The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker

Journal of Dermatological Science 61 (2011) 187–193

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The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker Hisashi Kanemaru, Satoshi Fukushima *, Junji Yamashita, Noritoshi Honda, Rie Oyama, Asako Kakimoto, Shinichi Masuguchi, Tsuyoshi Ishihara, Yuji Inoue, Masatoshi Jinnin, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 October 2010 Received in revised form 27 December 2010 Accepted 28 December 2010

Background: MicroRNA-221 (miR-221) is known to be abnormally expressed in malignant melanoma (MM) cells, and it favors the induction of the malignant phenotype through down-modulation of p27Kip1/CDKN1B and the c-KIT receptor. This suggests that the serum level of miR-221 might increase in patients with MM and thus could be used as a new tumor marker. Objective: To evaluate the possibility that the serum miR-221 level can be a marker of MM. Methods: Serum samples were obtained from 94 MM patients and 20 healthy controls. MicroRNAs were purified from serum, and miR-221 levels were measured by quantitative real-time polymerase chain reaction. Results: Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I–IV MM compared to those with MM in situ, and the levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. Conclusions: Serum levels of miR-221 were significantly increased in MM patients and may be useful not only for the diagnosis of MM, but also for the differentiating MM in situ from stage I–IV MM, and for evaluating tumor progression and monitoring patients during the follow-up period. In addition, considering that the serum levels of miR-221 were correlated with tumor thickness, miR-221 might also be useful as a prognostic marker for patients with MM. ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: MicroRNA-221 Melanoma Tumor marker Serum

1. Introduction Malignant melanoma (MM) is an aggressive neoplasm that can be fatal, especially at the metastatic stage. The incidence of MM has been increasing during the last decade [1], necessitating early diagnosis and better diagnostic tools. Since the serum is commonly used in clinical tests and can be well preserved to some extent, serum markers may therefore be useful for making a diagnosis. However, the serum levels of 5-S-cysteinyl-DOPA (5-S-CD), which is a common tumor marker for MM, sometimes remain within the normal limit, especially in early stages, so it is inadequate to use 5S-CD for the early detection of MM [2,3]. Therefore, the development of novel biomarkers of this tumor is urgently needed. Growing evidence has indicated the capability of using microRNAs (miRNAs) expression profiles to clearly distinguish between normal and neoplastic tissues, leading to the identification of new diagnostic and/or prognostic markers. MiRNAs are non-coding short

* Corresponding author. Tel.: +81 96 373 5233. E-mail address: [email protected] (S. Fukushima).

ribonucleic acid molecules an average of 22 nucleotides long, that are post-transcriptional regulators that bind to complementary sequences in the three prime untranslated regions (30 UTRs) of mRNAs, leading to gene silencing. They have been recently reported to play an important role in major cellular processes, including cancer development and progression (e.g., proliferation, differentiation, apoptosis, and angiogenesis) [4,5]. In addition, it has been shown that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity [6]. It has been suggested that measurement of tumor-derived miRNAs in serum or plasma may be an important approach for the blood-based detection of human cancer [7]. Thus, circulating miRNAs have the potential to serve as non-invasive diagnostic biomarkers for various cancers [8]. MiR-221, encoded on the X chromosome, is one of these miRNAs. It has been reported that miR-221 is overexpressed in glioblastoma [9,10], pancreatic cancer [11,12], papillary thyroid carcinoma [13], gastric cancer, colorectal carcinoma [14], hepatocellular carcinoma [8], and prostate carcinoma [15]. It was also reported that the miR-221 pathway controls the progression of melanoma [16,17] by down-regulating the c-Kit receptor and

0923-1811/$36.00 ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2010.12.010

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H. Kanemaru et al. / Journal of Dermatological Science 61 (2011) 187–193

p27Kip1/CDKN1B, leading to the blockage of differentiation and enhanced proliferation of the melanoma cells, respectively [15,18]. Furthermore, miR-221 is almost undetectable in normal human melanocytes, and is increasingly expressed throughout a stepwise MM transformation process [19]. Therefore, miR-221 may play a major role in the tumorigenesis of MM [19,20]. These reports inspired us to investigate whether melanoma patients have significantly higher levels of serum miR-221 compared to healthy controls, and whether the miRNA could be used as a new tumor marker of MM. In this study, we investigated the serum levels of miR-221 in MM patients and evaluated the possibility that serum levels of miR-221 can be a useful marker for the diagnosis, staging and monitoring of MM.

of gene expression between miR-221 and cel-miR-54 were calculated by the standard curve method.

