62 Multi-institutional药理药效研究 动物模型
医药实验常用动物模型及特殊实验技巧
大鼠淋巴插管模型（LVC）
大、小鼠鼻腔滴注实验
大鼠颈静脉、股静脉双插管模型（JVC&FVC）
大、小鼠空肠局部给药实验
大鼠颈静脉、肝门静脉双插管模型（JVC&PVC）
大、小鼠胶囊灌胃实验（肠溶，含进口仪器及胶囊）
大鼠颈静脉、颈动脉双插管模型（JVC&CAC）
家兔眼科组织取材：房水、角膜、结膜、泪腺、眼
体内蛋白结合，红细胞结合试验
啮齿类体内蛋白结合试验、红细胞结合试验
药理模型
炎症和免疫性疾病
大鼠II型胶原诱导的关节炎模型（CIA)
OVA诱导Balb/c小鼠过敏性哮喘
LPS诱导急性肺损伤
CCl4诱导急性肝损伤
咳嗽模型
Balb/c小鼠过敏
神经系统疾病模型
强迫游泳试验（抑郁）
悬尾试验（抑郁）
大鼠黑质纹状体损伤后的旋转行为试验（帕、大鼠、豚鼠、兔、药代动力学试验
生物利用度研究
小鼠、大鼠、豚鼠、兔、、腹腔、皮下等生物利用度试验
组织分布研究
小鼠、大鼠、豚鼠、兔、组织分布，血脑屏障，淋巴转运，肝提取试验
排泄研究
小鼠、大鼠、豚鼠尿液、胆汁和粪便药物分析
特殊给药制剂/给药途径药代动力学研究
啮齿类静脉滴注、鼻腔、十二指肠、门静脉、股静脉、侧脑室、眼部给药；片剂、胶囊给药
大、小鼠尾静脉插管infusion及其样品采集
大鼠颈动脉插管模型（CAC）
大、小鼠足背静脉给药及尾静脉采血
大鼠股静脉插管模型（FVC）
大、小鼠下颌静脉连续微量采血
大鼠肝门静脉插管模型（PVC）
大、小鼠perfusion及其脏器采集
大鼠十二指肠插管模型（DC）
药理学研究中的动物模型
药理学研究中的动物模型药理学是研究药物对生物系统的作用、药物相互作用及其在人体内的代谢和排泄等方面的科学。
而动物模型则是药理学研究的重要手段之一，是在药理学研究中广泛应用的实验方法。
本文将探讨动物模型在药理学研究中的运用以及存在的问题。
一、动物模型在药理学研究中的运用1.药效学研究药物剂量-反应关系是药一个药物治疗效果的关键参数。
通过对动物模型进行药效学研究，可以帮助研究者确定药物的剂量和给药方法，及其对特定疾病产生的治疗作用。
2.药代动力学研究药代动力学研究涉及药物在生物体内的吸收、分布、代谢及排泄等过程。
通过使用动物模型，研究者可以更好地理解人体内药物相互作用的机制，以及药物在体内的代谢及排泄吸收规律。
3.毒理学研究另外，动物模型也广泛应用于毒理学研究方面。
通过使用动物模型，研究者可以评估药物的毒性，并为药物的临床使用提供指导。
二、动物模型存在的问题1.模型转化性能的问题研究者对动物模型的应用需要深刻认识到动物模型本身具有的局限性。
动物模型无法完全反映人体内部各种细微变化的复杂性，因此，能否将动物的结果转化为人体的治疗方案存疑。
2.道德问题另外，动物模型研究也存在一定的道德问题。
因为动物在实验中往往会受到一定程度的折磨，所以必须确保实验的道德可接受，避免动物受到过度转化。
3.统计学意义上的问题最后，动物模型的应用还可能存在统计学意义上的问题，研究者必须严格控制实验中的各种环境因素，以确保研究结果的可靠性。
三、结语总体来说，动物模型在药理学研究中扮演着重要的角色。
尽管存在一些问题，但研究者仍需要认真对待这种研究方法，尽力避免它的局限性，改善其缺陷，并为临床应用提供可靠的依据。
同时，必须通过科学的伦理道德评估来确保研究过程的公正公平。
只有慎重对待，才能更好地补充人类已知的药理学知识，为发现从动物实验中发现的新药物奠定基础。
浅谈动物模型在中药药理研究的意义
２ ３ 中药 药理 动物 模型 是 实验 动物 学发 展的 新领 域 ．
药理 证 候 运 动 模型 是 指 在 中 医药 理论 指 导 下 ， 动物 身 上复 制 的 在 中 医药 证 候 ， 中药 药理 动 物 模 型独 具 一 格 的有 别 于 人类 疾病 动 是 物 模 型 的方 法 。 而 中 药 药 理 证 候 动 物模 型 的 研 究还 远 远 不 能 满
医药的现代化。
处 理 ， 自然情 况 下 ， 生 染 色 体 畸 变 ， 因 突 变 ， 通 过 定 向 培 在 发 基 并 育 而 保 留 下 来 的疾 病 模 型 ， 无胸 腺 裸 鼠 ， 症 肌 无 力 小 鼠 、 青 如 重 光 眼 免 、高 血 压 大 鼠 、肥 胖 症 小 鼠 等 。
足 中 药 药 理 学 发 展 的需 要 ， 待 增 加 研 究 投 入 ， 高 研 究 水 平 。 急 提 ３ ３ 中药药 理病证 动物模型 ． 中药药理病 证动物 模型 包括２ 面的 内容 ： ） 方 （ 用现 代医学的人类 １
３ ２ 中药药 理证 候动 物模 型 ．
中药 药 理 证 候 动 物 模 型 ， ６ 年 代 邝 安 建 立 第 一 个 类 “ 自 ０ 阳 虚 ” 动物 模 型 以来 ， 用 ２ ０多种 方法 ， 选 ０ 复制 建 立 了肾虚 证 、脾 虚 证 、肺 虚 让 、心 虚 证 、血 虚 证 、血 瘀 证 、肝 郁症 、寒 证 、热 证 、 Ｅ 痹 证 、里 实 证 、 厥 脱证 、 湿 阻 证 、温 病 等 证 候 动 物 模 型 。 中 药
中 医 中 药
浅 谈 动 物 模 型 在 中 药 药 理 研 究 的 意 义
于俊 敏 （ 吉林 省桦 中药的 药理研 究 自２年 代初 ， ０ 陈克恢研 究麻 黄 以来 ， 究方法逐 步完善 ， 究领域 日益扩大 ， 究水平不 断提 高 ， 研 研 研 形成 了自己的 学科体 系， 这就是 中药药理 学。 中一个重要 标志就是 中药药理 动物 模 型的研 究和应 用。 其 中药 药理 动物模 型是 中药药理 学独具一格 的研 究方 法， 它使 中药药理从 中 药和 药理学脱 胎而 出， 形成 了独特 的学科体 系。 【 键 词 】 中 药药理 疾 病 关 动物 模 型 【 中图 分类 号 】Ｒ２ ８５ 【 献标 识码 】Ａ 文 【 文章编 号 ｌｌ ７ — Ｉ （ｏ ｓ ｉ（） ０ ９ — ｌ ４ ０ ２ ０ ）２ｃ一 ０ ６ ０ ６ ７２
动物模型在药物研究中的应用
动物模型在药物研究中的应用药物研究是一个综合性强、需要借助各种手段和方法才能完成的科学研究。
其中，动物模型是一个重要的研究手段，在药物研究中应用广泛。
动物模型可以帮助研究人员更好地了解药物的药理作用、剂量、毒性等方面的信息，提高药物研究的效率和成功率。
本文将探讨动物模型在药物研究中的应用，并分析其优缺点。
一、1. 药物的药理作用研究动物模型可以帮助研究人员更好地了解药物的药理作用。
例如，针对肿瘤的化疗药物，研究人员可以通过动物模型来评估药物的抗肿瘤作用、毒性和耐受性。
动物模型可以模拟人体中的肿瘤环境，通过观察动物体内的肿瘤变化，了解药物的药理作用和剂量范围，为药物的临床应用提供依据。
2. 药效学研究动物模型可以帮助研究人员进行药效学研究。
例如，对于心血管疾病的药物，研究人员可以通过动物模型来评估药物的心血管效应、有效剂量、安全剂量等信息。
通过动物实验，研究人员可以获取药物在动物体内的体内药效学参数，了解药物的药效学特性，为药物的临床应用提供依据。
3. 毒理学研究动物模型可以帮助研究人员进行毒理学研究。
例如，在新药研究过程中，需要对药物的毒性进行评估。
通过动物模型，研究人员可以获取药物的毒性数据，包括剂量效应关系、生化毒性、组织学和病理学损伤等信息。
这些数据可以为药物的临床应用提供依据，帮助研究人员了解药物的毒性水平，以及如何使用药物时避免毒性损害。
4. 药物代谢动力学研究动物模型可以帮助研究人员进行药物代谢动力学研究。
例如，在新药研究过程中，需要了解药物的代谢途径、代谢产物和半衰期等信息。
通过动物实验，研究人员可以获得药物代谢动力学参数，如药物清除率、药物代谢酶的活性等，为药物的临床应用提供依据。
二、动物模型在药物研究中的优缺点1. 优点（1）相对真实：动物模型的研究结果相对比较真实，因为它可以模拟人体生理环境，给人类疾病的研究提供一定的可靠性。
（2）有利于筛选药效：动物模型有助于筛选药物的药效，检索药物的安全性、有效性等，从而为药物的研发和临床应用提供依据。
新药临床前药效学评价(下)
裸小鼠－突变系 主要生物学特性： 先天性胸腺发育缺陷 无被毛 细胞功能缺失 主要用于免疫学、肿瘤学研究和单克隆抗体的制备。
：白化大鼠－封闭群 主要生物学特性： 对疾病抵抗力较强 自发性肿瘤的发病率较低 对性激素敏感性高 广泛用于药理学和毒理学研究，如高血压、镇痛抗炎、 内分泌、药物依赖性等实验
短毛豚鼠 主要生物学特性 豚鼠嗅觉、听觉较发达 不能合成维生素 豚鼠易引发速发性变态反应 呼吸系统和消化系统抗病能力较差 应用于过敏反应、皮肤刺激试验、平喘、镇咳、 局部麻醉药等研究
新西兰兔 生物学特性 听觉和嗅觉灵敏 性成熟期因品种而异 对致热物质敏感 主要用于心血管病、热源、消化道炎症等实验
比格犬 国际上标准实验用犬。 性情温顺、易于驯服和抓捕 抗病能力强 遗传性能稳定，对实验反应一致。 常用于高血压、条件反射、消化系统、脑血管病等实验
猕猴 生物学特性
大脑发达 生理生殖与人非常接近
易感某些人类传染病 常用于药物依赖性、疫苗、行为科学和神经生理等实验
. 动物的预筛
大部分动物只要选用符合实验动物管理要求的动物就行。但 有些药理实验还必须首先将这些动物进行过筛，即预筛。选 择在一定药理反应指标内的动物，以免因个体差异太大而影 响结果的评判，然后进行随机分组试验。
如观察药物对小鼠游泳、学习记忆和抗癫痫药的转棒试验等， 都应先进行预筛。类似的还有镇痛试验用小鼠；镇咳试验用 豚鼠进行电刺激嗽时约有％的豚鼠可对刺激无反应或不能发 生复制反应，这些动物也应弃去；利尿药与抗慢性肾衰药试 验用大鼠也需预筛，测定无蛋白尿者方可入选使用。
. 性别的选择
临床前药效学试验通常用雌雄各半动物。
但不少药效学试验需用雄性动物:如抗癫痫药的癫痫模型动物；抗动 脉粥样硬化药和调血脂药的实验动物；精神药药效试验的动物；抗血 小板聚集药常用雄性兔和大鼠，以避免雌性动物的性周期对实验结果 的影响；抗凝血药最好用雄性动物；中毒性心肌炎在体模型用雄性大 鼠。
多发性硬化动物模型研究进展
多发性硬化动物模型研究进展彭真;张礼标;吴洁;孙云霄【摘要】多发性硬化(MS)是一种慢性中枢神经系统自身免疫性疾病,为临床神经系统的疑难重病,对其病理过程、发病机制的研究以及治疗药物的筛选和评价都需要合适的动物模型.论文从实验动物选择、几种重要的诱导方法及模型的行为学、影像学及病理评价等多方面对MS动物模型研究进展进行介绍.%Multiple sclerosis(MS)is a chronic autoimmune disease in the central nervous system,and a difficult case in clinical nervous system.Appropriate animal models play a key role for the research of the pathological process and mechanisms,drug screening and evaluation.In this review,we reviewed the progress on MS models from animal selection,induction methods and the evaluation of the behavior,imaging and pathological aspects.【期刊名称】《动物医学进展》【年(卷),期】2017(038)004【总页数】5页(P108-112)【关键词】多发性硬化;中枢神经系统;免疫;动物模型【作者】彭真;张礼标;吴洁;孙云霄【作者单位】中国药科大学生命科学与技术学院微基因药物实验室,江苏南京210009;广东省生物资源应用研究所/广东省动物保护与资源利用重点实验室/广东省野生动物保护与利用公共实验室,广东广州 510260;广东省生物资源应用研究所/广东省动物保护与资源利用重点实验室/广东省野生动物保护与利用公共实验室,广东广州 510260;中国药科大学生命科学与技术学院微基因药物实验室,江苏南京210009;广东省生物资源应用研究所/广东省动物保护与资源利用重点实验室/广东省野生动物保护与利用公共实验室,广东广州 510260【正文语种】中文【中图分类】S852.34多发性硬化(multiple sclerosis,MS)是一种慢性中枢神经系统(central nervous system,CNS)自身免疫性疾病，表现为局灶性炎性浸润(视神经、脑、脊髓)、脱髓鞘以及轴突损伤、胶质增生等，临床症状和病理特征极其易变。
中药在动物模型中的药效学研究
中药在动物模型中的药效学研究中药在动物模型中的药效学研究引言：中药作为中国传统医学的重要组成部分，具有悠久的历史和丰富的临床应用经验。
随着现代科学技术的发展，越来越多的研究开始关注中药在动物模型中的药效学研究。
通过动物模型的研究，可以更好地理解中药的药理学特性、药效机制以及临床应用的科学依据。
本文将介绍中药在动物模型中的药效学研究的重要性、方法和应用。
一、中药在动物模型中的药效学研究的重要性中药在动物模型中的药效学研究对于中药的临床应用具有重要意义。
首先，通过动物模型的研究，可以评估中药的药效和安全性。
动物模型可以模拟人体疾病的发生和发展过程，通过给予中药治疗，观察其对疾病的疗效以及对动物的毒副作用。
这对于评估中药的疗效和安全性具有重要意义，为临床应用提供科学依据。
其次，动物模型可以帮助揭示中药的药效机制。
通过对动物模型中中药的作用机制的研究，可以更好地理解中药的药理学特性，为中药的开发和应用提供理论基础。
此外，动物模型还可以用于筛选中药的活性成分和优化中药的配方，提高中药的疗效和安全性。
二、中药在动物模型中的药效学研究的方法中药在动物模型中的药效学研究主要包括药效评价、药代动力学研究和药物安全性评价。
药效评价是中药在动物模型中的主要研究内容之一，通过给予动物一定剂量的中药，观察其对疾病的疗效以及对动物的影响。
常用的药效评价方法包括行为学观察、生理指标检测和组织病理学分析等。
药代动力学研究是研究中药在动物体内的吸收、分布、代谢和排泄过程，通过测定中药在动物体内的浓度-时间曲线，了解其在体内的代谢和排泄动力学特性。
药物安全性评价是评估中药对动物的毒副作用的研究，通过观察动物在给予中药后的生理指标变化、组织病理学变化以及行为学变化等，评估中药的安全性。
三、中药在动物模型中的药效学研究的应用中药在动物模型中的药效学研究广泛应用于临床医学和药物研发领域。
在临床医学中，中药在动物模型中的药效学研究可以为中药的临床应用提供科学依据。
浅谈中药药理动物模型研究及其作用
Science &Technology Vision科技视界中药的药理研究从20年代初,陈克恢开始麻黄研究[1]以来,研究方法逐步完善,研究领域日益扩大,研究水平不断提高,形成了自己的学科体系,这就是中药药理学。
其中一个重要标志,就是中药药理动物模型的研究和应用。
中药药理动物模型是中药药理学独具一格的研究方法,它使中药药理学从中药和药理学脱胎而出,形成了独特的学科体系。
因此,有必要对中药药理动物模型进行整理、探索为进一步指导中药药理学发展、丰富实验动物学的内容起作用。
故本文较系统地论述了中药药理动物模型的概念、分类、现状和作用,探讨了中药药理动物模型的发展趋势。
1中药药理动物模型的概念中药药理动物模型是指根据中医药基本理论,为进行中药药理研究而对人类疾病原型的某些特征进行模拟复制,创造出的具有人类病证表现的动物实验对象及相关材料,包括人类疾病动物模型、人类证候动物模型、人类病证动物模型三部分的内容,它既是实验动物学的范畴,又是中药药理实验方法学的核心。
2中药药理动物模型的分类及现状中药药理动物模型的研究历经几十年的发展,已研制出百余种证型,其造模方法大致可归纳为以下三类:2.1依据中西医结合病因学说塑造动物模型:又称为中药药理病证动物模型[2]、病因病理结合型模型[3]这类模型的造模方法是既运用了中医的发病学说,又考虑了西医的致病原理,将现代医学的人类疾病动物模型与中医证候动物模型嫁接,建立病证结合动物模型。
如高脂性疾病血瘀证动物模型、失血性贫血血虚证动物模型、感染性休克厥脱证动物模型等,把现代医学的辨病论治与中医学的辨证论治结合起来,中西汇通[4]。
这方面的工作急待开展,以深化中药药理模型的研究,纠正证候动物模型难于深化、不好应用的不足。
2.2采用西医病因病理复制动物模型又称为中药药理疾病动物模型[2]、病理型模型[3],其可分为自发性的和诱发性的。
自发性疾病动物模型是指实验动物未经任何有意识的人工处理,在自然情况下,发生染色体畸变、基因突变,并通过定向培育而保留下来的疾病模型,如无胸腺裸鼠、重症肌无力小鼠、青光眼兔、高血压大鼠、肥胖症小鼠等;诱发性疾病动物模型是研究者通过使用物理、化学、生物等因素作用于动物,造成动物组织、器官或全身一定的损害,出现某些人类疾病的功能、代谢或形态结构方面的改变。
药理学研究中的动物模型选择
药理学研究中的动物模型选择概述：动物模型在药理学研究中发挥着重要的作用。
通过合适的动物模型可以更好地理解药物的机制、效果以及安全性。
然而，在选择适当的动物模型时需要考虑多个因素，包括相似性、成本和可行性等。
本文将探讨药理学研究中动物模型选择的原则和常见的应用。
一、相似性1.1 物种相似性：选择与人类生理相似度较高的动物作为研究对象，如大鼠和小鼠。
因为这些动物在生命活动、器官结构和代谢途径等方面与人类有较好的相似性，能够提供更可靠的数据。
1.2 疾病模型：根据所研究的药物治疗目标来选择与之相关的疾病模型。
例如，在心血管领域，可以使用高胆固醇饮食诱导小鼠产生高血压或者缺血再灌注损伤等模型来评估药物对这些情况下心脏功能改善效果。
二、成本和可行性2.1 成本：动物模型的建立和维护需要耗费大量资金，因此在选择时需要考虑经济实用性。
多个相关研究中常用的小鼠是较经济实惠的选择。
2.2 可行性：动物模型的选取还要根据实验室条件、技术设备和人员配备等方面进行考虑。
对于一些高度特化的模型，如果缺乏相关资源，则不宜选择。
三、常见应用3.1 药效学评价：通过动物模型可以评估药物在生物体内的药效学参数，包括药代动力学和药效学等。
这些数据对于了解药物吸收、分布、代谢和排泄过程非常重要。
3.2 治疗策略验证：在新药开发阶段，动物模型常被用来验证治疗策略的有效性。
例如，在癌症治疗研究中，使用小鼠移植瘤模型可以评估抗肿瘤药物的疗效。
3.3 安全评价：动物模型能够帮助检测潜在毒副作用，并评估药物在各种毒理学指标上的影响。
这对于药物安全性评价以及制定适当的用药指南至关重要。
四、局限性和新技术4.1 物种差异：尽管动物模型在探索药理学的研究中有很大帮助，但人类与动物之间仍然存在一定的生理、代谢差异。
因此，不能直接将动物实验结果推广到人体。
4.2 替代方法：随着科技的发展，出现了许多替代动物模型的技术，如体外细胞培养和计算机模拟等。
这些新技术可以减少对动物实验的需求，并提供更快速、精确、经济高效的研究手段。
银屑病药效学动物模型及其在中药新药研究中应用的思考
0061117栏目中药药物评价>>非临床安全性和有效性评价标题银屑病药效学动物模型及其在中药新药研究中应用的思考作者韩玲部门正文内容审评一部韩玲摘要：本文综述了银屑病药效学动物模型的进展，对治疗银屑病中药新药药效学研究方面存在的问题进行了分析，提出了应结合银屑病发病机制的研究进展完善药效学方法学研究的建议。
银屑病是一种慢性复发性皮肤病，其发病机制复杂，至今尚未明了。
可能是一种具有基因遗传特性的疾病，也与感染、内分泌机能障碍、免疫功能紊乱等有关。
中医学认为以风湿毒邪内侵为标，血虚、血燥为根源，血热、血虚、血瘀是本病的基本病机，故清热解毒、凉血、活血化瘀、祛风除湿成为治疗本病的基本法。
银屑病的组织病理学主要表现为角质形成细胞过度增生、炎症细胞聚集和真皮乳头部血管增生扩张三大特征，从而出现角化过度、角化不全、颗粒层消失、棘细胞层增厚等。
尽管许多不同的动物模型能复制银屑病的某个方面的病理特征，并能解释部分病因，但长期以来仍缺乏一种能够研究所有有关的潜在因素的动物模型。
即-缺乏理想的动物模型。
一、银屑病药效学动物模型银屑病动物模型是根据银屑病的特征，人为建立的一种与之相似的模型。
有人总结了银屑病动物模型的建立过程，包括以下4个不断完善的阶段：1．诱发性动物模型该模型是通过工方法在动物，如豚鼠、大鼠等皮肤上诱发银屑病样增生。
其方法包括紫外线照射、化学刺激剂外涂或药物外涂、缺乏必须脂肪酸的饲料喂养等。
这些方法均为过度增殖模型，可引起表皮增生加快。
但这些方法仅是针对表皮损伤的一种迟发反应，持续时间较短。
如缺乏必须脂肪酸饲料喂养的大鼠模型，因缺乏必须脂肪酸，故可使大鼠皮肤逐渐出现角化过度、棘层肥厚、基底细胞有丝分裂加快，DNA合成增加等变化，表皮细胞呈慢性增生状态，与药物诱发的急性表皮增生相比，其与银屑病更为相似。
如心得安诱导银屑病动物模型，是利用心得安特有的药理作用，即心得安可阻断角阮细胞的β-肾上腺能受体而降低了细胞内cAMP水平所致。
中医证候动物模型
针灸治疗学研究
针灸对寒证的疗效
通过针灸治疗寒证模型，观察针 灸对寒证症状的改善作用及机制 ，如针灸大椎、风池等穴位对感
冒、咳嗽等症状的改善作用。
针灸对热证的疗效
通过针灸治疗热证模型，观察针灸 对热证症状的改善作用及机制，如 针灸曲池、合谷等穴位对发热、咽 痛等症状的改善作用。
针灸对虚证的疗效
通过针灸治疗虚证模型，观察针灸 对虚证症状的改善作用及机制，如 针灸关元、足三里等穴位对乏力、 气短等症状的改善作用。
年龄
常用3-6月龄豚鼠，以模拟人类更年 期时期的生理状态。
造模方法
根据研究目的和中医证候，采用不同 的造模方法，如药物诱导、手术操作 等。
家兔模型
品种
多采用新西兰白兔。
性别
多选用雌性家兔，以满足中医“血瘀”等证候的研究需求。
年龄
常用6-12月龄家兔，以模拟人类老年时期的生理状态。
造模方法
根据研究目的和中医证候，采用不同的造模方法，如手术操作、药物诱导等。
稳定性和可靠性。
适用范围有限
中医证候动物模型主要适用于研 究某些特定的中医证候，对于其 他一些复杂的证候，如气血亏虚 证等，其适用性可能有所限制。
存在误差
由于造模过程中存在误差和变异 等因素，可能会影响中医证候动
物模型的可重复性和稳定性。
改进方向与建议
完善造模方法
01
进一步探索和研究更加稳定、可靠的中医证候动物模型造模方
促进中医药现代化进程
中医证候动物模型的应用有助于促进中医药的现 代化进程，为中医药的国际化发展提供有力的支 持。
为新药研发提供帮助
中医证候动物模型可以用于新药的研发和筛选， 为新药的研发过程提供有益的参考依据。
药理药效研究 动物模型subsequent erlotinib
Lung Cancer 73 (2011) 211–216Contents lists available at ScienceDirectLungCancerj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /l u n g c anDuration of prior gefitinib treatment predicts survival potential in patients with lung adenocarcinoma receiving subsequent erlotinibKazuhiro Asami a ,∗,Masaaki Kawahara b ,Shinji Atagi a ,Tomoya Kawaguchi a ,Kyoichi Okishio aa Kinki-chuo Chest Medical Center,1180Nagasone-cho,Kita-ku,Sakai City,Osaka 591-8555,Japan bOtemae Hospital,1-5-34Otemae,Chuo-ku,Osaka,Japana r t i c l ei n f oArticle history:Received 9August 2010Received in revised form 19October 2010Accepted 18December 2010Keywords:Gefitinib ErlotinibEGFR mutationResistance to EGFR-TKIsTime to progression of gefitiniba b s t r a c tPurpose:We investigated survival potential in patients receiving erlotinib after failure of gefitinib,focus-ing on response and time to progression (TTP)with gefitinib.Methods:We retrospectively reviewed lung adenocarcinoma patients who received erlotinib after expe-riencing progression with gefitinib.Our primary objective was to evaluate the prognostic significance of erlotinib therapy.Results:A total 42lung adenocarcinoma patients were included in this study.Overall disease control rate was 59.5%(partial response [PR],2.4%;stable disease [SD],57.1%).Median overall survival was 7.1months,and median progression-free survival was 3.4months.The number of patients who achieved PR and non-PR (SD+progressive disease [PD])with gefitinib were 22(52%)and 20(48%),respectively.Patients with PR for gefitinib showed significantly longer survival times than those with non-PR (9.2vs.4.7months;p =0.014).In particular,among PR patients,those with TTP <12months on gefitinib showed significantly longer survival times than those with TTP ≥12months (10.3vs.6.4months;p =0.04).Conclusions:Erlotinib may exert survival benefit for lung adenocarcinoma patients with less than 12months of TTP of prior gefitinib who achieved PR for gefitinib.© 2011 Elsevier Ireland Ltd. All rights reserved.1.IntroductionGefitinib and erlotinib are oral epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs).Gefitinib has been reported to be effective in limited populations such as never smokers,Asians,and patients with adenocarcinoma,and is particularly effective in patients with EGFR mutations [1–3].Erlotinib,which has a sim-ilar quinazoline frame to gefitinib,is the first EGFR-TKI shown to provide survival benefit in patients with non-small cell lung cancer (NSCLC)[4]:the BR.21trial revealed significantly longer sur-vival times among patients who received erlotinib compared with a placebo group [4].In addition,these two EGFR-TKIs have been found to occasionally induce a particularly significant response in EGFR-mutant patients.However,despite this documented efficacy,most cancer clones acquire resistance to these particular com-pounds over time [5].Previous studies have demonstrated that amplified MET onco-gene and secondary EGFR T790M mutations are most commonly responsible for resistance to gefitinib and erlotinib [6,7].Indeed,several previous studies showed that secondary EGFR T790M muta-tion and MET amplification occurred in nearly half and 20%of lung∗Corresponding author.Tel.:+81722523021;fax:+81722511372.E-mail address:kazu.taizo@ (K.Asami).cancer specimens that had become resistant to EGFR-TKIs,respec-tively [8–11].In addition,the majority of patients who showed secondary resistance had EGFR mutations such as exon 19dele-tion mutations or L858R point mutation,which have been found to be sensitive to EGFR-TKIs [12,13].Several reports have demonstrated clinical benefits when administering erlotinib to NSCLC patients following failure of gefi-tinib [14–18];in contrast,one previous report has suggested that no erlotinib-derived clinical benefit can be expected in patients who failed gefitinib [19].However,reports thus far have all had small sample sizes,and clear findings regarding efficacy of erlotinib in patients who failed gefitinib have yet to be obtained.Consequently,whether or not erlotinib is useful in these patients remains contro-versial.We hypothesize that tumor clones may require exposure to gefi-tinib treatment with a positive response for a specific duration to acquire secondary common resistance to EGFR-TKIs.Even if a patient experiences tumor progression on gefitinib therapy,sub-sequent erlotinib therapy may nevertheless still be able to inhibit progression,provided the tumor clones did not acquire secondary resistance.As such,in positive-responder patients with confirmed progression within a specific duration of gefitinib treatment,some tumor clones may remain sensitive to erlotinib,and therefore these patients may still experience survival benefit with erlotinib treat-ment.0169-5002/$–see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.lungcan.2010.12.014212K.Asami et al./Lung Cancer73 (2011) 