) expression and 5-year survival of primary melanoma patients. Patients with strong p-Akt expression have a significantly worse 5-year survival than those with negative to moderate p-Akt expression. (A) Overall 5-year survival (P < .05, log-rank test). (B) Disease-specific 5-year survival (P < .05, log-rank test). Cum., cumulative.

 


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  		Fig 6. Prognostic value of phospho-Akt (p-Akt) expression in subgroups of primary melanoma patients by log-rank test. Strong p-Akt expression only correlates with a worse patient 5-year survival in thin melanomas (thickness ≤ 1.5 mm). (A) Overall 5-year survival of patients with thin melanomas (≤ 1.5 mm; P < .05). (B) Disease-specific 5-year survival of patients with thin melanomas (P = .181). (C) Overall 5-year survival of patients with thick (> 1.5 mm) melanomas (P = .930). (D) Disease-specific 5-year survival of patients with thick melanomas (P = .239). Cum., cumulative.

 
Next, we examined whether strong p-Akt expression is an independent prognostic marker for melanoma. We performed a multivariate analysis including p-Akt expression, age, sex, thickness, ulceration, and location of the tumors for 170 primary melanomas. Our results indicate that p-Akt expression reached a borderline significance for predicting the patient outcome independently of other clinicopathologic parameters for both overall and disease-specific survival (P = .054 and P = .071, respectively; Table 2). Tumor thickness, location, and ulceration were the most significant prognostic markers for disease-specific survival (Table 2). We then performed a multivariate analysis including p-Akt expression, age, sex, tumor location, and AJCC stages. Our results indicated that strong p-Akt expression is an independent prognostic marker for both overall and disease-specific patient survival (P = .040 and P = .036, respectively; Table 3). However, AJCC stage and tumor location were more significant prognostic markers for disease-specific survival (Table 3). Since our Kaplan-Meier survival curve indicated that p-Akt expression correlates with a poor overall 5-year survival in 107 cases of low-risk melanomas (Fig 6A), we performed a further multivariate analysis including p-Akt, thickness, ulceration, age, sex, and location of the tumors in the low-risk melanoma group. Strikingly, our data showed that in thin tumors, strong p-Akt expression (P = .018), but not tumor thickness, was an independent prognostic factor of poor 5-year overall survival (Table 4). When we performed a multivariate analysis in high-risk melanomas (> 1.5 mm), we did not find p-Akt to be an independent prognostic marker (data not shown). Thus, p-Akt seems to represent a new independent prognostic marker for low-risk melanomas.


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  		Table 2. Multivariate Cox Regression Analysis of p-Akt and Tumor Thickness/Ulceration on 5-Year Patient Survival in 170 Primary Melanomas

 

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  		Table 3. Multivariate Cox Regression Analysis of p-Akt and American Joint Committee on Cancer Stages on 5-Year Patient Survival in 170 Primary Melanomas

 

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  		Table 4. Multivariate Cox Regression Analysis Based on 5-year Overall Survival in 107 Cases of Low-Risk (≤ 1.5 mm) Primary Melanomas

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
Authors' Disclosures of...
 REFERENCES
 
The role of Akt in tumorigenesis has been studied extensively in recent years. Elevated Akt activity has been observed in numerous types of human cancers.46 In this study, to better understand the role of Akt activity in melanoma progression, we used TMA technology and immunohistochemistry to investigate Akt activity in 292 cases of pigmented skin lesions at different stages using an antibody specific for phosphorylated Akt at Ser-473. Our results demonstrated that p-Akt expression increases as melanoma progresses (Fig 2). Furthermore, strong p-Akt expression is significantly correlated with tumor invasion (Fig 3) and is inversely correlated with patient 5-year survival in primary melanomas, especially for low-risk tumors (Figs 5 and 6 and Table 4). To our knowledge, this is the first study to analyze the phosphorylation of Akt in primary melanoma tumors, and its association with tumor progression and patient survival.