2. Patients and methods

3.1. miR-221 was present in the serum

2.1. Clinical assessment and patients

There have not been any reports demonstrating the expression of miR-221 in cell-free body fluid. To validate that the miRNA is indeed detectable in human serum, miRNA was extracted from the sera of healthy individuals, and the level of miR-221 was determined by quantitative real-time PCR using a primer set specific for miR-221. The amplification of miR-221 was observed, and Ct values were increased by the serial dilution of the miRNA by as is, 10-fold and 100-fold dilutions (29.2, 32.4 and 36.5, respectively) (Fig. 1). There was a good linearity between the log of sample input and Ct values (y = 3.69x + 28.997, R = 0.9997). Therefore, miR-221 was thought to be detectable and quantifiable in the serum using our method.

Serum samples were obtained from 90 patients with MM (45 males and 45 females; age range, 22–91 years). Eighteen patients had nodular melanoma (NM), 13 had superficial spreading melanoma (SSM), 35 had acral lentiginous melanoma (ALM), 11 had lentigo maligna melanoma (LMM), and 13 had mucosal melanoma. Additionally, we investigated the serum samples obtained from 8 patients who had experienced excisions of primary melanoma, but had later experienced a recurrence. The profiles of all 90 MM patients are shown in Table 1, and those of the eight patients with postoperative recurrence are shown in Table 2. The clinical and laboratory data reported in this study were obtained at the time of serum sampling. Control serum samples were also collected from healthy age- and sex-matched volunteers. Institutional review board approval and written informed consent were obtained before patients and healthy volunteers were entered into this study, according to the Declaration of Helsinki. All serum samples were stored at 80 8C prior to use. 2.2. miRNA extraction MiRNA isolation from serum samples was performed with the miRNeasy RNA isolation kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions with minor modifications [21]. Briefly, 100 ml of serum were supplemented with 5 ml of 5 fmol/ml synthetic non-human miRNA (C. elegans miR-54, Takara Bio Inc, Shiga, Japan) as controls, providing an internal reference for normalization of technical variations between samples. After Qiazol solution (1 ml) was added and mixed well by vortexing, the samples were incubated at room temperature for 5 min. Aqueous and organic phase separation was achieved by the addition of chloroform. The aqueous phase was applied to an RNeasy spin column and RNeasy MinElute spin column. The microRNA was eluted from the column with nuclease-free water. 2.3. Quantitative real-time polymerase chain reaction (PCR) The cDNA was synthesized from miRNA with a Mir-X miRNA First Strand Synthesis and SYBR qRT-PCR Kit (Takara Bio Inc). Quantitative real-time PCR with a Takara Thermal Cycler Dice (TP800)1 used primers and templates mixed with the SYBR Premix. The sequence of the miR-221 primer was designed based on miRBase (http:// www.mirbase.org): AGCTACATTGTCTGCTGGGTTTC. This primer was pre-validated to generate single amplicons. DNA was amplified for 50 cycles of denaturation for 5 s at 95 8C and annealing for 20 s at 60 8C. The data generated from each PCR reaction were analyzed using the Thermal Cycler Dice Real Time System ver 2.10B (Takara Bio Inc.) software package. The specificity of reactions was determined by melting curve analysis. The relative fold changes

2.4. Statistical analysis The statistical analysis was carried out with a Mann–Whitney test for the comparison of means between two groups, a Kruskal– Wallis test was used for the comparison among more than two groups, and Fisher’s exact probability test was performed for the analysis of frequency. P values < 0.05 were considered to be significant. 3. Results

