【作者】 蒲玉梅；

【导师】 胡勤刚；

【作者基本信息】 南京大学 ， 口腔临床医学， 2017， 博士

【摘要】 [研究背景]手术一次性完整切除肿瘤是控制口腔鳞癌术后复发的关键环节,而对手术切缘安全性的评估(包括粘膜切缘(mucosal margin)和深部软组织切缘(deep soft tissue margin)两个方面)是判断手术彻底性的重要指标。然而,外科安全手术切缘的标准并未被所有医师及学者所接受,仍需不断完善。就粘膜切缘而言,黏膜的异常增生程度对口腔鳞癌预后的影响尚需深入探讨。肿瘤深部即浸润前沿的处理是口腔鳞癌手术的难点。即使是整体切除的病灶,也不足以保证肿瘤深部的安全切缘,这可能是肿瘤的不同浸润方式需要不同的安全范围的缘故。肿瘤浸润方式是肿瘤侵袭特性的表现,也是肿瘤微环境,特别是炎症微环境与肿瘤相互作用的结果。因此,分析手术安全切缘和肿瘤浸润方式与口腔鳞癌预后的关系,探讨浸润方式与肿瘤炎症微环境的相互影响,对于提高口腔鳞癌手术治疗效果具有直接的临床指导价值和重要的理论意义。本研究着重分析了切缘状态、浸润方式和肿瘤相关中性粒细胞的关系,及其对口腔鳞癌患者预后的影响。一、不同切缘状态对口腔鳞癌预后的影响[目的]观察口腔鳞癌不同切缘状态对其患者生存率的影响,分析不同处理方式对切缘异常增生患者预后的影响。[方法]收集2005-2014年南京大学医学院附属口腔医院颌面外科收治的T1-4N0M0 口腔鳞癌患者的临床病例资料,资料分析内容包括切缘状态及处理方式在内的临床与病理学参数。采用电话、信访和复诊方式随访,获得患者的生存状况和肿瘤术后情况。以单因素和COX多因素统计学方法分析,分析各参数与口腔鳞癌患者的总生存率(OS)、无复发生存率(RFS)和无病生存率(DFS)的关系。[结果]符合研究标准者,共539例。临床资料分析表明,20%的患者中出现了切缘的异常,包括轻度异常增生、中度异常增生、重度异常增生及癌残留,异常切缘的发生率在不同术者之间无明显差异(p>0.05)。切缘为中度、重度异常增生及癌残留者的OS(p分别为 0.0028,0.0082),RFS(p 分别为 0.049,0.006)和 DFS(p 分别为 0.0043,0.0005)显著低于切缘阴性者。切缘轻度异常增生者,其术后OS(p==0.206),RFS(p=0.316)及DFS(p=0.183)与切缘阴性者无明显差异;但切缘轻度异常增生而未补充切除者的OS(p = 0.006),RFS(p=0.010),DFS(p=0.014)显著低于切缘阴性者;而切缘轻度异常增生行补充切除患者的OS(p = 0.006),RFS(p = 0.020),DFS(p = 0.020)明显高于未行补充切除者。[结论]切缘中、重度异常增生及癌残留是口腔鳞癌患者生存预后的独立影响因素;应作为高危因素列入阳性切缘的标准;手术切缘为轻度异常增生时,建议进一部补充切除直至为阴性,可提高生存率。二、浸润方式对切缘状态及颈淋巴结微转移的影响及其预后分析[目的]1.观察口腔鳞癌最差浸润方式(WPOI)对其患者术后生存的影响,分析WPOI与切缘状态、BPOI及CD1a+DC浸润密度的关系,探讨BPOI和CD1a+DC术前评估WPOI的可行性。2.分析口腔鳞癌患者WPOI与颈部淋巴结微转移的相关性,探讨微转移患者术后放疗的必要性。[方法]1、收集2005-2014年-南京大学医学院附属口腔医院颌面外科收治的T1-2N0M0 口腔鳞癌患者的临床资料,采用电话、信访和复诊方式随访,获得患者的生存状况和肿瘤术后情况,通过统计学相关性、单因素及多因素方法,分析内容包括切缘状态与肿瘤WPOI的相关性,以及包括WPOI和BPOI在内的临床病理学参数,与口腔鳞癌患者的OS,RFS和DFS的关系。免疫组化技术标志口腔鳞癌组织中CD1a+DC,进而采用相关性及ROC曲线方法,分析BPOI及CD1a+DC细胞数与WPOI的关系,以及其术前评估WPOI的特异性和敏感性。2、收集2010-2013年南京大学医学院附属口腔医院颌面外科收治的口腔鳞癌患者的临床资料,通过电话、信访和复诊方式随访,获得患者的生存状况和肿瘤术后情况,采用HE染色和CK免疫组化染色,观察淋巴结转移情况,并将其分为三组:淋巴结阴性组,微转移组及大灶转移组,分析包括肿瘤WPOI,淋巴结转移在内的临床与病理学参数对生存率的影响,进而分析WPOI与颈部淋巴结微转移的相关性,以及术后放疗对颈部淋巴结微转移患者生存率的影响。[结果]1、符合研究标准的口腔鳞癌患者,共444例。统计分析显示,WPOI对口腔癌患者的OS(p=0.002),RFS(p=0.022)和DFS(p=0.002)均有显著影响。对相同T分期患者,WPOI为4-5级的浸润深度(DOI)明显大于WPOI为1-3者(T1:p=0.006;T2:p<0.0001;T1+T2:p<0.0001)。肿瘤BPOI与患者预后无关,与WPOI中度一致(加权Kappa值:0.5468)。ROC曲线分析显示:浸润前沿CD1a+DC细胞数的线下面积为0.769,阈值为21.833,敏感度73.0%,特异度74.4%;联合口腔鳞癌活检组织中肿瘤浸润前沿CD1a+DC细胞数为指标细胞数,可以提高术前评估肿瘤WPOI的灵敏度(88.2%)及特异度(84.6%)。2、符合研究标准的口腔鳞癌患者,共311例。经CK免疫组化染色,发现12位淋巴结阴性患者(4.94%)的颈部淋巴结中有微小的转移灶;淋巴结微转移与WPOI显著相关(p=0.004);术后接受放疗的微转移组和术后未接受放疗微转移组的术后的生存率无明显统计学差异(OS:p=0.843,MFS:p=0.962 DFS:p=0.879)。[结论]WPOI与口腔鳞癌患者的生存预后显著相关,是OS,RFS和DFS独立预后因素;在相同T分期下,口腔鳞癌的WPOI分级越高,预示着肿瘤的DOI越深;肿瘤BPOI能在一定程度上反映WPOI;以肿瘤活检组织中浸润前沿的CD1a+DC细胞数为观察指标,结合活检组织BPOI检测结果,可以明显提高利用活检组织样本对WPOI的预测准确性。CK免疫组化染色能提高颈淋巴结微转移的检出率;WPOI与颈部淋巴结微转移密切相关;术后辅助放疗在提高颈部微转移口腔鳞癌患者术后生存率方面并没有显著的优势。三、肿瘤相关中性粒细胞对口腔鳞癌预后的影响及其与浸润方式的相关性[目的]分析口腔鳞癌中肿瘤相关中性粒细胞(TANs)的浸润情况;探讨TANs与肿瘤浸润方式的关系以及其对生存预后的影响,并初步探讨CXCL5和IL-17在口腔鳞癌TANs与WPOI相互影响中的作用。