LC3A在食管鳞癌中的放化疗敏感性预测作用
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摘要
第一部微管相关蛋白LC3A在食管鳞癌中的表达及意义
研究背景及目的：自噬是指细胞内需降解的长寿命蛋白质和细胞器等胞浆成分被包裹,并运送至溶酶体降解的过程,自噬性降解产生的氨基酸和其他一些小分子物质可被循环利用或产生能量。巨自噬即通常所指自筮。自噬从酵母到人是高度保守的。自噬这一从酵母到哺乳动物高度保守细胞死亡方式的基础作用在于维持细胞内环境稳定,但在生长因子缺乏、非折叠蛋白聚集、营养缺乏等不良刺激下,自噬作用被诱导增强。自噬常常作为不良应激的反应机制发挥有利于细胞存活的保护作用。大量研究已证实自噬参与了肿瘤发生、发展,其在肿瘤治疗中的作用越来越受到重视。目前研究发现自噬在肿瘤细胞“存与亡”中发挥着重要作用,但肿瘤细胞自噬激活后导致完全不同的结果,有时导致肿瘤细胞存活,而有时促进肿瘤细胞死亡,其原因可能是由于诱导自噬增强的应激类型的不同和肿瘤类型的差异。然而,多数研究认为自噬发挥着使肿瘤细胞抵抗不良应激,有助于肿瘤细胞存活的作用。进一步研究显示自噬激活也是肿瘤细胞抵抗药物毒性、抵抗电离照射的重要机制之一。所以,抑制自噬则成为抗肿瘤治疗的潜在靶点。微管相关蛋白轻链3(Microtubule-associated protein light chain3,MAPLC3A/LC3A)是自噬体重要组成部分,LC3蛋白在细胞内存在两种形式：LC3-Ⅰ和它的蛋白水解物LC3-Ⅱ,LC3在自噬过程中发挥重要作用,其表达及由LC3-Ⅰ到LC3-Ⅱ的转化与自噬活性相关,且与肿瘤预后相关。LC3有三种亚型,LC3A,LC3B,LC3C。LC3A作为LC3的重要亚型,被证明是自筮激活的重要标志且与肺癌、结直肠癌等多种肿瘤预后相关。LC3A在食管鳞癌(ESOphagea(?) squamous cell carcinoma,ESCC)的表达及意义未见报道。该研究通过分析LC3A在ESCC组织中表达及其与临床病理因素、预后的关系,旨在探索ESCC预后指标并为制定ESCC个体化治疗提供依据,进一步提高食管鳞癌治疗效果。
资料与方法：根据病案资料选取滨州医学院附属医院2005年1月至2008年12月间收治的食管鳞癌根治术病例243例。所有患者手术前未行任何抗肿瘤治疗,手术为根治性手术。其中Ⅱ/Ⅲ(TanyN+MO or T3,4NanyMO)期患者209例,158例术后行以放疗为主的辅助治疗。对于同步放化疗患者,顺铂联合氟尿嘧啶(Cisplatin and fluorouracil, DF)方案为同步化疗方案。放疗/放化疗后序贯化疗方案包括DF方案和紫杉类药物联合顺铂(Taxanes and cisplatin, TP)方案。免疫组化方法(Immunohistochemistry, IHC)检测石蜡包埋肿瘤组织中LC3A及P53蛋白表达,根据LC3A蛋白表达强弱,分为高表达及低表达。分析LC3A蛋白在ESCC中表达模式及其重要自噬凋亡共同调节基因p53蛋白表达关系,以及LC3A表达在ESCC临床病理因素及预后中的作用。
结果：1.LC3A在食管鳞癌组织中呈现胞浆内弥漫性表达,LC3A阳性率为52.7%。
2.LC3A表达与年龄、性别、体重减轻及肿瘤位置、肿瘤侵犯深度、分期及淋巴结转移无明显相关性。
3.LC3A与p53蛋白在ESCC中表达为独立事件,无关联性(p=0.9772)。
4.LC3A低表达ESCC组3、5年局部控制率分别为58.2、47.0%,而LC3A高表达组3、5年局部控制率则分别为46.1%、39.5%。LC3A低表达组与高表达局部控制率差异无统计学意义(p=0.082),但LC3A低表达组局部控制率有高于C3A高表达组倾向。
5.LC3A低表达组3、5年总生存率分别为49.2%、30.5%,LC3A高表达组3、5年总生存率分别为41.7%、26.5%,LC3A表达同预后无关；COX单因素及多因素分析显示T分期、N分期、临床分期、手术根治性及术后辅助治疗与预后相关,LC3A非独立预后因素。
6.将243例患者中,209例Ⅱ/Ⅲ期(TanyN+MO or T3,4NanyMO)患者进行亚组分析发现,对于未行术后放疗51例患者,LC3A高低表达两组预后差异无统计学意义。而对于158例行术后放疗为主辅助治疗者,LC3A低表达者预后明显优于LC3A高表达者(p<0.05)。158例术后行放疗为主辅助治疗Ⅱ/Ⅲ期病例例进一步亚组分析发现,LC3A High+RT组局部控制率低于LC3A Low+RT组,差异有统计学意义(p=0.01)。
结论：LC3A在ESCC组织中呈弥漫胞浆表达,其表达与浸润深度、淋巴结转移情况及分期无关。放疗为主的术后辅助治疗在LC3A低表达患者中获益更大,其局部控制率及总生存率高于LC3A高表达ESCC,提示LC3A表达水平与术后放疗疗效相关。
第二部分DC方案联合放疗治疗非手术ESCC疗效评价及微管相关蛋白LC3A对放化疗敏感性的预测作用
研究背景及目的：食管癌为最常见恶性肿瘤之一,其发病率高,预后差。包括我国在内的发展中国家中,食管癌更为常见,我国每年大约15万人死于食管癌,占世界食管癌死亡病例40%以上。早期食管癌给以根治性手术后可以获得较好的预后,而对于多数中晚期食管癌患者而言,单纯手术5年生存率仅20%左右。同步放化疗是非手术局部晚期食管癌标准治疗模式。作为治疗非手术食管癌的重要初始治疗手段,其5年生存率仅为30%左右,局部复发为最常见治疗失败原因,局部未控和复发率高达20-80%。且单一提高放射治疗剂量并未改善患者预后,优化同步化疗方案是进一步提高疗效的重要途径。近年来基于第三代化疗药物作为同步化疗方案的治疗模式得到逐步探索。研究已初步证实紫杉类联合铂类作为同步化疗方案治疗食管癌的可行性,初步研究显示有效,但耐受性不一,故化疗药物剂量及用法尚需进一步探讨。同一部位肿瘤,甚至相同分化程度、相同组织来源肿瘤对于放化疗敏感性存在差异,说明肿瘤细胞内在分子生物学特性影响了放射敏感性。肿瘤生物学行为预测指标是目前肿瘤研究的热点之一。尽管控制食管癌发病、决定其生物学行为的具体基因及确切机制尚未明确,但大量研究已证实涉及细胞周期调节、凋亡、DNA损伤修复机制、生长因子的许多基因突变及表达异常与肿瘤发生、发展密切有关,如p53、p21、Cyclin D、BRCA1、HIF-1等。近年来,自噬在食管鳞癌中的作用备受关注。体外实验研究显示抑制自噬功能,能够提高食管鳞癌细胞的放射敏感性,证实自噬在肿瘤细胞抵抗照射中起到保护肿瘤细胞的作用。研究显示LC3A作为重要自噬相关蛋白,在包括肺癌、结直肠癌在内多种肿瘤中广泛高表达,且与肿瘤预后相关。第一部分研究发现微管相关蛋白LC3A高表达食管鳞癌患者术后放化疗/放疗后局部控制率低于LC3A低表达者,提示LC3A表达同食管癌对放化疗敏感性有关。该部分旨在通过研究LC3A表达与食管鳞状细胞癌放化疗疗效之间的关系,探索LC3A蛋白表达在食管鳞癌放化疗敏感性中的作用。同时观察多西他赛顺铂(Docetaxel and cisplatin,DC)方案小剂量多次化疗联合放疗治疗食管鳞癌的可行性。
