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Keywords:

  • laryngeal neoplasms;
  • squamous cell carcinoma;
  • metadherin;
  • metastasis;
  • recurrence

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Metadherin (MTDH) is involved in tumourigenesis and cancer progression in multiple human malignancies. However, the MTDH protein has rarely been reported in laryngeal squamous cell carcinoma (LSCC). The expression pattern of the MTDH protein in 176 primary archival LSCC and 27 corresponding adjacent noncarcinoma specimens was detected by immunohistochemistry and further correlated with clinicopathological parameters. The results demonstrated that 161 (91.48%) primary LSCC samples stained positive for MTDH; however, staining was barely detectable in all adjacent noncarcinoma samples. Moreover, the expression of the MTDH protein was significantly associated with the primary tumour site (p = 0.021), T classification (p = 0.002), clinical stage (I + II/III + IV; p < 0.001), lymph node metastasis (p < 0.001) and postoperational recurrence (p < 0.001). Kaplan-Meier analysis revealed that MTDH expression was significantly associated with worse disease-free survival (DFS) and overall survival (OS) rates in patients with LSCC (both p < 0.001). When lymph node metastasis and MTDH expression were considered together, patients with lymph node metastasis and high MTDH expression had both poorer DFS and OS rates than others (both p < 0.001). Finally, multivariate analysis demonstrated that MTDH expression was an independent prognostic factor for both DFS and OS rates in patients with LSCC. Strong MTDH expression was negatively correlated with a canonical epithelial–mesenchymal transition molecule E-cadherin (p < 0.001) and positively associated with proangiogenic protein vascular endothelial growth factor (p < 0.001). MTDH overexpression was tightly associated with more aggressive tumour behaviour and a poor prognosis, indicating that MTDH is a valuable molecular biomarker for LSCC progression.

Laryngeal squamous cell carcinoma (LSCC), accounting for ∼1.5% of all cancers, is the most common malignancy arising in the upper airway in adults.[1, 2] Although patients with LSCC benefit from advanced diagnostic and therapeutic management, including routine surgery, chemotherapy, radiotherapy and biological therapy, outcomes for these patients have remained unsatisfactory during the last few decades. Cervical lymph node metastasis and postoperational recurrence have been reported to be the most decisive factors causing the dismal prognosis in patients with LSCC after surgical resection.[3, 4] However, the molecular mechanism of cancer initiation and progression remains unclear. Therefore, identifying sensitive and specific molecular markers that can predict patients with biologically aggressive neoplasms is of great significance for developing prospective strategies for the prevention and management of patients with LSCC.

Metadherin (MTDH, also identified as astrocyte-elevated gene-1/AEG-1 and lysine-rich CEACAM-1-associated protein/Lyric) was originally identified as a protein induced in primary human fetal astrocytes infected with HIV-1 or treated with HIV envelope glycoprotein (gp120) or tumour necrosis factor-alpha.[5-8] MTDH functions by regulating the NF-κB signalling pathway.[9] MTDH is also a downstream target molecule of Ha-ras and c-myc and mediates their pro-tumour effects, suggesting its potential roles in tumour initiation and development.[10, 11] In normal immortal cloned rat embryo fibroblast cells, the ectopic overexpression of MTDH has induced morphological transformation and enhanced invasion and anchorage-independent growth in soft agar and has formed aggressive tumours in nude mice, demonstrating that MTDH alone can function as an oncogene for rodent cells.[12] MTDH protein has been reported to be directly regulated by miR-375 (Ref. [13]) or miR-136 (Ref. [14]), and MTDH itself is present in the RNA-induced silencing complex and regulates the mRNA expression of various tumour suppressors.[15] Aberrant MTDH expression is prevalent in various human solid neoplasms, including those of breast cancer,[16] prostate,[17] glioma,[18] oesophageal squamous cell carcinoma,[19] hepatocellular carcinoma[20] and gastric cancer.[21] Moreover, high MTDH expression is associated with aggressive cancer phenotypes, such as advanced clinical stage, increased metastatic ability and decreased patient survival, revealing MTDH as a potential prognostic biomarker in a panel of human malignancies. Recent in vitro studies revealed that MTDH is significantly increased in nasopharyngeal carcinoma (NPC) samples.[13] LSCC is distinct from NPC by its epidemiology, histopathology, clinical characteristics, methods of treatment and patterns of failure.[22] The expression pattern of MTDH protein in LSCC and especially the prognostic significance of MTDH protein in LSCC thus remain to be elucidated.