3.2. Significant differences in miR-221 between MM patients and healthy controls The serum miR-221 levels in patients with MM and in the healthy control subjects are shown in Fig. 2. The mean serum miR221 levels were significantly higher in MM patients than in normal subjects (NS) (P < 0.0001). In addition, when MM patients were classified into five groups; NM (n = 18), SSM (n = 13), ALM (n = 35), LMM (n = 11) and mucosal MM (n = 13) as described in Section 2, the serum levels in each group were significantly higher than those of healthy controls (P < 0.0001, <0.0001, <0.0001, <0.01 and <0.0001, respectively). When the cut-off value was set at the mean + 2SD of the healthy controls, increased serum miR-221 concentrations over the cut-off value were found in 65 of the 90 MM patients (72.2%). 3.3. Significant differences in miR-221 level between MM in situ and the other stages The serum miR-221 levels in MM patients classified based on AJCC staging are shown in Fig. 3 [22]. These data do not include patients with mucosal melanoma. Eight patients had MM in situ, whereas the other stages of MM (stage I, II, III and IV) were found in 20, 19, 17, and 15 patients, respectively. There was a statistically significant difference in the values between the patients with MM in situ and those with stage I–IV MM (P < 0.0001). On the other hand, we could not find a significant difference between MM in situ and healthy controls (P = 0.46). In addition, the serum levels of miR-221 in MM patients in each stage (I, II, III and IV) were significantly higher than those of healthy controls (P < 0.0005, <0.0001, <0.0001 and <0.0001, respectively), however, we did not find any significant differences in the values among the patients with stage I–IV MM (P = 0.28). The serum miR-221 levels were increased in MM patients with stage I–IV disease, but not in patients with MM in situ. When the cut-off value was set at the mean + 2SD of healthy controls, the values in all patients with MM in situ were below the cut-off line,

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Table 1 The profiles of 90 Japanese patients with malignant melanoma and quantification of miR-221 in sera of patients. ND, not determined. Patients ID

Pathologic staging (T,N,M)

Stage

Age

Sex

Type

Relative serum concentrations of miR-221

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

is,0,0 is,0,0 is,0,0 is,0,0 is,0,0 is,0,0 is,0,0 is,0,0 1a,0,0 2a,0,0 1a,0,0 1b,0,0 1a,0,0 2a,0,0 1b,0,0 1a,0,0 1a,0,0 2a,0,0 1a,0,0 1a,0,0 2a,0,0 1a,0,0 1a,0,0 1a,0,0 1b,0,0 1b,0,0 1b,0,0 1a,0,0 4a,0,0 4b,0,0 2b,0,0 3b,0,0 3b,0,0 2b,0,0 3b,0,0 3b,0,0 3a,0,0 4b,0,0 3b,0,0 3a,0,0 3b,0,0 4b,0,0 4b,0,0 3b,0,0 4b,0,0 2b,0,0 3b,0,0 2b,0,0 4b,1b,0 4b,1a,0 4b,2b,0 3b,2c,0 4a,1b,0 4b,1b,0 3a,1b,0 4b,2b,0 3b,3,0 4b,1b,0 4b,1b,0 4b,2b,0 4b,1b,0 4b,1b,0 4b,2a,0 3b,1b,0 3b,1a,0 4b,2b,1c x,2b,1c 4b,2b,1c x,x,1c x,x,1b 4b,x,1c 4b,3,1a 4a,x,1c 4b,x,1c 4b,3,1c

0 0 0 0 0 0 0 0 IA IB IA IB IA IB IB IA IA IB IA IA IB IA IA IA IB IB IB IA IIB IIC IIA IIB IIB IIA IIB IIB IIA IIC IIB IIA IIB IIC IIC IIB IIC IIA IIB IIA IIIC IIIB IIIC IIIC IIIB IIIC IIIB IIIC IIIC IIIC IIIC IIIC IIIC IIIC IIIB IIIC IIIB IV IV IV IV IV IV IV IV IV IV

66 69 66 75 40 65 56 73 71 36 63 80 82 80 87 80 74 34 51 36 52 80 52 90 62 81 54 76 54 82 70 87 84 53 69 44 89 62 72 71 84 85 72 79 72 73 71 87 59 91 73 64 54 42 71 60 70 85 68 48 56 78 67 69 42 77 50 47 32 22 61 36 56 52 78

