【作者】 李亮；

【导师】 王斌全；

【作者基本信息】 山西医科大学 ， 耳鼻咽喉科学， 2014， 博士

【摘要】 背景和目的：喉鳞癌组织学上以鳞状细胞癌最为常见。喉鳞癌目前的治疗手段仍以外科手术为主。现代喉外科学提倡：喉鳞癌治疗应在保证生存率的前提下，尽量保留喉的功能。但是如何既保证生存率，又最大限度地保留喉功能是目前提高喉鳞癌手术疗效的瓶颈。外科切缘不仅是保证患者术后生存率与生存质量的核心因素，同时也是实施喉功能保留手术时临床医师较难把握的手术关键之一。切缘过大则喉正常组织被过多地切除，术后喉功能受损，严重影响患者术后的生存质量；切缘过小则容易导致肿瘤细胞残留，引起患者术后的复发，降低了生存率，因此对于外科切缘量和性的把握是临床医师诊疗过程中面临的棘手问题。常规喉外科切缘是否安全，常以形态学及组织病理学为基础，但常规病理学检查为阴性时仍有一定局部复发率，这反映出病理学检查在手术切缘安全判断的局限性。“区域癌化”(field cancerization)是指在外界致癌因素影响下，特定器官的一个或一组细胞积累遗传学改变，转化为癌前细胞，在致癌因素的持续作用下，癌前细胞进一步累积基因改变而转变为具有恶性的肿瘤细胞，而那些发生了基因改变的癌前细胞可以存在于切缘组织病理正常的黏膜中。杂合性缺失（loss of heterozygosity，LOH）反映的是肿瘤发生中特征性的基因改变，是研究肿瘤区域癌化的一种常用技术手段，目前针对喉鳞癌“区域癌化”的研究尚少。本研究拟通过LOH分析，验证喉鳞癌患者癌化区域的存在，通过分析比较基因改变情况，探寻在喉鳞癌发生发展过程中可能涉及的位点，并对目前的外科切缘的安全性进行探讨。方法：调取35例喉鳞癌患者手术切除后石蜡包埋组织标本，其中5例为术后复发并行二次手术患者。依据第7版UICC标准及WHO标准对患者进行TNM分期及病理分级。石蜡包埋组织行7μm连续切片、HE染色，在显微镜下区分肿瘤、癌旁及切缘等不同细胞分区并对各分区进行筛查，挑选符合标准的标本；使用激光捕获显微切割技术（Laser Capture microdissection，LCM）分别在肿瘤中心(T)，癌旁（F1-F4：癌旁距离<5mm； F5：癌旁距离>5mm），以及切缘(Ma-Md)位置进行显微切割，提取细胞；分别检测各区域细胞的LOH情况；结合病案资料分析对比各分区的LOH情况。为了检验喉鳞癌中是否存在区域癌化，本研究中使用位于9p、3p、17p、18q以及8p染色体上的9个微卫星位点（microsatellite）。这些位点选取是来自于潜在或者已知的抑癌基因的连锁位点，同时根据文献报道，选择在头颈肿瘤中研究较多的位点。对于在肿瘤中检测到LOH的患者，根据LOH的模式差异判断复发喉癌的克隆来源，并使用微卫星位点分析癌旁黏膜及手术切缘的差异。结果：1.喉鳞癌患者中有25例在癌细胞中心位置(T)检出一个或多个位点的LOH，检出率83.3%（25/30）；2.9个微卫星位点在肿瘤中心的检出率分别为： D3S1284（31.6%）、D3S1300（44.4%）、D3S1234（55%）、D3S1568（47.1%）、D9S171（44.4%）、D9S1870（42.1%）、TP53（58.8%）、D18S1110（33.3%）以及D8S518（35.3%）。3.在5名复发患者中，复发肿瘤LOH的模式与原发肿瘤的LOH一致；4.抑癌基因TP53在肿瘤中心位置LOH检出与喉鳞癌患者的年龄、肿瘤发生的部位、淋巴转移、T分期均无关（P＞0.05），与病理分级相关（P=0.021<0.05）。其余各指标在肿瘤中心位置LOH检出与喉鳞癌患者的年龄、肿瘤发生的部位、淋巴转移、病理分级、T分期均无关（P＞0.05）；5.