资料与方法：2011年11月至2012年10月，滨州医学院附属医院肿瘤科收治非手术食管癌患者65例。所有入组患者胃镜活检病理确诊为食管鳞癌，KPS评分不小于80分，无手术指证或者患者拒绝手术，且无放化疗禁忌。根据中国非手术治疗食管癌临床分期专家小组制定的“非手术治疗食管癌临床分期”系统分期。该组患者中位年龄62.1岁(42～70岁)。三维适形技术常规分割放疗，放疗中位剂量56.5Gy (44Gy-60Gy)。同步行DC方案化疗，多西他赛20mg/m2，顺铂25mg/m2于d1、d8、d15、d26、d33、d40给药。根据进食困难缓解作为主观疗效评价，食管钡餐造影作为客观疗效评价。并根据CTCAE3.0评估治疗中毒副反应。免疫组化方法检测食管癌活检组织中LC3A蛋白表达情况。分析DC方案联合放疗在ESCC中的近期疗效及耐受性，并分析LC3A同ESCC放化疗近期疗效的关系。
结果：
1.(1) DC方案为基础的同步放化步疗治疗非手术ESCC患者，主观有效率即进食困难缓解率为88.7%(55/162)。评分为0分，即无进食吞咽困难患者由治疗前3.1%(2/65)增加至50.0%(32/64)。
(2)DC方案化疗联合放疗治疗ESCC患者，食管钡餐造影评价完全缓解率(CR)48.4%(31/64)，部分缓解率(PR)为42.2%(27/64)，客观有效率(ORR)为90.6%(58/64)。
2.放射性食管炎及粒细胞减少是基于DC方案同步放化疗的最常见副反应。95.4%(62/65)患者出现放射性食管炎，轻度(1/2度)发生率为73.9%(48/65),3度放射性食管炎发生率为21.5%(14/65)。粒细胞减少发生率为61.6%(40/65),但多数为轻度(1/2度)粒细胞减少症，重度(3度以上)粒细胞减少症发生率为3.1%(2/65)。
3. LC3A低表达组ESCC患者CR率为58.1%(18131),明显高LC3A高表达组38.2%(13/34)，二者差异有统计学意义(p<0.001)。
结论：基于DC方案同步放化疗治疗ESCC有效，且毒性可以耐受。LC3A蛋白可以作为放化疗敏感的预测因子。
Part Ⅰ:The Expression and Significance of LC3A in Esophageal Squamous Cell Carcinoma
Objective:
Autophagy is a selfdegradation process in which intracell membrane structures sequester the superfluous and damage long-life proteins and organelles that are then delivered to lysosomes for degradation. The selfdigestion surpplies materials(such as amino acids) and energy. Macroautophagy is the usually referred autophagy. Autophagy is an evolutionarily conserved process from yeast to mammalians. The basal role of autophagy is to maintain homeostasis. In growth factors deprivation, accumulation of unfolded proteins, hungery induced stresses, autophagy is up-regulated and acts as a cytoprotective mechanism. It has been shown that autophagy is involved in tumorigenesis, tumor development, and the role of it in tumor therapy has attracted more and more attention. A lot of evidence has proven that in some life-threatening stresses, autophagy is activated and the activation leads to different fates with the kinds of stresses and tumor types. The general notion is that autophagy contributes to tumor cell survival during stresses and anti-cancer therapies. The autophagy activation makes tumor cells more resistant to anti-cancer drugs and ionizing radiation. Autophagy inhibition is a potential anticancer strategy. Microtubule-associated protein light-chain3(LC3)is one important component of the autophagosomes which has three isoforms,LC3A,LC3B,and LC3C. When autophagy occurs, cytosolic form LC3-Ⅰ converts to membrane form LC3A-Ⅱ.This change in intacellular localization of LC3protein correlates with autophagy activity. As one of LC3three isoforms and marker of autophagy activity, LC3A correlates well with the prognosis of a lot kinds of malignancies, such as lung cancer, colon cancer. This study aims to analyze the relationships between LC3A expression and clinicopathological factors, prognosis, and explores the prognostic value of LC3A in esophageal squamous cell carcinoma(ESCC).