To clarify the clinical implication of MTDH in LSCC, we investigated the expression of MTDH protein in 176 LSCC tissue samples and further assessed whether the expression of MTDH protein was associated with clinicopathological variables and prognosis in patients with LSCC. Finally, considering that both epithelial–mesenchymal transition[23] (EMT) and angiogenesis[24] are well-known processes involved in tumour metastasis, we assessed the expression of E-cadherin (a typical molecular biomarker associated with EMT) and vascular endothelial growth factor (VEGF, a potent protein promoting angiogenesis) in the same patient cohort to potentially provide mechanistic insight into the roles of MTDH in LSCC.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

LSCC tissue specimens

A series of 176 primary LSCC tissue specimens and information from patients who received partial or total laryngectomy at the Department of Otolaryngology in Xiangya Hospital, Central South University, Changsha, Hunan, China, were available and collected. These cases ranged from January 2000 to October 2005. Patients with LSCC enrolled in our study had to follow these inclusion criteria: no history of previous radiotherapy or chemotherapy and primary squamous cell carcinoma of the larynx without other malignancies. The main clinical and pathological variables of the patients are described in Supporting Information Table S1. Informed consent was obtained from all patients with LSCC before surgery, and all experiments were conducted adhering to the bioethics rules issued by the Research Ethics Committee of Central South University, Changsha, China.

Immunohistochemistry

Immunohistochemical staining was performed as described previously.[25-27] In brief, antigen retrieval was carried out in 10 mmol/l citrate buffer (pH = 6.0) in a microwave oven for 15 min. The activity of endogenous peroxidase was exhausted with 3% hydrogen peroxide for 10 min at room temperature. Rabbit MTDH polyclonal antibody (Proteintech Group, Chicago, IL), rabbit VEGF monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit E-cadherin polyclonal antibody (Abcam, Cambridge, MA) were applied overnight at 4°C at optimal working concentration of 1:600, 1:200 and 1:400, respectively. After sufficient phosphate buffered saline rinses, sections were immunostained with horseradish peroxidase-labelled goat anti-rabbit polymers. Finally, positive staining of MTDH, VEGF and E-cadherin protein was visualised with diaminobenzidine, and the cell nucleus was counterstained with Mayer's hematoxylin. For negative controls, the primary antibody was replaced with normal goat serum (Santa Cruz Biotechnology) or the rabbit anti-MTDH antibody was blocked with a recombinant human MTDH protein (Abnova, Taipei City, Taiwan) by coincubation at 4°C overnight under the same experimental conditions.

Evaluation of staining of MTDH, VEGF and E-cadherin

All immunohistochemical slides were independently and blindly assessed and scored by investigators (G. Li and S. Fan). If a disagreement occurred, the slides were re-examined to obtain a final consensus. A staining index (SI) of MTDH ranged from 0 to 12 was finally determined as the product of staining intensity and proportion of immunopositive cancer cells according to previously published criteria.[19] Staining intensity was classified as 0 to 3: 0 point meant no intensity, 1 point meant weak intensity, 2 points meant moderate intensity and 3 points meant strong intensity. In addition, the proportion of immunopositive cancer cells was ranked into four groups: <10% = 1, 10–34% = 2, 35–70% = 3 and >70% = 4. The expression level of MTDH was categorised as low expression (SI = 0–5) and high expression (SI = 6–12) based on the final SI values, in which the cutoff value for high and low MTDH expression was determined by measuring heterogeneity with statistical analysis of log-rank test regarding the overall survival.