[方法]随机选择2005年～2010年南京大学医学院附属口腔医院收治的口腔鳞癌患者作为研究对象,以免疫组织化学染色方法标志肿瘤组织中CD15+中性粒细胞,分析其对口腔鳞癌患者生存预后的影响以及与肿瘤WPOI的相关;以癌旁组织和正常黏膜上皮组织为对照,流式细胞技术检测口腔鳞癌TANs的比例,并比较WPOI为4-5级和1-3级口腔鳞癌中TANs的比例;进而以免疫组化技术观察不同WPOI 口腔鳞癌中IL-7及CXCL5的表达情况,分析其对中性粒细胞募集的影响。[结果]单因素分析显示,TANs浸润密度对口腔鳞癌患者的OS(p=0.0003)、RFS(p=0.017)及DFS(p=0.003)有显著影响;WPOI为4-5级的肿瘤浸润前沿处(p=0.0132)及WPOI区(p=0.0027)浸润的TANs密度显著高于WPOI为1-3级者;流式细胞检测结果显示,肿瘤组织中TANs比例明显高于癌旁组织及正常组织(p<0.0001),而癌旁组织与正常组织中的比例无明显统计学差异;而WPOI为4-5级的口腔鳞癌肿瘤组织中TANs的比例明显多于WPOI为1-3级。免疫组化分析口腔鳞癌组织的IL-17及CXCL5表达结果显示,WPOI为1-3级的表达量低于WPOI为4-5级者,且IL-17、CXCL5高表达者TANs的浸润也增加。[结论]TANs主要集中在口腔鳞癌的浸润前沿和WPOI区,这可能是其发挥影响的主要部位;TANs与口腔鳞癌的浸润方式密切相关,可能对口腔鳞癌浸润具有影响,并而影响患者的生存预后;口腔鳞癌对中性粒细胞具有募集作用,并且肿瘤的浸润方式对这种募集作用具有明显的影响;肿瘤及其微环境内的CXCL5及IL-17可能参与了口腔鳞癌对TANs的募集。更多还原

【Abstract】 BackgroundThorough removal of rumor during initial surgical treatment is crucial for oral squamous cell carcinoma(OSCC)local control.To access the thoroughness of surgery,margin status,including margins at the epithelium and basement,is a valuable indicator.However,the criteria of surgical margins have not yet been uniformed.For epithelial margin,margins with medium-to-severe dysplasia and/or residual cancer are recognized prognostic factor OSCC,but the treatment of mild dysplasia margin is controversial.The management of margins at the invasion front is difficult during OSCC operation.At present margin more than 5mm from tumor is defined as negative margin.However,even for such negative margins good prognosis is not guaranteed.This may be related to the biological behavior of the tumor.The ability of tumor to infiltrate into deep normal tissue represents the invasiveness of tumor and is directly related to invasion pattern,which is also the result of interaction between tumor and tumor microenvironment.This study mainly focuses on margin status,worst pattern of invasion,and their prognostic value of OSCC,as well as the relation between infiltrating tumor-associated neutrophils(TANs)and WPOI.OneThe influence of margin status on the prognosis of oral squamous cell carcinoma[Objective]To evaluate the clinical relevance between different margin statuses,especially mild dysplasia,and prognosis in patients undergoing primary surgical treatment for oral squamous cell carcinoma(OSCC).[Methods]Five hundred and thirty-nine patients with T1-4NOMQ OSCC,who underwent surgery alone at a stomatology hospital in Nanjing,China during the years 2005—2014,were included.Tumor and margin status were assessed.Overall survival(OS),disease-free survival(DFS),and recurrence-free survival(RFS)were calculated by Kaplan-Meier method.Predictors of RFS,OS,and DFS were analyzed.