Materials/Methods:
From our clinical archives, I selected243ESCC patients who were treated with radical surgery in the Department of Oncology of the Affiliated Hospital of Binzhou Medical College between January2005and December2008.All patients had no any treatment before radical surgery.158patients with stage Ⅱ/Ⅲ (TanyN+Mo or T3.4NanyMo) ESCC underwent adjuvant concurrent chmoradiotherapy, sequential chemoradiotherapy, or radiotherapy alone after surgery. Tissue specimens which obtained from paraffin-embedded tumor tissues were assessed immunohistochemically for LC3A, P53. Based on the immunostaining,LC3A expression was separated into high and low groups. The associations of LC3A eexpression with p53expression and with clinicopathological factors, prognosis were sought.
Results:
1. Immunohistochemical analysis showed that LC3A overexpression was observed in52.7%(128/243) of tumors. Moreover, LC3A mainly localized in the cytoplasm of tumor cells. In contrast to previous reports, LC3A expression was not observed in perinuclear,'stone-like'structures (SLS) in ESCC samples.
2. EC3A immunostaining was not linked to clinicopathological factors, such as sex, weight loss, age, tumor location, and, cell differentiation.
3. Immunohistochemical analysis showed that p53nuclear overexpression was observed in55.6%(135/243) of tumors. The correlation coefficient between LC3A and p53was-0.0184(P=0.9772)
4. The3-year and5-year local control rates were58.2%and47.0%, respectively, in the LC3A lower expression group and46.1%and39.5%, respectively, in the LC3A overexpression group. The difference was not statistically significant(p=0.082).
5The survival analysis shows LC3A was not a prognostic marker, while the T stage, N stage, clinical stage, radical surgery and adjuvant therapy correlated with prognosis.
6. Of the209patients with stage Ⅱ/Ⅲ(TanyN+M0or T3,4NanyMO),158patients underwent radiotherapy-based therapy after surgery. For this subgroup, Kaplan-Meier curves indicated that the prognosis of the patients with LC3A overexpression was poorer than that of LC3A lower expression(p<0.05). Moreover, in the158patients with adjuvant radiotherapy, the local control rate(LCR) in patients with LC3A overexpression was lower than that with LC3A low expression (p=0.01)。
Conclusions:
LC3A is expressed in the cytoplasm in ESCC tissues. None of the factors(such as T stage, lymph node metastasis) correlates with LC3A immunostaining. The LCR in patients with LC3A overexpression and adjuvant radiotherapy is lower than that in patients with LC3A low expression and adjuvant radiotherapy. The results above indicate LC3A is a indicator of the sensitivity of concurrent chemoradiotherapy in esophageal squamous cell Carcinoma.