Our result revealing a significant correlation between increased p-Akt expression and melanoma progression is consistent with the findings by Dhawan et al47 who showed an increased phospho-Akt expression in more advanced melanocytic lesions. However, the authors in that study only included 16 cases of nevi, one case of lentigo maligna, and 12 cases of metastatic melanoma. Since no primary melanomas were included in their study, it is impossible to draw conclusions on the role of activated Akt in melanoma progression. In order to study the role of Akt in melanoma pathogenesis, we selected 292 cases of melanocytic lesions at different stages: normal nevi, dysplastic nevi, primary melanomas, and metastatic melanomas. A linear trend of increased Akt phosphorylation was observed following progression of melanocytic lesions, and significant differences were recorded between normal nevi and primary melanomas, and between primary and metastatic melanomas (Fig 2). These stage-specific expression patterns suggest that increased Akt activity might be a common requirement for the transformation from benign neoplasia to malignancies, as well as from primary tumors to metastatic disease in melanoma. Dysplastic nevus is often considered to represent intermediate steps in melanoma tumorigenesis and may share some of the genetic alterations with primary melanoma.48 In our study, there is a clear trend that more p-Akt is expressed in dysplastic nevi compared with normal nevi (43% v 17%; P = .087, {chi}2 test). This difference did not reach significance probably due to the small number of normal nevi cases. The increased p-Akt staining in dysplastic nevi as compared with normal nevi is not due to TMA sampling. Sixty-two percent of our dysplastic nevi samples contained both junctional and dermal components. Eighty-three percent of these samples showed identical p-Akt levels between two components, whereas only 11% of cases showed higher p-Akt expression in the junctional component, and 6% cases showed higher p-Akt expression in the dermal component. On the other hand, the p-Akt expression pattern in dysplastic nevi was similar to primary melanomas (43% v 49%, P = .406; {chi}2 test; Fig 2), suggesting that Akt activation is a very early event in melanoma tumorigenesis.

The correlation between strong p-Akt expression and tumor invasion (Fig 3) and a poorer 5-year patient survival (Figs 5 and 6 and Table 4) are concordant with the previous studies describing elevated Akt signaling in melanoma. We previously showed that the expression of integrin-linked kinase, a kinase that plays an important role in mediating Akt phosphorylation at Ser-473, was correlated with tumor invasion in primary melanomas.49 As a key player in PI3K cell survival pathway, activated Akt modulates the function of numerous substrates involved in the regulation of cell cycle progression and cell survival. In melanoma, constitutively activated Akt has been shown to lead to upregulation of NF{kappa}B.47 As the negative regulator of Akt pathway, PTEN expression was reduced in melanoma biopsies, and loss of PTEN can promote melanoma tumor growth in vivo.50,51 Moreover, blocking Akt activity can inhibit cell proliferation and reduces the sensitivity of melanoma cells to apoptosis both in vitro and in vivo.52–54 These pieces of evidence point to the key role of Akt in melanoma tumorigenesis.

Interestingly, there was a higher percentage of strong p-Akt expression in male patients rather than female patients (Fig 4). Despite the different p-Akt expression patterns observed between the sexes, our Kaplan-Meier survival curve showed that strong p-Akt expression has a similar effect on 5-year disease-specific survival in male and female groups (data not shown). One possible explanation for higher p-Akt expression in male patients is that male patients may have thicker tumors at diagnosis than female patients, as shown by many studies.55–57 However, in our study, there is no statistical difference on average tumor thickness between sexes (average thickness: 2.0 mm for male patients and 1.8 mm for female patients; P = .453, t test). Also, when we compared the number of tumors ≤ 1.5 mm or thicker than 1.5 mm, there was no difference between male and female groups (P = .566, {chi}2 test). Another possible reason for higher p-Akt expression in men is that sex-related hormones may contribute to PI3K signaling. It has been reported that both androgen and estrogen have the ability to activate PI3K through interaction between their receptors and the p85{alpha} subunit of PI3K,58,59 thus leading to the activation of Akt. However, it seems that the differential expression of p-Akt between the sexes is tissue-specific. For example, it has been shown that in male rats, Akt activity is higher in the lacrimal gland, but lower in neuronal cells derived from cortical plate compared with female rats.60,61 The mechanisms of higher p-Akt expression in male melanoma patients and its clinical significance remain to be determined.

The data presented in this report demonstrated that phospho-Akt expression is significantly increased with progression of human melanoma. Most strikingly, our data indicate that increased p-Akt expression correlates with a worse 5-year survival of primary melanoma patients and is an independent prognostic factor for low-risk melanomas (thickness ≤ 1.5 mm). These data, coupled with a number of functional studies demonstrating an essential role of Akt activity in melanomagenesis,53,62 imply that Akt may serve as a promising prognostic marker and therapeutic target for malignant melanoma.


   Authors' Disclosures of Potential Conflicts of Interest
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
REFERENCES
 
The authors indicated no potential conflicts of interest.


   Acknowledgment
 
We gratefully thank David Huntsman, Nikita Makretsov, and Hamid Masoudi for the technical assistance in tissue microarray construction. Dr Gang Li is a Research Scientist of the National Cancer Institute of Canada supported with funds by the Canadian Cancer Society.


   NOTES
 
Supported by the National Cancer Institute of Canada.

Authors' disclosures of potential conflicts of interest are found at the end of this article.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
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Submitted July 28, 2004; accepted December 13, 2004.




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