211–216Here,we conducted a retrospective study primarily aimed at assessing overall survival(OS)of patients who received erlotinib therapy after failure with gefitinib.We also attempted to charac-terize the clinical features of patients who benefited from erlotinib treatment.2.Patients and methodsWe retrospectively reviewed records for patients with histopathologically diagnosed lung adenocarcinoma who received erlotinib after experiencing progression on gefitinib at Kinki-chuo Chest Medical Center between December2008and October2009. Responses were evaluated based on patient records and radio-graphic studies,such as chest roentgenograms and computed tomographic(CT)and magnetic resonance imaging(MRI)scans. We examined EGFR mutation status using the PCR-invader method with paraffin sections of biopsy specimens from patients.Time to progression(TTP)with gefitinib was defined as the period from initiation of gefitinib therapy to the date when dis-ease progression was confirmed.Overall survival was defined as the period from initiation of erlotinib therapy to the date of death or last follow-up.Disease control rate(DCR)was defined as com-plete response(CR)plus partial response(PR)plus stable disease (SD).Evaluation of response to gefitinib and erlotinib therapy by CT scan was performed according to the response evaluation crite-ria in solid tumors(RECIST).Stable disease plus progressive disease (PD)with prior gefitinib treatment was defined as“non-PR.”Categorical outcomes,including DCRs,were compared using the 2test,and survival distribution was estimating using the Kaplan–Meier method.Overall survival and progression-free sur-vival(PFS)were compared with regard to demographic factors such as gender,performance status,EGFR mutation status,response to gefitinib,TTP with gefitinib,and toxicity grade of skin rash,which may be associated with survival,using the log-rank test.Values were considered statistically significant for p<0.05.A multivariate Cox-proportional-hazards model was used to determine the clini-cal variables which influenced OS.Statistical analyses were carried out using SPSS software ver.11.0for Windows(IBM,Chicago,IL, USA).3.Results3.1.Patient characteristicsForty-two patients with lung adenocarcinoma were reviewed in the present study.All patients became refractory to gefitinib dur-ing the course of treatment and were subsequently switched to erlotinib therapy.Patient characteristics are described in detail in Table1.Thirty patients(71%)had received1or2regimens before Table1Patient characteristics.Number(%)Median age,years(range)65(31–85) SexMale13(31)Female29(69)Smoking historyNever28(67)Former/current14(33)ECOG score0–124(57)2–418(43)Cancer stageIIIB8(19)IV34(81) Number of previous treatments with erlotinib1–230a(71)3≤12(29) EGFR mutationExon19deletion mutation14(33)L858R14(33)Exon18point mutation1(2)Wild13(32)TTP with gefitinib treatment,months(range)8.1(0.9–40.7) <1229(69)≤1213(31)Response to gefitinibCR0(0)PR22(53)SD17(40)PD3(7)EGFR,epidermal growth factor receptor;TTP,time to progression;CR,complete response;PR,partial response;SD,stable disease;PD,progressive disease;ECOG, Eastern Cooperative Oncology Group.a Two patients received gefitinib asfirst-line treatment.initiation of erlotinib and2(7%)had received gefitinib asfirst-line treatment.EGFR mutations were detected in29(69%)patients:14(33%) had exon19deletions,14(33%)had L858R mutations,and1(2%) had an exon18point mutation.The median TTP with gefitinib treatment was8.1months.Thirteen(31%)patients had TTPs of12 months or more,while29(69%)had TTPs of less than12months. Twenty-two(53%)patients receiving gefitinib achieved PR,and17 (40%)achieved SD.None achieved CR while receiving gefitinib ther-apy.The response rate(RR)and DCR for gefitinib were53%(22of 42patients)and93%(39of42patients),respectively.Of the22 patients who achieved PR with gefitinib,19(86%)were found to have EGFR mutations.Of the20patients who had SD or PD(non-PR)Table2Response to erlotinib according to the response to prior gefitinib and EGFR mutation status.EGFR mutation Response to gefitinibPR(n=22)Non-PR a(n=20)SD(n=17)PD(n=3)Positive,n(%)Negative b,n(%)Positive,n(%)Negative,n(%)Positive,n(%)Negative,n(%) Response to erlotinib PR(N=1)1(4.5)0(0)0(0)0(0)0(0)0(0) SD(N=24)14(64)1(4.5)3(18)5(29)0(0)1(33)PD(N=17)4(18)2(9)6(35)3(18)1(33)1(33)EGFR,epidermal growth factor receptor;PR,partial response;SD,stable disease;PD,progressive disease.Overall disease control rate(PR+SD)was73%(EGFR mutation-positive:15/22[68%],EGFR mutation-negative:1/22[5%])among patients who achieved PR with gefitinib and45%(EGFR mutation-positive:3/20[15%],EGFR mutation-negative:6/20[30%])among patients with non-PR(SD+PD for gefitinib)with gefitinib treatment.Overall disease control rate was62%(PR for gefitinib:15/29[52%],non-PR for gefitinib:3/29[10%])in EGFR mutation-positive patients and54%(PR for gefitinib:1/13[8%],non-PR for gefitinib:6/13[46%])in EGFR mutation-negative patients.a Defined as SD plus PD with prior gefitinib therapy.b EGFR wild-type.K.Asami et al./Lung Cancer73 (2011) 211–216213 Table3Response to erlotinib stratified by TTP with prior gefitinib treatment.(A)TTP with gefitinib<12monthsResponse to gefitinib TTP with gefitinib(months)<12(n=29)PR(n=11)n(%)Non-PR(n=18)SD(n=15)n(%)PD(n=3)n(%) Response to erlotinib PR(n=1)1b(9)0(0)0(0)SD(n=17)8a,b(73)8a(44)1(6)PD(n=11)2(18)7(39)2(11)B.TTP with gefitinib≥12monthsResponse to gefitinib TTP with gefitinib(months)≥12(n=13)PR(n=11)n(%)Non-PR(n=2)SD(N=2)n(%)PD(N=0)n(%) Response to erlotinib PR(n=0)0(0)0(0)0(0)SD(n=7)7(64)0(0)0(0)PD(n=6)4(36)2(100)0(0)EGFR,epidermal growth factor receptor;PR,partial response;SD,stable disease;PD,progressive disease;TTP,time to progression.Overall disease control rate was62%(PR for gefitinib:9/29[31%],non-PR for gefitinib:9/29[31%])in patients with TTP of gefitinib<12months.Overall disease control rate was54%(PR for gefitinib:7/13[54%], non-PR for gefitinib:0/13[0%])in patients with TTP of gefitinib≥12months.a Ten patients showed improvement of target lesions,but not to PR standards.Seven and three patients achieved PR and SD,respectively,with gefitinib treatment.b A second biopsy from progression lesions was performed in three patients(one had PR and2had SD with erlotinib)who achieved PR with gefitinib.Exon19deletion mutations which were the same pattern as detected infirst biopsy specimen for primary diagnosis of NSCLC were identified,whereas EGFR T790M mutation,which endowed secondary common resistance to EGFR-TKIs,was not identified in those biopsy specimens.with gefitinib,EGFR mutations were detected in10(50%).Among patients with EGFR mutations,only one showed PD with gefitinib therapy,and RR and DCR in this group were66%(19of29)and97% (28of29),respectively.3.2.ResponseOn erlotinib therapy,1of42patients achieved PR,and24had SD.No patients achieved CR with erlotinib.Overall RR and DCR for erlotinib were2.4%(one of42)and59.5%(25of42),respectively.Response to erlotinib categorized by response to prior gefi-tinib duration and EGFR mutation status is described in Table2. Among patients who achieved PR with gefitinib,one achieved PR and15patients achieved SD with erlotinib therapy.Patients who achieved PR with gefitinib showed higher DCRs with erlotinib than patients who had non-PR with gefitinib(16[73%]of22vs.9[45%] of20),albeit without statistical significance(p=0.07).In addi-tion,EGFR mutation status was not found to be associated with response to erlotinib;in terms of DCR,no significant difference was noted between EGFR-mutant patients(18/29)and EGFR non-mutant patients group(7/13)(62%vs.54%,p=0.616).Time to progression with prior administration of gefitinib was not found to be associated with achieving a response with subse-quent erlotinib.Details regarding response to erlotinib categorized by TTP with gefitinib are shown in Table3.DCR among patients experiencing progression after less than12months of gefitinib therapy was18/29(62%).In contrast,DCR among patients with TTPs of12months or more was7/13(54%).No statistical significant difference in DCR was noted between these two groups accord-ing to TTP with prior administration of gefitinib(p=0.62).Of the 24patients who achieved SD with erlotinib therapy,10showed improvement in target lesions which had been exacerbated during gefitinib treatment;all10were EGFR-mutant patients(4L858R, 5exon19deletion mutations,and1exon18point mutation),and TTPs with gefitinib were all less than12months.Of the two patients who received gefitinib asfirst-line treatment,one had an EGFR L858R mutation and showed responses to gefitinib and subsequent erlotinib of PR and SD,respectively.While this particular patient showed a relatively long TTP(39.5months)with gefitinib,disease progression was confirmed4months after initiation of erlotinib therapy,and OS was58.6months.The other patient who received gefitinib asfirst-line treatment had EGFR-wild type,and responses to both gefitinib and subsequent erlotinib treatment were PD.TTP and OS in this patient were3and7.4months,respectively.A second biopsy of the progressed lesions was performed in three patients after gefitinib therapy failed.While exon19deletion mutations of the same pattern as noted in thefirst biopsy specimen for primary diagnosis were also detected on this second biopsy, we noted no EGFR T790M mutations.Of note,however,was the fact that imagingfindings for lesions after erlotinib therapy were improved on the second biopsy(Table3).3.3.SurvivalMedian OS and median progression-free survival(PFS)were 7.1months(95%confidence interval[CI]:4.4–9.8months)and3.4 months(95%CI:1.1–5.7months),respectively(Fig.1).Multivariate analysis of prognostic factors was performed using a Cox propor-tional hazards model to determine which clinical variables were most strongly associated with OS(Table4).Response to gefitinibTable4Multivariate analysis of prognostic variables for OS by use of a Cox proportional-hazards model.Multivariate analysisp a Hazard ratio95%CISex0.51 1.350.55–3.31 ECOG score0.190.580.25–1.31 EGFR mutation0.78 1.130.48–2.70 Response to gefitinib0.0050.230.80–0.64 TTP of gefitinib0.050.340.12–1.01 Grade of skin rash0.290.640.27–1.47EGFR,epidermal growth factor receptor;TTP,time to progression;CI,confidence interval;ECOG,Eastern Cooperative Oncology Group;PR,partial response.Response to gefitinib was the only independent prognostic factor.TTP with gefitinib showed borderline significance.Variables were compared as paired categories:sex(female vs.male),ECOG score(0–1vs.2–4),response to gefitinib(PR vs.non-PR),TTP of gefitinib(<12months vs.≥12months),grade of skin rash(3vs.1–2).a p<0.05was considered significant.214K.Asami et al./Lung Cancer73 (2011) 211–216Fig.1.Kaplan–Meier plot of survival time with erlotinib.(A)Overall survival rates and(B)progression-free survival rates of42patients.MST:median survival time; mPFS:median progression-free survival.was found to be the only independent prognostic factor(hazard ratio=0.23;95%CI:0.08–0.64,p=0.005),and time to progression with gefitinib showed borderline significance(hazard ratio=0.34; 95%CI:0.12–1.01,p=0.05).Kaplan–Meier curves of survival time according to response to prior gefitinib therapy are shown in Fig.2.Patients who achieved PR while receiving gefitinib therapy showed significantly longer OS(p=0.014).However,no significant difference was noted in PFS between patients with PR for gefitinib and those with non-PR(4.7 months[95%CI:2.9–6.5months]vs.1.8months[95%CI:1.4–2.2 months];p=0.122).Time to progression with gefitinib showed a borderline significant impact on survival with erlotinib therapy. However,among patients who achieved PR with gefitinib,TTP with gefitinib therapy was strongly correlated with survival time. Kaplan–Meier curves of survival time for patients who achieved PR with gefitinib stratified according to TTP are shown in Fig.3. Patients with TTPs of less than12months with gefitinib ther-apy were found to have significantly longer OS(10.3months[95% CI:7.0–13.6months]vs.6.4months[95%CI:2.6–10.2months]; p=0.04)and longer PFS(6.4months[95%CI:3.6–9.2months]vs.3.4 months[95%CI:1.2–5.6months];p=0.19)than patients with TTPs of12months or more.However,no statistically significant differ-ence was noted between the two groups in terms of PFS(p=0.19).Fig.2.Kaplan–Meier plot of survival time with erlotinib.(A)Overall survival rates and(B)progression-free survival rates stratified by response to prior gefitinib.Non-PR is defined as SD plus PD with gefitinib therapy.In addition,we found that skin rash was not predictive of sur-vival with erlotinib therapy.All patients in the present study were affected by rash of some grade while receiving erlotinib.The degree of skin rash toxicity due to erlotinib exceeded the grade noted dur-ing gefitinib treatment in32patients.Seven patients required dose reduction of erlotinib due to grade3skin ing a Cox propor-tional hazard model,we determined that skin rash grade had no impact on survival(hazard ratio=0.64[95%CI:0.27–1.47];p=0.29).4.DiscussionHere,we investigated survival potential in patients receiv-ing erlotinib after failure of gefitinib,focusing on response and TTP with gefitinib.Ourfindings suggest that administration of erlotinib subsequent to gefitinib may exert survival benefit in for-mer gefitinib-positive responders.Further,among those former responders,most with TTP<12months may not yet have sec-ondary resistance to EGFR-TKIs.Ourfindings suggest little chance for patients to achieve a high response with erlotinib therapy after experiencing progression with gefitinib therapy.This observation may be due to these two EGFR TKIs sharing the same mechanism of EGFR blockade or to cross resistance[5].Our retrospective study showed that response achieved with prior administration of gefitinib was the only prognostic factor for subsequent erlotinib therapy after experiencing progression on gefitinib therapy.In particular,among patients who achieved PR with gefitinib,patients with TTPs of less than12months with gefitinib therapy were found to have significantly longer OS thanK.Asami et al./Lung Cancer73 (2011) 211–216215Fig.3.Kaplan–Meier plot of survival time for patients who achieve PR with gefitinib.