Part II:Outcome of concurrent chemoradiotherapy with docetaxel/cisplatin in esophageal squamous cell carcinoma and the predictive significance of LC3A in ESCC sensitivity to concurrent chemoradiotherapy.
Objective:
Esophageal carcinoma (EC) is one of the most common malignancies worldwide, with poor outcome. In developing countries including China, EC has a higher incidence. There are150,000patients who died of EC, accounting for40%patients in the world. Early EC patients with surgery get good prognosis, while for most patients with advanced EC, the treatment outcome of surgery only is poor, with20%of5-year overall survival rate. Concurrent chemoradiotherapy is the standard treatment modality for nonoperative locally advanced EC patients. For this modality, the5-year overall survival rate of patients with locally advanced EC is30%, and the local relapse is the most common cause. Moreover, the prognosis does not improve by increasing the radiation dose. So finding more efficient chemotherapy regimen is a important way to improve the treatment outcome. Recently, a lot of studies explored the therapeutic effect of the third generation chemotherapeutics in concurrent chemoradiotherapy patterns. Taxanes with platinum has been proven to be an effective regimen, while the the doses and administration methods needs further confirmation. The indicators of tumor biological characteristics are another research hotspot. Although the mechanisms of tumorigenesis and development in ESCC is unknown, a great deal of evidence has shown that the mutation and abnormal expression of cell cycle related, DNA repair related and apoptosis related genes, such as p53,p21,Cyclin D, are involved in the occurrence and development of tumors. In recent years, the role of autophagy in ESCC has attracted more and more attention. Chen et al found that autophagy inhibition improved the radiosensitivity of esophageal squamous cells in vitro. EC3A is an important component in autophagosome. and highly expressed in many types of tumors. The result of part one has shown that ECR in patients with LC3A overexpression and adjuvant radiotherapy was lower than that in patients with LC3A low expression and adjuvant radiotherapy. This demonstrated LC3A was a prognostic factor of radiosensitivity in ESCC. In the following study, the association between LC3A expression and the efficacy of concurrent chemoradiotherapy was explored, and the feasibility of DC chemotherapy combined with radiotherapy was evaluated in patients with ESCC.
Materials and methods:
Sixty-five patients with ESCC were selected in the Department of Oncology of the Affiliated Hospital of Binzhou Medical College between November2011and Octobor2012.All eligible ESCC patients had the following conditions:diagnosed by endoscopic biopsies; were not suitable or refused operation; KPS greater than80;no contraindication of chemoradiotherapy. The classification of clinical stage was determined according to the Chinese staging criteria for nonoperative esophageal carcinoma. The median age of these patients was62.1years(42～70years).Three-dimensional comformal technique was used in radiotherapy, and the median radiation dose was56.5Gy(44～60Gy). Synchronous chemotherapy was DC(docetaxel20mg/m2and cisplatin25mg/m2,IV dl,8,15,26,33,40).The subjective treatment response was evaluated according to dysphagia remission, and objective response was evaluated according to esophageal barium meal. Toxicities were graded according to the Common Terminology Criteria for Adverse Events v3.0. LC3A protein expression was assessed immunohistochemically. In this study, they were evaluated of the efficacy and the toxicities of the DC regimen combined with radiation and the association between LC3A immunohistochemical expression and the sensitivity toconcurrent chemoradiotherapy in ESCC patients.
Results:1.(1) Subjective Response:For ESCC patients with combined therapy of DC chemotherapy and synchronous radiotherapy, dysphagia relief was observed in55of62patients(88.7%).Seven of the55patients with dysphagia relief were able to drink liquid diet before treatment and eat solid without any dysphagia. After treatment, the rate of patients without dysphagia increased from3.1%(2/65)to50.0%(32/64). (2)Objective response:Based on barium meal, the CR rate was48.4%(31/64), PR rate42.2%(27/64), and the objective response rate(ORR)was90.6%(58/64).
2. Esophagitis and granulocytopenia were the most frequent acute toxicities in the concurrent chemoradiotherapy. Of the65patients,62/65patients in the study group had esophagitis,and mild esophagitis(grade1/2) was73.9%(48/65), and grade3was21.5%(14/65).With regard to granulocytopenia, it occurred in61.6%(40/65) patients,while only3.1%(2/65) patients had grade3granulocytopenia.
3.Based on LC3A expression, the CR rate of the patients with low LC3A expression was58.1%(18/31),while that of the patients with LC3A overexpression was38.2%(13/34).The difference between the two subgroup was statistically significant(p<0.001).
Conclusions:The combined treatment modality of DC chemotherapy with synchronous radiotherapy is effective and tolerable in ESCC patients.LC3A is an indicator of the CRT sensitivity.
