Identificating Cathepsin D as a Biomarker for Differentiation and Prognosis of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Proteomic Analysis Ai-Lan Cheng,†,‡,# Wei-Guo Huang,†,‡,# Zhu-Chu Chen,† Peng-Fei Zhang,† Mao-Yu Li,† Feng Li,† Jian-Ling Li,† Cui Li,† Hong Yi,† Fang Peng,† Chao-Jun Duan,† and Zhi-Qiang Xiao*,† Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China, and Cancer Research Institute, University of South China, Hengyang 421001, China Received December 16, 2007
In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC. Keywords: Nasopharyngeal carcinoma • biomarker • cathepsin D • laser capture microdissection • proteomics • prognosis • differentiation
Introduction
distribution of NPC indicates that the development of this cancer may be related to genetic and environmental factors.
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and Southeast Asia and poses a major public health problem in southern China.1 Cantonese are the most frequently affected population, and the incidence rate of NPC in Cantonese is nearly 100-fold higher than that in Caucasians.2 This remarkable geographic and racial
A strong association between Epstein-Barr virus and NPC has been widely accepted, which was initially suggested on the basis of serological studies and has been subsequently substantiated by the detection of viral genomes and gene products in the tumor cells.2,3 Multiple genetic and epigenetic alternations have been identified in NPC.4–8 Although numerous efforts have been made to reveal the molecular mechanisms of NPC carcinogenesis, it remains poorly understood. In this regard, the identification of NPC-associated proteins using a proteomic approach may be an alternative way for deciphering the molecular characteristics of the malignancy.
* To whom correspondence should be addressed. Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. Tel: (86)7314327239. Fax: (86)731-4327321. E-mail:
[email protected]. † Central South University. ‡ University of South China. # These authors contributed equally to this work. 10.1021/pr7008548 CCC: $40.75
2008 American Chemical Society
NPC is a markedly heterogeneous disease in biological characters. For the pathological diagnosis, the WHO developed Journal of Proteome Research 2008, 7, 2415–2426 2415 Published on Web 04/24/2008
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Figure 1. LCM of tissues. NNET before (A) and after (B) LCM, and captured normal cells (C). NPC tissue before (D) and after (E) LCM, and captured cancer cells (F).
a three-tier histological classification of NPC based on the degree of its differentiation.9 According to the WHO classification, NPC is classified into keratinizing squamous cell carcinoma (type I/well differentiated), differentiated nonkeratinizing carcinoma (type II/moderately differentiated), and undifferentiated carcinoma (type III/poorly differentiated). A number of studies showed that the biological behavior and prognosis could be significantly different in the NPC patients with the same stage, histologic type, or differentiation grade, suggesting that the presence of other factors, such as molecular variables10 and ethnicity,11 can affect the behavior and prognosis of NPC. Therefore, it is urgent to discover biomarkers for indicating NPC biological characters and predicting the outcome of patients with NPC. High-throughput technologies such as microarrays and proteomics offer the potential ability to find alterations previously unidentified in NPC. Analyses for gene expression profiles of NPC have been reported using a cDNA array, and found that genes with aberrant expressions possibly contributed to the pathogenesis of NPC.12–14 Proteomics has introduced a new approach to cancer research which aims at identifying differential expression proteins associated with the development and progression of cancer,15 providing new opportunities to uncover biomarkers and therapeutic targets for NPC as well as reveal the molecular mechanism underlying this disease. Using tissue samples from patients may be the most direct and persuasive way to find biomarkers and therapeutic targets for cancers by a proteomic approach. A major obstacle, however, to the analysis of tumor specimens is tissue heterogeneity, which is particularly relevant to NPC as it often includes numerous infiltrating lymphocytes and stroma. Moreover, normal nasopharyngeal epithelial cells, from which the cancer is believed to arise, represent as little as 10% of nasopharyngeal 2416
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mucosal tissue. Several approaches have been employed to obtain homogeneous cell populations from a heterogeneous tissue for proteomic analysis, such as short-term cell culture and laser capture microdissection (LCM). Since 1996, LCM has emerged as a good choice for obtaining homogeneous cell populations from a heterogeneous tissue.16 There have been reports concerning proteomic research of laser capture microdissected tumor cells such as breast,17 hepatocellular,18 and pancreatic cancers.19 In the present study, we used a proteomic approach to compare the protein expression profiles of LCM purified cells from NPC and NNET to identify differential expression proteins. The differential expression of cathepsin D was significantly down-regulated in NPC compared with NNET, and was confirmed and related to differentiation degree and metastatic potential of the NPC cell lines. The accumulating evidence indicated that cathepsin D was involved in the progression and metastasis of human malignant tumors,20–23 as well as its expression level was associated with the tumor differentiation and the patient prognosis.24–26 But the role and clinicopathologic significance of cathepsin D in NPC is unclear. Therefore, we tested whether cathepsin D might mediate the in vitro invasiveness of NPC, and evaluated clincopathologic significances of cathepsin D in NPC.