The SI of VEGF protein was determined by the sum of percentage of positive cytoplasmic cells (≤25% = 1, 26–50% = 2 and ≥51% = 3) and staining intensity (0 point = no intensity, 1 point = weak intensity, 2 points = moderate intensity and 3 points = strong intensity), which was described by Mattern et al.[28] and in our previous report.[29] Meantime, as reported previously,[30] the SI of membranous E-cadherin staining was obtained by the combination of percentage of positive cells (≤10% = 1, 11–50% = 2, 51–80% = 3 and ≥81% = 4) and staining intensity (0 point = no intensity, 1 point = weak intensity, 2 points = moderate intensity and 3 points = strong intensity). Low expression was classified as SI = 0–3 in VEGF evaluation and SI = 0–3 in E-cadherin results, whereas high expression was defined as SI = 4–6 in VEGF and SI = 4–7 in E-cadherin.[28, 30]

Follow-up

In this patient cohort, clinical physical examination, imaging MRI or CT evaluation and pathological investigations were used to confirm local recurrence and metastasis. One hundred seventy-three patients with LSCC had a full-range postoperational follow-up, and intact clinical information was obtained. Only three patients failed to follow-up because their contact information was changed. Overall survival time (OS) and disease-free survival time (DFS) calculation was based on the duration from the day of surgery to the day of death or tumour relapse. Death of patients with LSCC resulting from other causes was defined as censored cases.

Statistical analysis

All statistical analyses were performed with SPSS 17.0 software package. The χ2 test was used to evaluate the associations between various clinicopathological parameters and the expression of MTDH protein. Kaplan-Meier survival analysis was used to assess the significance of MTDH protein alone or together with other clinical factors. The significant differences between survival curves were determined by the log-rank test. Multivariate analysis for independent prognostic indicators was performed via establishing the Cox proportional hazards model. The correlations among the expression of MTDH, E-cadherin and VEGF were determined using Spearman's rank correlation coefficient. A p-value < 0.05 was considered to be statistically significant in a two-tailed test.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Expression pattern of MTDH protein in archival LSCC specimens

To clarify the distribution of MTDH protein in LSCC, 176 paraffin-embedded, archival primary LSCC specimens and 27 corresponding adjacent noncancerous specimens were first detected using immunohistochemistry. We used normal goat serum to replace MTDH antibody and coincubated MTDH antibody with recombinant MTDH polypeptide to confirm the specificity of the MTDH antibody applied in our experiment because negative MTDH staining was obtained. MTDH protein was detected in 161 (91.48%) primary LSCC tumour samples. However, MTDH protein was barely detectable in all the corresponding adjacent noncarcinoma samples (Fig. 1a). As demonstrated in Figure 1, positive MTDH protein staining was predominantly distributed in the cytoplasm of LSCC cancer cells. Among these tumour samples, negative MTDH protein staining was observed in 15 specimens (8.52%; Fig. 1b), low expression in 60 specimens (34.09%; Fig. 1c) and high expression in 101 specimens (57.39%; Fig. 1d). Although MTDH staining was mostly found in the cytoplasm of primary cancer cells, a minority of LSCC tumour cells in 12 specimens with cervical lymph node metastasis (6.82%) also stained in the nucleus (Fig. 1d, red arrows indicated) and in the perinuclear region (Fig. 1d, black arrows indicated). The interesting observations indicated a potential mechanism of MTDH protein nuclear translocation in LSCC metastasis.

image

Figure 1. Representative immunohistochemical staining for MTDH protein in primary LSCC tissues and adjacent noncarcinoma epithelial tissues. Negative expression of MTDH protein in (a) adjacent noncarcinoma epithelial tissues and (b) primary LSCC specimens. (c) Low expression of MTDH protein in primary LSCC specimens. (d) High expression of MTDH protein in primary LSCC specimens. Red arrows indicate nuclear staining, and black arrows indicate perinuclear staining. (a–d) Original magnification, ×400.

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Associations between the expression of MTDH protein and clinicopathological parameters

Based on the above finding that MTDH protein was overexpressed in a large proportion of LSCC tumours, the expression of MTDH protein was further correlated with major clinicopathological characteristics of patients with LSCC. As summarised in Table 1, MTDH protein expression level was positively correlated with primary tumour site (p = 0.021), T classification (T1 + T2 vs. T3 + T4; p = 0.002), clinical stage (I + II vs. III + IV; p < 0.001), lymph node metastasis (p < 0.001) and postoperational recurrence (p < 0.001). However, no significant correlation was observed between the expression of MTDH protein and parameters such as gender (p = 0.225), age (p = 0.326), alcohol consumption (p = 0.370), smoking history (p = 0.312) and histological grade (p = 0.783).

