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Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma
Oral Oncology 46 (2010) 166–171
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma q Marcilei Eliza Cavicchioli Buim a,*, Silvia Vanessa Lourenço b, Kátia Candido Carvalho a, Roberta Cardim c, Cláudia Pereira c, André Lopes Carvalho d, José Humberto Fregnani e, Fernando Augusto Soares a,b a
Department of Anatomical Pathology, Hospital A.C. Camargo, São Paulo, Brazil Department of General Pathology, Dental School, University of São Paulo, Brazil Department of Head and Neck Surgery, Hospital A.C. Camargo, São Paulo, Brazil d Department of Head and Neck Surgery, Barretos Cancer Hospital, São Paulo, Brazil e Department of Gynecological Oncology, Barretos Cancer Hospital, São Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 25 September 2009 Received in revised form 19 November 2009 Accepted 20 November 2009 Available online 8 January 2010 Keywords: Tetraspanin CD9 CD82 Oral squamous carcinoma Immunohistochemistry
s u m m a r y Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction One of the clinical characteristics of squamous cell carcinoma of the oral cavity (OSCC) is its capacity to invade locally and metastasize to regional lymph nodes. Identification of cervical lymph node metastasis is the main prognostic factor of patients with OSCC and has a major impact on treatment selection.1 Tetraspanins have been implicated in a wide array of cellular processes including cell adhesion, motility, intracellular signaling, cell matrix adhesion and proliferation.2–5 These molecules have also been considered as suppressors of metastasis in solid tumors.3,6
Reduced expression of tetraspanin proteins has been identified in many different types of carcinoma. Additionally, lack of expression of these proteins has been frequently reported in metastatic lesions, and patients with tumors that lack tetraspanins tend to have poorer prognoses and survival rates.7–11 The aim of this study was to analyze the patterns of expression of CD9, CD37, CD63, CD81 and CD82 antigens in OSCC and normal oral tissue by immunohistochemistry (IHC) and correlate this information with clinicopathological parameters. Based on the results from IHC, where OSCC expressed CD9 and CD82 exclusively, we also investigated the patterns of CD9 and CD82 gene expression by quantitative Real Time-polymerase chain reaction (qRT-PCR). Materials and methods
q
Supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. * Corresponding author. Address: Department of Anatomical Pathology, Hospital A.C. Camargo, 109 Professor Antônio Prudente St., Zip Code 01509-900, São Paulo, Brazil. Tel./fax: +55 11 21895185. E-mail address: [email protected] (M.E.C. Buim). 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.11.009
Tissue samples and patient characteristics Surgically resected primary OSCC were collected at the Department of Head and Neck Surgery, Hospital A.C. Camargo, São Paulo,
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Brazil, between 1992 and 2004. None of the patients had received radiotherapy or chemotherapy before surgical excision. Paraffinembedded specimens from 179 patients were sampled and used to construct two tissue microarray (TMA) blocks. These blocks were then used for IHC tetraspanins analyses. Additionally, qRTPCR was performed in 66 cases (from these, 50 cases were present in the TMA blocks and 16 were additional cases included in the study). Paraffin-embedded tissue from the 16 additional cases was not accessible for IHC analysis. Follow-up information was not available from these 66 samples. Fourteen samples of normal mucosa were used as controls, ten samples for IHC analysis, and four samples for the qRT-PCR experiments. The studies were performed according to local ethical committee approval (Study No. 986/07). The patients ranged in age from 30 to 90 years old (mean age of 57 years). The clinicopathological characteristics of patients with OSCC are summarized in Table 1.
with the indirect dextran polymer detection system (Novocastra Laboratories Ltd., Newcastle, UK). Staining was performed by incubating the slides in 3,30 -diaminobenzidine tetrachloride (Dako, Carpinteria, USA). The slides were then lightly counterstained with hematoxylin, dehydrated in absolute ethanol and xylene, and then mounted with coverslips using a permanent mounting medium and observed using an optical light microscope. All IHC reactions were carried out in quadruplicate (two cores per slide, with two stained slides). The semi-quantitative analysis of the results evaluated the average area of staining in all cores (four cores each case). We considered negative or downregulated cases up to 10% of tumor cells positively staining (downregulated/negative). Positive cases were identified where over 10% of the tumor cells were stained with the marker.