Materials and Methods Materials. Immobiline pH gradient (IPG) DryStrips (pH 3-10, length 24 cm), IPG buffer (pH3-10), DryStrip cover fluids, thiourea, urea, CHAPS, DTT, Pharmalyte (pH 3-10), bromophenol blue, Bis, TEMED, Commassie brilliant blue G-250, molecular weight marker, Tris-base, SDS, glycine, second antibodies conjugated with horseradish peroxidase, and
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Figure 2. Representative 2-DE maps of microdissected NNET (A) and NPC tissues (B). The protein spots marked with arrows were 36 differential protein spots identified by MS. (C) Close-up of the region of the gels showing partial differential expression proteins between the NNET and NPC tissues.
the enhanced chemiluminescence (ECL) system were purchased from Amersham Biosciences (Stockholm, Sweden). Sequencing-grade modified trypsin was obtained from Promega (Madison, WI). Polyvinylidene difluoride membrane and ZipTip C18 columns were obtained from Millipore (Boston, MA). Mouse monoclonal antibody against cathepsin D, cathepsin D siRNA, control siRNA, and mouse monoclonal antibody against SCCA1 (squamous cell carcinoma antigen 1) were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against CK8 (cytokeratin 8) was from Laboratory Vision Corp (Fremont, CA). Mercaptoethanol, iodoacetamide, R-cyano-4-hydroxycinnamic acid, and HCl were from SigmaAldrich (St. louis, MO). All buffers were prepared with Milli-Q water. Tissues. Fifty-four cases of fresh undifferentiated NPC (WHO type III) tissues and 54 cases of fresh NNET from healthy individuals were obtained from the First and Second Xiangya Hospitals of Central South University and the Cancer Hospital of Hunan Province, China, at the time of diagnosis before any therapy, and used for 2-DE and Western blotting. The patients and healthy individuals signed an informed consent form for the study which was reviewed by the Institutional Review Board. All tissue samples were verified by histopathology before microdissection. An additional group of formalin-fixed and paraffin-embedded archival tissue specimens including 72
cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastatic NPC (LMNPC) between 1996 and 2000 were obtained from the Cancer Hospital of Hunan Province, and used for immunohistochemistry. According to the 1978 WHO classification,9 72 cases of primary tumors were histopathologically diagnosed as keratinizing squamous cell carcinoma (WHO type I, 5 cases), differentiated nonkeratinizing squamous cell carcinoma (WHO type II, 9 cases), and undifferentiated carcinoma (WHO type III, 58 cases), and all LMNPC were undifferentiated carcinoma. The clinical stage of all the patients was classified or reclassified according to the 1992 NPC staging system of China.27 All the patients underwent radiotherapy treatment and were given follow-up. The follow-up period at the time of analysis was 6-72 months (average, 48 ( 14.5). The clinicopathologic features of the patients used in the present study are shown in Table 3. Tissue Processing and LCM. Eight-micrometer-thick frozen sections of fresh NPC and NNET were prepared using a Leica CM 1900 cryostat (Leica, Milton Keynes, U.K.) at -25 °C. The sections were placed on a membrane-coated glass slides (2.0 µm, 50 pieces, PEF Membrane; Leica), fixed in 75% alcohol for 30 s, and stained with 0.5% violet-free methyl green (Sigma). All solutions for staining were supplemented with protease inhibitor cocktail tablets (Roche Molecular Biochemicals, Indianapolis, IN). Following staining, the sections were air-dried Journal of Proteome Research • Vol. 7, No. 6, 2008 2417
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Table 1. Proteins with Different Expression Levels between NPC and Normal Control Identified by MALDI-TOF-MS and ESI-Q-TOF-MS spot no.