(A)Overall survival rates and(B)progression-free survival rates stratified by TTP with gefitinib.patients with TTPs of12months or more.In addition,most of these patients showed some degree of improvement in imagefind-ings after subsequent erlotinib therapy.We noted no EGFR T790M mutations in any of three patients who underwent a second biopsy of their progressed lesions after failure with gefitinib therapy.We therefore supposed that most patients with TTP<12months may have not yet acquired the EGFR T790M mutation.However,we only investigated the presence of a secondary EGFR T790M mutation in three patients in the present study.Validation of this hypothesis will require collection of more molecular information from patients who are no longer responsive to gefitinib in the future.Shepherd et al.demonstrated that TTP was2.6months in NSCLC patients who had previously been treated with docetaxel therapy [20].We observed that PFS was3.4months in patients with TTP≥12 months who achieved PR in our study,a duration which appears improved over that demonstrated by Shepherd et al.Given these findings,we posited that,regardless of duration of gefitinib ther-apy,subsequent erlotinib may be able to prolong PFS compared to chemotherapy with cytotoxic agent provided the patients demon-strated a positive response with gefitinib.However,given that our results were obtained in a retrospective study with an extremely small sample population,a prospective study is warranted to clar-ify whether or not erlotinib administered subsequent to gefitinib can elicit greater survival benefit in gefitinib-positive responders than chemotherapy with cytotoxic agents.We noted here that treatment with erlotinib following gefitinib resulted in more toxic grades of skin rash in patients,findings which suggest that erlotinib may have greater biological activity than gefitinib.Several other investigators have also suggested based on their ownfindings that erlotinib may have higher biological activity than gefitinib.Costa et al.showed that differing efficacy between gefitinib and erlotinib was due to differences in commonly admin-istered dosages between the two drugs[21].Gefitinib(250mg per day)is typically administered at one third of its maximum-tolerated dose,whereas erlotinib(150mg per day)is administered at its maximum tolerated dose.In vitro data showed that the mean concentration of gefitinib was0.24g/ml at the300-mg daily dose and1.1g/ml at1000mg/day.In contrast,median concentration of erlotinib at150mg/day was1.26g/ml.These previousfindings suggest that erlotinib(150mg/day)has a higher biological dose of EGFR inhibition than gefitinib(250mg/day).Recent studies have demonstrated that the increased biological activity of EGFR-TKIs is associated with control of tumor clones. Yoshimasu et al.reported observing a dose-response relationship between inhibition rates and gefitinib concentration[22].Clarke et al.reported that high-dose erlotinib was effective in controlling leptomeningeal metastases progression while receiving standard erlotinib therapy in EGFR-mutant patients[23].These authors demonstrated that a weekly1200-mg dose of erlotinib controlled leptomeningeal metastases in a patient who was no longer respon-sive to a standard daily dose of erlotinib(150mg).Ourfindings here suggest that a treatment duration of12 months of gefitinib therapy may be the borderline period for tumor clones to attain resistance to EGFR-TKIs.However,speculation as to whether or not previously EGFR-TKI-sensitive clones gradually grow resistant to EGFR-TKIs has not been resolved.Further studies are necessary to validate ourfindings.In conclusion,gefitinib responders may achieve survival ben-efits from erlotinib therapy after experiencing progression with gefitinib.Among patients who have been receiving gefitinib ther-apy for less than12months,tumor clones may not yet have acquired a secondary mutation.However,further studies are needed to clarify precisely how tumor clones attain such secondary resistance to EGFR-TKIs.Conflicts of interest statementNone declared.AcknowledgementsWe are grateful to the staff of Kinki-Chuo Chest Medical Cen-ter for their helpful comments.We are especially indebted to Dr. Masahiko Ando of Kyoto University for support on statistical anal-yses.References[1]Moscatello DK,Holgado-Madruga M,et al.Frequent expression of a mutantepidermal growth factor receptor in multiple human tumors.Cancer Res 1995;55(23):5536–9.[2]Janne PA,Engelman JA,et al.Epidermal growth factor receptor mutations innon-small-cell lung cancer:implications for treatment and tumor biology.J Clin Oncol2005;23(14):3227–34.[3]Baselga J,Arteaga CL.Critical update and emerging trends in epidermal growthfactor receptor targeting in cancer.J Clin Oncol2005;23(11):2445–59.[4]Pao W,Miller V,Zakowski M,et al.EGF receptor gene mutations are commonin lung cancers from“never smokers”and are associated with sensitivity of tumors to gefitinib and erlotinib.Proc Natl Acad Sci2004;101:13306–11. [5]Mitsudomi T,Kosaka T,Endoh H,et al.Mutations of the epidermal growth factorreceptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence.J Clin Oncol 2005;23(11):2513–20.[6]Mok TS,Wu Y-L,et al.Gefitinib or carboplatin–paclitaxel in pulmonary adeno-carcinoma.N Engl J Med2009;361(September(10)):947–57.[7]Shepherd FA,Rodrigues Pereira J,et al.Erlotinib in previously treated non-small-cell lung cancer.N Engl J Med2005;353(2):123–32.216K.Asami et al./Lung Cancer73 (2011) 211–216[8]Johnson JR,Cohen M,et al.Approval summary for erlotinib for treatmentof patients with locally advanced or metastatic non-small cell lung cancer after failure of at least one prior chemotherapy regimen.Clin Cancer Res 2005;11(18):6414–21.[9]Allan S,et al.Efficacy of erlotinib in patients with advanced non-small celllung cancer(NSCLC)relative to clinical characteristics:subset analysis from the TRUST study.In:Poster presented at ASCO.2008.[10]Baselga J,Rischin D,et al.Phase I safety,pharmacokinetic,and pharmaco-dynamic trial of ZD1839,a selective oral epidermal growth factor receptor tyrosine kinase inhibitor,in patients withfive selected solid tumor types.J Clin Oncol2002;20(November(21)):4292–302.[11]Hidalgo M,Siu LL,Nemunaitis J,Rizzo J,et al.Phase I and pharmacologic studyof OSI-774,an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies.J Clin Oncol2001;19(July(13)):3267–79.[12]Riely GJ,Pao W,et al.Clinical course of patients with non-small cell lung cancerand epidermal growth factor receptor exon19and exon21mutations treated with gefitinib or erlotinib.Clin Cancer Res2006;12(3(Pt1)):839–44.[13]Choong NW,Dietrich S,Seiwert TY,Tretiakova MS.Gefitinib response oferlotinib-refractory lung cancer involving meninges-role of EGFR mutation.Nat Clin Pract Oncol2006;3(January(1)):50–7[quiz1p following57].[14]Mitsudomi T,Kosaka T,Endoh H,Yoshida K.Mutational analysis of theEGFR gene in lung cancer with acquired resistance to gefitinib.J Clin Oncol 2006;24(18S):7074.[15]Pao W,Balak MN,Riely GJ,Li AR.Molecular analysis of NSCLC patients withacquired resistance to gefitinib or erlotinib.J Clin Oncol2006;24(18S):7078.[16]Bean J,Brennan C,Shih JY,Riely G,Viale A.MET amplification occurs with orwithout T790M mutations in EGFR mutant lung tumors with acquired resis-tance to gefitinib or erlotinib.Proc Natl Acad Sci USA2007;104(December(52)):20932–27.[17]Engelman JA,Zejnullahu K,Mitsudomi T.MET amplification leads to gefitinibresistance in lung cancer by activating ERBB3signaling.Science2007;316(May (5827)):1039–43.[18]Engelman JA,Zejnullahu K,Mitsudomi T,et al.MET amplification leads togefitinib resistance in lung cancer by activating ERBB3signaling.Science 2007;316(May(5827)):1039–43[Epub2007April26].[19]Balak MN,Gong Y,Riely GJ,et al.Novel D761Y and common secondary T790Mmutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.Clin Cancer Res2006;12(Nov(21)):6494–501.[20]Shepherd FA,Dancey J,Ramlau R,et al.Prospective randomized trial ofdocetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy.J Clin Oncol 2000;18(10):2095–103.[21]Costa DB,Schumer ST,Tenen DG,et al.Differenctial responses to erlotinib inepidermal growth factor receptor(EGFR)-mutated lung cancers with acquired resistance to gefitinib carrying the L747S or T790M secondary mutations.J Clin Oncol2008;26(7):1182–4.[22]Yoshimasu T,Ohta F,Oura S,et al.Histoculture drug response assay for gefi-tinib in non-small-cell lung cancer.Gen Thorac Cardiovasc Surg2009;57(March(3)):138–43[Epub2009March12].[23]Clarke JL,Pao W,Wu N,et al.High dose weekly erlotinib achieves thera-peutic concentrations in CSF and is effective in leptomeningeal metastases from epidermal growth factor receptor mutant lung cancer.J Neurooncol 2010;February.。
疼痛动物模型的建立及药效案例
疼痛一直是困扰人类的重要问题之一，而在医学研究中，疼痛动物模型的建立和药效案例的研究更是至关重要。
本文将对疼痛动物模型的建立和药效案例进行深入探讨，旨在为读者提供关于疼痛研究领域的全面了解和深入思考。
一、疼痛动物模型的建立疼痛动物模型的建立是疼痛研究的基础和关键。
在建立疼痛动物模型时，需要考虑以下几个方面：1.1 选择合适的动物在疼痛研究中，常用的动物模型包括小鼠、大鼠、猫、狗等，不同动物有着不同的疼痛传导机制和疼痛反应特点。
在建立疼痛动物模型时，需要选择合适的动物种类来模拟人类疼痛情况。
1.2 选择合适的疼痛诱导方法在建立疼痛动物模型时，通常会采用化学诱导、物理性刺激或者神经损伤等方法来诱发疼痛反应。
选择合适的疼痛诱导方法是建立有效的动物模型的关键。
1.3 疼痛行为评价在建立疼痛动物模型后，需要对动物进行疼痛行为评价，包括观察动物的疼痛行为表现、疼痛敏感性测试等。
通过疼痛行为评价，可以确定动物模型的有效性和可靠性。
二、药效案例研究在建立了有效的疼痛动物模型后，研究人员通常会进行药效案例研究，以评估不同药物对疼痛的治疗效果。
药效案例研究的关键内容包括：2.1 药物的选择疼痛治疗涉及多种药物，包括止痛药、抗炎药、镇痛药等。
在药效案例研究中，需要选择合适的药物，以评估其对疼痛的治疗效果。
2.2 药效评价方法药效案例研究需要借助合适的药效评价方法来评估药物的治疗效果，常用的药效评价方法包括疼痛行为测试、疼痛感知阈值测试、电生理学测试等。
2.3 药效案例研究结果的分析在药效案例研究中，研究人员需要对药物的治疗效果进行全面的分析和总结，以为临床治疗提供参考依据。
三、疼痛动物模型的意义和挑战疼痛动物模型的建立和药效案例研究具有重要的意义和挑战。
3.1 意义通过疼痛动物模型的建立和药效案例研究，可以更好地理解疼痛的发生机制、寻找新的治疗靶点和开发新的疼痛治疗药物，为临床疼痛治疗提供更多的选择和可能性。
3.2 挑战疼痛动物模型的建立和药效案例研究也面临着一些挑战，包括疼痛模型的有效性和可靠性、疼痛行为的客观评价等问题，需要研究人员加大研究力度，不断完善相关研究方法和技术手段。
CTR-S-14-00173药理药效研究 动物模型
Elsevier Editorial System(tm) for Cancer Treatment ReviewsManuscript DraftManuscript Number:Title: The Value of Elective Lymph Node Irradiation in Definitive Concurrent Chemoradiotherapy for Esophageal Cancer.Article Type: Review ArticleSection/Category: Anti-tumour TreatmentKeywords: Esophageal cancer; chemoradiotherapy; elective lymph node irradiation. Corresponding Author: Dr.Med. Shixiu Wu, M.D.Corresponding Author's Institution: Hangzhou cancer hospitalFirst Author: X.D. LiangOrder of Authors: X.D. Liang; R.F. Xie; T. Song; Y.B. Gao; Shixiu Wu, M.D.Abstract: Background: Esophageal cancer remains one of the most lethal carcinomas and concurrent chemoradiotherapy has been accepted as the standard non-surgical treatment. However, no consistent conclusions have been reached whether elective lymph node irradiation (ENI) should be delivered. Therefore, we performed a systematic review of the literature on the feasibility and value of ENI during definitive concurrent chemoradiotherpay for esophageal cancer.Methods: A search based on PubMed electronic databases was carried out to select studies including definitely concurrent chemoradiotherapy with ENI for esophageal cancer. All of the studies were evaluated carefully regarding with acute and late toxicities, treatment-related death, patterns of failure and overall survival.Results: Fourteen studies were identified with a total of 975 patients included. Concurrent chemoradiotherapy with ENI was feasible with acceptable acute and late toxicities. The localregional control rate seems to be higher with ENI, comparing with studies which omitted ENI. However, no obvious overall survival benefit with ENI was indicated in this review.Conclusion: the localregional control rate seems to be higher with ENI in concurrent chemoradiotherapy for esophageal cancer and no obvious better OS results were indicated in this review. Therefore, the value of ENI remains controversial and further prospective phase III trials in this setting are highly warranted.Suggested Reviewers:Conflict of Interest StatementCompeting interestsThe authors declare that they have no competing interests.Cover LetterJun 1, 2014Dear Editor:We would like to submit the manuscript entitled ‘The value of elective lymph nodeirradiation in definitive concurrent chemoradiotherapy for esophageal cancer.’ whichwe sincerely wish to be considered for publication in Cancer Treatment Reviews.Esophageal cancer remains one of the most lethal carcinomas and concurrentchemoradiotherapy has been accepted as the standard non-surgical treatment.However, no consistent conclusions have been reached whether elective lymph nodeirradiation (ENI) should be delivered. Therefore, we performed a systematic review ofthe literature on the feasibility and value of ENI during definitive concurrentchemoradiotherpay for esophageal cancer.We confirm that this paper has not been published elsewhere, nor is it currently underconsideration by any other journal. We hereby acknowledge that all mentionedauthors have contributed substantially to conception and design of the study, draftingor revising the paper as well as giving final approval for submission. We confirm thatwe had full access to all the data in the study and we take final responsibility for thedecision to submit the manuscript for publicationYours sincerely,S.X. Wu*Highlights (for review)Highlights.Definitive CCRT with ENI is feasible with acceptable acute and late toxicities foresophageal cancer. The local-regional control rate seems to be higher with ENI andimproved control of local and regional tumors could lengthen OS, Multicenter phaseIII clinical trials are urgently needed and our ongoing prospective randomized clinicaltrial (NCT00686114) is awaited.*ManuscriptClick here to view linked ReferencesArticle type: Review;Title: The Value of Elective Lymph Node Irradiation in DefinitiveConcurrent Chemoradiotherapy for Esophageal Cancer.Authors: X.D. Liang1, R.F. Xie1, T. Song1, Y.B. Gao1, S.X. Wu1*Author’s affiliation:1. Department of Radiation Oncology, Hangzhou Cancer Hospital,Hangzhou 310000, Zhejiang, P. R. China.