| High incidence and mortality rate of lung cancer in all malignant tumors has been attracted extensive attention of the international community. In the United States, more than 200,000 cases were diagnosed each year. And the incidence and mortality of lung cancer in China is creasing, at present ranking the first in all malignant tumors. The treatment of lung cancer up to now is still combined with surgery, radiotherapy and chemotherapy, but the treatment effect is poor. About half of the total patients were in advanced stage when diagnosed, and the median survival time is only 4 months. For non-small cell lung cancer (NSCLC) patients in early stage, surgery can significantly prolong the survival period. Even though the patients in early stage have better prognosis, a high risk of recurrence still exists in these patients. If we can find some predictors of recurrence, then find these patients out and give them more effective treatment and follow up, we can extend the period of recurrence or detect the recurrence earlier. It has important significance for these patients.Cancer stem cell theory proposes that only a very small part of the tumor tissue, called tumor-initiating cells, has the ability to form tumors. These tumor-initiating cells have the ability of unlimited proliferation, self-renewal and multilineage differentiation. The tumor stem cell is the key to tumor’s growth and metastasis. The first and most important question is how to recognize cancer stem cells. Cancer stem cells markers are widely used to help researchers identify them in current studies. CD133 and ABCG2 are the most common surface molecular markers of CSC, and widely used in a variety of cancer stem cell researches, such as brain tumor stem cells, pancreatic stem cells, colon cancer stem cells, et al. In this study, these two markers were used to detect the resected tissue of NSCLC patients in stage I.In the first part of this study,145 patients with NSCLC were enrolled. These patients’tissues were pathologically proven lung cancer, and identified as stage I according to their clinical records. These patients were followed up until relapse occurred and the time was recorded. At the same time, CD133 and ABCG2 were identified in these patients’resected tissues by immunohistochemical staining, and analyzed combined with their clinical data. Recurrence-free survival time was record on a basis of’"month", and the maximum time was obtained at the last follow-up. Postoperative recurrence time was evaluated using the Kaplan-Meier method with log-rank analysis. T-test and chi-square test were used to evaluate the difference between groups. Multiple risk factors were analyzed by COX proportional hazards analysis.The analysis of these patients’clinical data showed in this group the recurrence postoperative has no relationship with the patients’different sex, age, smoking history, tumor pathology or tumor staging. CD133 and ABCG2 were detected positive in 31.7% and 37.9% of total surgical samples respectively. Neither of the two markers’expression has obvious correlation with patients’clinicopathological features including recurrence. Thus, both of them are not independent risk factors which can affect prognosis. However, if the patients were divided according to whether CD133 and ABCG2 expressed simultaneously, the results were found as following:CD133 and ABCG2 co-expressed in 33 patients and 112 patients remained as non-co-expression, including CD133+/ABCG2-(13 cases), CD133-/ABCG2+(22 cases) and CD133-/ABCG2-(77 cases). In co-expression group,18 cases were found relapsed during follow-up period and 15 cases not; among 112 cases of non-co-expression group,38 cases relapsed and 74 not. Between the two groups there was a significantly statistic difference. The average recurrence time of co-expression group (33 months) is obviously shorter than that of non-co-expression group (51 months). COX proportional hazards analysis reveals that the co-expression of CD 133 and ABCG2 is an independent risk factor which can affect prognosis. The risk of recurrence in patients with co-expression of CD 133 and ABCG2 is 4.925 times than that in patients with non-co-expression.Part I suggests NSCLC patients with CD133/ABCG2 co-positive have a shorter time to relapse. Therefore, if all NSCLC patients in stage I were routinely detected CD 133 and ABCG2 expression, and give them more intensive treatment and follow up, the prognosis of patients prone to relapse should be improved greatly.While the first part of this research suggests that patients with CD133+/ABCG2+ more likely relapsed, the pathological mechanism is unknown. Present studies reveal that new blood vessels play