M F F F F M F F F M F M F M M M F M F M F M F F M M M M F F F M F F F F M M M M F M M M M F F M M F F F M M M M F F F F M F M M F M M F M M M M F M F

LMM ALM LMM ALM ALM ALM ALM ALM ALM ALM ALM ALM LMM LMM LMM LMM NM NM SSM SSM SSM SSM ALM LMM ALM ALM LMM ALM ALM ALM ALM ALM ALM LMM LMM NM NM SSM ALM ALM ALM NM ALM ALM ALM ALM ALM ALM ALM ALM ALM ALM LMM NM NM SSM SSM SSM SSM SSM NM NM ALM ALM SSM ALM SSM NM NM NM NM NM NM NM NM

0.096 0.759 0.204 0.446 0.013 0.026 0.098 0.015 0.186 0.032 0.636 0.142 0.087 0.423 4.513 71.357 162.53 91.573 177.759 20.441 103.973 120.279 346.928 36.427 45.384 236.505 46.056 27.536 63.349 52.428 550.108 38.9 246.711 13.91 0.605 2.84 3.203 9.847 73.509 205.714 37.655 168.049 491.484 124.973 179.372 95.839 220.785 0.755 7.473 1.431 6.485 24.215 42.091 24.193 9.797 20.201 5.049 35.193 2.63 102.95 58.984 119.057 114.275 76.208 23.201 3.051 57.206 39.523 70.313 29.993 6.16 31.413 7.416 121.689 20.319

H. Kanemaru et al. / Journal of Dermatological Science 61 (2011) 187–193

190 Table 1 (Continued ) Patients ID

Pathologic staging (T,N,M)

Stage

Age

Sex

Type

Relative serum concentrations of miR-221

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

3b,2c,1c 4b,3,1c ND ND ND ND ND ND ND ND ND ND ND ND ND

IV IV ND ND ND ND ND ND ND ND ND ND ND ND ND

65 68 71 25 53 66 61 58 58 66 65 75 75 78 58

M F F F M F M M F F F M F F M

NM SSM Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal Mucosal

141.913 53.677 72.463 1.682 3.96 6.702 57.601 6.243 78.033 62.757 74.759 82.657 90.917 195.962 77.184

Table 2 The profiles of eight MM patients with postoperative recurrence. Patients ID

Pathologic staging (T,N,M)

Stage

Age

Sex

Type

Relative serum concentrations of miR-221 Preoperation

Postoperation

recurrence

39

3b,0,0

IIB

72

M

ALM

1.000

0.067

0.047

40 62 89 91 92

3a,0,0 4b,1b,0 4b,2a,0 2a,0,0 3b,0,0

IIA IIIC ND IB IIB

71 78 78 40 87

M F F M M

ALM NM Mucosal ALM ALM

1.000 1.000 1.000 1.000 1.000

0.948 0.026 0.307 0.509 0.129

1.736 0.575 0.854 0.659 0.323

93 94

4b,0,0 4b,0,0

IIC IIC

83 42

F M

ALM SSM

1.000 1.000

0.099 0.146

0.291 0.627

3.4. The correlation between serum miR-221 levels and tumor thickness

Fig. 1. MiR-221 is present in serum samples. Serial dilution of serum-derived miRNA (as is, 10-fold, 100-fold dilutions and water) was used as the template for real-time PCR as described in Section 2. Amplification curves of gene-specific transcripts are shown to illustrate the process of exponential increase of fluorescence. Horizontal dotted line indicates the threshold.