TMA and immunohistochemistry
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and first-strand cDNA was synthesized from 1 lg of total RNA using High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA), according to the protocol provided by the manufacturer. Sixty-six OSCC samples and four samples of normal control oral mucosal tissue (grouped into one pool) were evaluated by qRTPCR. The CD9 (Genbank Accession Number NM_001769.2) and CD82 (Genbank Accession Number NM_002231) sequences were used as a template for the construction of the primer pairs using the 3.0 version of the PrimerExpress software (Applied Biosystems, Foster City, CA). The PCR primer set sequences were designed as follows: CD9 forward primer 50 -GCA TTG CCG TGG TCA TGA T-30 and CD9 reverse primer 50 -TGC GGA TAG CAC AGC ACA AG-30 ; CD82 forward primer 50 -CGT GGG TGT GGC CAT CAT-30 and CD82 reverse primer 50 -TTG CTG TAG TCT TCG GAA TGG-30 ; the internal control was the b-actin forward primer 50 - GCA CCC AGC ACA ATG AAG-30 and the reverse primer 50 -CTT GCT GAT CCA CAT CTG C-30 . PCR amplification was performed in duplicate in an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR reactions were performed in 20 lL volumes with 10 ng of the cDNA sample, 0.2 lM of each of the primers and 1X of the Syber-Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA). The amplification program consisted of one cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The same PCR conditions were used to amplify the b-actin gene. The relative amount of PCR product generated from each primer set was determined on the basis of the Ct value and relative quantification was calculated by a mathematical model, previously described by Pfaffl.12
The selected blocks of each sample of tumor and normal tissue were used for the construction of the TMA using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments Microarray Technology, Silver Spring, MI, USA). The sampling consisted of 2 mm cores from different areas of the tumor from a single case of OSCC, placed at a specified coordinated place. Each case and tissue of normal mucosa was spotted in duplicate. Four micrometer sections were deparaffinized and rehydrated. Antigen retrieval was performed with citrate buffer solution of sodium citrate (pH 6.0) and boiled in pressure cooker for 15 min. Slides were placed in 3% hydrogen peroxide 10 V three times for 5 min. The sections were blocked with protein block serum-free (Dako, Carpinteria, USA) at room temperature for 20 min. The sections were incubated with the primary antibodies (all from NeoMarkers-Lab Vision-Corporation, USA) at room temperature for 2 h. After being washed with phosphate buffered solution three times for 5 min each; sections were then incubated for 1 h
Table 1 Clinicopathological characteristics of patients with OSCC for IHC and qRT-PCR analysis. Clinicopathological parameters
IHC
qRT-PCR
Total (n) (%)
Total (n) (%)
Sex Male Female
145 (81) 34 (19)
52 (79) 14 (21)
Clinical stage I and II III and IV
53 (30) 126 (70)
30 (45) 36 (55)
Lymph nodes N0 N1–3 No lymph nodes dissection
73 (44) 92 (56) 14
27 (45) 32 (54) 7
Perineural invasion No Yes No available
91 (55) 74 (45) 14
33 (55) 27 (45) 6
Vascular embolization No Yes Not available
109 (65) 58 (35) 12
53 (84) 10 (16) 3
Histological grade Well-differentiated Poorly/moderately differentiated Not available
127 (72) 49 (28) 03
39 (59) 27 (41)
Isolation of RNA and qRT-PCR
Statistical analysis Association between protein expression and clinicopathological parameters were assessed by the v2-test. The disease-free survival and the overall survival curves were constructed according to the Kaplan–Meier statistical method. The Mann–Whitney test was used to determine the association between the expression of the CD9 and CD82 genes and the pathological parameters. The relationship between protein levels and genes expression was evaluated using the Mann-Whitney test. All p-values were based on two-tailed statistical analysis and p = 0.05 was considered to be statistically significant. Statistical analysis was performed using the SPSS 13.0 statistical package (SPSS Inc, Chicago, IL).
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in patients with CD9-positive expression (71.6%) (p = 0.018) (Fig. 2a). The 5-year overall survival rate of patients with downregulated/absent CD9 expression was 44% versus 64% of CD9-positive cases (p = 0.071) (Fig. 2b).
Results Immunohistochemistry analysis There was no expression of CD63, CD37 and CD81 proteins in all 179 tumors and in all 10 of the normal oral mucosa control samples analyzed. Expression of CD9 and CD82 and their relationship with the clinicopathological parameters are shown in Table 2. In normal oral mucosa, CD9 and CD82 were observed on the cellular membrane of the supra-basal and intermediate layers cells of the squamous epithelium. Expression of CD9 and CD82 was not observed on the cells of the basal lamina surface and the most superficial cells (Fig. 1G, H, O and P). Most OSCC did not express CD82; the protein was downregulated/absent in 127 of 179 (80%) OSCC cases analyzed (Fig. 1K– N). In Fig. 1I and J, CD82 was positively expressed by the cell membrane. There was no correlation between the expression of CD82 and all clinicopathological parameters tested (Table 2). Diseasefree survival of patients with downregulated/negative levels of CD82 in tumors were similar when compared to CD82 positive tumors (63.3% versus 64.4%; p = 0.705), the same results was observed in 5-year overall survival, 57.7% survival of patients with downregulated/negative CD82 expression and 58.3% survival of patients with positive CD82 expression (p = 0.549). The results of CD9 expression in OSCC are also demonstrated in Table 2. Among the 179 cases of OSCC, 75 (42%) were downregulated/negative for CD9 expression (Fig. 1C–F). Membranous expression of the marker was observed in 104 (58%) cases (Fig. 1A/B). Downregulation/absent CD9 expression was observed in lymph node metastasis; tumors that had already metastasized to lymph nodes had an increased frequency of downregulated/negative CD9 expression than tumors without lymph node metastasis (p = 0.017). The significance between CD9 expression and other clinicopathological features was not statistically established (Table 2). The disease-free survival rate of patients with downregulated/ absent CD9 expression (48.6%) was significantly lower than that
Quantitative RT-PCR analysis qRT-PCR allowed mRNA quantification of CD9 and CD82 expression in 66 OSCC cases and four normal oral mucosa (grouped into one pool). There was no statistical correlation between CD9 and CD82 mRNA expression and clinicopathological features (gender, histological grade, lymph node status, perineural invasion and vascular embolization) (Table 3). However, a marginal, but statistically insignificant correlation (p = 0.056) was found between CD82 mRNA expression and the histological grade. On the other hand, well-differentiated tumors were observed to have increased levels of CD82 mRNA expression compared to moderate or poorly differentiated tumors. Correlation between mRNA and protein expression Associated analysis of IHC and RT-PCR was possible in 50 cases. Relationship was observed between mRNA and protein expression for CD9 (p = 0.015). The mRNA expression no associated with protein expression for CD82 (p = 0.156). The comparisons between IHC and qRT-PCR evaluation of CD9 and CD82 are shown in Fig. 3.