[Results]Positive or dysplastic margins were found in the initial specimen in 20.0%of the cases.On multivariate analysis,there was no significant difference between RFS(hazard ratio(HR)1.379,p = 0.361)or DFS(HR 1.452,p = 0,183)of those with mild dysplasia and those with negative margins.However,patients with mild dysplasia who did not undergo re-excision demonstrated significantly worse RFS(HR 2.286,p = 0.010)and DFS(HR 2.070,p = 0.014)than those with negative margins.[Conclusions]There appears to be a correlation between initial mild dysplastic margins that are not subjected to re-excision and inferior RFS and DFS.Additional attention should be drawn to mild dysplasia at the initial margin in OSCC,and extended excision is suggested.TwoThe influence of pattern of invasion on the prognosis of oral squamous cell carcinoma[Objective]To evaluate the prognostic value of worst pattern of tumour invasion(WPOI)and biopsy pattern of invasion(BPOI)of patients with early-stage oral squamous cell carcinoma(OSCC),To assess the ability of CD1a+DC and BPOI to predict WPOI preoperationly.To evaluate OSCC patients with only micrometastasis and inferior worst pattern of invasion(WPOI),as well as those with Ⅳ/Ⅴlevels of micrometastasis,need postoperative radiotherapy is unclear.[Methods]1.This retrospective study included 444 patients with pTl-2N0M0 OSCC who had undergone surgical treatment only in the Department of Oral and Maxillofacial Surgery,Nanjing Stomatology Hospital from 2005 to 2014.The value of WPOI and BPOI in predicting prognosis and the ability of CD1a+DC in predicting the WPOI preoperationly were assessed.Statistical analyses were performed using the χ2 test,Kaplan-Meier method,and Cox regression model.Predictors of outcome were identified using multivariate analysis.2.OSCC patients(n=311)with negative(n=247),only micrometastasis(n=44)and macrometastasis(n=20)were detected and selected by HE staining.Micrometastasis was re-assessed using immunohistochemical staining of cytokeratin(CK)in HE negative patients to find out the false negative cases.The incidence and clinical features of the primary tumour in relation to the only micrometastatic status,and the value of the postoperative radiotherapy on the only micrometastasis patients were evaluated.[Results]1.The outcome(including overall survival,recurrence-free survival and disease-free survival 1)of patients with WPOI 4-5 is worse than WPOI 1-3(p=0.002,p=0.022,p=0.002,respectively).However,BPOI was not significantly associated with OS,RFS,or DFS(p=0.979,p=0.422,p=0.440,respectively).But among the 80 BPOI type 4 cases,their matching WPOI were all type 4-5.And WPOI 4-5 was significantly associated with a deeper invasion which was also significantly associated with OS,RFS,or DFS.The areas under ROC were 0.769 for CD1a+DC/HP,the sensitivity and specificity were 