分次放射治疗的生物学基础.ppt

分次放疗的生物学基础第一课中南大学湘雅医院肿瘤科周卫兵问题分次方案选用的理由？放射生物学分次方案选用的理由？放射生物学=药理学药理学是否有更合理的分次方案？是否有更合理的分次方案？肿瘤特征 生物分子指标 周边危及器官 患者合并疾病和一般状况 治疗目的 射线，设备，技术 综合方案怎样提高放疗疗效？怎样提高放疗疗效？其他放疗中断或更改分次方案 的依据不同放疗手段的相加性？再程放疗剂量的给予IMRT、IGRT、SRS等剂量的特殊性，与常规剂量分割模式的矛盾研究分次效应的艰巨性和重要性第一课大纲（一）放射生物学基本概念放射生物学基本概念细胞死亡 细胞存活曲线LET RBE OER LQ模型/Tpot（二）放射生物学中的放射生物学中的4Rs 及临床应用价值及临床应用价值Repair 再修复Redistribution 细胞周期再分布Reoxygenation 乏氧细胞的再氧合Repopulation 再群体化第二课大纲（一）常规分割放疗常规分割放疗定义 优缺点 注意事项提高放射治疗疗效的途径：（二）非常规分割放疗非常规分割放疗超分割 定义 临床应用加速分割 定义 临床应用（三）未来研究方向未来研究方向IMRT面临的问题靶向治疗 保护剂 增敏剂（同步化疗）放射生物基本概念Gy cGy RAD REM Sv LETOERBEDEUDNTCPTCP放射生物基本概念射线作用原理 作用的靶：DNA,双链断裂（DSB）有 1520 核苷酸大小，可造成：微核 染色体畸形 失去持续增殖能力死亡 射线作用原理物理化学反应影响因素物理化学反应影响因素细胞内环境：细胞内环境：自由基 水 85 .OH（生物分子损伤的65）O2：参与形成自由基；固定自由基损伤；限制化学修复。电子亲和物电子亲和物 巯氢基分子：巯氢基分子：作用相反射线的质射线的质，剂量和剂量率剂量和剂量率射线作用原理物理化学反应影响因素物理化学反应影响因素细胞的敏感性生化结构和基因构成细胞的敏感性生化结构和基因构成 特定分子 线粒体 细胞膜脂质 生长因子受体 信号传导通路（外部 内部）（乏氧，细胞因子 细胞连接，细胞外基质），导致：细胞死亡或存活细胞周期阻滞或前进DNA损伤修复或不稳定（细胞感知放射损伤的方式再决定细胞最终反应中起重要作用）直接 vs 间接作用间接作用间接作用:电子作用于水，形成.OH 自由基(2/3 x-线 生物损伤).能被增敏剂和防护剂修饰能被增敏剂和防护剂修饰直接直接:电子直接作用于靶分子(高LET).不能被增敏剂和防护剂修不能被增敏剂和防护剂修饰饰射线作用原理射线作用的交叉性与肿瘤起源与应激 乏氧 炎症 化疗 热疗 氧化射线作用的特殊性：往往致死性；DNA-DSBDSB双链断裂是电离辐射在染色体上所致的最关键损伤.单击损伤DNA MutationCell survives but mutatedCancer?Cell deathMutation repairedUnviable CellViable Cell 基本放射生物损伤Cell death or dysfunctionNeoplastic diseaseImmune relatedGenetic defectsSystemic diseaseReproductiveO2H20Free radicals formed(OH-,H2O2,HO2)Free Radicals Attack macro-moleculesThe higher the dose,the more severe the early effects and the greater the possibility of delayed effectsChemical Damage10-10 SecondsBiologicalMolecularDamageSymptomsHours to yearsSeconds to hours物理阶段单次4Gy的X线人全身照射=致死(多数情况下)=70Kg的正常人,能量吸收67卡(cal)=热量温度升高0.002=喝一口热咖啡=一个人从地面举起16英寸物理阶段X射线的作用不在于所吸收的总能量的大小,而在于单个能量包的大小(packets of energe)-LET-化学键生物学事件放射敏感性内在敏感性：分子 细胞因素组织相关特性临床特征其他 有再增殖能力的细胞数目 功能亚单位的再增殖的潜力 凋亡细胞依赖性淋巴细胞肿瘤细胞-丧失再增殖完整性=细胞死亡?放射可治愈性=肿瘤不能增殖克隆源细胞的存活比例 SF放射敏感性组织的反应细胞的放射反应局部控制之一要求：杀灭所有肿瘤干细胞局部控制的其他要求：血管损伤（联合生物化学治疗）放疗修饰剂（肿瘤与宿主的关系，如细胞浸润，促进或抑制肿瘤生长）旁观细胞：信号修复 细胞因子，生物分子，引起正常组织的重塑组织反应细胞内的信号网络：物理剂量生物剂量共性 可预测的方式 个体间相似细胞周期（周转）增殖储备正常组织的变化小：时间 程度肿瘤的差异大：一些可治愈 一些不可治愈细胞的死亡是放射线对细胞的遗传物质和DNA造成不可修复的损伤所致正常组织的放射反应死亡类型 分裂性死亡分裂性死亡（增殖性死亡,mitotic death）：2Gy照射，细胞23次分裂后一部分死亡一部分进入增殖池，导致复发 间期死亡间期死亡(interphase death)：照射后26小时放射敏感细胞，具有凋亡前趋势（淋巴细胞；GBM）快速凋亡分裂性死亡机制M期 纺锤体形成失败G2检查点丢失，导致分裂大灾难，或者不正常的染色体隔离（损伤和丢失了基因）染色体畸变结果凋亡坏死其他特征：延迟，分裂性死亡放射敏感损伤的结局凋亡：I型程序性死亡，不完全依赖P53坏死：病理过程，炎症反应自我吞噬：II型程序性死亡分化，衰老，静止：保留部分功能存活与细胞微环境生长因子，细胞接触，细胞外基质放射抵抗：生长因子激活的淋巴细胞促有丝分裂原纤维母细胞生长因子提高内皮细胞的放射抵抗性放疗增敏：靶向药物存活与细胞微环境PLDR：接触抑制TNF凋亡与反应凋亡前细胞过多（超过需要）时，放疗引起的快速凋亡，将对个体影响很小。如 WP53和P53裸鼠的肠道照射研究正常组织反应一种组织可含有多种细胞类型，可表现急性和晚期反应。而且急性与晚期反应有一定相关，如纤维化或坏死与湿性脱皮和急性溃疡，溃疡与狭窄等。正常组织反应发生在标准放疗（68周）内。干细胞。可矫正。亚急性反应：放疗后几个月如：Lhermittes syndrome,嗜睡，亚急性肺炎。可逆晚期反应：受多种因素影响（手术，化疗，外伤，感染）。难逆转，不可矫正。反应程度与疗效不一定相关急性反应不一定与晚期反应相关（同步化疗可利用）正常组织反应的决定因素FSU:功能亚单位的排列方式，串联或并联；FSU损伤的数目；FSU储备的数目。微血管损伤支持组织的损伤（基质或间质细胞）细胞增殖动力学和总剂量损伤的耐受严重受损：发生率5%重度损伤：发生率1025%损伤对患者的威胁？