| ObjectiveThe clinical significance of vasculogenic mimicry (VM) was investigated. The related mechanisms of epithelial-to-mesenchymal transition (EMT) in VM formation and worsening malignant degree of pulmonary sarcomatoid carcinoma (PSC) were examined.MethodsClinical characteristics and prognosis of22PSC patients and79lung squamous cell carcinoma (LSCC) patients who had not undergone chemotherapy or radiotherapy prior to surgery between1997and2007were reviewed. Formalin-fixed and paraffin-embedded samples from the patients were obtained from the Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, China. The expression of VM, Twistl, E-cadherin, Vimentin in pulmonary sarcomatoid carcinoma tissues and lung squamous cell carcinoma were detected by HE, CD31/periodic acid Schiff double-staining and Immunohistochemistry. Moreover, the essential immunostainings, such as CK8/18, CK5/6, p63and Vimentin, were detected to ensure the origin of pulmonary sarcomatoid carcinoma. We further confirmed the correlation of Twistl with VM by comparing Twistl expression and the positive rates of VE-cadherin, EphA2and MMP-2. The clinical significance of VM and the mechanism of EMT promoting VM formation was explored.Results1. The origin of pulmonary sarcomatoid carcinomaIn the carcinomatous components of pulmonary sarcomatoid carcinoma, positive CK5/6or p63expression was observed in18cases, CK8/18expression in2cases, both p63and CK8/18at different locations in2cases, and vimentin in9cases.. In the sarcomatous components, positive expressions of CK5/6(or p63) and Vimentin were found in17and22cases, respectively. Positive squamous carcinoma makers were found in most sarcomatous components. 2. Expressions of EMT-associated makers were examined in PSC and LSCC.In PSC tissue samples, the positive rate of Twistl, E-cadherin, Vimentin was50%,36.4%and90.9%, respectively. However, in LSCC tissue samples, the positive rate of Twistl, E-cadherin, Vimentin was35.6%,75.9%,20.3%, respectively. Statistical analysis revealed a significant difference in the Twistl, E-cadherin and Vimentin expressions between the PSC and LSCC tissue samples (P=0.037, P=0.000, P=0.000). Comparing to LSCC group, vimentin and Twistl expressions were found to be higher in the PSC group, and the down-expression of E-cadherin was more frequently detected in the PSC group.3. Relationship between VM, Twistl and clinical and pathological features in PSC.We identified VM both in PSC and LSCC patients. A Kaplan Meier survival analysis revealed that patients with VM had a shorter survival period than those without VM expression (Log-rank x2=4.544, P=0.033; x2=5.846, P=0.016). However, no correlation was found between the presence of VM and patient characteristics, such as age, gender, tumor size, lymth node metastasis, recurrence (P=0.622, P=0.226, P=0.368, P=0.073, P=0.818). The VM-positive group had a higher rate of hematogenous metastasis (2/4,50%) than the VM-negative group (0/18,0%)(P=0.026). VM expression was also significant in TNM stages III and IV (4/10,40%) than in TNM stages â… and â…¡,(0/12,0%)(P=0.029). No correlation was found between the Twistl expression and patient characteristics, such as age, gender, tumor size, hematogenous metastasis, recurrence and TNM stage (P=0.330, P=0.500, P=0.500, P=0.238, P=0.500, P=0.528). Importantly, Twistl overexpression correlated significantly with lymph node metastasis (P=0.040). We identified VM in4(18.2%) out of22PSC patients. The frequency of VM was significantly higher in PSC (4/22,18.2%) than in lung squamous cell carcinoma (LSCC;4/79,5.1%) as control (X2=4.06, P=0.044).4. Vasculogenic mimicry (VM) was associated with the expression of Twistl and VM relevant makers.The expressions of Twistl and VE-cadherin, EphA2, MMP-2, VEGF, Hif-la were examined using IHC to investigate whether a differential expression exists between the VM positive and VM negative in PSC tissue samples. Statistical analysis revealed a significant difference (P=0.045, P=0.029. P=0.045, P=0.046) in the Twistl, VE-cadherin, EphA2, MMP-2, expressions of the VM-positive (4/11,36.4%;4/10,40%;4/11,36.4%and3/6,50%;respectively) and VM-negative groups (0/11,0%;0/12,0%;0/11,0%and1/15,6.7%, respectively). However, no correlation was found in the VEGF and Hif-la expressions of the VM-positive (3/4,75%and0/4,0%) and VM-negative groups (10/18,55.6%and2/18,11.1%.)(P=0.616; P=0.662).5. Twistl expression was correlated to VE-cadherin, EphA2, and MMP-2in PSC.We further confirmed the correlation of Twistl with VM by comparing Twist expression and positive rates of VE-cadherin, EphA2and MMP-2and found a significant value (Positive rates:11/22,50%;10/22,45.5%;11/22,50%;6/22,27.3%. Spearman correlation test; rs=0.638, P<0.05, rs=0.694, P<0.05, rs=0.669, P<0.05). The correlation between VE-cadherin and EphA2was positive (rs=0.470, P<0.05). In addition, the correlation between EphA2and MMP-2was positive (rs=0.548, P <0.05).Conclusion1. Of the22sections of PSC, most carcinomatous components were squamous carcinoma and squamous carcinoma makers were positive in many sarcomatous components. Therefore, spindle cell morphology of sarcomatous-like components may have originated form EMT.2. EMT processes are crucial for the progress of embryonic development and VM channels form patterns similar to those of the embryonic vasculogenic network. Using hematoxylin-eosin (HE) staining and CD31/PAS double-staining, VM was distinguished by channels lined with tumor cells instead of shuttle-like endothelial cells. The total survival period of patients with VM in PSC was found to be significantly shorter than that of patients without VM. The VM-positive group had a higher rate of hematogenous metastasis than the VM-negative group. Possiblely because of the EMT, the frequency of VM was significantly higher in PSC than in lung squamous cell carcinoma. Coincident with Twistl induced-EMT in PSC, VM formation increased compared with LSCC, indicating that the interaction between VM and EMT leads to more malignant PSC. Importantly, Twistl overexpression correlated significantly with lymph node metastasis in PSC. EMT has been associated with invasion and metastasis rate. Our results confirm that transcription factor Twistl induced EMT by up-regulation of Vimentin and down-regulation of E-cadherin. EMT regulator Twistl can stimulate carcinoma metastasis by up-regulation of Vimentin and down-regulation of E-cadhern.3. Vasculogenic mimicry (VM) was associated with VE-cadherin, EphA2, MMP-2high expression in PSC. VE-cadherin/EphA2/PI3K/MMP was a important pathway of VM formation. Twistl expression was correlated to VE-cadherin, EphA2, and MMP-2in PSC. After detection, our results confirm that EMT regulator Twistl induced VM changes by enhancing the expressions of the VE-cadherin, EphA2and MMP-2. |