73.0%and 74.4%,the optimal threshold values was 21.833.When combined the two methods the sensitivity and specificity were 88.2%and 84.6%.2.Among the negative lymph node cases(n=247),the positive rate of CK was 4.94%(n=12).Patients with only micrometastasis had higher T stage and inferior WPOI than patients without micrometastasis,but they had longer overall survival(OS),metastasis-free survival(MFS)and Disease-free survival(DFS)than macrometastasis patients.Micrometastasis is an independent diagnostic indicator for MFS.However,the survival time of only micrometastasis patients with or without postoperative radiotherapy was comparable,even in patients with inferior WPOI.Radiotherapy,however,may only benefit patients with IV/V levels of micrometastasis.[Conclusions]The WPOI was significant indicators of prognosis.BPOI type 4 indicates WPOI type 4-5 invasion and is predictive of a poor outcome,thus more extensive resection should be considered for such patients.The number of CD1a +DC cells infiltrating the front of tumor biopsy tissue as an observational index which can improve the accuracy of assessing WPOI when combined with BPOI.There appears to be a correlation between WPOI and micrometasitasis.Postoperative radiotherapy is dispensable for micrometastasis.ThreeEffects of Different WPOI on Recruitment of Tumor-Associated Neutrophils[Objectives]To study infiltrating tumor-associated neutrophils(TANs)in OSCC microenvironment and their prognostic value;the distribution of TANs in different WPOI;effects of different types of WPOI on recruitment of TANs.[Methods]OSCC patients with complete follow-up data who received primary surgical treatment in Affiliated Stomatological Hospital of Nanjing University Medical School from 2005 to 2010 were chosen for the study.Difference of numbers of neutrophils in OSCC tissue between WPOI 1-3 and WPOI 4-5 were determined with immunohistochemistry and flow cytometry.The ability of IL-7 and CXCL5 to recruit neutrophils in different types of WPOI was accessed with immunohistochemistry.[Results]Univariate analysis showed that neutrophil density has significant influence in OS(p=0.0003),RFS(p=0.017)and DFS(p=0.003);CD15+ neutrophils at the invasion front were significantly different between WPOI 1-3 and WPOI 4-5(p= 0.0132,p=0.0027).Flow cytometry showed the same result.This difference may be related to high expression of IL-17 and CXCL5 at WPOI 4-5 invasion front.[Conclusions]Though the density of CD15+ neutrophils was not an independent prognostic factor of OSCC,it influenced the survival rate of OSCC patients.In WPOI 4-5,TANs increased and mainly distributed in the invasion front,especially in worse pattern of invasion.CXCL5 and IL-17 participated in recruitment of TANs.更多还原