accession number
1
P17987
2 3 4
P43487 P10809 P06576
5 6 7 8
P13647 P31947 Q53HK9 P15531
9 10 11 12 13 14 15 16
P08670 P05109 P04792 Q96CE4 P37802 P06702 Q6NTA2 P47756
17 18 19 20
O15540 P62937 Q5VWK3 Q53HU2
21 22
P14550 Q9NR45
23 24 25
P04632 P30084 P40121
26
Q9UBS4
27 28 29 30 31 32 33 34 35
P07339 Q5TZZ9 Q86W04 P05787 Q53HR3 P00352 P05783 Q13938 P55072
36
P34931
a
protein name
mass weight
pI
sequence coverage (%)
scores
expression in NPC/NNET
t-complex-type molecular chaperone TCP1 Ran-binding protein 1 Chaperonin GroEL precursor H+-transporting two-sector ATPase beta chain precursor Keratin 5 Enolase 1 Acidic ribosomal protein P0 Metastasis inhibition factor/ nm23 protein Vimentin Calgranulin A HSP27 protein Stathmin Transgelin-2 MRP-14 protein HNRPL protein Actin-capping protein beta chain Fatty acid binding protein Peptidylprolyl isomerase A VDAC2 protein Guanine nucleotide binding protein Alcohol dehydrogenase N-acetylneuraminic acid phosphate synthase Calpain small chain Enoyl-CoA hydratase Human Macrophage Capping Protein ER-associated Hsp40 co-chaperone Cathepsin D Annexin I SCCA1 cytokeratin 8 14-3-3 σ Aldehyde dehydrogenase keratin 18 Calcyphosine Transitional endoplasmic reticulum ATPase Heat shock 70 kDa
60356
6.03
43
570
v2.00
34252 61187 56525
5.71 5.7 5.26
38 49 73
298 203 262
v2.00 v2.01 v2.01
54478 47139 34423 20398
6.53 7.01 5.71 7.07
21 57 49 32
83 826 163 188
v2.04 v2.09 v2.11 v2.25
53653 10885 22313 17326 24400 12770 60719 30952
5.03 6.51 7.83 5.76 8.44 5.55 6.65 5.69
43 77 67 18 36 55 33 64
908 87 392 152 335 73 166 196
v2.29 v2.51 v2.57 v3.15 v3.24 v3.24 v3.56 v5.09
15497 17999 30329 35055
6.6 7.85 8.00 7.60
57 39 32 18
101 78 138 186
a a a a
36761 40267
5.11 6.29
30 28
150 154
V2.02 V2.03
28469 31351 38500
5.05 8.34 5.32
31 43 26
74 262 362
V2.03 V2.11 V2.64
40774
5.81
39
110
V3.08
26229 38690 44507 53529 27757 54827 47305 20954 89266
5.31 6.57 6.35 5.52 4.68 6.30 5.27 4.74 5.14
48 45 37 36 58 36 33 37 35
304 946 541 765 552 550 394 391 594
V3.17 V3.42 V4.08 V4.42 V5.22 V7.07 V7.50 V10.01 b
70854
5.37
3
62
a, only expression in NPC; b, only expresson in NNET.