研究背景及目的：自噬是指细胞内需降解的长寿命蛋白质和细胞器等胞浆成分被包裹,并运送至溶酶体降解的过程,自噬性降解产生的氨基酸和其他一些小分子物质可被循环利用或产生能量。巨自噬即通常所指自筮。自噬从酵母到人是高度保守的。自噬这一从酵母到哺乳动物高度保守细胞死亡方式的基础作用在于维持细胞内环境稳定,但在生长因子缺乏、非折叠蛋白聚集、营养缺乏等不良刺激下,自噬作用被诱导增强。自噬常常作为不良应激的反应机制发挥有利于细胞存活的保护作用。大量研究已证实自噬参与了肿瘤发生、发展,其在肿瘤治疗中的作用越来越受到重视。目前研究发现自噬在肿瘤细胞“存与亡”中发挥着重要作用,但肿瘤细胞自噬激活后导致完全不同的结果,有时导致肿瘤细胞存活,而有时促进肿瘤细胞死亡,其原因可能是由于诱导自噬增强的应激类型的不同和肿瘤类型的差异。然而,多数研究认为自噬发挥着使肿瘤细胞抵抗不良应激,有助于肿瘤细胞存活的作用。进一步研究显示自噬激活也是肿瘤细胞抵抗药物毒性、抵抗电离照射的重要机制之一。所以,抑制自噬则成为抗肿瘤治疗的潜在靶点。微管相关蛋白轻链3(Microtubule-associated protein light chain3,MAPLC3A/LC3A)是自噬体重要组成部分,LC3蛋白在细胞内存在两种形式：LC3-Ⅰ和它的蛋白水解物LC3-Ⅱ,LC3在自噬过程中发挥重要作用,其表达及由LC3-Ⅰ到LC3-Ⅱ的转化与自噬活性相关,且与肿瘤预后相关。LC3有三种亚型,LC3A,LC3B,LC3C。LC3A作为LC3的重要亚型,被证明是自筮激活的重要标志且与肺癌、结直肠癌等多种肿瘤预后相关。LC3A在食管鳞癌(ESOphagea(?) squamous cell carcinoma,ESCC)的表达及意义未见报道。该研究通过分析LC3A在ESCC组织中表达及其与临床病理因素、预后的关系,旨在探索ESCC预后指标并为制定ESCC个体化治疗提供依据,进一步提高食管鳞癌治疗效果。
资料与方法：根据病案资料选取滨州医学院附属医院2005年1月至2008年12月间收治的食管鳞癌根治术病例243例。所有患者手术前未行任何抗肿瘤治疗,手术为根治性手术。其中Ⅱ/Ⅲ(TanyN+MO or T3,4NanyMO)期患者209例,158例术后行以放疗为主的辅助治疗。对于同步放化疗患者,顺铂联合氟尿嘧啶(Cisplatin and fluorouracil, DF)方案为同步化疗方案。放疗/放化疗后序贯化疗方案包括DF方案和紫杉类药物联合顺铂(Taxanes and cisplatin, TP)方案。免疫组化方法(Immunohistochemistry, IHC)检测石蜡包埋肿瘤组织中LC3A及P53蛋白表达,根据LC3A蛋白表达强弱,分为高表达及低表达。分析LC3A蛋白在ESCC中表达模式及其重要自噬凋亡共同调节基因p53蛋白表达关系,以及LC3A表达在ESCC临床病理因素及预后中的作用。
结果：1.LC3A在食管鳞癌组织中呈现胞浆内弥漫性表达,LC3A阳性率为52.7%。
2.LC3A表达与年龄、性别、体重减轻及肿瘤位置、肿瘤侵犯深度、分期及淋巴结转移无明显相关性。
3.LC3A与p53蛋白在ESCC中表达为独立事件,无关联性(p=0.9772)。
4.LC3A低表达ESCC组3、5年局部控制率分别为58.2、47.0%,而LC3A高表达组3、5年局部控制率则分别为46.1%、39.5%。LC3A低表达组与高表达局部控制率差异无统计学意义(p=0.082),但LC3A低表达组局部控制率有高于C3A高表达组倾向。
5.LC3A低表达组3、5年总生存率分别为49.2%、30.5%,LC3A高表达组3、5年总生存率分别为41.7%、26.5%,LC3A表达同预后无关；COX单因素及多因素分析显示T分期、N分期、临床分期、手术根治性及术后辅助治疗与预后相关,LC3A非独立预后因素。
6.将243例患者中,209例Ⅱ/Ⅲ期(TanyN+MO or T3,4NanyMO)患者进行亚组分析发现,对于未行术后放疗51例患者,LC3A高低表达两组预后差异无统计学意义。而对于158例行术后放疗为主辅助治疗者,LC3A低表达者预后明显优于LC3A高表达者(p<0.05)。158例术后行放疗为主辅助治疗Ⅱ/Ⅲ期病例例进一步亚组分析发现,LC3A High+RT组局部控制率低于LC3A Low+RT组,差异有统计学意义(p=0.01)。
结论：LC3A在ESCC组织中呈弥漫胞浆表达,其表达与浸润深度、淋巴结转移情况及分期无关。放疗为主的术后辅助治疗在LC3A低表达患者中获益更大,其局部控制率及总生存率高于LC3A高表达ESCC,提示LC3A表达水平与术后放疗疗效相关。
第二部分DC方案联合放疗治疗非手术ESCC疗效评价及微管相关蛋白LC3A对放化疗敏感性的预测作用
研究背景及目的：食管癌为最常见恶性肿瘤之一,其发病率高,预后差。包括我国在内的发展中国家中,食管癌更为常见,我国每年大约15万人死于食管癌,占世界食管癌死亡病例40%以上。早期食管癌给以根治性手术后可以获得较好的预后,而对于多数中晚期食管癌患者而言,单纯手术5年生存率仅20%左右。同步放化疗是非手术局部晚期食管癌标准治疗模式。作为治疗非手术食管癌的重要初始治疗手段,其5年生存率仅为30%左右,局部复发为最常见治疗失败原因,局部未控和复发率高达20-80%。且单一提高放射治疗剂量并未改善患者预后,优化同步化疗方案是进一步提高疗效的重要途径。近年来基于第三代化疗药物作为同步化疗方案的治疗模式得到逐步探索。研究已初步证实紫杉类联合铂类作为同步化疗方案治疗食管癌的可行性,初步研究显示有效,但耐受性不一,故化疗药物剂量及用法尚需进一步探讨。同一部位肿瘤,甚至相同分化程度、相同组织来源肿瘤对于放化疗敏感性存在差异,说明肿瘤细胞内在分子生物学特性影响了放射敏感性。肿瘤生物学行为预测指标是目前肿瘤研究的热点之一。尽管控制食管癌发病、决定其生物学行为的具体基因及确切机制尚未明确,但大量研究已证实涉及细胞周期调节、凋亡、DNA损伤修复机制、生长因子的许多基因突变及表达异常与肿瘤发生、发展密切有关,如p53、p21、Cyclin D、BRCA1、HIF-1等。近年来,自噬在食管鳞癌中的作用备受关注。体外实验研究显示抑制自噬功能,能够提高食管鳞癌细胞的放射敏感性,证实自噬在肿瘤细胞抵抗照射中起到保护肿瘤细胞的作用。研究显示LC3A作为重要自噬相关蛋白,在包括肺癌、结直肠癌在内多种肿瘤中广泛高表达,且与肿瘤预后相关。第一部分研究发现微管相关蛋白LC3A高表达食管鳞癌患者术后放化疗/放疗后局部控制率低于LC3A低表达者,提示LC3A表达同食管癌对放化疗敏感性有关。该部分旨在通过研究LC3A表达与食管鳞状细胞癌放化疗疗效之间的关系,探索LC3A蛋白表达在食管鳞癌放化疗敏感性中的作用。同时观察多西他赛顺铂(Docetaxel and cisplatin,DC)方案小剂量多次化疗联合放疗治疗食管鳞癌的可行性。
