结直肠癌是胃肠道的主要恶性疾病，发病率在肿瘤中位于第三，也是全球范围内癌症致死的第二大原因。局部进展期直肠癌的治疗方案主要包括术前同步放化疗后行直肠癌根治术和手术切除后行同步放化疗。然而患者的个体差异性导致对放化疗的敏感性和产生副反应及生存预后差异。本研究采用候选基因策略，分析凋亡通路关键基因的遗传变异与直肠癌术前同步放化疗敏感性、术后同步放化疗中重度不良反应发生，以及两批患者生存预后相关的位点之间的关联，寻找具有预测价值的遗传变异。

本研究选取了17个凋亡通路关键基因包括凋亡蛋白活性因子1(apoptotic protease activating factor-1, APAF1)，Bcl2拮抗剂(BCL2 homologous antagonist/killer, BAK)，Bcl2相关X凋亡调控因子(BCL2 associated X, apoptosis regulator, BAX)，B细胞淋巴瘤/白血病-2(B cell lymphoma/leukemia2, BCL2)，BH3相互作用域死亡激动剂(BH3 interacting domain death agonist, BID)，半胱天冬酶3 (caspase 3, CASP3)、半胱天冬酶6(caspase 6, CASP6)、半胱天冬酶7(caspase 7, CASP7)、半胱天冬酶8(caspase 8, CASP8)、半胱天冬酶9(caspase 9, CASP9)、半胱天冬酶10(caspase 10, CASP10)，Fas细胞表面死亡受体(Fas cell surface death receptor, FAS)，Fas配体(Fas ligand, FASL)，肿瘤坏死因子受体超家族成员1a (tumor necrosis factor receptor superfamily, member 1a , TNFR)，TNF超家族成员10 (TNF superfamily member 10, TRAIL)，TNF受体超家族成员10a(TNF receptor superfamily member 10a, TRAILR1)和TNF受体超家族成员10b (TNF receptor superfamily member 10b, TRAILR2)的56个标签单核苷酸多态（tagging single nucleotide polymorphism, tagSNP）位点。先后入组了两批直肠癌患者，分别为术前同步放化疗后行直肠癌根治术的患者136例和手术切除后行同步放化疗的患者362例。同步放化疗前抽取静脉血2 ml，采用Sequenom MassARRAY检测凋亡通路中56个tagSNP基因型。对于术前同步放化疗的患者，使用logistic 回归模型校正临床因素，进行56个tagSNP与直肠癌患者术前同步放化疗敏感性的关联研究，发现位点FAS rs4934431、BID rs8190256和BAX rs905238显著影响放化疗敏感性，加性模型计算得到OR值分别为2.25（95% CI= 1.28-3.95，P = 0.005）、0.36（95% CI = 0.15-0.87，P = 0.023）和0.58（95% CI = 0.34-0.99，P = 0.048）。Cox风险回归模型计算tagSNP与术前同步放化疗直肠癌患者总生存的关联，发现位点CASP3 rs116609630、CASP7 rs12415607和FAS rs1800682与这批患者的总生存相关，显性模型计算得到HR值分别为2.31（95% CI = 1.00-5.34，P = 0.050）、0.48（95% CI = 0.24-0.96，P = 0.037）和2.59（95% CI = 1.07-6.28，P = 0.035）。CASP7 rs12415607和FAS rs1800682两个位点进行联合分析，发现相比于携带0～2个风险等位基因，携带3～4个风险等位基因的患者死亡风险显著增加（HR = 2.84，95% CI = 1.46-5.53，P = 0.002）。