Discussion We hypothesized that downregulation of tetraspanins could interfere with the tumorigenic and/or metastatic potential of OSCC. Our results have shown that the tetraspanins CD37, CD63 and CD81 were not expressed by cell membranes in either normal or neoplastic mucosal keratinocytes, suggesting that these proteins have no participation in the mechanisms of OSCC growth and metastasis. Other authors have reported no relationship between
Table 2 Correlation between the expression of CD9 and CD82 and clinicopathological parameters in patients with OSCC by IHC. CD9 expression Clinicopathological parameters
CD82 expression
Total (n)
Downregulated (No. of patients; %)
Positive (No. of patients; %)
p (value)
Total (n)
Downregulated (No. of patients; %)
Positive (No. of patients; %)
Gender Male Female
145 34
58 (40) 17 (50)
87 (60) 17 (50)
0.287
145 34
102 (70) 25 (74)
43 (30) 09 (26)
0.73
Clinical stage I and II III and IV
53 126
19 (36) 56 (44)
34 (64) 70 (56)
0.287
53 126
35 (66) 92 (73)
18 (34) 34 (27)
0.294
Lymph nodes N0 N1–3 Not dissected
73 92 14
23 (31) 46 (50) –
50 (69) 46 (50) –
0.017
73 92 14
48 (66) 71 (77) –
25 (34) 21 (23) –
0.104
Perineural invasion No Yes Not available
91 72 16
35 (39) 31 (43) –
56 (61) 41 (57) –
0.553
91 74 14
62 (68) 55 (74) –
29 (32) 19 (26) –
0.384
Histological grade Well differentiated Poorly/moderated differentiated Not available
127 49 3
49 (39) 24 (49) –
78 (61) 25 (51) –
0.21
127 49 3
89 (70) 36 (73) –
38 (30) 13 (27) –
0.657
Vascular embolization No Yes Not available
109 58 12
45 (41) 23 (40) –
64 (59) 35 (60) –
0.838
109 58 12
73 (67) 44 (76) –
36 (33) 14 (24) –
0.232
p (value)
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Figure 1 Immunohistochemical analysis of CD9 and CD82 expression in oral squamous cell carcinoma (OSCC) tissue and normal oral mucosa. A and B: invasive OSCC with positive CD9 expression; C and D: invasive OSCC with downregulated CD9 expression; E and F: invasive OSCC with negative CD9 expression; G and H: normal oral epithelium with strong CD9 protein expression; I and J: invasive OSCC with positive CD82 expression; K and L: invasive OSCC with downregulated CD82 expression; M and N: invasive OSCC with negative CD82 expression; O and P: normal oral epithelium with positive CD82 expression.
CD63 and the progression of lung, breast, thyroid and pancreatic tumors.13–15 On the other hand, this molecule has been implicated in the tumor progression of melanomas in which high levels of CD63 are detected in early stages of the disease, whereas the molecules are hardly detectable or absent in later stages of tumor progression.16,17 All these reports are from tumors derived from tissues that originally did not have a stratified epithelium, which therefore differ from our present study. Tetraspanin molecules have been considered as suppressors of metastasis in solid tumors.3,6 Based on the fact that experimental transfection of CD9, CD82 and CD63 has been shown to inhibit cell motility in vitro and tumor metastasis in vivo, it is believed that these tetraspanins cause cells to become more static by modulating the function of integrins, and thus prevent the tumor invasion and metastasis.2,17–19 The CD82 protein was originally identified as metastasis suppressor gene for prostate cancer.19 Additional studies have
shown downregulation of CD82 expression in a variety of cancers, including bladder, colon, breast, gastric, endometrial and esophageal.4,20–24 Consistent with previous studies25,26 our IHC results detected a frequent detection of CD82 protein in the cellular membranes of the squamous epithelium in normal oral mucosa, but not in OSCC. Reduction/absence of CD82 protein expression was a feature observed in OSCC. In spite of these results, analysis of CD82 mRNA expression did not agree with IHC finding. The discrepancy between the results may be due to post-translational modifications or protein degradation, as the protein levels is not directly proportional to gene expression. Downregulation or loss of CD82 protein and gene expression was not associated with increased malignant behavior, diseasefree survival or overall survival. Only a marginal correlation was found between CD82 mRNA expression and the histological tumor grade, suggesting that expression of CD82 gene could be lost in
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Figure 3 Comparison between protein levels by IHC and gene expression by qRTPCR. 3a, CD9; 3b, CD82. Figure 2 Kaplan–Meier curves: (a) disease-free survival, (b) 5-year overall survival rate of patients with OSCC in relation to CD9 expression status analyzed by IHC.
