【作者】 李锋；

【导师】 应可净；

【作者基本信息】 浙江大学 ， 内科学， 2011， 博士

【摘要】 在所有恶性肿瘤中,肺癌的高发病率及高死亡率已引起国际社会的广泛重视。在美国肺癌每年新发逾20万,在中国肺癌标化发病率和死亡率均居各种恶性肿瘤之首。对肺癌的治疗现在仍然是以手术、放疗、化疗为主的综合治疗,总体而言治疗效果较差。大约一半的肺癌病人在确诊时已进入晚期,平均中位生存期只有4个月。对于早期的非小细胞肺癌患者,手术可以明显提高其生存期限。但是,即使是预后最好的Ⅰ期肺癌患者,仍然有很高的复发危险。对于这一最有可能治愈的肺癌患者群体而言,仍然有较高的术后复发率是令人非常遗憾的。若能寻找到对此类患者术后复发的某些预测因素,从而对这类患者进行筛选,采取更有力的治疗并重点监控,便可及早发现其复发或延长其术后复发的时间,对延长此类患者的生命有重要意义。肿瘤干细胞理论的提出让人们对肿瘤的发生机制有了新的认识,目前认为在全部肿瘤组织中,只有极小部分肿瘤起源细胞才具有形成肿瘤的能力。这些肿瘤起源细胞具有无限增殖、自我更新以及多向分化的能力,是肿瘤起源和形成的种子。研究肿瘤干细胞成为了解肿瘤发生、生长及转移的关键。而要研究某种类型的肿瘤干细胞,首先是需识别出这种肿瘤干细胞。目前对肿瘤干细胞的研究均使用干细胞标记物来帮助人们识别。CD133与ABCG2是广泛运用于各种肿瘤干细胞研究的表面分子标记物,如脑肿瘤干细胞、胰腺癌干细胞、结肠癌干细胞等。本研究即采用这两种标记物来研究Ⅰ期肺癌患者的手术切除组织。在本研究的第一部分中,共入选145例Ⅰ期非小细胞肺癌患者。这些患者手术切除组织均经病理证实为肺癌,且综合患者情况认定为Ⅰ期患者,对这些患者进行跟踪随访,记录其术后复发的时间。同时对这类患者的手术切除样本进行CD133、ABCG2免疫组织化学染色,结合患者的临床资料进行分析。生存时间按月计算,以末次随访所获得的结尾时间为准。术后复发时间采用Kaplan-Meier法进行单因素生存分析,log-rank法计算不同组间的生存差异。用COX风险回归进行多因素生存分析。分析该组患者的临床资料,结果发现该组患者的不同性别、发病年龄、吸烟史特点、肿瘤病理类型及分期等对患者术后复发没有明显影响。对这种患者切除组织中的GD133、ABCG2的表达情况进行分析,发现CD133有31.7%的样本中检测出阳性,ABCG2在37.9%的样本中检测出阳性。该两种标记物与患者的临床病理学特征无明显相关,而且这两种标记物的单独表达与患者的术后复发没有明显相关性,即不是对术后复发有影响的独立因素。但是如果以"CD133和ABCG2是否共同表达”为变量对患者进行重新分组,有33例患者为CD133和ABCG2共同阳性表达,其余112例患者为非共同表达,包括CD133+/ABCG2-(13例)、CD133-/ABCG2+(22例)、CD133-/ABCG2-(77例)。共同表达组在随访期内复发的有18人,未复发的有15人,非共同表达组112人中在随访期内复发的有38人,未复发的有74人。两组之间比较有显著统计学差异。并且共同表达组中,非小细胞肺癌患者平均复发时间(33月)比非共同表达组的非小细胞肺癌患者平均复发时间(51月)明显缩短,两组之间具有显著的统计学差异。对不同危险因素进行的COX分析发现CD133与ABCG2共同表达是对预后有影响的独立危险因素。CD133和ABCG2共同表达的Ⅰ期非小细胞肺癌患者术后复发的风险是非共同表达组患者的4.925倍。这一部分的研究提示Ⅰ期非小细胞肺癌患者中CD133/ABCG2共同表达阳性这一群体有更短的复发时间。因此,如果能对所有Ⅰ期非小细胞肺癌患者的切除组织进行常规CD133、ABCG2表达的检测,及早发现这一群体并给予更多的关注,包括更积极的治疗及随访,对这一有更高复发危险的患者群体有较大的临床意义。尽管第一部分的研究提示CD133+/ABCG2+的肺癌患者更易出现早期复发,但相关的病理机制尚不得而知。现有研究认为在肿瘤的侵袭转移中新生血管的形成扮演了重要角色,是肿瘤生长和转移的先决条件。肿瘤新生血管不仅提供了肿瘤生长所必需的营养,而且为转移提供了通路。动物实验显示新发生的肿瘤细胞呈线性生长,向组织浸润却很少转移。