急性反应：头3个月晚期反应：放疗后3个月并发症急性组织反应晚期组织反应急性反应发生的时间依赖于细胞周期时间粘膜反应放疗第2周皮肤反应 放疗第5周放疗结束后数周消退RTOG 急性反应 vast majority dieFactors Affecting Tumor Growth肿瘤氧合低氧合=低增殖低氧合=细胞死亡增加随着肿瘤体积增大，肿瘤氧合下降慢性和暂时的乏氧都会产生效应.放射生物放射生物（一）细胞存活曲线细胞存活和敏感性间接反应链电离，激发电离，激发:H2OH2O+e-H2O+is an Ion Radical自由基形成自由基形成:H2O+H+OH 存活的定义 非增殖，分化好的细胞非增殖，分化好的细胞:丧失特殊功能例如：神经和肌肉细胞 增殖细胞增殖细胞:丧失完整的增殖能力(增殖性死亡)存活,必须能增殖(克隆).(相对于整个器官)损伤的细胞可能分裂几次,但最终死亡-因为不能存活细胞存活的检测平板培养体外的存活曲线人类细胞的存活曲线细胞存活曲线的描述细胞存活分数(对数坐标)vs 剂量高密度电离辐射(-粒子,中字)起始就一直为线性;1个参数:（斜度 Do)稀疏密度电离辐射:(x 和 电子线)初始斜度 D1终末斜度 D2中间的肩区,n or Dq描述 资料采用线性二次方程拟合:S=exp(-D-DS=exp(-D-D2 2)在几十Gy的剂量范围内拟合很好提示有两种杀灭方式:to D,to D2 Meaning of and Linear-Quadratic ModelCell Survival (1 fraction):单击损伤单击损伤单击损伤单击损伤 不可修复不可修复不可修复不可修复 无分隔效应无分隔效应无分隔效应无分隔效应 亚损伤相互作用亚损伤相互作用亚损伤相互作用亚损伤相互作用 部分损伤修复部分损伤修复部分损伤修复部分损伤修复 有分隔效应有分隔效应有分隔效应有分隔效应Surviving 1:单击单靶模型(A),单击多靶模型(B)指数“单击”曲线 细胞（或生物大分子）的存活分数为辐射剂量的简单细胞（或生物大分子）的存活分数为辐射剂量的简单函数。函数。见于病毒或酶的灭活，以及少数哺乳动物细胞的杀灭。见于病毒或酶的灭活，以及少数哺乳动物细胞的杀灭。属于单击单靶模型。属于单击单靶模型。D37的倒数即为存活曲线斜率。的倒数即为存活曲线斜率。D37：引起细胞（或酶分子）引起细胞（或酶分子）63死亡（或失活）的死亡（或失活）的照射剂量。照射剂量。多靶“单击”模型 假设在细胞死亡前有两个或多个靶受到假设在细胞死亡前有两个或多个靶受到一次击中。一次击中。剂量存活曲线的直线部分斜率的倒数为剂量存活曲线的直线部分斜率的倒数为D0-平均致死剂量。平均致死剂量。曲线初始斜率不等于曲线初始斜率不等于0，即在很低剂量时，即在很低剂量时仍有一些细胞死亡。仍有一些细胞死亡。这个模型对许多哺乳动物细胞都适用。这个模型对许多哺乳动物细胞都适用。Surviving 2:Two-component model(A),LQ-model(B)Effect of fractionation:LQ model used D0值：细胞的平均致死剂量（细胞的平均致死剂量（mean lethal dosemean lethal dose）剂量存活曲线的剂量存活曲线的直线部分斜率的倒数直线部分斜率的倒数D D0 0愈小，斜率愈大愈小，斜率愈大D D0 0值大小代表细胞放射敏感性的高低值大小代表细胞放射敏感性的高低从存活曲线对数坐标从存活曲线对数坐标0.10.1和和0.0370.037各作一条与横坐标相平行的各作一条与横坐标相平行的线与曲线相交，从这两个交点分别作垂直线与剂量轴相交。线与曲线相交，从这两个交点分别作垂直线与剂量轴相交。相交点剂量之差即为相交点剂量之差即为D D0 0值，多在值，多在1 12 2GyGy细胞存活曲线的参数细胞存活曲线的参数 Dq值值准阈剂量准阈剂量（quasithreshould dose）细胞积累亚致死性损伤的能力，与损伤修复有细胞积累亚致死性损伤的能力，与损伤修复有关。克服肩区所需的剂量关。克服肩区所需的剂量由纵坐标由纵坐标1.01.0处作一条与横坐标的平行线，与处作一条与横坐标的平行线，与外推线的交点在横坐标上投影点的数值即为外推线的交点在横坐标上投影点的数值即为DqDq多在多在0.50.52.5Gy2.5Gy n值值代表细胞内靶的个数或所需击中靶的次数代表细胞内靶的个数或所需击中靶的次数将直线部分外推与纵坐标相交点的数值即为外将直线部分外推与纵坐标相交点的数值即为外推推n n值（值（extrapolation number)extrapolation number)多为多为1 13 3 D37值引起细胞（或酶分子）引起细胞（或酶分子）6363死亡的照射剂量死亡的照射剂量D D3737D D0 0+Dq+Dq单靶单击时，剂量存活曲线无肩区，单靶单击时，剂量存活曲线无肩区，DqDq为为0 0，此时此时D D3737就等于就等于D D0 0Classical description of survival curveLow doses:“shoulder”region,where survival falls slowly with doseHigher doses:region where survival curve bends and survival shows greater change with increasing doseHigh doses:region where survival falls rapidly with dose Most models used to fit survival curves are based on curves with this general shape.Curves based on Target TheorySingle target-single hitMulti-target single hitComposite curves(erroneously called multitarget in some editions of Halls book)Curves based on linear-quadratic modelBoth types of models are clearly over-simplifications of the true dose-response curves for real cell populationsThis is becoming increasingly evident as more precise measurements of cell survival curves are made using microbeam irradiators that can irradiate individual cells with precisely known numbers of photons or particles.When cells are irradiated with very low doses of radiation,When cells are irradiated with very low doses of radiation,the initial