Table 3 The relationship between CD9 and CD82 gene expression measured by RT-PCR and the clinicopathological features of OSCC. Clinicopathological parameters
Patients
CD9
(n)
Mean
SD
p value
CD82 Mean
SD
p value
Gender Male Female
52 14
33.75 32.57
2.38 1.36
0.838
33.94 33.94
1.48 1.31
0.718
Clinical stage I/II III/IV
30 36
32.80 34.08
2.58 2.37
0.787
35.13 32.14
2.79 2.59
0.528
Lymph node status No Yes Not dissection
27 32 07
30.59 29.50
2.09 2.47
0.808
28.33 31.41
1.69 1.35
0.494
Perineural infiltration No Yes Not available
33 27 06
33.42 26.93
2.63 1.74
0.152
31.79 28.93
1.76 0.90
0.528
Histological grade Well differentiated Poorly/moderately differentiated
39 27
29.85 38.78
1.08 3.06
0.063
37.26 28.07
1.49 1.33
0.056
Vascular embolization No Yes Not available
53 10 03
30.15 41.80
2.26 2.10
0.065
31.98 32.10
1.57 0.67
0.985
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moderated/poor differentiated tumors. Our data partially agree with others studies in which no statistical correlation was found between CD82 expression and prognosis factors of OSCC.26,27 However, Farhadieh et al.26 demonstrated a significant correlation between positive CD82 protein expression with a better diseasefree survival and overall survival, Imai et al.27 observed that gene CD82 expression was associated with the histological grade. A study by Uzawa et al.25 showed an association between regional lymph node metastasis and downregulation of CD82 protein expression in OSCC. The score used was distinct to that used in our study, and the author used a cut-off of 30% of tumors cells with loss of antigen expression to define the cases as negative. We believe that this cut-off for determining prognosis would be difficult to apply in clinical practice. Therefore, we think that, for clinical purposes, the value of 10% of cells expressing the CD82 protein as shown by IHC studies is a more appropriate cut-off, because it clearly indicates a downregulation of protein expression. Our data revealed that reduction or loss of CD9 protein expression in OSCC was associated with lymph node metastasis. OSCC with lymph node metastasis showed a greater frequency of downregulation. We could not reproduce the same result in gene expression by qRT-PCR. However, we found higher similarity between protein levels and gene expression of CD9 determined by qRT-PCR. Reduction or loss of CD9 expression correlates with poor prognosis in parotid gland tumors,28 endometrial carcinoma,29 urothelial bladder carcinoma,9 and esophageal squamous cell carcinoma,22 small cell lung cancers30 and prostate tumors.31 Corroborating our results, Kusukawa et al.11 observed that downregulation/negative CD9 protein expression in OSCC was correlate with a high incidence of lymph node metastasis and advanced tumor status, as well as correlating with the histological grade of the tumor, Erovic et al.3 showed that high expression of CD9 protein by head and neck squamous cell carcinomas (SCC) was associated with lower rates of neck lymph node metastasis and less disease recurrence. Our data suggests that downregulation/negative CD9 expression in OSCC is related to a shorter disease-free survival and overall survival when compared with patients who had positive CD9 tumors. Kusukawa et al.11 and Erovic et al.3 support this finding in patients with OSCC and head and neck SCC, respectively. In conclusion, our results demonstrate that CD37, CD63 and CD81 are not expressed in normal oral mucosa or in OSCC, and therefore they may not interfere with the tumorigenic or metastatic properties of OSCC. Additionally, the tetraspanin CD82 cannot be considered as a useful prognostic marker for OSCC. Aberrant expression of CD9 protein was found to be significantly associated with the incidence of cervical lymph node metastasis and survival. CD9 may be a supplementary tool for identifying patients who are at risk of disease progression. Conflicts of Interest Statement None declared. References 1. Kawano K, Yanagisawa S. Predictive value of laminin-5 and membrane type 1matrix metalloproteinase expression for cervical lymph node metastasis in T1 and T2 squamous cell carcinomas of the tongue and floor of the mouth. Head Neck 2006;28(6):525–33. 2. Longo N, Yanez-Mo M, Mittelbrunn M, de la Rosa G, Munoz ML, SanchezMadrid F, et al. Regulatory role of tetraspanin CD9 in tumor–endothelial cell interaction during transendothelial invasion of melanoma cells. Blood 2001;98(13):3717–26. 3. Erovic BM, Pammer J, Hollemann D, Woegerbauer M, Geleff S, Fischer MB, et al. Motility-related protein-1/CD9 expression in head and neck squamous cell carcinoma. Head Neck 2003;25(10):848–57.