*Full address for correspondence:Department of Radiation Oncology, Hangzhou Cancer Hospital, No.34, Yanguan Lane, Shangcheng District, Hangzhou 310000, P. R. China,TEL: +86-571-86826086; FAX: +86-571-86062281; E-mail:wushixiu@The authors indicated no potential conflicts of interest.AbstractBackground: Esophageal cancer remains one of the most lethal carcinomas and concurrent chemoradiotherapy has been accepted as the standard non-surgical treatment. However, no consistent conclusions have been reached whether elective lymph node irradiation (ENI) should be delivered. Therefore, we performed a systematic review of the literature on the feasibility and value of ENI during definitive concurrent chemoradiotherpay for esophageal cancer.Methods: A search based on PubMed electronic databases was carried out to select studies including definitely concurrent chemoradiotherapy with ENI for esophageal cancer. All of the studies were evaluated carefully regarding with acute and late toxicities, treatment-related death, patterns of failure and overall survival. Results: Fourteen studies were identified with a total of 975 patients included. Concurrent chemoradiotherapy with ENI was feasible with acceptable acute and late toxicities. The localregional control rate seems to be higher with ENI, comparing with studies which omitted ENI. However, no obvious overall survival benefit with ENI was indicated in this review.Conclusion: the localregional control rate seems to be higher with ENI in concurrent chemoradiotherapy for esophageal cancer and no obvious better OS results were indicated in this review. Therefore, the value of ENI remains controversial and further prospective phase III trials in this setting are highly warranted.Key Words: Esophageal cancer; chemoradiotherapy; elective lymph node irradiation.IntroductionEsophageal carcinoma is the eighth most common cancer and sixth cause of cancer death with approximately 480 000 new cases and 400 000 deaths annually worldwide 1-3. Esophageal cancer remains one of the most lethal carcinomas and the prognosis is dismal with surgery or radiotherapy (RT) alone. Surgery is the standard treatment for patients with resectable esophageal cancer currently. Concurrent chemoradiotherapy (CCRT) has been considered as the standard non-surgical treatment for esophageal cancer based on the results of Radiation Therapy Oncology Group (RTOG) 85-01 trial 4and 94-05 trial 5. However, there were disagreements between two trials such as elective lymph node irradiation (ENI) was used in RTOG 85-01 but was omitted in RTOG 94-05. Subsequently, the incidence of local-regional failure rate (44.3%) was apparently decreased in RTOG 85–01 than the standard arm in RTOG 94-05 (55%), which suggested that ENI could improve local-regional control rate. Better local-regional control rate with ENI was also reported in some studies 4, 6-10but in other studies 11-16, and thus no consistent conclusions could be reached. To date, only one small sample sized prospective clinical trial 17 has been focused on the value of ENI in CCRT for cervical and upper-thoracic esophageal cancer and the conclusions were inconclusive. Therefore, the role of ENI in CCRT for esophageal cancer remains controversial.Esophageal carcinomas are prone to spread axially to regional lymphatics and high incidence of occult regional lymph node metastasis was revealed by surgery 18-21. A total of 20 patients (26%) were found to have node metastasis in the dissected neck lymph nodes while the primary lesions were located at upper/middle/lower esophagus (7/42/28 patients respectively) 18. Better overall survival (OS) with prophylactic three-field lymph node dissection had been reported in esophageal cancer 18, 19, 22. In theory, CCRT with ENI may improve local control and thus improve OS in line with the benefit of three-field lymph node dissection in curative surgery.The purpose of this study was to review the value of ENI in CCRT for esophageal cancer regarding with toxicities，local-regional control rate and OS. Only articleswith ENI in definitely CCRT were reviewed.Methods and materialsThis review was designed to investigate the feasibility and value of ENI in definitively CCRT for patients with esophageal cancer. The literature search was conducted with assistance from a research librarian in the database of PubMed, to identify all clinical trials since 1980 including definitely CCRT with ENI in esophageal cancer. The following terms were explored and used for search: (esophageal OR esophagus OR oesophageal) AND (cancer OR carcinoma OR neoplasm) AND chemotherapy AND radiotherapy. Studies were excluded as following: neoadjuvant or adjuvant chemoradiotherapy combined with surgery; combined with target therapy; not published in English; not published in full text; phase I study; with other site of cancers. The abstracts of articles were reviewed by the two investigators. Irrelevant citations were removed according to the criteria mentioned above, thus creating a preliminary set of potentially relevant publications. Secondly, the full text articles were distributed to the two reviewers along with an evaluation form customized for reviewing the treatment outcomes for ENI in CCRT. Two reviewers independently evaluated a number of allocated articles and extracted information regarding study design, study population, methods, outcome measures, results and conclusions for each article. The evaluation results were compared and re-evaluated until consensus was reached between two reviewers or decided by the corresponding author. When complete information was not available, attempts were made to contact the corresponding authors of the studies for additional information. Fourteen studies with a total of 975 patients were included and the characteristic was shown in table 1.Results1.Clinical target volume (CTV) for ENI.There are some slight differences in literature regarding the CTV of ENI. At large, the CTV of ENI included the whole thoracic esophagus and periesophageal lymph nodes. For the boost field, margins were usually at least 2-5 cm in thecraniocaudal direction for the primary tumor, and 1-2 cm in the lateral direction for adjacent mediastinum.For cervical esophageal cancer, supraclavicular region were included in the CTV of ENI. However, lower mediastinal lymph nodal region was excluded by some authors 23.For upper thoracic esophageal carcinoma, the CTV of ENI included superior mediastinal lymph nodes alone by some authors7. Supraclavicular fossa nodes were included in the CTV for ENI in most studies 4, 6, 9, 10, 12, 13, 24.The CTV of ENI included mediastinal and perigastric lymph nodes for carcinoma of the middle thoracic esophagus7. Supraclavicular fossa was also included in the CTV of ENI in many studies 4, 9, 10, 12, 24.The supraclavicular nodes were not included while lesions were located in the lower third of the esophagus 4, 6, 16, 24. Perigastric nodes were included in ENI for lower thoracic esophageal carcinoma 7, 10, 23 and celiac lymph nodal region were also included in many studies 6, 7, 10, 13, 23.2.Feasibility of CCRT with ENI2.1 acute toxicities.Although acute toxicities of grade 3 or higher were frequently reported (table 2), CCRT applied with ENI was feasible. Leukopenia and esophagitis were the most common toxicities noted in literature. The incidence of leukopenia (grade 3 or higher) varied from 24% to 82.3%, and that of esophagitis varied from 6% to 35%. Other common toxicities included nausea, vomitting, anemia, infection, stomatitis, thrombocytopenia and cardiac ischemia.2.2 late toxicities.Only 6 papers detailed late toxicities (table 3). Cardiac related toxicities were the most common late toxicities and the incidence varied from 0 to 16%, most of patients suffered from pericardial effusion. The incidence of grade 3 or greater esophagus-related toxicities (dysphagia, stenosis, fistula) varied from 0 to 13%. The incidence of pneumonitis varied from 0 to 5.9%. Pleural effusion occurred in 0 to 5% patients. One case of gastrointestinal hemorrhage was reported.2.3 treatment-related death.A total of 25 deaths were reported to be associated with treatment and the incidence of treatment-related death varied from 0 to 12.5% (table 4) with pooled estimates of 2.6% 4, 6, 8, 10-12, 15, 24. On the whole, eight patients died from pneumonitis, 6 died from esophagus related (mainly fistula), 4 from upper-gastrointestinal hemorrhage. Each one patient died from pericarditis, pleural effusion, myocardial infarction, renal failure and neutropenic sepsis respectively. In addition, two patients died at home without defined cause 4 and 10 weeks after treatment 24.plete response rateComplete remission (CR) rate with ENI in this review varied from 33% to 75%. It seems that CR rates were comparable between higher dose (around 60Gy) and lower dose (around 50Gy) with ENI.4.Patterns of failure with ENIIn order to evaluate the patterns of failure, localregional failure was defined as the first site of failure (local persistence plus localregiongal recurrence) in this review. Distant failure was defined as failure in any site beyond the primary tumor and regional lymph nodes accordingly. Some authors defined localregional failure as localregional failure without distant failure, while other authors defined localregional failure as localregional failure both with and without distant failure. We defined localregional failure rate 1 (LRFR1) as the incidence of localregional failure without distant failure and localregional failure rate 2 (LRFR2) as the incidence of localregional failure both with and without distant failure. Localregional failure pattern in all of the articles included in this review was shown in table 5. For all of the studies included in this review (patients in stage I-IV), LRFR1 ranged from 26.5% to 54.68% and LRFR2 ranged from 29.4% to 61%. We analyzed these articles as a whole group, the pooled estimates of LRFR1 and LRFR2 were 45.6% and 52.3% respectively. A total of 6 studies were identified which including patients in stage I-III only (mainly stage II-III) 4, 6, 7, 9, 15, 16, the LRFR1 and LRFR2 were 27% to 47.8% and 35% to 61% respectively and the failure pattern was shown in table 5. LRFR1 and LRFR2 were 37.7% and 46.2% for a pooled analysis of all of the 6 studies.5.OS with ENIThe mean and median OS were 62.5% and 59% at 1 year; 41.5% and 41% at 2 year; 40.7% and 43% at 3 year; 29.2% and 27.5% at 5 year respectively (table 6). It was comparable between higher dose group (around 60 Gy) and lower dose group (around 50 Gy) with ENI regarding OS (table 7).6.Dose of ENI and boostIn RTOG 8501 and another report 16, 24, the dose of ENI was 30Gy in 15 fractions, and additional 20 Gy was delivered to the boost field. Kodaira et al 11delivered 36 Gy for ENI and the total dose to the boost field was 63Gy. The highest dose delivered to the CTV of ENI was 50.4 Gy and no boost was delivered in that article 10. The dose of ENI varied from 40 to 41.4Gy and the total dose varied from 50.4 to 60 Gy in the other studies 6-9, 12-14, 23.7.Chemotherapy regimenNo difference was documented between protracted infusion chemotherapy and standard short-term infusion chemotherapy for esophageal cancer 23. Two cycles of chemotherapy were concurrently administered in all of the studies and additional two cycles of chemotherapy were delivered in some studies 7, 12, 24. 5-Fu combined with cisplatin was used both in CCRT and consolidated chemotherapy by most authors. Some authors replaced cisplatin with nedaplatin 10, 11.DiscussionThere is a lack of data consistently support or against the delivering of ENI in patients with esophageal cancer treated with CCRT, and the value of ENI in CCRT remains uncertain. Different UICC stage system and toxicities evaluation criterion were used, different races and stages were included in this review, it is difficult to compare the results because of heterogeneity of these studies. However, based on these results included in this review, the localregional control rate seems to be higher with ENI compared with that in RTOG 94-05 which omitted ENI. Nevertheless, we suggest that there is not a large OS difference between the treatment approaches with or without ENI.In this review, LRFR1 and LRFR2 were comparable with those in the combined modality arm of RTOG 8501. However, LRFR1 and LRFR2 with stage I-III (37.7% and 46.2%) were much lower than those in RTOG 94-05 (53.2% and 55%) while ENI was omitted in that study, which indicates that ENI could improve localregional control. However, in the RTOG 95–04 trial, patients with T1-4N0-1 were included (7 patients with T4), the prognosis could be worse than that in the RTOG 8501, in which patients with T1-3N0-1 were included. In addition, the regional failure rate (7% and 9%) in RTOG 94-05 was consistent with other report 25. Only one patient of elective nodal failure was noted in patients with ENI by Onozawa et al., therefore ENI is effective for localregional controlling 13.Studies of CCRT without ENI 26-33 have yielded an OS of 28–56% at 2 years, and 20–35% at 3-5 years, including the trial RTOG 95-04, in which 2 year OS of 31% and 40% were reported. So the results of OS are comparable to that in this review and it seems that a better localregional control could not translate into an obvious OS benefit. Due to the difference of study design, the relatively small number of enrolled patients, it was difficult to compare the OS results between studies with ENI and without ENI. On the other hand, the main failure after CCRT was local failure and distant metastases. Therefore, it is difficult to translate a better regional control with ENI into an obvious OS benefit although an OS benefit may exist. Dissection of regional lymph nodes seems to improve the OS of patients for esophageal cancer 19. In addition, the 5-year OS in this review was comparable to that (15–40%) reported in patients with resectable esophageal cancer who underwent surgery 34, though many patients with unresectable disease were included in this review.The OS results were comparable between higher dose group (around 60 Gy) and lower dose group (around 50 Gy) with ENI in this review. However, the median 5 year OS was 35% in the higher dose group was much higher comparing with historical data and further research is needed. We suggest that total radiation dose of 50 Gy was reasonable in CCRT and 50-64.8 Gy was also acceptable.Herskovic et al 4 noted the LRFR2 of 44% with 30 Gy of ENI and Yamashita etal 10 noted the LRFR2 of 47% with 50-50.4 Gy of ENI, which indicates that 30 Gy of ENI may be comparable with 50-50.4Gy of ENI and that 30 Gy could be enough dose for ENI. The incidence of grade 3 or greater acute toxicities was high in the study by Yamashita et al 10, which indicates more severe acute toxicities with higher radiation dose (50-50.4 Gy) for ENI. However, the late toxicities and death rate in that study were comparable to those studies with lower dose of ENI. The 5-year OS was also satisfactory with 30 Gy dose of ENI in RTOG 8501 10. Therefore, 30 Gy of radiation dose could be adequate for ENI and 30 to 50 Gy of ENI was acceptable.Studies in this review showed that CCRT with ENI was feasible with acceptable acute and late toxicities. Acute and late toxicities were comparable between high dose group (total dose of 60Gy) and low dose group (total dose of 50Gy) with ENI. However, most studies with high total dose had a 1-2 week treatment break. In RTOG 9504, more treatment-related deaths (11 patients, 10%) occurred in the high-dose arm without ENI. However, 7 of these 11 treatment-related deaths occurred in patients who had received 50.4 Gy or less. Therefore, it seems that more deaths were