shape of the survival curve is found to be the initial shape of the survival curve is found to be complex,with a region of hypersensitivity at very low dosescomplex,with a region of hypersensitivity at very low dosesThere is increasing evidence for hypersensitivity and There is increasing evidence for hypersensitivity and other anomalous effects at very low radiation dosesother anomalous effects at very low radiation doses“Low dose hypersensitivity”Hypersensitivity at very low single doses“Adaptive response”Seen in fractionated and protracted treatmentsLow initial dose of radiation induces resistance to subsequent irradiation“Bystander effects”Unirradiated cells are affected by irradiation of neighboring cellsContact effects requiring intimate cell-cell contactEffects from diffusible factorsCan affect survival,mutation,transformation,etc.Vary with cell type,dose and other factorsReminder:All curves are plotted on semi-log plotsTarget theory:single target,single hit survival curveTarget theory:single target,single hit survival curveAssumptionsCell contains a single targetIf this target is hit with at least one photon(or particle)the cell diesIf this target is not hit,the cell livesProbability of cell surviving=probability that cell is not hitNo shoulder survival decreases exponentially with doseD0=dose required to produce an average of 1 hit per target(and per cell)=1/e=measure of sensitivity of target(cell)Each increment of dose is equally effectiveMulti-target-single hit survival curveMulti-target-single hit survival curveAll cells are identicalEach cell contains several targetsn=number of targetsIf all n targets are hit,the cell diesIf one or more targets remain unhit,the cell survivesThe survival curve begins with initial slope of zero(flat)because the probability that all targets are hit is zero at very low doses Then it curves Then it becomes exponential at high doses,because only one unhit target remains in each cell.Parameters used to describe these curvesD0=dose needed to reduce survival by 1/e on linear portion of survival curve(i.e.when only one target remains)=mean dose needed to hit and inactivate one target=measure of sensitivity of targetn=extrapolation number=number of targets=measure of width of shoulderDq=quasi-threshold dose=measure of width of shoulderLn(n)=Dq/D0 D10=dose to reduce survival by 1/10=2.3 D0(used in calculations of tumor cell survival in Hall and Giaccias book)Effect of changing DEffect of changing Do o and n and nImplicationsCells can accumulate sublethal damage(SLD)SLD is not lethalSLD lesion can combine with other SLD lesion to produce lethal damageIf this damage can be repaired after irradiation,then fractionating the irradiation results in less cell killingIf this damage can be repaired during irradiation,protracting the irradiation results in less cell killingSmall doses(on the shoulder)have less effect per Gy than larger dosesEffect of fractionation(solid lines)or protraction Effect of fractionation(solid