当肿瘤形成自身血管系统之后,生长开始接近指数性生长,并且癌细胞表现出明显的转移倾向,可进入血管或淋巴管,从而发生转移。尽管新生血管的形成机制较为复杂,尚未完全明晰,但是诸多的研究均观察到在肿瘤新生血管的形成中有大量促血管生成因子的释放,包括：血管内皮生长因子(VEGF)、血小板衍生生长因子(PDGF)等。许多学者的研究均报道VEGF和PDGF在肿瘤的转移及患者预后方面有重要意义。并且观察到微血管密度(MVD)在肿瘤转移及预后中的重要意义。理论上认为肿瘤微血管密度表明单位肿瘤组织中新生血管形成数量的多少,直接提示肿瘤的生长速度以及发生转移的可能性。本研究的第二部分继续以第一部分中145例Ⅰ期非小细胞肺癌患者的手术切除组织为研究对象,检测样本中VEGF与PDGF的表达差异,并通过CD34染色结果计数微血管密度。探讨不同组间的表达差异,并探讨影响患者预后的机制。结果发现在145例患者的切除组织中的VEGF在65例标本中表达,表达的阳性率为44.8%,PDGF在58例标本中表达,表达的阳性率为40%。结合患者的临床资料进行考察发现VEGF以及PDGF的表达与不同的患者性别,吸烟史的特点,肿瘤病理类型,分期以及是否复发没有明显相关性。若以CD133、ABCG2是否共表达重新分组,分为共同表达组与非共同表达组,在共同表达组的33例患者中,VEGF表达阳性有21例,占63.6%,表达阴性有12例,占36.4%,在非共同表达组的112例患者中,VEGF表达阳性的44例,占39.3%,表达阴性的68例,占60.7%,VEGF表达在两组之间具有显著的统计学差异。PDGF的检查结果发现在共同表达组中表达阳性的19例,占57.6%,表达阴性的14例,占42.4%,在非共同表达组中,表达阳性的39例,占34.8%,表达阴性的73例,占65.2%。PDGF的表达在两组之间同样具有显著的统计学差异。MVD的检测结果发现在总共145例切除组织中MVD值为68.4·23.6。统计表明MVD与不同的患者性别,吸烟史的特点,肿瘤病理类型,分期以及是否复发没有明显相关性。但在CD133/ABCG2共同表达组的33例患者中,MVD值为85.4±31.7,在非共同表达组的112例患者中,MVD值为55.34±24.5。两组之间具有显著的统计学差异。研究的第二部分是在第一部分结果的基础上从病理的角度探讨CD133/ABCG2共同表达组患者具有较短复发时间的原因,结果发现共同表达组具有更高的VEGF、PDGF及MVD检测值。这提示CD133/ABCG2共同表达组患者之所以较易复发,可能与此有关,并且从临床角度提示该组患者可能会受益于抗血管生成治疗。通过本实验研究,我们得出了如下结论：1、在本研究的145例Ⅰ期非小细胞肺癌患者的手术切除组织中CD133、ABCG2、VEGF、PDGF、CD34均有部分表达。2、结合患者的临床资料,发现在这组患者中不同的患者性别,发病年龄,吸烟史特点,肿瘤病理类型,分期,CD133表达、ABCG2表达、VEGF表达、PDGF表达以及微血管密度等对术后复发均无明显提示意义。3、以CD133和ABCG2是否共同表达对患者重新分组,发现CD133+/ABCG2+共同表达组患者的术后复发时间比非共同表达组明显缩短。多变量COX回归分析表明,CD133和ABCG2联合阳性表达是非小细胞肺癌患者术后复发的独立危险因素。4、在CD133+/ABCG2+共同表达组中,检测出VEGF、PDGF、MVD表达均显著高于非共同表达组(non-CD133+/ABCG2+).这提示CD133/ABCG2共同表达组患者之所以较易复发,可能与高表达VEGF、PDGF、MVD有关。5、本研究的临床意义在于若能对Ⅰ期非小细胞肺癌患者的切除组织进行常规CD133、ABCG2表达的检测,便能及早发现CD133+/ABCG2+这一患者群体,由于其有更短的复发时间,临床应当给予更多的关注。从病理角度也证实CD133+/ABCG2+组患者的肿瘤组织中具有更高的VEGF、PDGF及MVD检测值,从而提示该组患者可能会受益于抗血管生成治疗。更多还原

【Abstract】 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.更多还原