and microdissected using a Leica AS LMD system (Leica). Approximately 200 000-250 000 microdissected cells were required for each 2-DE, and 20 000-25 000 microdissected cells were required for each Western blotting. Each cell population was determined to be 95% homogeneous by microscopic visualization of the captured cells (Figure 1). The microdissected cells were dissolved in lysis buffer (7 mol/L urea, 2 mol/L thiourea, 100 mmol/L DTT, 4% CHAPS, 0.5 mmol/L EDTA, 40 mmol/L Tris, 2% NP40, 1% Triton X-100, 5 mM PMSF, and 2% Phamarlyte) at 4 °C for 1 h, and then centrifuged at 12 000 rpm for 30 min at 4 °C. The supernatant was transferred to a fresh tube and stored at -80 °C until 2-DE. The concentration of the total proteins was measured by 2-D Quantification kit (Amersham Biosciences). Two-Dimensional Gel Electrophoresis. 2-DE was done to separate proteins from three sets of pooled microdissected NPC 2418
b
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and NNET as previously described by us.28 Briefly, 650 µg of protein samples were diluted to 450 µL with a rehydration solution [7 mol/L urea, 2 mol/L thiourea, 0.2% DTT, 0.5% (v/ v) pH3-10 IPG buffer, and trace bromophenol blue], and applied to IPG strips (pH 3-10L, 24 cm) by 14 h rehydration at 30 V. The proteins were focused successively for 1 h at 500 V, 1 h at 1 000 V and 8.5 h at 8 000 V to give a total of 68 kVh on an IPGphor (Amersham Biosciences). Focused IPG strips were equilibrated for 15 min in a solution [6 mol/L urea, 2% SDS, 30% glycerol, 50 mmol/L Tris-HCl (pH 8.8), and 1%DTT], and then for an additional 15 min in the same solution except that DTT was replaced by 2.5% iodoacetamide. After equilibration, SDS-PAGE was done on Ettan DALT II system (Amersham Biosciences). After SDS-PAGE, the Blue Silver staining method, a modified Neuhoff’s colloidal Coomassie blue G-250 stain, was used to visualize the protein spots in the 2-DE gels.29
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Figure 3. MALDI-TOF-MS and ESI-Q-TOF-MS analysis of differential protein spot 27. (A) The MALDI-TOF-MS mass spectrum of spot 27 identified as the cathepsin D according to the matched peaks. (B) Protein sequence of cathepsin D was shown, and matched peptides were underlined. (C) The ESI-Q-TOF-MS sequenced spectrum of spot 27. The amino acid sequence of a doubly charged peptide with m/z 801.4439 was identified as LVDQNIFSFYLSR from mass differences in the y-fragment ions series, and matched with residues 223-235 of cathepsin D. (D) Protein sequence of cathepsin D was shown. MS/MS fragmentation matched peptide was underlined.
Image Analysis. The stained 2-DE gels were scanned with MagicScan software on an Imagescanner (Amersham Biosciences) and analyzed using a PDQuest system (Bio-Rad Laboratories) according to the protocols provided by the manufacturer. Three separate gels were prepared for each microdissected tissue. The gel spot pattern of each gel was
summarized in a standard after spot matching. Thus, we obtained one standard gel for each tissue. Spot intensities were quantified by calculation of spot volume after normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. The change index was defined as the ratio between the spot percentage relative Journal of Proteome Research • Vol. 7, No. 6, 2008 2419
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Figure 4. Representative results of Western blotting of the partial identified proteins in the tissue and cell samples. (A) Western blotting shows changes in the expression levels of cathepsin D, SCCA1, and CK8 in microdisscted NPC tissue (T) and NNET (N). (B) Histogram shows relative changes in the expression levels of the three proteins in the tumor and normal tissues as determined by densitometric analysis. (C) Western blotting shows changes in the expression level of cathepsin D in NPC cell lines 6-10B, 5-8F, CNE2, and CNE1. (D) Histogram shows relative changes in the expression level of cathepsin D in the 4 NPC cell lines as determined by densitometric analysis.