an important role in tumor growth and metastasis. Tumor angiogenesis not only provides necessary nutrients for tumor growth, but also provides a pathway for metastasis. In animal experiments, the linear growth form of tumor cells was observed before new blood vessels formation and rarely transferred. Once tumor tissues formed their own vascular system, they began to approach an exponential growth form, and the cancer cells showed obvious tendency to enter the blood or lymphatic vessels, which occurred in the metastasis.Although the mechanism of tumor angiogenesis is more complex and not fully clear yet, a lot of studies observed a large number of angiogenic factors released in tumor angiogenesis, including vascular endothelial growth factor (VEGF); platelet derived growth factor (PDGF) and so on.It has been reported by many scholars that VEGF and PDGF have important significance in tumor metastasis and are related with prognosis. Microvessel density (MVD) is usually used to research with VEGF and PDGF. The density of microvessel in tumor tissue directly prompts the transfer speed and the possibility of recurrence.The second part of this study is to investigate why the CD133+/ABCG2+ patients have shorter postoperative relapse time. The expression of VEGF and PDGF were detected by immunohistochemical staining and microvessel density was evaluated by CD34 staining. The differences between different groups were statistically analyzed. It was found that VEGF was detected in 65 cases in all 145 specimens and PDGF was positive in 58 specimens, positive rate 44.8% and 40%, respectively. The expression of VEGF and PDGF did not correlate with patients’clinicopathological characteristics including gender, onset age, smoking history, histological tumor type, stage, and recurrence rate in NSCLC. In CD133+/ABCG2+ co-expression group, VEGF was positive in 21 cases and negative in 12 cases. In the non co-expression group, VEGF was positive in 44 cases, accounting for 39.3% of total 112 patients, negative in 68 cases, accounting for 60.7%. The expression of VEGF was statistically different between the two groups. This result was same to the expression of PDGF. PDGF was detected positive in 19 cases of all CD133+/ABCG2+ co-expression patients, accounting for 57.6%, negative in 14 cases, accounting for 42.4%; in the non-CD 133+/ABCG2+ group, the examination was positive in 39 cases, accounting for 34.8%, negative in 73 cases, accounting for 65.2%. The expression of PDGF equally was statistically different between the two groups. MVD evaluated by CD34 staining was calculated in all 145 specimens and the average value was 68.4±23.6. Statistics analysis showed that the MVD did not correlate with patients’gender, onset age, smoking history, histological tumor type, stage, and recurrence rate. However, in CD133+/ABCG2+group, MVD was 85.4±31.7, significantly different from its value 55.3±24.5 in non-co-expression group.PartⅡof this study investigated the reason that the patients with CD133/ABCG2 co-expression have a shorter time to recurrence. The results showed that the patients in co-expression group were found higher VEGF, PDGF and MVD level. This suggests that stage I NSCLC with dual overexpression of CD 133 and ABCG2 may benefit from anti-angiogenic therapy.Conclusions:1. In this study,145 patients with stage I NSCLC were enrolled and CD133, ABCG2, VEGF, PDGF, CD34 were detected positive in part of their tissues.2. The expression of single variant, including CD133, ABCG2, VEGF, PDGF, and MVD, did not correlate with any patients’clinicopathological characteristics including gender, onset age, smoking history, histological tumor type, stage, and recurrence rate.3. If the patients were categorized into two subgroups, that is CD133+/ABCG2+ NSCLC and non-CD133+/ABCG2+NSCLC, the recurrence time was obviously shorter in co-expression group than in non-co-expression group. And multivariate COX analysis revealed conjoined positive expression of CD133 and ABCG2 is an independent predictor of postoperative recurrence for patients with stage I NSCLC.4. Further analysis demonstrated that CD133+/ABCG2+NSCLC patients had a significantly higher microvessel density (MVD) and higher frequencies of VEGF and PDGF overexpression than non-CD133+/ABCG2+NSCLC patients.5. Our findings suggest that synchronous co-expression of CD133 and ABCG2 in stage I NSCLC predicts early recurrence and high levels of angiogenesis. And suggest that stage I NSCLC with dual overexpression of CD133 and ABCG2 deserves more intensive anti-cancer therapy, and further angiogenesis-targeted therapy in this subtype should be considered. |
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