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma q Marcilei Eliza Cavicchioli Buim a,*, Silvia Vanessa Lourenço b, Kátia Candido Carvalho a, Roberta Cardim c, Cláudia Pereira c, André Lopes Carvalho d, José Humberto Fregnani e, Fernando Augusto Soares a,b a
Department of Anatomical Pathology, Hospital A.C. Camargo, São Paulo, Brazil Department of General Pathology, Dental School, University of São Paulo, Brazil Department of Head and Neck Surgery, Hospital A.C. Camargo, São Paulo, Brazil d Department of Head and Neck Surgery, Barretos Cancer Hospital, São Paulo, Brazil e Department of Gynecological Oncology, Barretos Cancer Hospital, São Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 25 September 2009 Received in revised form 19 November 2009 Accepted 20 November 2009 Available online 8 January 2010 Keywords: Tetraspanin CD9 CD82 Oral squamous carcinoma Immunohistochemistry
s u m m a r y Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction One of the clinical characteristics of squamous cell carcinoma of the oral cavity (OSCC) is its capacity to invade locally and metastasize to regional lymph nodes. Identification of cervical lymph node metastasis is the main prognostic factor of patients with OSCC and has a major impact on treatment selection.1 Tetraspanins have been implicated in a wide array of cellular processes including cell adhesion, motility, intracellular signaling, cell matrix adhesion and proliferation.2–5 These molecules have also been considered as suppressors of metastasis in solid tumors.3,6
Reduced expression of tetraspanin proteins has been identified in many different types of carcinoma. Additionally, lack of expression of these proteins has been frequently reported in metastatic lesions, and patients with tumors that lack tetraspanins tend to have poorer prognoses and survival rates.7–11 The aim of this study was to analyze the patterns of expression of CD9, CD37, CD63, CD81 and CD82 antigens in OSCC and normal oral tissue by immunohistochemistry (IHC) and correlate this information with clinicopathological parameters. Based on the results from IHC, where OSCC expressed CD9 and CD82 exclusively, we also investigated the patterns of CD9 and CD82 gene expression by quantitative Real Time-polymerase chain reaction (qRT-PCR). Materials and methods
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Supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. * Corresponding author. Address: Department of Anatomical Pathology, Hospital A.C. Camargo, 109 Professor Antônio Prudente St., Zip Code 01509-900, São Paulo, Brazil. Tel./fax: +55 11 21895185. E-mail address: [email protected] (M.E.C. Buim). 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.11.009
Tissue samples and patient characteristics Surgically resected primary OSCC were collected at the Department of Head and Neck Surgery, Hospital A.C. Camargo, São Paulo,
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Brazil, between 1992 and 2004. None of the patients had received radiotherapy or chemotherapy before surgical excision. Paraffinembedded specimens from 179 patients were sampled and used to construct two tissue microarray (TMA) blocks. These blocks were then used for IHC tetraspanins analyses. Additionally, qRTPCR was performed in 66 cases (from these, 50 cases were present in the TMA blocks and 16 were additional cases included in the study). Paraffin-embedded tissue from the 16 additional cases was not accessible for IHC analysis. Follow-up information was not available from these 66 samples. Fourteen samples of normal mucosa were used as controls, ten samples for IHC analysis, and four samples for the qRT-PCR experiments. The studies were performed according to local ethical committee approval (Study No. 986/07). The patients ranged in age from 30 to 90 years old (mean age of 57 years). The clinicopathological characteristics of patients with OSCC are summarized in Table 1.
with the indirect dextran polymer detection system (Novocastra Laboratories Ltd., Newcastle, UK). Staining was performed by incubating the slides in 3,30 -diaminobenzidine tetrachloride (Dako, Carpinteria, USA). The slides were then lightly counterstained with hematoxylin, dehydrated in absolute ethanol and xylene, and then mounted with coverslips using a permanent mounting medium and observed using an optical light microscope. All IHC reactions were carried out in quadruplicate (two cores per slide, with two stained slides). The semi-quantitative analysis of the results evaluated the average area of staining in all cores (four cores each case). We considered negative or downregulated cases up to 10% of tumor cells positively staining (downregulated/negative). Positive cases were identified where over 10% of the tumor cells were stained with the marker.