资料与方法：2011年11月至2012年10月，滨州医学院附属医院肿瘤科收治非手术食管癌患者65例。所有入组患者胃镜活检病理确诊为食管鳞癌，KPS评分不小于80分，无手术指证或者患者拒绝手术，且无放化疗禁忌。根据中国非手术治疗食管癌临床分期专家小组制定的“非手术治疗食管癌临床分期”系统分期。该组患者中位年龄62.1岁(42～70岁)。三维适形技术常规分割放疗，放疗中位剂量56.5Gy (44Gy-60Gy)。同步行DC方案化疗，多西他赛20mg/m2，顺铂25mg/m2于d1、d8、d15、d26、d33、d40给药。根据进食困难缓解作为主观疗效评价，食管钡餐造影作为客观疗效评价。并根据CTCAE3.0评估治疗中毒副反应。免疫组化方法检测食管癌活检组织中LC3A蛋白表达情况。分析DC方案联合放疗在ESCC中的近期疗效及耐受性，并分析LC3A同ESCC放化疗近期疗效的关系。
结果：
1.(1) DC方案为基础的同步放化步疗治疗非手术ESCC患者，主观有效率即进食困难缓解率为88.7%(55/162)。评分为0分，即无进食吞咽困难患者由治疗前3.1%(2/65)增加至50.0%(32/64)。
(2)DC方案化疗联合放疗治疗ESCC患者，食管钡餐造影评价完全缓解率(CR)48.4%(31/64)，部分缓解率(PR)为42.2%(27/64)，客观有效率(ORR)为90.6%(58/64)。
2.放射性食管炎及粒细胞减少是基于DC方案同步放化疗的最常见副反应。95.4%(62/65)患者出现放射性食管炎，轻度(1/2度)发生率为73.9%(48/65),3度放射性食管炎发生率为21.5%(14/65)。粒细胞减少发生率为61.6%(40/65),但多数为轻度(1/2度)粒细胞减少症，重度(3度以上)粒细胞减少症发生率为3.1%(2/65)。
3. LC3A低表达组ESCC患者CR率为58.1%(18131),明显高LC3A高表达组38.2%(13/34)，二者差异有统计学意义(p<0.001)。
结论：基于DC方案同步放化疗治疗ESCC有效，且毒性可以耐受。LC3A蛋白可以作为放化疗敏感的预测因子。
Part Ⅰ:The Expression and Significance of LC3A in Esophageal Squamous Cell Carcinoma
Objective:
Autophagy is a selfdegradation process in which intracell membrane structures sequester the superfluous and damage long-life proteins and organelles that are then delivered to lysosomes for degradation. The selfdigestion surpplies materials(such as amino acids) and energy. Macroautophagy is the usually referred autophagy. Autophagy is an evolutionarily conserved process from yeast to mammalians. The basal role of autophagy is to maintain homeostasis. In growth factors deprivation, accumulation of unfolded proteins, hungery induced stresses, autophagy is up-regulated and acts as a cytoprotective mechanism. It has been shown that autophagy is involved in tumorigenesis, tumor development, and the role of it in tumor therapy has attracted more and more attention. A lot of evidence has proven that in some life-threatening stresses, autophagy is activated and the activation leads to different fates with the kinds of stresses and tumor types. The general notion is that autophagy contributes to tumor cell survival during stresses and anti-cancer therapies. The autophagy activation makes tumor cells more resistant to anti-cancer drugs and ionizing radiation. Autophagy inhibition is a potential anticancer strategy. Microtubule-associated protein light-chain3(LC3)is one important component of the autophagosomes which has three isoforms,LC3A,LC3B,and LC3C. When autophagy occurs, cytosolic form LC3-Ⅰ converts to membrane form LC3A-Ⅱ.This change in intacellular localization of LC3protein correlates with autophagy activity. As one of LC3three isoforms and marker of autophagy activity, LC3A correlates well with the prognosis of a lot kinds of malignancies, such as lung cancer, colon cancer. This study aims to analyze the relationships between LC3A expression and clinicopathological factors, prognosis, and explores the prognostic value of LC3A in esophageal squamous cell carcinoma(ESCC).