Brain, liver, both adrenal glands, spleen, right rib (X), celiac lymph nodes Skin Lung, axillary lymph nodes External iliac lymph nodes Lung, mediastinal lymph nodes Stomach, liver, external iliac lymph nodes Lung External iliac lymph nodes

from this analysis. There was a statistically significant difference in the values between these two groups. The serum miR-221 levels in patients with a tumor thickness > 1 mm (n = 48) were significantly higher than those of patients with a tumor thickness  1 mm (n = 25). Similarly, there was also a statistically significant difference between the patients with a tumor thickness > 2 mm and those with a tumor thickness  2 mm (P = 0.0257, data not shown). In addition, we found that MM patients with elevated serum miR-221 levels had >1 mm, >2 mm, or >4 mm tumor thicknesses

whereas increased serum miR-221 concentrations over the cut-off value were found in 56 of the 71 (78.9%) patients with stage I–IV (13 of 20 stage I patients (65.0%), 15 of 19 stage II patients (79.0%), 14 of 17 stage III patients (82.4%), 14 of 15 stage IV patients (93.3%), respectively).

The serum miR-221 levels in MM patients divided into two groups according to their tumor thickness (1 mm or >1 mm) are [()TD$FIG]shown in Fig. 4. Patients with mucosal melanoma were excluded

Anatomic sites of recurrence

[()TD$FIG]

Fig. 2. The serum concentrations of miR-221 in patients with nodular melanoma (NM), superficial spreading melanoma (SSM), acral lentiginous melanoma (ALM), lentigo maligna melanoma (LMM) or mucosal melanoma and in normal subjects (NS). MiR-221 levels were measured with real-time PCR as described in Section 2. MiR-221 concentrations are shown on the ordinate. The horizontal dotted line indicates the cut-off levels. Bars show means. P values between two groups were determined by a Mann–Whitney test and those among more than two groups were determined by a Kruskal–Wallis test.

[()TD$FIG]

[()TD$FIG]

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Fig. 5. The relationship between tumor thickness and measured levels of miR-221. Individual findings are plotted and both scales are logarithmic. MiR-221 concentrations are shown on the ordinate. Fig. 3. The serum concentrations of miR-221 in patients with malignant melanoma in situ, the other stages (I–IV) and in normal subjects (NS). Staging was based on AJCC classification. MiR-221 levels were measured with real-time PCR as described in Fig. 2.

at a significantly higher incidence than those with normal serum levels (P = 0.0029, 0.0047 or 0.016, respectively). These results indicate that there is a significant correlation between the serum miR-221 levels and tumor thickness. Fig. 5 shows the relationship between tumor thickness and the levels of miR-221 in stage I–IV with logarithmic scales.

Table 3 The rates of increased serum miR-221 concentrations over the cut-off value (the mean + 2SD of healthy controls) and those of serum 5-S-CD concentrations over 10 nmol/L.

3.5. Comparison of serum miR-221 levels with 5-S-CD in MM patients Considering that 5-S-CD is commonly used as a tumor marker for MM, we expected that there might be a correlation between serum 5-S-CD levels and miR-221 levels. However, we could not find any significant correlations between 5-S-CD and miR-221. Rather, the rates of increased serum miR-221 concentrations above the cut-off value were higher than those of serum 5-S-CD concentrations in patients with stage I, II, III and IV disease and in all MM patients (as a single group) (Table 3). The sensitivities of increased serum miR-221 in each stage, I–IV, and for all MM patients were 65.0%, 79.0%, 82.4%, 93.3% and 72.8%, respectively.

Increased miR-221 (%)

Increased 5-S-CD (%)

In situ I II III IV I–IV All MM

0/8 (0.0) 13/20 (65.0) 15/19 (79.0) 14/17 (82.4) 14/15 (93.3) 56/71 (78.9) 67/92 (72.8)

1/8 (12.5) 4/20 (20.0) 1/19 (5.3) 1/17 (5.9) 6/15 (40.0) 12/71 (16.9) 18/92 (19.6)

Therefore, the serum miR-221 levels appear to be decreased or increased in proportion to the tumor activity. 4. Discussion It has been proposed that there are more than 1000 miRNAs in the human genome, which may target about 60% of all mammalian genes [23]. These miRNAs have recently been reported to play important roles in vivo, including roles in cancer development, angiogenesis and the immune response [24–28]. It was also reported that some unique microRNA signatures were associated with prognostic factors and disease progression in several cancers [29,30]. Therefore, we hypothesized that the serum miRNA level can also be a marker for MM, and that the serum miR-221 level would increase in the sera of MM patients because miR-221 is over-expressed in melanoma cells. We found that MM patients had significantly higher serum miR-221 levels than control subjects (Fig. 2). These data suggest that miR-221 has potential as a useful diagnostic marker for MM. However, miR-221 is over-expressed in various other malignancies, so this marker may not have high specificity for melanoma.