lines)or protraction(dotted line)of radiation(dotted line)of radiationEffect of shoulder width(n)on fractionationComposite survival curve:Single-target,single-hit survival curve(D1)+multitarget single hit survival curve(D0)Initial slope=1/D1Final slope=1/D0Used to get a finite initial slope for the survival curveNot widely used in modeling difficult to handle and not especially useful in any practical contextLinear quadratic survival curveInitial component for which survival falls exponentially with dose(linear component)Later component for which survival falls with square of dose(quadratic component)Several different theoretical basis have been used to justify these curves:shapes of dose response curves for induction of chromosome damagemodels considering nonrepairable and repairable damage models considering“one hit”and“two hit”damageLinear quadratic survival curve:implicationsAny dose of radiation,no matter how small,has a finite chance of killing a cellThe effect of radiation increases continually with increasing dose at high doses(this is not in agreement with the radiobiological data)Protraction,fractionation increase survival only if the dose is high enough that you are on the curving part of the survival curve Effect of fractionation varies with/ratio)/ratioAt D=/,damage from linear killing=damage from quadratic killingI.e.Equal amounts of one hit/two hit damage or non-repairable/repairable damage(depending on underlying biological model assumed)Ratio defines“curviness”of survival curveRatio has been used to predict/compare effects of different fractionated regimensSurvival curves with different/ratiosLarge /ratio Early responding tissuesMarked initial slopeBends at higher doses Flatter survival curve over range of clinical fractionsMulti-hit(or repairable)damage becomes important only at higher dosesLess effect of fractionation or protractionLess effect of fraction sizeCan calculate/ratio without determining the cell survival curve and without identifying the critical cell populationCalculating Calculating /ratios from fractionation ratios from fractionation isoeffectisoeffect data:data:plot=plot=totaltotal doses needed to give equal early skin reactions in micedoses needed to give equal early skin reactions in mice intercept/slope=intercept/slope=/(see Hall and (see Hall and GiacciaGiaccia p 322-323 for derivation)p 322-323 for derivation)Fractionation Fractionation isoeffectisoeffect data for some mouse tissues data for some mouse tissuesfrom Withers,et al.Most data are for functional endpoints,not cell from Withers,et al.Most data are for functional endpoints,not cell survival.survival.Late responses=solid lines;Late responses=solid lines;early responses=dashed linesearly responses=dashed linesCalculated/ratios for some tissues细胞存活曲线:小结Cultured cells which retain reproductive integrity grow separate colonies at a rate depending on plating efficiency.Fraction of seeded cells that survive(irradiation)and grow into colonies are plotted on a log scale vs radiation dose.Plot for high LEThigh LET rad is straight line-exponentialstraight line-exponential responseFor x-rays,curve has initial slope,followed by a shoulder of varying width,then final slope.Reasonably estimated by linear-quadraticlinear-quadratic relationship:S=exp(-S=exp(-D-DD-D2)2)Good evidence that nucleus(i.e.,DNA)is primary target,due to induced chromosomal aberrations.