Table 1. Correlations between the expression of MTDH protein and clinicopathological parameters in patients with LSCC
 Number ofMTDH expression  
ParameterspatientsLowHighχ2 valuep-valuea
  1. a

    p ≤ 0.05 was considered to be statistically significant, in which significant p-values were indicated in bold.

  2. b

    Three patients lost to follow-up because of telephone number changes or home moving.

Age     
<588533520.9660.326
≥58914249  
Gender     
Male17071991.4700.225
Female642  
Alcohol consumption    
Yes8632540.8030.370
No703139  
Unknown20128  
Smoking history    
Yes10343601.0220.312
No582929  
Unknown15312  
Histological grade    
G111046640.0760.783
G2 + G3662937  
Primary tumour site    
Glottic11757605.3180.021
Others591841  
T classification     
T1 + T29149429.7210.002
T3 + T4852659  
Clinical stage     
I–II72442817.048<0.001
III–IV1043173  
Lymph node status    
N0110624822.678<0.001
N+661353  
Recurrenceb     
Yes89533621.075<0.001
No842163  

Prognostic significance of MTDH protein in the survival of patients with LSCC

Of the 176 patients with LSCC, 173 had complete clinical outcome information available for further survival analysis. On the basis of the final SI, we categorised the whole cohort of patients with LSCC into two groups: high (n = 99) and low (n = 74) MTDH expression. Kaplan-Meier survival analysis demonstrated that patients with high MTDH expression had increased risk of overall recurrence (p < 0.001; Fig. 2a) and mortality (p < 0.001; Fig. 2b) in patients with LSCC. Log-rank tests further confirmed that the difference between 5-year DSF and OS rates in these two groups was statistically significant. Univariate Cox regression analyses determined that LSCC cancer site, clinical stage, T classification, metastasis, MTDH protein, VEGF protein and E-cadherin protein were significantly associated with both DSF and OS. Additionally, alcohol intake was also correlated with OS (Table 2). However, only metastasis and MTDH protein were finally determined to be independent factors with prognostic value for both DSF (p = 0.008, P = 0.019) and OS (p = 0.003, p = 0.016) in patients with LSCC via the multivariate Cox analyses (Table 2).

image

Figure 2. Kaplan-Meier survival analysis of (a) DSF and (b) OS in all patients, according to the expression of MTDH protein. The log-rank test was used to calculate the p value.

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Table 2. The disease-free survival and overall survival Cox regression analysis
 Disease-free survivalOverall survival
 Relative risk (95% CI)p-valueaRelative risk (95% CI)p-valuea
  1. a

    p-values in bold were statistically significant. Abbreviation: 95% CI: 95% confidence interval.

Univariate    
Gender0.596 (0.147–2.424)0.5230.658 (0.162–2.680)0.559
Age1.108 (0.721–1.703)0.6400.966 (0.619–1.507)0.880
Smoking history1.014 (0.640–1.605)0.9541.047 (0.649–1.687)0.852
Alcohol intake1.377 (0.871–2.178)0.1711.726 (1.046–2.799)0.027
Histological grade1.017 (0.653–1.584)0.9421.007 (0.636–1.594)0.975
Primary tumour site0.314 (0.203–0.484)<0.0010.284 (0.181–0.446)<0.001
T classification1.755 (1.135–2.712)0.0112.040 (1.294–3.215)0.002
Clinical stage3.175 (1.915–5.263)<0.0013.999 (2.301–6.950)<0.001
Metastasis4.821 (3.068–7.576)<0.0015.668 (3.511–9.150)<0.001
MTDH expression0.333 (0.202–0.546)<0.0010.304 (0.179–0.517)<0.001
VEGF expression2.058 (1.244–3.405)0.0051.942 (1.157–3.260)0.012
E-cadherin expression0.523 (0.340–0.805)0.0030.517 (0.330–0.809)0.004
Multivariate    
Metastasis2.622 (1.282–5.362)0.0083.209 (1.483–6.947)0.003
MTDH expression0.507 (0.288–0.894)0.0190.452 (0.237–0.864)0.016