Materials/Methods:
From our clinical archives, I selected243ESCC patients who were treated with radical surgery in the Department of Oncology of the Affiliated Hospital of Binzhou Medical College between January2005and December2008.All patients had no any treatment before radical surgery.158patients with stage Ⅱ/Ⅲ (TanyN+Mo or T3.4NanyMo) ESCC underwent adjuvant concurrent chmoradiotherapy, sequential chemoradiotherapy, or radiotherapy alone after surgery. Tissue specimens which obtained from paraffin-embedded tumor tissues were assessed immunohistochemically for LC3A, P53. Based on the immunostaining,LC3A expression was separated into high and low groups. The associations of LC3A eexpression with p53expression and with clinicopathological factors, prognosis were sought.
Results:
1. Immunohistochemical analysis showed that LC3A overexpression was observed in52.7%(128/243) of tumors. Moreover, LC3A mainly localized in the cytoplasm of tumor cells. In contrast to previous reports, LC3A expression was not observed in perinuclear,'stone-like'structures (SLS) in ESCC samples.
2. EC3A immunostaining was not linked to clinicopathological factors, such as sex, weight loss, age, tumor location, and, cell differentiation.
3. Immunohistochemical analysis showed that p53nuclear overexpression was observed in55.6%(135/243) of tumors. The correlation coefficient between LC3A and p53was-0.0184(P=0.9772)
4. The3-year and5-year local control rates were58.2%and47.0%, respectively, in the LC3A lower expression group and46.1%and39.5%, respectively, in the LC3A overexpression group. The difference was not statistically significant(p=0.082).
5The survival analysis shows LC3A was not a prognostic marker, while the T stage, N stage, clinical stage, radical surgery and adjuvant therapy correlated with prognosis.
6. Of the209patients with stage Ⅱ/Ⅲ(TanyN+M0or T3,4NanyMO),158patients underwent radiotherapy-based therapy after surgery. For this subgroup, Kaplan-Meier curves indicated that the prognosis of the patients with LC3A overexpression was poorer than that of LC3A lower expression(p<0.05). Moreover, in the158patients with adjuvant radiotherapy, the local control rate(LCR) in patients with LC3A overexpression was lower than that with LC3A low expression (p=0.01)。
Conclusions:
LC3A is expressed in the cytoplasm in ESCC tissues. None of the factors(such as T stage, lymph node metastasis) correlates with LC3A immunostaining. The LCR in patients with LC3A overexpression and adjuvant radiotherapy is lower than that in patients with LC3A low expression and adjuvant radiotherapy. The results above indicate LC3A is a indicator of the sensitivity of concurrent chemoradiotherapy in esophageal squamous cell Carcinoma.