直肠癌术后同步放化疗的患者，进行56个tagSNP与直肠癌患者术后急性不良反应的关联研究，加性模型计算发现与中重度骨髓抑制相关3个位点包括APAF1 rs11296996（OR = 0.69，95% CI = 0.49-0.98，P = 0.039）、BAX rs4645904（OR = 0.69，95% CI = 0.50-0.97，P = 0.030）、FAS rs1468063（OR = 1.51，95% CI = 1.07-2.15，P = 0.020）；与中重度腹泻的发生相关5个位点包括APAF1 rs11296996（OR = 1.42，95% CI = 1.02-2.00，P = 0.040）和rs74619561（OR = 2.16，95% CI = 1.27-3.68，P = 0.005）、CASP7 rs12263370 （OR = 1.67，95% CI = 1.05-2.66，P = 0.029）和rs12247479（OR = 1.85，95% CI = 1.12-3.08，P = 0.017）、TRAIL rs112822654（OR = 0.68，95% CI = 0.48-0.69，P = 0.027）。Cox风险回归模型计算56个tagSNP与术后同步放化疗直肠癌患者总生存的关联，发现6个与这批患者的总生存相关位点，即BAK rs74499662（HR = 0.57，95% CI = 0.36-0.91，P = 0.019），BCL2 rs34811186（HR = 1.91，95% CI = 1.13-3.19，P = 0.014）、BID rs148719901（HR = 0.52，95% CI 0.31-0.86，P = 0.011）、CASP9 rs1052576（HR = 0.52，95% CI 0.32-0.83，P = 0.006）和rs12741552（HR = 0.54，95% CI 0.34-0.88，P = 0.013）以及FASL rs763110（HR = 0.61，95% CI0.37-0.99，P = 0.044）。CASP9 rs1052576、PMS1 rs4920657和hsa-miR-4274 rs202195689三个位点联合分析显示，相比于携带0～1个风险基因型，携带2～3个风险基因型的患者死亡风险显著增加（HR = 2.07，95% CI = 1.30-3.30，P = 0.002）。

我们的研究发现凋亡通路基因FAS、BID、BAX的遗传变异与直肠癌患者术前同步放化疗的敏感性显著相关；FAS、APAF1和BAX 的遗传变异与直肠癌患者术后同步放化疗发生中重度骨髓抑制有关；APAF1、CASP7及TRAIL的遗传变异与患者术后同步放化疗中重度腹泻的发生有关；CASP3、CASP7和FAS的遗传变异与直肠癌术前同步放化疗患者的总生存显著相关；BAK、BCL2、BID、CASP9以及FASL的遗传变异与术后同步放化疗直肠癌患者的总生存显著相关。这些基因遗传变异位点可能作为直肠癌个体化治疗的潜在遗传生物标志物。

Colorectal cancer is the main malignant disease of the gastrointestinal tract. Its incidence is the third in cancer, and it ranks the second leading cause of cancer death worldwide. The treatment options for locally advanced rectal cancer include preoperative concurrent chemoradiotherapy combined with total mesorectal excision surgery and concurrent chemoradiotherapy after surgical resection. Patients vary in their response to the preoperative chemoradiotherapy or the postoperative chemoradiotherapy. A lot of them suffer from adverse events or tumor progression or even death. Inherited genetic factors may explain some of the variability of patients’ response and prognosis. In this study, candidate gene strategy was used to investigate the associations between the genetic variations of apoptosis genes and the sensitivity, acute adverse events and survival prognosis of concurrent chemoradiotherapy in patients with rectal cancer.

In this study, two groups of patients with rectal cancer were enrolled in succession, including 136 patients who underwent radical resection of rectal cancer after preoperative concurrent chemoradiotherapy and 362 patients who underwent concurrent chemoradiotherapy after surgical resection. Whole blood sample (2 ml) was collected from patient at the time of enrollment before therapy. Sequenom MassARRAY was used to detect the genotypes of 56 tagSNPs in seventeen apoptosis genes, including APAF1, BAK, BAX, BCL2, BID, CASP3, CASP6, CASP7, CASP8, CASP9, CASP10, FAS, FASL, TNFR, TRAIL, TRAILR1 and TRAILR2. Logistic regression model was used to correct clinical factors, and 56 tagSNPs were associated with the sensitivity of preoperative concurrent chemoradiotherapy or the risk of acute adverse events of postoperative concurrent chemoradiotherapy in rectal cancer patients. FAS rs4934431, BID rs8190256 and BAX rs905238 significantly affected the sensitivity of chemoradiotherapy, odds ratios (ORs) values ​​calculated by the additive model were 2.25 (95% CI = 1.28-3.95, P = 0.005), 0.36 (95% CI = 0.15-0.87, P = 0.023), 0.58 (95% CI = 0.34-0.99, P = 0.048), respectively. The Cox proportional regression model calculated the association between 56 tagSNPs and the overall survival of patients undergoing preoperative concurrent chemoradiotherapy for rectal cancer. CASP3 rs116609630, CASP7 rs12415607 and FAS rs1800682 were associated to the overall survival of this group of patients, and hazard ratios (HRs) and 95% confidence intervals (CIs) were 2.31 (95% CI = 1.00-5.34, P = 0.050), 0.48 (95% CI = 0.24-0.96, P = 0.037), 2.59 (95% CI = 1.07-6.28, P = 0.035), respectively. A combined analysis of CASP7 rs12415607 and FAS rs1800682 found that patients with 3 to 4 risk alleles had a significantly increased risk of death compared with 0 to 2 risk alleles (HR = 2.84, 95% CI = 1.46-5.53, P = 0.002).