细胞周期细胞周期Cell cycle phases:Mitosis:1 hourSynthesis:6-15 hoursG2:3-4 hoursG1:Very variableRadiation Sensitivity versus cycle phase:Studied by producing synchronously dividing cells cultures Hydrodyurea:imposes block after G1细胞周期与细胞死亡Most radiosensitive phases:M,G2,Most radioresistant phase:S,Checkpoints:G1/S,intra-S,G2/M,DNA damage:cell cycle arrest repair or loss of function(diff.cells),loss of reproductive integrity(stem cells).细胞存活 vs 细胞周期细胞存活 vs 细胞周期细胞周期放射敏感性-小结Cell cycle phases:mitosis(M),G1,DNA synthesis(S),G2Cycle time varies widely,almost entirely due to varying G1May be studied using techniques of cell cycle synchronizationIn general,cells are most sensitive in M and G2Highest resistance in late S-partly since genome is doubledFor long cycle cells,a 2nd resistance peak occurs in late G1Resistance pattern and sensitivity correlates with levels of sulfhydryl compounds (natural radioprotectors)in cell.分次照射效应分次照射,损伤修复和 剂量率效应照射剂量 和细胞修复PLDR：Survival of density-inhibited stationary phase cells sub-cultured immediately after irradiation or after delay(6 or 12 hrs).Stationary phase time inhibits growth,thus allowing time for damage repair before attempting complex mitosis process分次照射:存活 vs 分次间隔时间Hamster cell survival increases four-fold if held at room tempbetween doses for 1(to prevent movement through cell cycle).分次,修复 和 再分布Since most cells surviving 1st dose fraction are in resistant cell phases,a sensitive period for the 2nd dose occurs as cells move into G2-M phase.剂量率效应Idealized fractionation experiment:A:A:survival curve for single acute exposure.F:F:dose given in several small fractions with sufficient inrterval for sub-lethal repair.With several small fractions,F approximates a curve for continuous,low dose rate exposure 剂量率效应的各种因素因素放射损伤&剂量率效应Types of damage:Types of damage:Lethalsub-lethal:normally repairable within hour if no further damagePotentially lethal:can be modified by post-radiation conditions (if conditions suboptimal for growth,inhibiting mitosis)Repair is studiedRepair is studied via split-dose experiments with varying intervalSplit dose effectsSplit dose effects for mammal cells follow cycle related to cell cycle and 4 Rs:repair,reassortment,repopulation,reoxygenationSub-lethal repair and survival curve shoulder width correlatedSub-lethal repair and survival curve shoulder width correlatedHigh LET radiation:High LET radiation:exhibits no apparent repair of damageAs dose rate lowersAs dose rate lowers(OER remains unchanged for ALL dose levels in a given phase of cell cycle.氧效应-小结Presence of OxygenPresence of Oxygen dramatically influences bi