TMA and immunohistochemistry
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and first-strand cDNA was synthesized from 1 lg of total RNA using High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA), according to the protocol provided by the manufacturer. Sixty-six OSCC samples and four samples of normal control oral mucosal tissue (grouped into one pool) were evaluated by qRTPCR. The CD9 (Genbank Accession Number NM_001769.2) and CD82 (Genbank Accession Number NM_002231) sequences were used as a template for the construction of the primer pairs using the 3.0 version of the PrimerExpress software (Applied Biosystems, Foster City, CA). The PCR primer set sequences were designed as follows: CD9 forward primer 50 -GCA TTG CCG TGG TCA TGA T-30 and CD9 reverse primer 50 -TGC GGA TAG CAC AGC ACA AG-30 ; CD82 forward primer 50 -CGT GGG TGT GGC CAT CAT-30 and CD82 reverse primer 50 -TTG CTG TAG TCT TCG GAA TGG-30 ; the internal control was the b-actin forward primer 50 - GCA CCC AGC ACA ATG AAG-30 and the reverse primer 50 -CTT GCT GAT CCA CAT CTG C-30 . PCR amplification was performed in duplicate in an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR reactions were performed in 20 lL volumes with 10 ng of the cDNA sample, 0.2 lM of each of the primers and 1X of the Syber-Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA). The amplification program consisted of one cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The same PCR conditions were used to amplify the b-actin gene. The relative amount of PCR product generated from each primer set was determined on the basis of the Ct value and relative quantification was calculated by a mathematical model, previously described by Pfaffl.12
The selected blocks of each sample of tumor and normal tissue were used for the construction of the TMA using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments Microarray Technology, Silver Spring, MI, USA). The sampling consisted of 2 mm cores from different areas of the tumor from a single case of OSCC, placed at a specified coordinated place. Each case and tissue of normal mucosa was spotted in duplicate. Four micrometer sections were deparaffinized and rehydrated. Antigen retrieval was performed with citrate buffer solution of sodium citrate (pH 6.0) and boiled in pressure cooker for 15 min. Slides were placed in 3% hydrogen peroxide 10 V three times for 5 min. The sections were blocked with protein block serum-free (Dako, Carpinteria, USA) at room temperature for 20 min. The sections were incubated with the primary antibodies (all from NeoMarkers-Lab Vision-Corporation, USA) at room temperature for 2 h. After being washed with phosphate buffered solution three times for 5 min each; sections were then incubated for 1 h
Table 1 Clinicopathological characteristics of patients with OSCC for IHC and qRT-PCR analysis. Clinicopathological parameters
IHC
qRT-PCR
Total (n) (%)
Total (n) (%)
Sex Male Female
145 (81) 34 (19)
52 (79) 14 (21)
Clinical stage I and II III and IV
53 (30) 126 (70)
30 (45) 36 (55)
Lymph nodes N0 N1–3 No lymph nodes dissection
73 (44) 92 (56) 14
27 (45) 32 (54) 7
Perineural invasion No Yes No available
91 (55) 74 (45) 14
33 (55) 27 (45) 6
Vascular embolization No Yes Not available
109 (65) 58 (35) 12
53 (84) 10 (16) 3
Histological grade Well-differentiated Poorly/moderately differentiated Not available
127 (72) 49 (28) 03
39 (59) 27 (41)
Isolation of RNA and qRT-PCR
Statistical analysis Association between protein expression and clinicopathological parameters were assessed by the v2-test. The disease-free survival and the overall survival curves were constructed according to the Kaplan–Meier statistical method. The Mann–Whitney test was used to determine the association between the expression of the CD9 and CD82 genes and the pathological parameters. The relationship between protein levels and genes expression was evaluated using the Mann-Whitney test. All p-values were based on two-tailed statistical analysis and p = 0.05 was considered to be statistically significant. Statistical analysis was performed using the SPSS 13.0 statistical package (SPSS Inc, Chicago, IL).
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in patients with CD9-positive expression (71.6%) (p = 0.018) (Fig. 2a). The 5-year overall survival rate of patients with downregulated/absent CD9 expression was 44% versus 64% of CD9-positive cases (p = 0.071) (Fig. 2b).
Results Immunohistochemistry analysis There was no expression of CD63, CD37 and CD81 proteins in all 179 tumors and in all 10 of the normal oral mucosa control samples analyzed. Expression of CD9 and CD82 and their relationship with the clinicopathological parameters are shown in Table 2. In normal oral mucosa, CD9 and CD82 were observed on the cellular membrane of the supra-basal and intermediate layers cells of the squamous epithelium. Expression of CD9 and CD82 was not observed on the cells of the basal lamina surface and the most superficial cells (Fig. 1G, H, O and P). Most OSCC did not express CD82; the protein was downregulated/absent in 127 of 179 (80%) OSCC cases analyzed (Fig. 1K– N). In Fig. 1I and J, CD82 was positively expressed by the cell membrane. There was no correlation between the expression of CD82 and all clinicopathological parameters tested (Table 2). Diseasefree survival of patients with downregulated/negative levels of CD82 in tumors were similar when compared to CD82 positive tumors (63.3% versus 64.4%; p = 0.705), the same results was observed in 5-year overall survival, 57.7% survival of patients with downregulated/negative CD82 expression and 58.3% survival of patients with positive CD82 expression (p = 0.549). The results of CD9 expression in OSCC are also demonstrated in Table 2. Among the 179 cases of OSCC, 75 (42%) were downregulated/negative for CD9 expression (Fig. 1C–F). Membranous expression of the marker was observed in 104 (58%) cases (Fig. 1A/B). Downregulation/absent CD9 expression was observed in lymph node metastasis; tumors that had already metastasized to lymph nodes had an increased frequency of downregulated/negative CD9 expression than tumors without lymph node metastasis (p = 0.017). The significance between CD9 expression and other clinicopathological features was not statistically established (Table 2). The disease-free survival rate of patients with downregulated/ absent CD9 expression (48.6%) was significantly lower than that
Quantitative RT-PCR analysis qRT-PCR allowed mRNA quantification of CD9 and CD82 expression in 66 OSCC cases and four normal oral mucosa (grouped into one pool). There was no statistical correlation between CD9 and CD82 mRNA expression and clinicopathological features (gender, histological grade, lymph node status, perineural invasion and vascular embolization) (Table 3). However, a marginal, but statistically insignificant correlation (p = 0.056) was found between CD82 mRNA expression and the histological grade. On the other hand, well-differentiated tumors were observed to have increased levels of CD82 mRNA expression compared to moderate or poorly differentiated tumors. Correlation between mRNA and protein expression Associated analysis of IHC and RT-PCR was possible in 50 cases. Relationship was observed between mRNA and protein expression for CD9 (p = 0.015). The mRNA expression no associated with protein expression for CD82 (p = 0.156). The comparisons between IHC and qRT-PCR evaluation of CD9 and CD82 are shown in Fig. 3.