not resulted from higher dose of irradiation.On contrary, Zhao et al 25argued that ENI was unnecessary. However, regional nodal recurrence (out-of-field) was 8% in that study consistent with that in RTOG 95-04, which could be prevented with ENI. ENI may be necessary in definitive CCRT for esophageal cancer in order to reach a higher localregional control.We did not include the only one published prospective randomized trial 17 in this review. The causes are that, firstly, the localregional failure rate of 13.6% with ENI and 17.6% without ENI was much lower than those included in this review; secondly, the 1 year and 2 year OS was much higher than that in this review, however, the 4 year OS fall to nearly zero abruptly; thirdly, patients with cervical and upper-thoracic esophageal cancer were included only, superior mediastinal lymphatic regions could receive 60-70% of the prescribed dose in the group without ENI 17; so we did not consider the patients characteristic in that study were consistent with those in this review.ConclusionsIn conclusion, definitive CCRT with ENI is feasible with acceptable acute and late toxicities for esophageal cancer. The localregional control rate seems to be higher with ENI and improved control of local and regional tumors could lengthen OS, although no obvious better OS results were indicated in this review. It seems appropriate to enlarge the radiation field to cover the lymph node areas at high risk and lower the radiation dose to 30 Gy for ENI in CCRT for esophageal cancer. The incidence and severity of toxicities will be improved with modern radiation technique and better supportive care. Consequently, the treatment related death rate may be reduced further. To date, there is no large phase III study has been published evaluating the role of ENI in definitive CCRT for esophageal cancer and its role has not been defined. Therefore, multicenter phase III clinical trials are urgently needed and our ongoing prospective randomized clinical trial (NCT00686114) is awaited. DisclosureThe authors have declared no conflicts of interest.Reference1. Parkin DM, Bray F. Evaluation of data quality in the cancer registry: principles and methods Part II. Completeness. Eur J Cancer. 2009;45:756-64.2. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. 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HOTAIR药理药效研究 动物模型
HOTAIR,a prognostic factor in esophageal squamous cell carcinoma,inhibits WIF-1expression and activates Wnt pathwayXiao-Song Ge,2Hua-Juan Ma,2Xiao-Hui Zheng,2Hong-Lian Ruan,Xiao-Yu Liao,Wen-Qiong Xue,Yuan-Bin Chen, Ying Zhang and Wei-Hua Jia1Department of Experimental Research,State Key Laboratory of Oncology in South China,Sun Yat-Sen University Cancer Center,Guangzhou,China (Received June7,2013⁄Revised September9,2013⁄Accepted September19,2013⁄Accepted manuscript online October1,2013)Long non-coding RNAs(LncRNAs)have been recently found to be pervasively transcribed in the genome and critical regulators of the epigenome.HOTAIR,as a well-known LncRNA,has been found to play important roles in several tumors.Herein,the clini-cal application value and biological functions of HOTAIR were focused and explored in esophageal squamous cell carcinoma (ESCC).It was found that there was a great upregulation of HOTAIR in ESCC compared to their adjacent normal esophageal tissues.Meanwhile,patients with high HOTAIR expression have a significantly poorer prognosis than those with low expression. Moreover,HOTAIR was further validated to promote migration and invasion of ESCC cells in vitro.Then some specific molecules with great significance were investigated after HOTAIR overex-pression using microarray and quantitative real time-polymerase chain reaction(qPCR).WIF-1playing an important role in Wnt⁄b-catenin signaling pathway was selected and further tested by immunehistochemistry.Generally,inverse correlation between HOTAIR and WIF-1expression was demonstrated both in ESCC cells and tissues.Mechanistically,HOTAIR directly decreased WIF-1expression by promoting its histone H3K27methylation in the promoter region and then activated the Wnt⁄b-catenin signaling pathway.This newly identified HOTAIR⁄WIF-1axis clarified the molecular mechanism of ESCC cell metastasis and represented a novel therapeutic target in patients with ESCC.(Cancer Sci,doi:10.1111/cas.12296,2013)E sophageal cancer is one of the most common cancersworldwide.(1,2)Esophageal squamous cell carcinoma (ESCC)is the most prevalent type in eastern countries,includ-ing China.(3)Despite the wide application of radical esophag-ectomy and systemic chemo-radiotherapy,the overall5-year survival rate of patients with ESCC remains extremely low.(4) Therefore,it is essential not only to quickly identify clinically applicable biomarkers for ESCC prognosis but also to deter-mine the crucial molecular mechanisms associated with this disease.Long non-coding RNAs(LncRNAs)are a new class of tran-scripts recently discovered to be pervasively transcribed in the genome and are critical regulators of the epigenome.(5)As with microRNAs,LncRNAs may be useful in predicting tumor prognosis and in regulating tumorigenesis.(6)As a result, LncRNAs have gained attention worldwide.HOTAIR,a widely focused LncRNA,was initially proposed to be involved in primary breast cancer and breast cancer metastasis.(7)HOTAIR overexpression induces genome-wide targeting of the polycomb repressive complex2(PRC2),lead-ing to an altered methylation of histone H3lysine27(H3K27) and gene expression.Clinical studies demonstrated that HOTAIR overexpression is a potential candidate biomarker for predicting tumor recurrence in hepatocellular carcinoma patients who have undergone liver transplant therapy and may be a potential therapeutic target.(8)Consistent with the role in breast tumors,HOTAIR upregulation was also discovered to be a critical element in metastatic progression in colorectal cancer(CRC).(9)Furthermore,frequent HOTAIR upregulation was discovered to be associated with the malignant behavior of gastrointestinal stromal tumors.(10)Overall,numerous stud-ies have clearly demonstrated the importance of HOTAIR in tumors.(7–13)HOTAIR overexpression correlates with poor prognosis and promotes metastasis of the ESCC cell line.(14) However,few studies have examined in detail the molecular mechanisms of HOTAIR in ESCC.The Wnt⁄b-catenin signaling pathway is an evolutionarily conserved pathway required for adult tissue maintenance in bone,heart,muscle,and other tissues.In addition,the pathway plays an important role in regulating cell proliferation and migration and in controlling tumor progression.Aberrant acti-vation of Wnt⁄b-catenin signaling,generally caused by genetic and epigenetic alterations,has been linked to several types of tumors,including ESCC.(15,16)Common epigenetic alterations include DNA hypermethylation in the promoter region of APC,Axin2,SFRPs,Wnt inhibitory factor1(WIF-1),etc.(17) WIF-1,as a key inhibitor of the Wnt⁄b-catenin signaling pathway,binds directly to extracellular Wnt ligands,prevent-ing their interaction with the receptors and leading to degrada-tion of cytosolic b-catenin by the APC⁄Axin1destruction complex.(18)Previous studies have determined that the epige-netic silence of WIF-1due to promoter hypermethylation is a frequent mechanism that causes aberrant activation of the Wnt⁄b-catenin pathway in several human cancers,as well as in ESCC.(19,20)Generally,WIF-1downregulation is a promi-nent characteristic of tumor progressions.However,the epige-netic regulator of WIF-1and the regulatory mechanism is poorly understood.In this study,the prognosis value of HOTAIR in ESCC was further measured in a larger clinical cohort.More impor-tantly,it was observed that altered HOTAIR expression was involved in repressing the transcription of WIF-1,thus acti-vating the Wnt⁄b-catenin signaling pathway.This is thefirst report that HOTAIR increases the H3K27methylation in the WIF-1promoter and induces its silence.Apart from the DNA hypermethylation in the WIF-1promoter region,trimethyla-tion of H3K27represented a novel mechanism for epigenetic regulation of WIF-1.This regulatory mode mediated by LncRNA may illustrate the epigenetic alteration of other key molecules.1To whom correspondence should be addressed.E-mail:jiaweih@2These authors contributed equally to this work.doi:10.1111/cas.12296Cancer Sci|2013Materials and MethodsESCC samples and cell lines.A total of137ESCC tumor tissues and matched adjacent normal esophageal tissues were obtained from patients at the Sun Yat-Sen University Cancer Center between the years2004and2007.The tumor stage was classified according to the tumor node metastasis(TNM)clas-sification of the6th edition The American Joint Committee on Cancer.The patients included94men and43women with age ranging from34to80(mean age:56years).All patients recruited to this study did not receive any pre-operative treat-ments.This study was approved by the Human Ethics Com-mittee of the Sun Yat-Sen University Cancer Center and,all patients signed an informed consent form.The ESCC cell lines KYSE30,KYSE140,KYSE180, KYSE410and KYSE510were obtained from DSMZ,the Ger-man Resource Center for Biological Material and grown using standard condition(Data S1).RNA preparation,reverse transcription and qPCR.Total RNA was extracted from the cell lines and frozen ESCC samples using the TRIzol reagent(Invitrogen,Carlsbad,CA,USA). PrimeScript RT reagent Kit with gDNA Eraser(Takara, Dalian,China)and SYBR Premix Ex TaqII kit(Takara)were used to perform reverse transcription and qPCR(Data S1).RNAi and overexpression HOTAIR,migration and invasion assay.siRNA oligonucleotides targeting HOTAIR or the nega-tive control were transfected into KYSE180and KYSE140 cells,respectively,as described in the Data S1.Overexpression HOTAIR in KYSE180and KYSE410cells was performed by retrovirus mediated gene transfer.For migration and invasion assay,Chamber from Becton Dickson(Bedford,MA,USA) (8l m pore size)with or without matrigel were used (Data S1).cDNA microarray and bioinformatic analysis.The cDNA was labeled and hybridized to the4944K human gene expression microarray(Agilent Technologies,Santa Clara,CA,USA). The threshold set for up-and downregulated genes was a2.0-fold change.The microarray results are indicated in Table S1.A gene ontology(GO)annotation for Biological Process and Pathway analysis was conducted using DAVID online tools (/).(21)A selection of the topfive enriched the GO biological process for genes with a P-value lower than0.05.The data were sorted by the number of genes that associated with each GO term and pathway.Nuclear protein extraction,Western blot,and chromatin immu-noprecipitationassay.The nuclear protein fraction was extracted using the NE-PER Nuclear and Cytoplasmic Extrac-tion Kit(78835;Pierce Biotechnology,Rockford,IL,USA) from KYSE180-vector cells and KYSE180-HOTAIR cells, according to the manufacturer’s instructions.For Western blot, antibodies against WIF-1(sc-373780;Santa Cruz,CA,USA), b-catenin(06-734;Millipore,Bedford,MA,USA)and GAP-DH(D16H11,CST)were used.A chromatin immunoprecipitation(CHIP)assay was conducted using EZ-ChIP(17-371;Millipore),according to the manufacturer’s instruction.Retrieved DNA was detected by PCR reaction. The primers for the WIF-1promoter region are listed in Data S1.Tissue microarray construction and immunohistochemistry (IHC).The tissue samples(98of the137ESCC tissues that mentioned before)were collected,fixed in formalin and embedded in paraffin.The HE-stained sections from a single random block of each specimen were reviewed by a senior pathologist to define the representative tumor.Two cores of each sample were obtained using a tissue array instrument (ALPHELYS,Plaisir,France).Immunohistochemistry(IHC) was performed based on a previously described method.(22) Immunofluorescent confocal analysis.The cells were probed using a b-catenin primary antibody(06-734;Millipore)and Alexa Fluor488secondary antibodies(Invitrogen).Nuclear DNA were stained with DAPI.Confocalfluorescent images were captured using an Olympus FV500microscope under a 609oil objective.Statistical analysis.Statistical analysis was performed using the Stata10.0statistical software package(Stata;College Station,TX,USA).Receiver operating characteristic(ROC) curves were used to determine the cutoff value for the HOTAIR high and HOTAIR low groups in this study.The gene expression levels of HOTAIR in tumors were compared with normal adjacent mucosa using the Wilcoxon test,whereas the associations between HOTAIR expression and clinical characteristics were evaluated using the chi-square test.Sur-vival curves were estimated using the Kaplan–Meier method. The log-rank test was used to estimate the significant differ-ences between the survival curves.A Cox proportional hazards analysis was performed to calculate the hazard ratio(HR)and the95%confidence interval(CI)to evaluate the association between HOTAIR expression and survival.In addition,a mul-tivariate Cox regression was performed to adjust for other co-variates.A two-tailed P-value of0.05or less was considered to be statistically significant.ResultsHOTAIR expression and clinicopathologic factors in ESCC.Upregulation of HOTAIR was frequently detected in ESCC tissues(Fig.1a).Approximately79%(108⁄137)of the tumor tissue expression of HOTAIR was>1.5-fold higher than the corresponding normal tissues.There was no significant association of the expression with gender,age,tumor location (upper⁄middle⁄lower),T status as indicated in Table1.How-ever,an association between HOTAIR expression and histo-logic grade(G1⁄G2⁄G3)or Nodal status approached statistical significance(P=0.080and P=0.074for histologic grade and Nodal status,respectively;Table1).The137patients were then divided into HOTAIR-high(n=90)and low groups(n=47),(a)(b)(c)Fig.1.HOTAIR is upregulated in esophageal squamous cell carcinoma(ESCC)tissues and have prognostic value for metastasis and death.(a)Box plot analysis,based on quantitative reverse transcription-polymerase chain reaction(qRT-PCR)analysis expression of HOTAIR in137 paired ESCC tumor samples and corresponding normal esophageal tissues.Kaplan–Meier curves for metastasis-free survival(b)or overall survival (c)of the same137ESCC tissues measured in(a).