Stratified survival analyses according to MTDH expression

We further analysed the prognostic significance of MTDH in patients with different T classifications and different clinical stages. Our data revealed that the DSF and OS time in patients with LSCC with high MTDH expression was obviously shorter than that of patients with relatively low MTDH expression, in the T1–T2 patient group (p = 0.001, p = 0.002; Figs. 3a and 3b), the T3–T4 patient group (p = 0.012, p = 0.010; Figs. 3c and 3d) and the early (p = 0.006, p = 0.008; Figs. 3e and 3f) and advanced (p = 0.017, p = 0.012; Figs. 3g and 3h) clinical stage groups. In this respect, MTDH protein seems to be a valuable molecular marker for prognosis in both early- and late-stage patients with LSCC.

image

Figure 3. Stratified survival analysis according to T classification and clinical stage. DSF analysis in (a) T1 + T2 and (c) T3 + T4 patients, according to the expression of MTDH protein. OS analysis in (b) T1 + T2 and (d) T3 + T4 patients, according to the expression of MTDH protein. DSF analysis in (e) early clinical stage and (g) advanced clinical stage patients, according to the expression of MTDH protein. OS analysis in (f) early clinical stage and (h) advanced clinical stage patients, according to the expression of MTDH protein. The log-rank test was used to calculate the p value.

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Combined survival analyses according to MTDH expression

It has been widely accepted that lymph node metastasis is a known unfavourable predictor for patients with LSCC.[26, 31] Our data also demonstrated the prognostic value of lymph node metastasis, which was negatively correlated with both DSF and OS time (both p < 0.001; Figs. 4a and 4b and Table 2). Therefore, we combined the expression of MTDH protein and lymph node metastasis status to enhance the capacity to prognosticate. Our data revealed that patients with LSCC with a phenotype of high MTDH expression and positive lymph node metastasis had worse DSF and OS than others (both p < 0.001; Figs. 4c and 4d).

image

Figure 4. Kaplan-Meier survival analysis of (a) DFS and (b) OS in all patients, according to the lymph node metastasis status. Analysis of (c) DFS and (d) OS in all patients, according to the expression of MTDH and the lymph node metastasis status. The log-rank test was used to calculate the p value.

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Expression of VEGF, E-cadherin and their correlations with MTDH

To further study the potential roles of MTDH protein in LSCC cancer progression, the expression patterns of VEGF and E-cadherin were analysed in the above-mentioned archival LSCC specimens. As shown in Supporting Information Figure 5a, VEGF protein was mainly observed in the tumour cell cytoplasm, and E-cadherin was stained predominantly in tumour cell membranes or cytoplasm. Furthermore, MTDH expression was found to be positively correlated with the expression of VEGF (r = 0.309, p < 0.001) and negatively associated with E-cadherin expression (r = −0.329, p < 0.001) by the Spearman's rank test (Supporting Information Figs. 5b and 5c). These results support the potential roles of MTDH in the processes of LSCC angiogenesis and EMT.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The aberrant expression of MTDH has been previously reported in diverse human malignancies, including breast, glioma, prostate, melanoma and oesophageal cancers.[16-21] MTDH is a versatile molecule, implicated in multiple cancer malignant processes including proliferation,[32] chemoresistance,[33, 34] protective autophagy[35] and metastasis.[5, 34] To the best of our knowledge, this is the first study to explore the prognostic value of MTDH protein in LSCC and to report that the upregulated expression of MTDH protein is a predictor of different aggressive tumour behaviours such as advanced clinical stage and metastasis and post-operational recurrence in patients with LSCC. The significant association between MTDH protein expression and tumour origin sites has never been reported previously, which was not the case in our study. A plausible explanation for this result is that superaglottic and subglottic carcinoma tends to be much more susceptible to cervical lymph node metastasis,[36] which is consistent with the positive correlation between MTDH expression and metastasis.