Discussion We hypothesized that downregulation of tetraspanins could interfere with the tumorigenic and/or metastatic potential of OSCC. Our results have shown that the tetraspanins CD37, CD63 and CD81 were not expressed by cell membranes in either normal or neoplastic mucosal keratinocytes, suggesting that these proteins have no participation in the mechanisms of OSCC growth and metastasis. Other authors have reported no relationship between
Table 2 Correlation between the expression of CD9 and CD82 and clinicopathological parameters in patients with OSCC by IHC. CD9 expression Clinicopathological parameters
CD82 expression
Total (n)
Downregulated (No. of patients; %)
Positive (No. of patients; %)
p (value)
Total (n)
Downregulated (No. of patients; %)
Positive (No. of patients; %)
Gender Male Female
145 34
58 (40) 17 (50)
87 (60) 17 (50)
0.287
145 34
102 (70) 25 (74)
43 (30) 09 (26)
0.73
Clinical stage I and II III and IV
53 126
19 (36) 56 (44)
34 (64) 70 (56)
0.287
53 126
35 (66) 92 (73)
18 (34) 34 (27)
0.294
Lymph nodes N0 N1–3 Not dissected
73 92 14
23 (31) 46 (50) –
50 (69) 46 (50) –
0.017
73 92 14
48 (66) 71 (77) –
25 (34) 21 (23) –
0.104
Perineural invasion No Yes Not available
91 72 16
35 (39) 31 (43) –
56 (61) 41 (57) –
0.553
91 74 14
62 (68) 55 (74) –
29 (32) 19 (26) –
0.384
Histological grade Well differentiated Poorly/moderated differentiated Not available
127 49 3
49 (39) 24 (49) –
78 (61) 25 (51) –
0.21
127 49 3
89 (70) 36 (73) –
38 (30) 13 (27) –
0.657
Vascular embolization No Yes Not available
109 58 12
45 (41) 23 (40) –
64 (59) 35 (60) –
0.838
109 58 12
73 (67) 44 (76) –
36 (33) 14 (24) –
0.232
p (value)
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Figure 1 Immunohistochemical analysis of CD9 and CD82 expression in oral squamous cell carcinoma (OSCC) tissue and normal oral mucosa. A and B: invasive OSCC with positive CD9 expression; C and D: invasive OSCC with downregulated CD9 expression; E and F: invasive OSCC with negative CD9 expression; G and H: normal oral epithelium with strong CD9 protein expression; I and J: invasive OSCC with positive CD82 expression; K and L: invasive OSCC with downregulated CD82 expression; M and N: invasive OSCC with negative CD82 expression; O and P: normal oral epithelium with positive CD82 expression.
CD63 and the progression of lung, breast, thyroid and pancreatic tumors.13–15 On the other hand, this molecule has been implicated in the tumor progression of melanomas in which high levels of CD63 are detected in early stages of the disease, whereas the molecules are hardly detectable or absent in later stages of tumor progression.16,17 All these reports are from tumors derived from tissues that originally did not have a stratified epithelium, which therefore differ from our present study. Tetraspanin molecules have been considered as suppressors of metastasis in solid tumors.3,6 Based on the fact that experimental transfection of CD9, CD82 and CD63 has been shown to inhibit cell motility in vitro and tumor metastasis in vivo, it is believed that these tetraspanins cause cells to become more static by modulating the function of integrins, and thus prevent the tumor invasion and metastasis.2,17–19 The CD82 protein was originally identified as metastasis suppressor gene for prostate cancer.19 Additional studies have
shown downregulation of CD82 expression in a variety of cancers, including bladder, colon, breast, gastric, endometrial and esophageal.4,20–24 Consistent with previous studies25,26 our IHC results detected a frequent detection of CD82 protein in the cellular membranes of the squamous epithelium in normal oral mucosa, but not in OSCC. Reduction/absence of CD82 protein expression was a feature observed in OSCC. In spite of these results, analysis of CD82 mRNA expression did not agree with IHC finding. The discrepancy between the results may be due to post-translational modifications or protein degradation, as the protein levels is not directly proportional to gene expression. Downregulation or loss of CD82 protein and gene expression was not associated with increased malignant behavior, diseasefree survival or overall survival. Only a marginal correlation was found between CD82 mRNA expression and the histological tumor grade, suggesting that expression of CD82 gene could be lost in
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Figure 3 Comparison between protein levels by IHC and gene expression by qRTPCR. 3a, CD9; 3b, CD82. Figure 2 Kaplan–Meier curves: (a) disease-free survival, (b) 5-year overall survival rate of patients with OSCC in relation to CD9 expression status analyzed by IHC.