**P<0.005.2doi:10.1111/cas.12296according to the ROC curve method.A high expression level of HOTAIR is a significant predictor of subsequent metastasis and death(P<0.0001for both metastasis and death,Fig.1b, c).Compared with the low expression group,the5-year overall survival rates were46%in the high expression group and81% in the low expression group.Moreover,multivariate analysis indicated that HOTAIR expression was an independent prog-nostic indicator for metastasis and death(P<0.001and P=0.002for metastasis and death,respectively,Table2).HOTAIR promoted the migration and invasion of ESCC cell lines in vitro.Given upregulation of HOTAIR was significantly associated with distant metastasis in patients with ESCC,we inhibited HOTAIR function via small interfering RNAs(siR-NAs)in KYSE180and KYSE140,two cell lines that express HOTAIR(Fig.S1).HOTAIR siRNAs decreased their migra-tion and invasion ability(Fig.2a,c).Conversely,the ESCC cell lines KYSE180and KYSE410(KYSE410cells has rela-tive low HOTAIR expression,Fig.S1)with stable HOTAIR expression demonstrated greater cancer cell migration and invasion(Fig.2d–h)without affecting the cell proliferation of both cell lines(Fig.S2).Based on our results,the overexpres-sion of HOTAIR promoted the migration and invasion of ESCC cells in vitro.A wide range of gene expressions was altered after HOTAIR overexpression.Gene expression profiling of HOTAIR overex-pression in KYSE180cells on cDNA microarrays(44K human microarrays;Agilent Technologies)indicated that HOTAIR modulates the transcriptional regulation of847genes(427 upregulated,420downregulated;Table S1).Gene ontology analysis indicated that significant numbers of upregulated genes were associated with biological processes such as intra-cellular signaling and cell adhesion,whereas downregulated genes were clustered in biological processes such as ion trans-port,homeostatic processes,immune response,response to organic substance and cell proliferation(Fig.3a).Consistently, pathway analysis revealed that11genes were enriched in the WNT⁄b-catenin signaling pathway,which is usually activated and promotes metastasis of ESCC(Fig.3b).To further identify the possible target genes of HOTAIR,we downloaded and analyzed microarray data collected for HOTAIR RNAi gastro-intestinal stromal tumor(GIST)cells(GIST-T1)from Gene Expression Omnibus(GEO).Forty-three potential target genes overlapped in the two microarray data(Fig.3c),among which WNT5B and WIF-1are important regulatory molecules in the WNT⁄b-catenin signaling pathway.In addition,seven genes, which have been reported to be related to ESCC,were also validated by qRT-PCR.WIF-1had the most significance dif-ference(fold change13.9,P<0.005)and the only one that expression been suppressed by HOTAIR overexpression (Fig.S3).HOTAIR promoted H3K27trimethylation in the WIF-1promoter region and was inversely correlated with WIF-1expression.Con-sistent with the qRT-PCR results,the protein level of WIF-1 was greatly decreased after HOTAIR overexpression(Fig.4a). Conversely,the knock down of HOTAIR by siRNA restored the mRNA level of WIF-1;however,the protein level slightly changed(Fig.4b).A Dual Luciferase reporter assay was performed to further demonstrate the inverse correlation between HOTAIR and pared to the KYSE180-vector group,a significant decrease of luciferase fluorescence intensity was observed in the KYSE180-HOTAIR group(Fig.S4).Besides,it has been reported that EZH2 was the main component of PRC2,and HOTAIR exerting its function was PRC2dependent.After PRC2depletion,there was a great increase of WIF1mRNA expression.Correspond-ingly,migration ability was greatly decreased(Fig.S5).After adding recombinant human WIF-1,it could significantly inhi-bit HOTAIR-induced migration and invasion.These results strongly indicated that HOTAIR-induced activity was WIF-1 dependent(Fig.S6).The inverse correlation between HOTAIR and WIF-1expression was further validated in16ESCC clini-cal samples using real time qRT-PCR(Fig.4c).Furthermore, immunohistochemistry analysis of WIF-1expression in98 ESCC tissues revealed that WIF-1expression was significantly inversely correlated with HOTAIR expression(Fig.4d,e). Spearman correlation analysis indicated there was a negative correlation between HOTAIR expression and WIF-1mRNA levels(r=À0.365,P<0.001).Based on the regulatory mechanism in breast and colorectal cancer,the levels of histone H3K27trimethylation in KYSE180-Vector andTable1.HOTAIR expression and clinicopathological characteristics in esophageal squamous cell carcinoma(ESCC)Characteristics Case numberHOTAIRexpression P-value High LowGenderMale9470240.497Female433310Age≤569472220.324>56433112T statusT1-2332670.775T3-41047727N statusN07352210.074N1⁄2⁄3645113Histologic gradeG1*******.080G2662442G332626Tumor locationUpper13850.843Middle896029Lower352213Table2.Multivariate analysis of risk factor for death and metastasisas thefirst recurrence event in esophageal squamous cell carcinoma(ESCC;Cox proportional hazards regression model)Risk factorsDeath MetastasisHR P-value95%CI HR P-value95%CIHOTAIRexpression(low⁄high)3.160.002 1.53–6.524.47<0.001 1.99–10.06Age(≤56⁄>56)1.430.2010.83–2.48 1.170.580.67–2.06Gender(male⁄female)0.980.9550.55–1.750.800.470.44–1.47Histologicgrade(G1⁄G2⁄G3)1.070.7320.74–1.53 1.240.260.85–1.79Tumorlocation0.780.3190.47–1.28 1.040.890.62–1.73T status(T1-2⁄T3-4)0.900.7280.50–1.640.890.730.47–1.69N status(N0⁄N1,2,3)3.18<0.001 1.76–5.77 2.190.008 1.23–3.93HR,hazards ratio;95%CI,95%confidence interval.Ge et al.Cancer Sci|2013|3KYSE180-HOTAIR cells were measured.Chromatin immuno-precipitation (CHIP)clearly demonstrated that there was a sig-nificant increase in H3K27trimethylation in the WIF-1promoter (Fig.4f).HOTAIR,targeting WIF-1,activated Wnt ⁄b -catenin signaling pathway.After HOTAIR overexpression,the presence ofb -catenin was reduced on cell membrane and increaseaccumulation in the cell nucleus (Fig.5a).The concentration of b -catenin in the cell nucleus and cytoplasma plus cell mem-brane were measured,and increased b -catenin expression was observed in the nucleus after HOTAIR overexpression by Western blot analysis (Fig.5b).Accumulation of b -catenin in cell nucleus indicated the activation of canonical Wnt ⁄b -cate-nin pathway.Then expressions of some downstream target(a)(b)(c)(d)(g)(h)(e)(f)Fig.2.HOTAIR promotes migration and invasion of esophageal squamous cell carcinoma (ESCC)cells in vitro (a),siRNA mediated knock down of HOTAIR in KYSE140and KYSE180cells transfected with negative control (siRNA-NC)or siRNA target HOTAIR (siRNA-HOTAIR)(error bars =SD,n =3).The relative mRNA level was normalized to the endogenous genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH).(b,c)Migration (b)and invasion (c)assay in the KYSE140and KYSE180cells that transfected with siRNA in (a)(error bars =SD,n =3).HPF,high power field.(d)qPCR of relative mRNA level of HOTAIR in retrovirus infected KYSE180cells and KYSE410cells.(error bars =SD,n =3).(e –h).Migration (e,g)and invasion (f,h)assay after enforced HOTAIR expression in KYSE180and KYSE410cells (error bars =SD,n =3).*P <0.05.(a)(c)(b)Fig.3.The expression of target genes regulated by HOTAIR.(a)Gene ontology analysis of HOTAIR overexpression microarray data in KYSE180Cells using the DAVID program.(b)Pathway analysis of HOTAIR overexpression microarray data in KYSE180Cells using the DAVID program.(c)Schematic depicting integrative approach that led to identification of putative HOTAIR target genes.Transcriptomic profiling of genes upregulated by greater than twofold upon HOTAIR overexpression in KYSE180cells were overlapped with genes down-regulated by more than twofold upon HOTAIR RNAI in GIST cells.4doi:10.1111/cas.12296genes such as ZEB1,SNAIL and MMP13,playing important roles in tumor progression,were all increased in the HOTAIR overexpressed cells.These results further demonstrated the activation of Wnt⁄b-catenin pathway(Fig.5c).Based on our results,it was suggested that HOTAIR,along with PRC2, inhibited WIF-1expression by increase H3K27trimethylation in the WIF-1promoter and then activated Wnt⁄b-catenin sig-naling pathway(Fig.5d).DiscussionIn this study consisting of137patients and in a previous study consisting of78patients,(14)HOTAIR was demonstrated as an independent prognostic indicator of ESCC(Fig.1).Compared with the previous study,evaluating HOTAIR in another clini-cal cohort in our study will greatly increase its value in clini-cal application.In fact,HOTAIR overexpression was also discovered to be associated with poor prognosis in other can-cers.(7–13)It was believed that this tendency would be demon-strated in more cancers.This common feature strengthened the clinical value of HOTAIR.It was reported that HOTAIR,in collaboration with PRC2, reprogrammed the chromatin state and regulated the expression of hundreds of genes by epigenetic regulation to promote can-cer metastasis.Considering the regulatory complexity and diversity in different types of tumors,it was necessary to con-duct a systematic study in ESCC.Consistent with the previous report in ESCC,HOTAIR overexpression promotes migration and invasion in ESCC cell lines(KYSE180and KYSE410). Compared with the single knockdown experiment,stable over-expression and a corresponding knockdown experiment in this study fully validated this phenomenon(Fig.2).The expression of847genes changed after HOTAIR overex-pression in ESCC cells(Fig.3a).Pathway analysis based on these genes by DAVID online tools revealed11genes were involved in WNT signaling(Fig.3b).Alteration of WNT sig-naling regulators including WIF-1and WNT5B were both demonstrated by microarray and qRT-PCR(Figs3c,S3).These results suggest that HOTAIR may affect the Wnt⁄b-catenin signaling cascade by epigenetic regulation.In addition, FOXQ1,FGF12and CXCR7,which contribute to ESCC progression,were also detected and validated in our study.(a) (c)(d) (f)(e) (b)Fig.4.The mechanism of HOTAIR that promotescell migration and invasion(a,b)Analysis mRNAlevel of WIF-1by qRT-PCR and protein level byWestern blot in HOTAIR overexpression KYSE180cells(a)and HOTAIR RNAI KYSE180cells(b)(errorbars=SD,n=3).(c)Analysis mRNA level of bothWIF-1and HOTAIR by qRT-PCR in16esophagealsquamous cell carcinoma(ESCC)tumor tissues.TheRelative mRNA level was normalized to theendogenous genes GAPDH.(d)WIF-1expression byimmunohistochemical staining(IHC).The imagesshow four different tumor samples with differentcytoplasmatic staining intensities gainst the WIF-1protein.The IHC-score of0,1,2or3is shown atthe lower right corner of ach image.The examplesare representative for the whole set of samples(magnification:200).(e)The results were calculatedon the basis of analyses performed using98ESCCissue samples.The relationship between WIF-1andHOTAIR expression was compared sing Spearman’scorrelation coefficient.(f)CHIP assay in KYSE180-Vector or HOTAIR ells.Primers locations areindicated in the WIF-1schematic.The PCR productswere analyzed by gel electrophoresis andquantitated by Gel Analyzer(error bars=SD,n=3).*P<0.05,**P<0.005.Ge et al.Cancer Sci|2013|5Epigenetic disruptions,including promoter CpG island meth-ylation and histone modification of tumor-related genes,have been identified as key events in cancer development.So far, many critical genes silenced by promoter methylation in ESCC have been reported.(23)It is worth noting that the expression of WNT signaling-related genes such as APC,SFRP1⁄2,WNT5A and WIF-1were all changed after promoter region methyla-tion.(17,24–27)However,the mechanism leading to this abnor-mality is unclear.The inverse correlation between HOTAIR overexpression and decreased WIF-1expression was validated in both ESCC cells and tissues(Fig.4a–e).In this study,we further confirmed that HOTAIR overexpression promoted H3K27methylation in promoter region of WIF-1(Fig.4f). H3K27trimethylation is characteristic of Polycomb group (PcG)target genes and is associated with transcriptional repression.(28)The PcG protein such as PRC2complex cata-lyzes H3K27methylation.(29)We suggest that HOTAIR bound by PRC2inhibits the expression of WIF-1.Although a direct interaction between HOTAIR and PRC2in ESCC cells is not yet reported,the abnormal PRC2expression has been associ-ated with various cancers including ESCC,(30,31)and HOTAIR was proved to directly interact with PRC2in types of tumors.(7,9,32)Consistently,a significant increase of WIF-1 mRNA expression and decrease of migration ability were observed in our experiment(Fig.S5).The HOTAIR⁄WIF-1 regulatory model identified in this study provides a novel view on WIF-1regulation.In fact,WIF-1downregulation has been reported to be involved in progression in types of tumors including ESCC.(27,33,34)Furthermore,Rubin et al.(35)have reported that inhibition of WIF-1could trigger Wnt⁄b-catenin signaling and thereby promotes tumor invasion and migration. In our experiments,exogenously added recombinant human WIF-1protein in HOTAIR overexpression ESCC cells inhib-ited the migration and invasion ability(Fig.S6).However,we did notfind the correlation between WIF-1protein level and prognosis of patients with ESCC.Based on this regulation model,further functional investigation is required for exploring the epigenetic alterations of other genes related to ESCC and exploring these genes in other HOTAIR-overexpression cancers.b-catenin,as a significant transcription factor,acts as the main effector of the canonical WNT signaling cascade.(36) Cytoplasmic b-catenin is degraded by a multiprotein degrada-tion complex when the WNT signaling pathway is inactive. Conversely,b-catenin evades degradation,accumulates in the cytoplasm andfinally translocates to the nucleus during WNT signaling pathway activity,thus exerting its transcriptional activity.It was hypothesized that HOTAIR overexpression activated the WNT signaling pathway by decreasing WIF-1 expression.As expected,there was a great increase in the amount of b-catenin in the cell nucleus after HOTAIR overex-pression(Fig.5a,b).Increased expression of some target genes such as ZEB1,SNAIL and MMP13further illustrated the acti-vation of canonical WNT⁄b-catenin signal(Fig.5c).Our results demonstrated the activation of the Wnt⁄b-catenin sig-naling pathway by inhibiting WIF-1expression after HOTAIR overexpression.Despite limitations,our results provided convincing insight into the mechanisms underlying HOTAIR-promoted migration and invasion of ESCC cells (Fig.5d).It had been reported that non-canonical Wnt(a)(b) (d)(c)Fig.5.HOTAIR promoted accumulation ofb-catenin in cell nucleus.(a)Immunofluorescencestaining of b-catenin in KYSE180-Vector cells andKYSE180-HOTAIR cells.Blue,DAPI;green,b-catenin;960.(b)Western-blot analysis of b-catenin in cellnucleus and cytoplasma plus cell membrane inKYSE180-Vector cells and KYSE180-HOTAIR cells.GAPDH was used as the control in cytoplasmand histone-H3was used as the control innucleus.Results in three experiments are similar.