to volumes in the microdessected tumor and normal tissues. Proteins were classified as being differentially expressed between the two types of tissues when spot intensity showed a difference of g2-fold variation in tumor tissue in comparison to normal tissue. Significant differences in protein expression levels were determined by Student’s t test with a set value of P < 0.05. Protein Identification by MS. All the differential protein spots were excised from stained gels using a punch, and ingel trypsin digestion was done as previously described by us.28 The tryptic peptide was mixed with an R-cyano-4-hydroxycinnamic acid matrix solution. One microliter of the mixture was analyzed with a Voyager System DE-STR 4307 MALDI-TOF Mass Spectrometer (ABI) to obtain a peptide mass fingerprint (PMF). In peptide mass fingerprint map database searching, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss-Prot database were done using the Mascot search engine (http://www.matrixscience. com/) with a mass tolerance of (50 ppm. The protein spots identified by MALDI-TOF-MS were also subjected to analysis of ESI-Q-TOF-MS (Micromass; Waters). Briefly, the samples were loaded onto a precolumn (320 µm × 50 mm, 5 µm C18 sillica beads; Waters) at 30 µL/min flow rates for concentrations and fast desalting through a Waters CapLC autosampler, and then eluted to the reversed-phase column (75 µm × 150 mm, 5 µm, 100 Å; LC Packing) at a flow rate of 200 nL/min after flow splitting for separation. MS/MS spectra were done in datadependent mode in which up to four precursor ions above an intensity threshold of 7 counts/s were selected for MS/MS analysis from each survey “scan”. In tandem mass spectrometry data database query, the peptide sequence tag (PKL) format 2420
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file that was generated from MS/MS was imported into the Mascot search engine with a MS/MS tolerance of (0.3 Da to search the Swiss-Prot database. Western Blotting. Twenty-four pairs of microdissected fresh NPC and NNET and four NPC cell lines (6-10B, 5-8F, CNE1, and CNE2) were used for Western blotting as previously described by us.28 6-10B without metastatic potential and 5-8F with high metastatic potential were kindly provided by Dr. H. M. Wang of the Cancer Center, Sun Yat-sen University, China.30 Well-differentiated CNE1 and poorly differentiated CNE2 are established NPC cell lines.31 Briefly, 40 µg of lysates was separated by 8% or 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Blots were blocked with 5% nonfat dry milk for 2 h at room temperature, and then incubated with primary anti-cathepsin D, anti-SCCA1 or antiCK8 antibody for 2 h at room temperature, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent and quantitated by densitometry using ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics). The mouse anti-β-actin antibody (1:5000, Sigma) was detected simultaneously as a loading control. Administration of Cathepsin D siRNA to 5-8F Cells. The cells were transfected with cathepsin D siRNA or control siRNA (Santa Cruz Biotechnology) according to the siRNA transfection protocol provided by the manufacturer. Briefly, the day before transfection, 5-8F cells were plated into 6-well plates at the density of 105 cells/mL in RPMI-1640 medium (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen). When the cells were 60-80% confluent, they were transfected with 10 nmol/L of cathepsin D siRNA or control siRNA after a preincubation
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Figure 5. In vitro cell invasion assay of the cathepsin D siRNA-transfected and control 5-8F cells. (A) Western blotting showing the expression level of cathepsin D in cathepsin D siRNA-transfected and control 5-8F cells. (B) The invasion of cathepsin D siRNAtransfected and control 5-8F cells was measured by using transwell chambers. Tumor cells penetrating the precoated polycarbonate membrane were photographed. (C) The numbers of invasive tumor cells per field in cathepsin D siRNA-transfected and control 5-8F cells.