Table 3 The relationship between CD9 and CD82 gene expression measured by RT-PCR and the clinicopathological features of OSCC. Clinicopathological parameters
Patients
CD9
(n)
Mean
SD
p value
CD82 Mean
SD
p value
Gender Male Female
52 14
33.75 32.57
2.38 1.36
0.838
33.94 33.94
1.48 1.31
0.718
Clinical stage I/II III/IV
30 36
32.80 34.08
2.58 2.37
0.787
35.13 32.14
2.79 2.59
0.528
Lymph node status No Yes Not dissection
27 32 07
30.59 29.50
2.09 2.47
0.808
28.33 31.41
1.69 1.35
0.494
Perineural infiltration No Yes Not available
33 27 06
33.42 26.93
2.63 1.74
0.152
31.79 28.93
1.76 0.90
0.528
Histological grade Well differentiated Poorly/moderately differentiated
39 27
29.85 38.78
1.08 3.06
0.063
37.26 28.07
1.49 1.33
0.056
Vascular embolization No Yes Not available
53 10 03
30.15 41.80
2.26 2.10
0.065
31.98 32.10
1.57 0.67
0.985
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moderated/poor differentiated tumors. Our data partially agree with others studies in which no statistical correlation was found between CD82 expression and prognosis factors of OSCC.26,27 However, Farhadieh et al.26 demonstrated a significant correlation between positive CD82 protein expression with a better diseasefree survival and overall survival, Imai et al.27 observed that gene CD82 expression was associated with the histological grade. A study by Uzawa et al.25 showed an association between regional lymph node metastasis and downregulation of CD82 protein expression in OSCC. The score used was distinct to that used in our study, and the author used a cut-off of 30% of tumors cells with loss of antigen expression to define the cases as negative. We believe that this cut-off for determining prognosis would be difficult to apply in clinical practice. Therefore, we think that, for clinical purposes, the value of 10% of cells expressing the CD82 protein as shown by IHC studies is a more appropriate cut-off, because it clearly indicates a downregulation of protein expression. Our data revealed that reduction or loss of CD9 protein expression in OSCC was associated with lymph node metastasis. OSCC with lymph node metastasis showed a greater frequency of downregulation. We could not reproduce the same result in gene expression by qRT-PCR. However, we found higher similarity between protein levels and gene expression of CD9 determined by qRT-PCR. Reduction or loss of CD9 expression correlates with poor prognosis in parotid gland tumors,28 endometrial carcinoma,29 urothelial bladder carcinoma,9 and esophageal squamous cell carcinoma,22 small cell lung cancers30 and prostate tumors.31 Corroborating our results, Kusukawa et al.11 observed that downregulation/negative CD9 protein expression in OSCC was correlate with a high incidence of lymph node metastasis and advanced tumor status, as well as correlating with the histological grade of the tumor, Erovic et al.3 showed that high expression of CD9 protein by head and neck squamous cell carcinomas (SCC) was associated with lower rates of neck lymph node metastasis and less disease recurrence. Our data suggests that downregulation/negative CD9 expression in OSCC is related to a shorter disease-free survival and overall survival when compared with patients who had positive CD9 tumors. Kusukawa et al.11 and Erovic et al.3 support this finding in patients with OSCC and head and neck SCC, respectively. In conclusion, our results demonstrate that CD37, CD63 and CD81 are not expressed in normal oral mucosa or in OSCC, and therefore they may not interfere with the tumorigenic or metastatic properties of OSCC. Additionally, the tetraspanin CD82 cannot be considered as a useful prognostic marker for OSCC. Aberrant expression of CD9 protein was found to be significantly associated with the incidence of cervical lymph node metastasis and survival. CD9 may be a supplementary tool for identifying patients who are at risk of disease progression. Conflicts of Interest Statement None declared. References 1. Kawano K, Yanagisawa S. Predictive value of laminin-5 and membrane type 1matrix metalloproteinase expression for cervical lymph node metastasis in T1 and T2 squamous cell carcinomas of the tongue and floor of the mouth. Head Neck 2006;28(6):525–33. 2. Longo N, Yanez-Mo M, Mittelbrunn M, de la Rosa G, Munoz ML, SanchezMadrid F, et al. Regulatory role of tetraspanin CD9 in tumor–endothelial cell interaction during transendothelial invasion of melanoma cells. Blood 2001;98(13):3717–26. 3. Erovic BM, Pammer J, Hollemann D, Woegerbauer M, Geleff S, Fischer MB, et al. Motility-related protein-1/CD9 expression in head and neck squamous cell carcinoma. Head Neck 2003;25(10):848–57.
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