Although close links between the expression of MTDH and lymph node metastasis in patients with LSCC has been established in current investigation, determining possible molecular mechanisms will require deeper investigation. Our data clearly revealed that MTDH expression was found to be negatively associated with E-cadherin expression (a typical molecular biomarker associated with EMT) and positively correlated with the expression of VEGF (a potent protein promoting angiogenesis). The result was consistent with recent publications in breast[37] and hepatocellular carcinoma,[38] in which ectopic MTDH overexpression could significantly enhance the invasion and migration of tumour cells by inducing EMT. Additionally, MTDH has been reported to be involved in the process of tumour angiogenesis in both in vitro and in vivo conditions.[12, 39, 40]

In our investigation, MTDH is predominantly cytoplasmic in tumour cells except for that a minority of LSCC specimens (12/176, 6.82%) presented with nuclear and perinuclear staining of MTDH protein. The function of MTDH in different cellular locales remains unclear. Recent report demonstrates that cytoplasmic MTDH can act as an RNA-binding protein to regulate the expression of drug resistance-associated proteins.[41] As to nuclear MTDH, we have to note that all samples with nuclear and perinuclear staining in our study were metastatic. This observation coincides with previous publications in breast cancer, in which advanced clinical stage sections display noticeably increased MTDH nuclear translocation and a large proportion of cancer cells in liver metastases reveal MTDH translocation to the nucleus.[16] Similar positive correlations between MTDH nuclear staining and both advanced stages and poor prognosis were also reported for hepatocellular carcinoma and melanoma.[42] MTDH was found to directly interact with p65 subunit of NF-κB complex and to modulate the function of NF-κB in nucleus, indicating that MTDH protein is decisively involved in the regulating process of gene expression mediated by the NF-κB signalling pathway.[9, 43] Paradoxically, the fact that nuclear staining of MTDH correlates with a favourable prognosis in prostate cancer was also reported.[44] The authors of the previous study hypothesised that nuclear MTDH might have a function in normal prostate epithelial cells that is lost during tumourigenesis.[44] These controversial results reflect the complexity of regulation function of nuclear MTDH protein. It is possible that nuclear MTDH protein performs context-specific functions depending on the microenvironment of cells and the type of the tumour. These hypotheses need to be elucidated in future.

Currently, outcome prediction is still based on critical clinical parameters, such as the clinical phase, tumour sites and histopathologic study. Recent advances in molecular biology of human malignancies provide a hint that the discovery molecular abnormalities may facilitate early diagnosis and prognosis prediction.[45] Our study revealed that MTDH protein level was significantly negatively associated with the postoperational prognosis in patients with LSCC. When stratified according to tumour T classification and clinical stage, MTDH protein expression also had good prognostic capability in both early- and late-stage patients with LSCC.

Our current data, together with previous reports,[3, 4] suggested that lymph node metastasis was inversely correlated with the prognosis for patients with LSCC. Survival analysis via combining cervical lymph node metastasis and MTDH protein status showed that patients with LSCC with both high MTDH expression and positive lymph node metastasis had worse prognosis than that of patients with LSCC with other phenotypes. These results suggested that much more powerful prognosis predictions for patients with LSCC can be obtained by considering lymph node metastasis and MTDH protein together, which may contribute to appropriate decisions about management strategies.

In summary, our data demonstrated that aberrant MTDH protein expression was tightly associated with aggressive clinical behaviours in human LSCC, indicating MTDH as a useful biomarker for prognosis in LSCC. Additionally, based on in vitro and in vivo studies and numerous expression analysis using patient specimens, emerging evidence demonstrates that MTDH plays diverse roles in the process of tumourigenesis in multiple organs, indicating that MTDH presents as an attractive therapeutic target. Therefore, further intensive research in both in vitro and in vivo conditions is being conducted in our laboratory to identify the exact functional mechanisms of MTDH in the process of malignant transformation in LSCC.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ijc28071-sup-0001-suppfig5.jpg2144KSupplementary Figure 5. Expression of VEGF, E-cadherin in LSCC samples and their correlations with MTDH. (a) Representative immunohistochemical staining for VEGF and E-cadherin in LSCC samples. Original magnification, ×200 (b) Correlations between MTDH expressions in LSCC and VEGF expression. (c) Correlations between MTDH expressions in LSCC and E-cadherin expression.
ijc28071-sup-0002-supptab1.doc42KSupporting information Table 1. Clinicopathological features of the studied 176 cases of LSCC.

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