In patients with rectal cancer who were undergoing postoperative concurrent chemoradiotherapy, three SNPs were associated with the risk of grade ≥ 2 myelosuppression by the additive model, including APAF1 rs11296996 (OR = 0.69, 95% CI = 0.49-0.98, P = 0.039), BAX rs4645904 (OR = 0.69, 95% CI = 0.50-0.97, P = 0.030), FAS rs1468063 (OR = 1.51, 95% CI = 1.07-2.15, P = 0.020). Five SNPs that significantly associated with risk of grade ≥ 2 diarrhea, includingAPAF1 rs11296996 (OR = 1.42, 95% CI = 1.02-2.00, P = 0.040) and rs74619561 (OR = 2.16, 95% CI = 1.27-3.68, P = 0.005), CASP7 rs12263370 (OR = 1.67, 95% CI = 1.05-2.66, P = 0.029) and rs12247479 (OR = 1.85, 95% CI = 1.12-3.08, P = 0.017), TRAIL rs112822654 (OR = 0.68, 95% CI = 0.48-0.69, P = 0.027). The Cox proportional regression model calculated the association between 56 tagSNPs and overall survival of patients with rectal cancer undergoing concurrent postoperative chemoradiotherapy, and found six SNPs that significantly associated with overall survival of the patients, including BAK rs74499662 (HR = 0.57, 95% CI = 0.36-0.91, P = 0.019), BCL2 rs34811186 (HR = 1.91 , 95%CI = 1.13-3.19, P = 0.014), BID rs148719901 (HR = 0.52, 95% CI 0.31-0.86, P = 0.011), CASP9 rs1052576 (HR = 0.52, 95% CI 0.32-0.83, P = 0.006), rs12741552 (HR = 0.54, 95% CI 0.34-0.88, P = 0.013) and FASL rs763110 (HR = 0.61, 95% CI 0.37-0.99, P = 0.044). The combined analysis of the three SNPs including CASP9 rs1052576, PMS1 rs4920657 and hsa-miR-4274 rs202195689 showed that patients with 2 to 3 risk genotypes had a significantly increased risk of death compared with patients carrying 0 to 1 risk genotype (HR = 2.07, 95% CI = 1.30-3.30, P = 0.002).

Our study found that the genetic variations of the apoptosis pathway genes FAS, BID, BAX are significantly associated with the sensitivity of preoperative chemoradiotherapy in patients with rectal cancer; the genetic variations of FAS, APAF1 and BAX are associated with the occurrence of severe bone marrow suppression in rectal cancer patients undergoing postoperative chemoradiotherapy. The genetic variations of APAF1, CASP7 and TRAIL are associated with the occurrence of severe diarrhea in patients undergoing concurrent radiotherapy and chemotherapy. The genetic variations of CASP3, CASP7 and FAS are significantly associated with the overall survival of patients with rectal cancer preoperative concurrent radiotherapy and chemotherapy. The genetic variations of BAK, BCL2, BID, CASP9 and FASL are significantly associated with the overall survival of rectal cancer patients who had postoperative chemoradiotherapy. These genetic variations may be potential genetic biomarkers for individualized treatment of rectal cancer.