Part II:Outcome of concurrent chemoradiotherapy with docetaxel/cisplatin in esophageal squamous cell carcinoma and the predictive significance of LC3A in ESCC sensitivity to concurrent chemoradiotherapy.
Objective:
Esophageal carcinoma (EC) is one of the most common malignancies worldwide, with poor outcome. In developing countries including China, EC has a higher incidence. There are150,000patients who died of EC, accounting for40%patients in the world. Early EC patients with surgery get good prognosis, while for most patients with advanced EC, the treatment outcome of surgery only is poor, with20%of5-year overall survival rate. Concurrent chemoradiotherapy is the standard treatment modality for nonoperative locally advanced EC patients. For this modality, the5-year overall survival rate of patients with locally advanced EC is30%, and the local relapse is the most common cause. Moreover, the prognosis does not improve by increasing the radiation dose. So finding more efficient chemotherapy regimen is a important way to improve the treatment outcome. Recently, a lot of studies explored the therapeutic effect of the third generation chemotherapeutics in concurrent chemoradiotherapy patterns. Taxanes with platinum has been proven to be an effective regimen, while the the doses and administration methods needs further confirmation. The indicators of tumor biological characteristics are another research hotspot. Although the mechanisms of tumorigenesis and development in ESCC is unknown, a great deal of evidence has shown that the mutation and abnormal expression of cell cycle related, DNA repair related and apoptosis related genes, such as p53,p21,Cyclin D, are involved in the occurrence and development of tumors. In recent years, the role of autophagy in ESCC has attracted more and more attention. Chen et al found that autophagy inhibition improved the radiosensitivity of esophageal squamous cells in vitro. EC3A is an important component in autophagosome. and highly expressed in many types of tumors. The result of part one has shown that ECR in patients with LC3A overexpression and adjuvant radiotherapy was lower than that in patients with LC3A low expression and adjuvant radiotherapy. This demonstrated LC3A was a prognostic factor of radiosensitivity in ESCC. In the following study, the association between LC3A expression and the efficacy of concurrent chemoradiotherapy was explored, and the feasibility of DC chemotherapy combined with radiotherapy was evaluated in patients with ESCC.
Materials and methods:
Sixty-five patients with ESCC were selected in the Department of Oncology of the Affiliated Hospital of Binzhou Medical College between November2011and Octobor2012.All eligible ESCC patients had the following conditions:diagnosed by endoscopic biopsies; were not suitable or refused operation; KPS greater than80;no contraindication of chemoradiotherapy. The classification of clinical stage was determined according to the Chinese staging criteria for nonoperative esophageal carcinoma. The median age of these patients was62.1years(42～70years).Three-dimensional comformal technique was used in radiotherapy, and the median radiation dose was56.5Gy(44～60Gy). Synchronous chemotherapy was DC(docetaxel20mg/m2and cisplatin25mg/m2,IV dl,8,15,26,33,40).The subjective treatment response was evaluated according to dysphagia remission, and objective response was evaluated according to esophageal barium meal. Toxicities were graded according to the Common Terminology Criteria for Adverse Events v3.0. LC3A protein expression was assessed immunohistochemically. In this study, they were evaluated of the efficacy and the toxicities of the DC regimen combined with radiation and the association between LC3A immunohistochemical expression and the sensitivity toconcurrent chemoradiotherapy in ESCC patients.
Results:1.(1) Subjective Response:For ESCC patients with combined therapy of DC chemotherapy and synchronous radiotherapy, dysphagia relief was observed in55of62patients(88.7%).Seven of the55patients with dysphagia relief were able to drink liquid diet before treatment and eat solid without any dysphagia. After treatment, the rate of patients without dysphagia increased from3.1%(2/65)to50.0%(32/64). (2)Objective response:Based on barium meal, the CR rate was48.4%(31/64), PR rate42.2%(27/64), and the objective response rate(ORR)was90.6%(58/64).
2. Esophagitis and granulocytopenia were the most frequent acute toxicities in the concurrent chemoradiotherapy. Of the65patients,62/65patients in the study group had esophagitis,and mild esophagitis(grade1/2) was73.9%(48/65), and grade3was21.5%(14/65).With regard to granulocytopenia, it occurred in61.6%(40/65) patients,while only3.1%(2/65) patients had grade3granulocytopenia.
3.Based on LC3A expression, the CR rate of the patients with low LC3A expression was58.1%(18/31),while that of the patients with LC3A overexpression was38.2%(13/34).The difference between the two subgroup was statistically significant(p<0.001).
Conclusions:The combined treatment modality of DC chemotherapy with synchronous radiotherapy is effective and tolerable in ESCC patients.LC3A is an indicator of the CRT sensitivity.
引文
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