(c)Expressions of some downstream target genessuch as ZEB1,SNAIL and MMP13were measured byQ-PCR.(d)Proposed model illustrating the effect ofHOTAIR in the esophageal squamous cell carcinoma(ESCC).6doi:10.1111/cas.12296signaling through Wnt5B promoted cell migration in a previ-ous study.(37)Actually,we did observe that a significant increase of WNT5B expression after HOTAIR overexpression in KYSE180-HOTAIR cells(Fig.S3).However,further study showed that WNT5B depletion did not have an effect on cell migration(Fig.S7).Therefore,it was thought that HOTAIR induced cell metastasis was through canonical Wnt pathway. Apart from promoting migration and invasion,increased lev-els of b-catenin can also initiate transcriptional activation of proteins such as cyclin D1and c-myc,which control the G1to S phase transition in the cell cycle.However,we did not observe a significant increase of proliferation after HOTAIR overexpression(Fig.S2).In mechanism,the activity of HO-TAIR is at least in part due to interaction with PCR2,which enhances H3K27trimethylation to decrease expression of mul-tiple genes.(7)Previously,studies of HOTAIR in breast cancer, colon cancer and pancreatic cancer and so on indicated that HOTAIR-dependent gene regulation differed significantly among different cancer cells and was very complex.For exam-ple,an expanded function of HOTAIR in pancreatic cancer cells involving cell cycle progression was observed,compared to its function in breast cancer.(30)However,the regulation of HOTAIR on cell cycle in ESCC cells was not very clear.It was concluded that a balance underlying cell proliferation may not be broken.However,further functional investigation is needed to be conducted in the future.AcknowledgmentsThis work was supported by the National Basic Research Program of China(2011CB504303)and the Ministry of Science and Technology of China(2011ZX09307-001-04).We thank Dr Howard Y.Chang from the Howard Hughes Medical Institute(USA)for providing the PLZRS-HOTAIR vector.Disclosure StatementThe authors have no conflict of interest.References1Parkin DM,Bray F,Ferlay J,Pisani P.Global cancer statistics,2002.CA Cancer J Clin2005;55:74–108.2Jemal A,Bray F,Center MM,Ferlay J,Ward E,Forman D.Global cancer statistics.CA Cancer J Clin2011;61:69–90.3Umar SB,Fleischer DE.Esophageal cancer:epidemiology,pathogenesis and prevention.Nat Clin Pract Gastroenterol Hepatol2008;5:517–26.4Jemal A,Siegel R,Ward E et al.Cancer statistics,2008.CA Cancer J Clin 2008;58:71–96.5Guttman M,Amit I,Garber M et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs 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中医证候动物模型复制管见-2019年精选文档
中医证候动物模型复制管见随着科学的发展，中医药学的研究已经完全突破了长期以来以经典校注、引证发挥和临床诊治观察为主的传统模式，实验研究已成为中医科研方法体系的一个重要组成部分。
而在实验研究当中，中医证候动物模型的复制更是开展中医证候及中药新药实验研究的一个重要途径，以其实证性研究方法为广大中医药和中西医结合工作者采用。
近年来，为了探索中医证候动物模型的研制，诸多医家在此方面进行了广泛的研究，取得了较为理想的效果，然而仍存在许多问题需深入探讨，有待进一步的充实完善，笔者不揣浅陋，试探讨建立中医证候动物模型需要注意的有关问题。
1中医证候动物模型需要在中医基础理论体系的指导下研制中医证候动物模型是在中医学整体观念及辨证施治思想指导下，运用脏象学说和病因病机理论，把人类疾病原型的某些特征在动物身上加以模拟复制而成，是中医学人体证候的具体再现。
而中医的证是一个综合的症状群，它是疾病发生、发展过程中特定阶段的病理变化，有着深刻的病因病机理论。
是辨证论治的起点和核心，也是中医证候动物模型的灵魂。
只有在中医理论体系指导下研制的中医证候动物模型，才可以从根本上基本等同于人体临床证候，才具有现实意义。
而目前部分中医证候动物模型的复制。
不是在中医理论体系的指导下，而是纯粹利用化学药物造成一种类似中医的某一“证”的病理状态。
这种与中医临床貌合神离的模型或许实际上只是某些西药引起的毒性反应或机械损伤，出现的病理状态和中医的证候生拉硬套，难免有牵强附会之嫌。
比较典型的如采用注射大剂量外源性醋酸氢化可的松制作阳虚动物模型，四氯化碳肝毒性制作肝阴虚动物模型，利血平毒性制作脾虚动物模型，肾皮质电灼伤诱发“肾阳虚”动物模型等等。
笔者认为，中医证候动物模型所揭示的应该是中医学的基本内涵，这种利用西药造成的中毒反应和病理状态来苟合中医证候之表象，是难以模拟出人体证候之本质的，是不可取的，更是经不起推敲的。
我们应当在中医理论的指导下从本质上对证候进行复制，从而使实验模型和临床人体达到尽可能的一致，为中医证候的实验研究提供真实而可靠的依据。
33 Definitive Chemoradiotherapy 药理药效研究 动物模型
Copyright � The Korean Academy of Medical Sciences
Definitive Chemoradiotherapy with Capecitabine and Cisplatin in Patients with Esophageal Cancer: A Pilot Study
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patients were 20-75 yr of age with a performance status of 0-2 on the Eastern Cooperative Oncology Group (ECOG) scale. Plus, adequate hematological (WBC count ≥4×109/L, platelet count ≥100×109/L, hemoglobin ≥9 g/dL), renal (serum creatinine ≤1.5 mg/dL and creatinine clearance ≥ 50 mL/min), and hepatic (total bilirubin ≤2.0 mg/dL and serum transaminase level ≤3 times the upper limit of the normal range) levels were also required. Patients were ineligible if they had previously received chemotherapy or radiation therapy, or had other severe medical illnesses, distant metastasis, another active malignancy in the last 5 yr, except treated nonmelanoma skin cancer or cervical dysplasia, or a history of anaphylaxis to drugs. The institutional review board of the authors’ institution approved the protocol, and written informed consent was obtained from all patients before enrollment.
浅谈中药药理动物模型研究及其作用
浅谈中药药理动物模型研究及其作用作者：陈永惠王小林来源：《科技视界》2013年第30期【摘要】中药药理动物模型是中药药理学独具一格的研究方法，它使中药药理从中药和药理学中脱胎而出，形成了独特的学科体系。
本文系统论述了中药药理动物模型的定义、分类、作用；详细地分析了中药药理动物模型的现状和今后一个时期内的发展趋势；指出中药药理动物模型作为一种独特的研究方法，将为中药药理学和实验动物学的发展起到重要作用。
【关键词】中药药理；动物模型；病症中药的药理研究从20年代初，陈克恢开始麻黄研究[1]以来，研究方法逐步完善，研究领域日益扩大，研究水平不断提高，形成了自己的学科体系，这就是中药药理学。
其中一个重要标志，就是中药药理动物模型的研究和应用。
中药药理动物模型是中药药理学独具一格的研究方法，它使中药药理学从中药和药理学脱胎而出，形成了独特的学科体系。
因此，有必要对中药药理动物模型进行整理、探索为进一步指导中药药理学发展、丰富实验动物学的内容起作用。
故本文较系统地论述了中药药理动物模型的概念、分类、现状和作用，探讨了中药药理动物模型的发展趋势。
1 中药药理动物模型的概念中药药理动物模型是指根据中医药基本理论，为进行中药药理研究而对人类疾病原型的某些特征进行模拟复制，创造出的具有人类病证表现的动物实验对象及相关材料，包括人类疾病动物模型、人类证候动物模型、人类病证动物模型三部分的内容，它既是实验动物学的范畴，又是中药药理实验方法学的核心。
2 中药药理动物模型的分类及现状中药药理动物模型的研究历经几十年的发展，已研制出百余种证型，其造模方法大致可归纳为以下三类：2.1 依据中西医结合病因学说塑造动物模型：又称为中药药理病证动物模型[2]、病因病理结合型模型[3]这类模型的造模方法是既运用了中医的发病学说，又考虑了西医的致病原理，将现代医学的人类疾病动物模型与中医证候动物模型嫁接，建立病证结合动物模型。
如高脂性疾病血瘀证动物模型、失血性贫血血虚证动物模型、感染性休克厥脱证动物模型等，把现代医学的辨病论治与中医学的辨证论治结合起来，中西汇通[4]。
2022年实验动物模型及相关信息表
实验动物模型及相关信息表一、简表实验动物模型信息采集简表二、主要材料1、命名。
参考国际实验动物命名通用原则，结合动物模型类型的I、II和III级分类法进行联合命名，即“疾病+动物品种+造模方法〞进行命名，命名时应尽量细化。
中医药动物模型应表达中医药特点。
需用中英两种文字命名〔见〕。
2、制备方法包含实验材料、实验环境、实验操作规程等内容。
对涉及的实验动物、实验试剂、检测仪器应标明详细信息，并符合国家相关规定。
3、动物模型的评价与验证基于模型制备三原则〔表观效度、预测效度以及结构效度〕对模型指标进行评价，其中包含整体行为特征、组织器官、细胞和分子等在内的指标评价体系。
评价方法包含以整体实验为主，涵盖行为、影像、生理生化和组织切片等技术方法进行详细介绍。
采纳的仪器设备、试剂应满足模型评价的要求，指标完善，条件稳定。
评价指标包含核心指标〔如特异的基因改变、特异的染毒病原株或高载量病毒核酸、特异性免疫抗体〕；主要指标〔模拟人类疾病临床病症和体征、易感器官的特征性改变和特异性生物标志物〕；重要指标〔模拟疾病某些方面的改变的指标〕和辅助指标。
应包含阳性药物对其指标的证实效应。
应说明重复验证的批数。
中医药模型应有表达中医药特点的评价指标。
4、动物模型的生物安全性。
动物模型的制备和应用实验必须在具备相应资质的实验室开展。
动物模型的制备、应用过程中的监督治理、处置措施、对环境和生态影响等应符合国家相关法律规定。
5、商量和结论总结该模型鉴定和评价的技术方法和指标体系；分析该模型与国内外现有模型的异同；商量该模型的技术难点、创新性和应用价值。
6、有助于动物模型鉴定和评价的其它材料其它有助于评价的材料，包含第三方应用机构的证明、在行业一流学术刊物上发表学术文章和引用情况等材料。
三、承诺函本人保证提交的上述材料真实客观，全部数据来源符合我国的法律法规。
承诺人〔签字〕单位〔公章〕。
新药临床前药效学评价下
如豚鼠常用作过敏试验及维生素C缺乏症试验；家兔用于皮肤及眼的刺激 试验；鸭用于引起白内障的药物及病毒性肝炎治疗的模型。
5.3 品系之间的差异
不同种属动物对药物反应有差异性，就是同一种动物的不同 品系有时也有药物反应的不同。这种反应的差异是由遗传因 素决定的。
SD：白化大鼠－封闭群 主要生物学特性： • 对疾病抵抗力较强 • 自发性肿瘤的发病率较低 • 对性激素敏感性高 广泛用于药理学和毒理学研究，如高血压、镇痛抗炎、 内分泌、药物依赖性等实验
短毛豚鼠 主要生物学特性 • 豚鼠嗅觉、听觉较发达 • 不能合成维生素C • 豚鼠易引发速发性变态反应 • 呼吸系统和消化系统抗病能力较差 应用于过敏反应、皮肤刺激试验、平喘、镇咳、 局部麻醉药等研究
1、避免了人体实验造成的风险和伦理问题 2、利于研究发病率低，病程长的疾病 3、可严格控制实验条件，排除复杂因素 4、样品收集方便，结果分析容易
❖ 实验动物的三个特殊要求 • 人工培育，遗传背景清楚 • 生活环境设施应达国标中的等级标准 • 专门用于科研、教学、生产、检定、实验等
❖ 常用实验动物 • 小鼠、大鼠、豚鼠、地鼠（仓鼠）、 • 兔、犬、猴、猫、小型猪、鸡等。
3. 年龄的选择
一般选用成年动物。但有些实验要选用年龄不同的动物。
如观察药物对生长、发育、内分泌等作用时，以幼年动物为好； 而观察抗衰老作用时，则宜用老年动物或衰老过程较快的模型 动物。病毒性心肌炎体外模型的心肌细胞需用新生3～4 天SD大 鼠的心脏所得的细胞悬液，病毒性心肌炎在体模型宜用3～4周 龄BABL／C 雄性小鼠，ip柯萨奇B3病毒感染而成；豚鼠致喘不 仅要用纯种，而且体重宜小于200g的豚鼠引喘较敏感；抗肿瘤 的药物中细胞毒类药物药敏试验用大鼠宜用3～4周龄，约50～ 70g较宜；鸭乙型肝炎病毒试验需用1日龄北京雏鸭；减肥药试 验用刚断乳的SD大鼠，约50g，用于营养性肥胖模型。若是下 丘脑性肥胖模型则用新生乳鼠，自出生头一天起sc l5％谷氨酸 钠造模。
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PII S0360-3016(99)00253-9CLINICAL INVESTIGATION EsophagusMULTI-INSTITUTIONAL RANDOMIZED TRIAL OF EXTERNALRADIOTHERAPY WITH AND WITHOUT INTRALUMINAL BRACHYTHERAPYFOR ESOPHAGEAL CANCER IN JAPAN T OMOHIKO O KAWA ,M.D.,*T AKUSHI D OKIYA ,M.D.,†M ASAMICHI N ISHIO ,M.D.,‡Y OSHIOH ISHIKAWA ,M.D.,§K OZO M ORITA ,M.D.,AND J APANESE S OCIETY OF T HERAPEUTIC R ADIOLOGYAND O NCOLOGY (JASTRO)S TUDY G ROUP*Department of Radiology and Oncology,Tokyo Women’s Medical University,Tokyo,Japan;†Department of Radiology,Tokyo Medical Center,Tokyo,Japan;‡Department of Radiology,Sapporo National Hospital,Hokkaido,Japan;§Health and WelfareDepartment,Hyogo Prefectural Government,Hyogo,Japan;and Department of Radiation Oncology,Aichi Cancer Center,Aichi,JapanPurpose:With the aim of improving the results of treatment of esophageal cancer,we designed this multi-institutional,randomized trial to establish the optimal irradiation method in radical radiation therapy for esophageal cancer by clinically evaluating external irradiation alone and in combination with intraluminal brachytherapy.Methods and Materials:The study population consisted of patients with squamous cell carcinoma who were expected to be successfully treated with radical radiation therapy.The patients who could be given intraluminal brachytherapy at the end of external irradiation of 60Gy were stratified into 2groups.Patients assigned to receive external irradiation alone received boost irradiation of 10Gy/week on a schedule similar to the previous one,and with the same or smaller irradiation field.Intraluminal brachytherapy was performed,as a rule,with the reference dose point set at a depth of 5mm of the esophageal submucosa,and a total of 10Gy was irradiated at a daily dose of 5Gy,on a once-weekly schedule with low-dose-rate or high-dose-rate brachytherapy equipment.Results:A total of 103patients were registered,94of whom were analyzable,with 8ineligible,and 1for whom complete information was unavailable.The overall cumulative survival rate was 20.3%at 5years.The cause-specific survival rate was 31.8%at 5years.The cause-specific survival rate at 5years was 27%in the external irradiation alone group and 38%in intraluminal brachytherapy combined group.There was no significant difference between the 2groups (p ؍0.385).However,in the patients with 5cm or less tumor length,the cause-specific survival rate was 64%at 5years in the intraluminal brachytherapy combined group,which showed a significant improvement over 31.5%in the external irradiation alone group (p ؍0.025).In the patients with Stage T1and T2disease,cause-specific survival rates tended to be better in the intraluminal brachytherapy combined group than in the external irradiation alone group (p ؍0.088).In the patients with more than 5cm tumor length or Stage T3–4disease,there were no significant differences between the two groups by treatment methods (p ؍0.290).The incidence of early and late complications did not differ according to whether intraluminal brachytherapy was used.Conclusion:For the purpose of establishing the usefulness of intraluminal brachytherapy,further prospective randomized studies are necessary to evaluate the efficacy in tumors with short length and those with shallow invasion,or to assess the usefulness of intraluminal brachytherapy,as additional irradiation in large advanced tumors have been shown to have disappeared by diagnostic imaging after chemoradiotherapy with 60Gy/6w external irradiation.©1999Elsevier Science Inc.Esophageal cancer,Radiotherapy,Intraluminal Brachytherapy.INTRODUCTIONTo improve the results of treatment of patients with esoph-ageal cancer,it is important to achieve good local control.Because the esophagus is adjacent to highly radiation-sen-sitive organs such as the lungs,bone marrow,etc.,it is difficult to irradiate tumors with high doses.Although irra-diation techniques have improved as a result of advances in treatment planning equipment and irradiation equipment,there are still some patients in whom radical radiation therapy within doses that the bone marrow and lung can tolerate is difficult with external irradiation alone.On the other hand,intraluminal brachytherapy allows high-dose irradiation of esophageal cancer,with little exposure ofReprint requests to:Tomohiko Okawa,M.D.,Department of Radiology and Oncology,Tokyo Women’s Medical University,8-1Kwada-cho,Shinjuku Ku Tokyo,162-8666,Japan.Tel:03-3353-8111;Fax:035269-7355.Acknowledgments —We wish to express our sincere thanks to the medical institutions and physicians who kindly contributed to this investigation by registering cases and recording case report forms.Accepted forpublication 28April 1999.Int.J.Radiation Oncology Biol.Phys.,Vol.45,No.3,pp.623–628,1999Copyright ©1999Elsevier Science Inc.Printed in the USA.All rights reserved0360-3016/99/$–see front matter623adjacent organs at risk.However,intraluminal brachyther-apy,in which the dose sharply declines with the distance from the radiation source,is suggested to be a useful tech-nique for tumors with relatively shallow invasion.With the aim of improving the results of treatment of esophageal cancer,we designed this study to establish the optimal irradiation method in radical radiation therapy for esopha-geal cancer by clinically evaluating external irradiation alone and in combination with intraluminal brachytherapy.METHODS AND MATERIALSThe study population consisted of patients with squamous cell carcinoma who were expected to be successfully treated with radical radiation therapy.The patients entered into the study satisfied all of the following criteria as a rule:(a)patients who had received no prior treatment;(b)patients with squamous cell carcinoma which had primarily devel-oped in the intrathoracic esophagus,18–24cm from incisor;(c)the tumor was 10cm or less in length;(d)no distant metastasis;(e)aged 80years or less;(f)no serious compli-cation;(g)performance status (PS)was 3or less;(h)no active double/multicentric cancer;(i)patients whose pre-treatment laboratory test data fulfilled the normal condi-tions.We performed the pre-and post-treatment work-up of barium swallow,endoscopy,and CT scan in all cases,and in some cases,endoesophageal ultrasound (EUS).We used TNM (UICC,1987)for staging.The patients entered the study after informed consent to participate had been ob-tained from the patient or his or her family.We evaluated whether intraluminal brachytherapy (ILBT)was able to perform after 60Gy by barium swallow and endoscopic findings.Patients were allocated 2groups,external radiotherapy (ERT)alone or ERT with ILBT.About two-thirds of patients were entered into this trial at each institution.After the stratification,the central statistical board randomly assigned the patients to receive external irradiation alone or in combination with intraluminal brachytherapy,using a telephone call or facsimile;and treatment after an external irradiation of 60Gy was per-formed according to their assignment.After radiotherapy,maintenance chemotherapy with etoposide (25mg/day,3cycles every 2weeks)was performed in each institution (Fig.1).Radiation therapy was started with external irradiation with linac