3.6. Serum miR-221 levels in patients with preoperative, postoperative and recurrent status Fig. 6 shows a longitudinal study of serum miR-221 levels in eight MM patients who had experienced excisions of primary melanoma but who had developed a recurrence during the followup period. The serum levels of miR-221 were significantly reduced after the surgery (P < 0.001). In addition, we found in 7 of the 8 patients, that the serum levels were increased again at recurrence.

[()TD$FIG]

[()TD$FIG]

Fig. 4. Comparison of the serum miR-221 levels between MM patients with a tumor thickness  1 mm and those with a thickness > 1 mm. MiR-221 levels were measured with real-time PCR as described in Section 2. MiR-221 concentrations are shown on the ordinate. Bars show means. P values were determined by a Mann– Whitney test.

Stage

Fig. 6. The longitudinal study of serum miR-221 levels in eight MM patients before the operation (preoperation), after operation (postoperation), and at recurrence (recurrence). The values in preoperative status were set at 1. P values were determined by a Mann–Whitney test.

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MiR-222, which is also clustered on the X chromosome, and may possibly be transcribed from a common precursor, has a coordinate functional role with miR-221 [14,16]. In our preliminary study, miR-222 also showed almost the same expression pattern as miR221 (data not shown). Therefore, miR-222 also could represent a diagnostic marker. However, in contrast to miR-221, miR-222 did not show an expression pattern which decreased or increased in proportion to the tumor activity. In addition, we also found that MM patients with stage I–IV disease had significantly increased miR-221 levels compared to those with MM in situ (Fig. 3). The recommended surgical margin is different between MM in situ and the other stages of MM, therefore it is beneficial to distinguish MM in situ from the other cases using non-invasive examinations. The tumor progression of MM is defined according to its TNM classification. It has been shown that as the tumor thickness increases, there is a highly significant decrease in 5- and 10-year survival rates (P < 0.0001) [22]. The 10-year survival was 92% among 11,841 patients with T1 melanomas (1.00 mm thickness), while it was 80% in 8046 patients with T2 melanomas (1.01– 2.00 mm), 63% in 5291 patients with T3 melanomas (2.01– 4.00 mm), and 50% in 2461 patients with T4 melanomas (>4.00 mm) [22]. Since we found that there was a correlation between the miR-221 level and tumor thickness (Figs. 4 and 5), we can assume that miR-221 might be correlated directly or indirectly with patients. Therefore, further studies are needed to elucidate whether a direct relationship exists between miR-221 and the prognosis of MM. Finally, the miR-221 levels tended to decrease after the surgical removal of the primary tumor, and to increase again when the tumor recurred (Fig. 6). These data suggest that miR-221 may be useful not only for the diagnosis of the disease, but also for monitoring and follow-up of patients with MM. Additionally, these data support the idea that serum miR-221 levels are derived from melanoma cells. MiR-221 has been reported to be increasingly upregulated throughout a stepwise transformation process [17]. However, we did not find any significant correlations of the miR221 levels between the excised tumors and serum samples when we investigated tissue samples from 10 melanoma patients (data not shown). Therefore, further studies with a larger sample number are needed to confirm whether the increased serum miR221 levels are derived from melanoma cells. Although we could not find any significant correlations between the serum 5-S-CD levels and miR-221 levels, this may be explained by the fact that miR-221 levels were increased even in early stage disease (Fig. 3) while the 5-S-CD level usually starts to increase in metastatic stages, as described above (Table 3). Furthermore, considering that the sensitivities of increased serum miR-221 were higher than those of 5-S-CD in stage I–IV patients, miR-221 may be an ideal tumor marker, especially for earlier stages of the disease. In addition, miR-221 seems to behave in a completely different way than already established melanoma markers like protein S100b (S100), melanoma inhibitory activity (MIA) and glypican-3 (GPC3). While GPC3 could diagnose about 40% of melanomas [3], the sensitivity of miR-221 was 72.8% (Table 3). Furthermore, in general, the tumor markers like S100b and MIA are not elevated in melanoma patients during early stages, but are increased in the serum levels during the later metastatic stage [31]. MiR-221, however, is found to already be increased in earlier stages with a smaller tumor thickness, and there was no clear increase in the levels in metastatic disease (Figs. 3 and 6) in our study. This suggests that miR-221 plays an important role especially during the earlier stages of the multi-step tumorigenesis of MM. The c-Kit and p27 molecules, which are down-regulated by miR-221, were reported to be decreased in the vertical growth