for 20 min with siRNA transfection reagent in siRNA transfection medium (Santa Cruz Biotechnology). Four hours after the beginning of the transfection, the medium was replaced with in RPMI-1640 medium containing 10% FBS and we continued to culture the cells for an additional 44 h. At the end of the
transfection, cathepsin D expression level in the cells was determined by Western blotting. In Vitro Cell Invasion Assay. The invasiveness of 5-8F cells transfected with cathepsin D siRNA or control siRNA was evaluated in 24-well transwell chambers (Costar, Cambridge, Journal of Proteome Research • Vol. 7, No. 6, 2008 2421
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Figure 6. Representative results of immunohistochemistry of cathepsin D in the tissue specimens. Immunohistochemiscal staining of cathepsin D in NNET (A), WHO type I (B), NNET and type II (C), type III (D), NNET and type III (E) primary NPC, and LMNPC (F). Original magnification, ×200. Table 2. The Difference of Cathepsin D Expression among NNET, NPC, and LMNPC Score
NNET NPC LMNPC
n
low (0-2)
moderate (3-4)
high (5-6)
P
28 72 20
1 24 3
12 32 5
15 16 12
0.000a 0.004b
a P < 0.01 by Mann-Whitney U test, NNET vs NPC. Mann-Whitney U test, NPC vs LMNPC.
b
P < 0.01 by
MA) as directed by the manufacturer. Briefly, the upper and lower culture compartments of each well are separated by polycarbonate membranes (8 µm pore size). The membranes were precoated with 100 µg/cm2 of collagen matrix (Matrigel; Collaborative Biomedical Products, Bedford, MA), which was reconstituted by adding 0.5 mL of serum-free RPMI-1640 medium to the well for 2 h. To assess the ability of the cells to penetrate the precoated polycarbonate membrane, 1.25 × 104 cells in 0.5 mL of RPMI-1640 medium containing 1% FBS was placed into the upper compartment of wells, and 0.75 mL of RPMI-1640 medium containing 10% FBS was placed into the lower compartment. The transwell chambers were incubated for 24 h at 37 °C in humidified 5% CO2 atmosphere. Invaded cells attached underneath the chamber membrane were stained with a Diff-Quik stain kit (Dade Behring, Newark, DE) and counted in eight random fields with an inverted microscope (at 200× magnification). Invasive ability was defined as the average cell numbers that penetrated the matrix-coated membrane per field. The experiment was performed in triplicate. Immunohistochemistry. Immunohistochemistry was done on formalin-fixed and paraffin-embedded tissue sections using a standard immunohistochemical technique. Four-micrometerthick tissue sections were deparaffinized in xylene, rehydrated 2422
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in a graded ethanol series, and treated with an antigen retrieval solution (10 mmol/L sodium citrate buffer; pH 6.0). The sections were incubated with mouse monoclonal anti-cathepsin D antibody (dilution 1:100) overnight at 4 °C, and were then incubated with a 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) according to the manufacturer’s instructions. Finally, tissue sections were incubated with 3′, 3′-diaminobenzidine (SigmaAldrich) until a brown color developed, and counterstained with Harris’ modified hematoxylin. In negative controls, primary antibodies were omitted. Evaluation of Staining. Sections were blindly evaluated by two investigators in an effort to provide a consensus on staining patterns by light microscopy (Olympus). Cathepsin D staining was assessed according to the methods described by Hara and Okayasu32 with minor modifications. Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were chosen randomly, and >1000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, 60% stained positive. The minimum score when summed (extension + intensity) was, therefore, 0, and the maximum, 6. A combined staining score (extension + intensity) of e2 was considered to be a negative staining (low staining); a score between 3 and 4 was considered to be a moderate staining; whereas a score between 5 and 6 was considered to be a strong staining. Statistical Analysis. Statistical analysis was done using SPSS (version 13.0). The significant difference of cathepsin D expres-
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Proteomics and Biomarkers of NPC Table 3. Relationships between Cathepsin D Expression and Clinicopathologic Factors Score
Gender Male Female Age (y) g50