X-rays or 60Co ␥-rays,and the patients received,as a rule,an external irradiation equivalent to 60Gy/6weeks at a daily dose of 2Gy,on a 5-day weekly schedule.After 60Gy irradiation,either external irradiation or intralu-minal brachytherapy was performed according to the as-signment.Patients assigned to receive external irradiation alone received boost irradiation of 10Gy/week on a sched-ule similar to the previous one,and with the same or smaller irradiation field.Intraluminal brachytherapy was performed,with the reference dose point set at a depth of 5mm of the esophageal submucosa,because all of the institutions used this reference point.A total of 10Gy was irradiated at a daily dose of 5Gy,on a once-weekly schedule with low-dose-rate (LDR)or high-dose-rate (HDR)brachytherapy equipment.Definition of the HDR or LDR (50–150cGy/h)is according to ICRU Report 38.Ra and Cs were used in LDR and Ir was used in HDR.Diameter of the intra-esophageal brachytherapy applicator is 1cm with balloon,which surface touches the esophageal tumor surface after insertion.The survival rate was calculated by the Kaplan-Meier method,counting from the first irradiation day to May 1997,as the final follow-up day.The log-rank test was used to test for significant differences,and p Ͻ0.05was judged to be significantly different.The generalized Wil-coxon test was also used if necessary.Fisher’s exact prob-ability was used for testing between the two groups.RESULTSThis study was performed as a multi-institutional coop-erative study designated by The Japanese Society for Ther-apeutic Radiology and Oncology (JASTRO).The registra-tion of patients was conducted between May 1,1991and May 31,1995,at 22medical institutions in various regions of Japan.A total of 103patients were registered,94of whom were analyzable,with 8ineligible,having more than 10cm in tumor length and neoadjuvant chemotherapy be-fore radiotherapy cases,and 1for whom complete informa-tion was unavailable.The follow-up period ranged from 10to 82months,the median being 24months.The median follow-up period in the survivors was 48months,with 4alive for 5years or longer.The characteristics of the analyzable patients were as follows:73men and 21women,aged 41–83years (median 73),PS 0in 39patients,1in 34,2in 15,and 3in 6.Five patients exceeded 80years of age,but were included in the analysis because they fulfilled all of the other selection criteria.The main location of the lesions was upper thoracic in 11patients,middle thoracic in 59,and lower thoracic in 24,with middle thoracic accounting for 62.8%.The major axis of the lesion was 5.5cm in median length,ranging from 0.8to 10cm.In the TNM classification,the T-stage was T1in 20patients,T2in 25,T3in 36,T4in 10,and Tx in 3;N-stage was N0in 68patients,N1in 23,and Nx in 3.The response at the time of registration,(i.e.,the response to the external irradiation of 60Gy)was a complete response(CR)Fig.1.Randomization and treatment schedule.624I.J.Radiation Oncology●Biology●Physics Volume 45,Number 3,1999by barium swallow and endoscopicfindings in28patients, partial response(PR)in59,no change(NC)in4,and unknown in3.Fifty-one patients received external irradia-tion alone,and43received external irradiation with intralu-minal brachytherapy.The characteristics or response to the 60Gy external irradiation did not differ between the exter-nal irradiation-alone group and the intraluminal brachyther-apy combined group.(Table1).Among the94patients who completed radiation therapy, the response at1month after radiotherapy was evaluated as CR in49,PR in43,NC in1,and unknown in1;the response rate(CRϩPR)being97.9%,and the CR rate 52.1%.The median duration of response,taking all relapses and metastases into consideration by every1-month fol-low-up study,was134days(27–971days).Overall cumulative survival rate with actuarial method, and cause-specific survival rate in the analyzable patients are presented in Fig.2.Cause-specific survival rate was corrected by handling the14patients who died of other diseases under no-cancer-bearing conditions as“discontin-ued”at the time of death.The overall cumulative survival rate was32.9%at2years and20.3%at5years.The cause-specific survival rate was40.7%at2years and31.8% at5years.The complete response rate at the completion of irradia-tion was49%for external irradiation alone and55.8%for intraluminal brachytherapy combined.Intraluminal brachy-therapy made no difference in tumor response(pϭ0.327).The cause-specific survival rate was27%in the external irradiation alone group and38%in intraluminal brachytherapy combined group.There was no significant difference between the two groups(pϭ0.385,Fig.3). However,in the patients with5cm or less tumor length,the cause-specific survival rate was74.6%at2years and64% at5years in the intraluminal brachytherapy combined group,which showed a significant improvement over the 39.4%and31.5%in the external irradiation alone group(pϭ0.025,Fig.4).In the patients with Stage T1and T2 disease,the relapse-free and cause-specific survival rates tended to be better in the intraluminal brachytherapy com-bined group than in the external irradiation alone group (pϭ0.088,Fig.5).In the patients with more than5cm tumor length or stage T3-4disease,there were no significant differences between the two groups by treatment methods(cause specific:pϭ0.290,relapse-free:pϭ0.743).In the combined group of external irradiation and intralu-minal brachytherapy,28institutions used LDR and15HDR brachytherapy.The results of tumor response,survival rate, and complications were not different in the two brachyther-apy types.The complications that occurred from start of treatment to 6months after the completion of treatment were regarded as “early complications,”and those noted thereafter,as“late complications.”Special attention was paid to pain,nausea/ vomiting,appetite loss,and general fatigue.All types of complications were counted in patients with more than one complication during the observation period,and the grade included in the analysis was the most severe one during that period.Findings clearly judged to result from the aggrava-tion of the underlying disease or from other complications were not included.In the early period,pain,nausea/vomit-ing,appetite loss,and general fatigue were recorded as subjective manifestations.Early complications of grade3or more were found in8patients,with an incidence of5.9% for external irradiation alone and11.6%for intraluminal brachytherapy combined.The incidence of early complica-tions did not differ between the two groups(pϭ0.266). Late complications of Grade3or more were found in8.9% of the patients receiving external irradiation alone(ulcer2, pneumonitis1)and8.3%of those receiving combined in-traluminal brachytherapy(ulcer2,stenosis1),showing no difference according to the irradiation method(pϭ0.625).Two patients underwent an operation for esopha-geal stenosis after radiation therapy,one treated with exter-nal irradiation alone and the other treated with intraluminal brachytherapy combined.Table1.Patient characteristics according to external irradiation with/without intraluminal brachytherapy Characteristic ERT ERTϩILBT Total no.5143 Male:Female40:1133:10 Median age(years)7275 Range41–8350–82 Performance status02118 12113 269 333 Tumor locationLU65LM3029EI159 Tumor lengthMedian 6.35 Range0.8–102–10Ϲ5cm2420Ͼ5cm2723 TNM stageT1137T21213T31917T455Tx21N03830N11310Nx03 Effect at60GyCompleteresponse1414 Partial response3326No change13N/A30 ERTϭexternal radiotherapy;ILBTϭintraluminal brachyther-apy;IUϭupper intrathoracic esophagus;IMϭmiddle intratho-racic esophagus;EIϭlower intrathoracic esophagus;N/Aϭcomplete information not available.625RadiotherapyϮintraluminal brachytherapy for esophageal cancer●T.O KAWA et al.These results showed that the incidence of early and late complications did not differ whether intraluminal brachy-therapy was used.DISCUSSIONIn the treatment of unresectable advanced esophageal cancer,radiation therapy is reported to have a 5-year sur-vival rate of about 10%(1,2).Such unfavorable treatment results may be caused by the inclusion of patients who have large tumors,distant metastases,or poor medical condition in the cases of indication for radiotherapy.In our prospec-tive randomized study,we excluded patients with distant metastases,stratified the enrolled patients in advance by prognostic factors such as sex and tumor length,and then assigned the patients who could receive intraluminal brachytherapy after 60Gy external irradiation to either of the two treatment groups.Intraluminal brachytherapy as part of the radiation therapy for esophageal cancer has been reported to be useful,because its combined use results in the improvement of primary response and survival rate (3–6);however,all of these reports are retrospective studies.In addition,because these studies included all patients having CR or PR after ERT,and who could receive intraluminal brachytherapy,it is undeniable that the selection of patients was greatly biased,which makes it difficult to simply eval-uate the treatment results compared with external irradiation cases.In other words,it seems likely that favorable resultswere obtained,because the subjects were limited to those who responded to external irradiation and who could be equipped with an applicator for intraluminal brachytherapy.The statistical analysis of all of the patients enrolled in our study revealed no significant differences in the primary response at the end of treatment,or the survival rate be-tween the external irradiation alone group and the intralu-minal brachytherapy combined group.However,in rela-tively small tumors with shallow invasion (tumor length of 5cm or less,T1or T2stage),the intraluminal brachyther-apy combined group yielded significantly better ly,it is suggested that the tumor with short length and shallow invasion is a proper indication for intraluminal brachytherapy after external irradiation.This is because intraluminal brachytherapy,in which the dose sharply de-clines with the distance from the radiation source,enables a uniform irradiation of small tumors with shallow invasion but does not irradiate large tumors with deep invasion (7–9).Recently,it has been reported that in superficial (namely,stage T1)tumors,the 2-year survival rate was higher with intraluminal brachytherapy combined than with external irradiation alone (10),and that even intraluminal brachy-therapy alone is expected to be effective in small superficial tumors (11).In Japan,the following schedule has been generally used as the optimal irradiation method for radical radiation ther-apy in combination with intraluminal brachytherapy for esophageal cancer:after the patient shows a remarkable response as potentially CR or PR at 50ϳ60Gy external irradiation,intraluminal brachytherapy is employed as boost therapy by LDR irradiation at 10ϳ20Gy/2ϳ3fractions using the mucosa plane as the reference dose point (12,13).Hishikawa et al.have reported that HDR intraluminal brachytherapy following 60Gy external irradiation is at a high risk of causing fistula formation unless the dose is 20Gy or less (14).Yorozu et al.have reported that,in view of esophageal ulcer,HDR intraluminal brachytherapy is safe up to a total dose of 16Gy (4Gy/fraction,twice-weekly schedule)after 50Gy external irradiation (15).The high incidence of complications with intraluminal brachytherapy may be explained by the fact that the areas close to the radiation source are irradiated at an extremely high dose,while the dose sharply declines in some areas whichareFig.2.Overall survival curves of esophagealcancer.Fig.3.Cause-specific survival by treatmentmethod.Fig.4.Cause-specific survival (tumor length Ϲ5cm).626I.J.Radiation Oncology●Biology●Physics Volume 45,Number 3,1999distant,even if slightly,from the radiation source.Calling the area given more than twice as high dose as the reference dose point “hyperdose sleeve,”Marinello et al.have de-scribed that the tissue included in this area is at a high risk of developing radiation complications such as necrosis (16).In our study,8.3%of the patients developed late complica-tions of Grade 3or more,but the incidence did not differ between the external irradiation alone group and the intralu-minal brachytherapy combined group.This may be because “hyperdose sleeve”resulting from intraluminal brachyther-apy was limited,with the dose set at 10Gy in total (5Gy/fraction)at a depth of 5mm under the esophageal submucosa.As the definitive treatment for tumors 10cm or less in length,the guidelines presented by the American Brachytherapy Society suggest that after 45–50Gy irradia-tion,intraluminal brachytherapy at a total dose of 10Gy with two fractions in HDR or a single irradiation of 20Gy in LDR brachytherapy (17).As the palliation of advanced esophageal cancer,Sur et al.reported that dose of optimalbrachytherapy without external radiation ranges between 16Gy in two fractions and 18Gy in three fractions given a week apart (18).In our study of radiotherapy with curative intent,after 60Gy external irradiation,10Gy/2fractions,for both LDR and HDR,were added to make a total dose of 70Gy.As a result,intraluminal brachytherapy proved ef-fective for small and shallow tumors.Therefore,in small and shallow tumors,we consider it appropriate to achieve complete response using 50ϳ60Gy external irradiation and then performing intraluminal brachytherapy at 10ϳ15Gy/2ϳ3fractions.In this case,it is very important that the total dose should not exceed 70Gy.For large and deep tumors,(tumor length more than 5cm,T3or T4stage),for multi-modal treatment including chemotherapy,it is necessary to achieve complete tumor response and to improve the sur-vival rate.For the purpose of establishing the usefulness of intraluminal brachytherapy,prospective randomized studies seem necessary to evaluate the efficacy in tumors with short length and those with shallow invasion,or to assess the usefulness of intraluminal brachytherapy,as additional ir-radiation in large advanced tumors have shown to have disappeared by diagnostic imaging after chemoradiotherapy with 60Gy/6W external irradiation.Multidisciplinary stud-ies (19–23)on the combination of hyperfractionation radio-therapy and various anticancer drugs are also valuable.Finally,reference dose point is a problem to be solved in intraluminal brachytherapy.In general,the reference dose point is set at a depth of 5mm of the submucosa;however,some institutions use the mucosal surface or a certain dis-tance from the 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