phase compared with the radial growth phase [32]. Therefore, miR221 might be important in earlier stage of tumorigenesis of MM because of its down-regulation of c-Kit and p27. However, it is well known that certain types of melanoma, like mucosal and acral melanoma, frequently show genetic amplification of the c-Kit locus and activating mutations of the gene [33,34]. In our present study, the average of miR-221 level in ALM patients (93.5) was relatively higher than that of NM, SSM, LMM and mucosal patients (61.6, 56.3, 19.6 and 62.4, respectively), although we did not find any significant differences among patients with the different types of melanoma. Our results have some limitations because of the small number of patients examined by this study. Therefore, further studies with a larger sample number are needed in order to confirm whether miR-221 can be used as a new tumor marker. Acknowledgements This study was supported in part by the Japan Society for the Promotion of Science (Grant-in-Aid for Research Activity Start-up No. 21890199), by Management Expenses Grants from the Government to the National Cancer Center (21S-7[TD$INLE] ) and by the Lydia O’Leary Memorial Foundation. References [1] Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature 2007;445:851–7. [2] Mouawad R, Spano J, Khayat D. Old and new serological biomarkers in melanoma: where we are in 2009. Melanoma Res 2010;20:67–76. [3] Ikuta Y, Nakatsura T, Kageshita T, Fukushima S, Ito S, Wakamatsu K, et al. Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels. Clin Cancer Res 2005;11:8079–88. [4] Calin G, Croce C. MicroRNA signatures in human cancers. Nat Rev Cancer 2006;6:857–66. [5] Esquela-Kerscher A, Slack F. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 2006;6:259–69. [6] Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008;105:10513–8. [7] Lawrie CH, Gal S, Dunlop HM, Pushkaran B, Liggins AP, Pulford K, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 2008;141:672–5. [8] Osaki M, Takeshita F, Ochiya T. MicroRNAs as biomarkers and therapeutic drugs in human cancer. Biomarkers 2008;13:658–70. [9] Ciafre` SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G, et al. Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem Biophys Res Commun 2005;334:1351–8. [10] le Sage C, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A, et al. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO J 2007;26:3699–708. [11] Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, et al. Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer 2007;120:1046–54. [12] Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 2007;297:1901–8. [13] He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, et al. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA 2005;102:19075–80. [14] Chun-Zhi Z, Lei H, An-Ling Z, Yan-Chao F, Xiao Y, Guang-Xiu W, et al. MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN. BMC Cancer 2010;10:367. [15] Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafre` SA, et al. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1. J Biol Chem 2007;282:23716–24. [16] Felicetti F, Errico MC, Segnalini P, Mattia G, Care` A. MicroRNA-221 and -222 pathway controls melanoma progression. Expert Rev Anticancer Ther 2008;8:1759–65. [17] Mueller D, Bosserhoff A. Role of miRNAs in the progression of malignant melanoma. Br J Cancer 2009;101:551–6. [18] Igoucheva O, Alexeev V. MicroRNA-dependent regulation of cKit in cutaneous melanoma. Biochem Biophys Res Commun 2009;379:790–4. [19] Felicetti F, Errico MC, Bottero L, Segnalini P, Stoppacciaro A, Biffoni M, et al. The promyelocytic leukemia zinc finger-microRNA-221/-222 pathway controls melanoma progression through multiple oncogenic mechanisms. Cancer Res 2008;68:2745–54.

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