微卫星位点在癌旁黏膜（F1-F4，F5）有不同程度检出，各微卫星位点检出率分别是：D3S1284（83.3%）、D3S1300（75%）、D3S1234（72.7%）、D3S1568（75%）、D9S171（87.5%）、D9S1870（75%）、TP53（80%）、D8S518（33.3%）以及D18S1110（33.3%）；6.微卫星位点在切缘（Ma-Md）有不同程度LOH检出，各微卫星位点检出率分别是：D3S1284（83.3%）、D3S1300（37.5%）、D3S1234（54.5%）、D3S1568（50%）、D9S171（62.5%）、D9S1870（62.5%）、TP53（70%）、D8S518（16.7%）以及D18S1110（16.7%）；7.18名患者(60%)癌旁黏膜存在至少一个微卫星位点的LOH，其中10名患者（编号：1、4、6、9、10、12、14、23、26、27）不同区域的癌旁黏膜表现为相同的基因改变，6名患者（编号：11、17、19、20、22、25）癌旁黏膜表现为不同的基因位点的丢失。此外还有2名患者只有一个区域发生LOH（编号：3、8）；8. D8S518位点LOH在17、23患者癌旁黏膜检出，尤其是17号患者，该位点的改变扩展到了手术的切缘。D18S1110在3、20号患者的癌旁黏膜中检出，其中20号患者在手术后8个月发生了喉癌的复发（编号33）；;9.15名患者(50%)切缘检测到至少1个位点的LOH，其中6名患者（编号：4、8、9、10、12、22）不同区域的切缘黏膜表现为相同的基因改变，1名患者（编号：3）切缘黏膜表现为不同的基因位点的丢失，其余8名患者（编号：6、11、14、17、19、23、26、27）只有一个区域的切缘检测到了LOH；10.8名患者(编号：1、6、10、11、14、19、26、27)肿瘤中心（T）与癌旁区域表现为相同的基因改变，10名患者的肿瘤中心（T）较癌旁黏膜有额外的基因改变(编号：3、4、8、9、11、12、17、20、23、25)。此外，有6名患者(编号：3、6、10、11、14、19)肿瘤中心（T）与切缘表现为相同的基因改变，还有10名患者(编号：3、4、8、9、12、17、22、23、26、27)切缘黏膜表现不同于肿瘤中心（T）的基因改变；11.12名患者（编号：3、4、6、8、9、10、11、12、14、17、19、26）同一标本中手术切缘与癌旁黏膜具有LOH的一致性，这两个区域具有共同的基因改变过程。而3号患者切缘（Mb）、22（Mb、Md）、27号患者切缘（Ma）表现出与癌旁黏膜的LOH模式的差异；12.5名复发肿瘤患者的癌旁黏膜都检测到了微卫星的LOH,3名患者(编号：31、32、34)癌旁黏膜与肿瘤LOH模式一致，33和35号患者肿瘤中心（T）较癌旁黏膜有额外的基因改变。4名复发患者(编号：32、33、34、35)在切缘检测到LOH，发生LOH的位点较肿瘤中心（T）减少。与原发肿瘤相比复发肿瘤的切缘和癌旁黏膜的LOH模式存在差异，31号患者未在切缘检测到LOH，32、33、34、35号患者癌旁黏膜的LOH模式不同于相对应的原发肿瘤癌旁黏膜，32、33、34、35号患者切缘检出的LOH模式也与原发肿瘤存在差异；13.癌旁黏膜（F1-F4，F5）LOH检出与喉鳞癌患者的年龄、肿瘤发生的部位、T分期、病理分级均无关（P＞0.05），与淋巴转移相关（P=0.008<0.05）；14.切缘（Ma-Md）LOH检出与喉鳞癌患者的年龄、肿瘤发生的部位、T分期、病理分级均无关（P＞0.05），与淋巴转移相关（P=0.003<0.05）；15. TP53在癌旁黏膜（F1-F4，F5）检出与喉鳞癌患者的年龄、肿瘤发生的部位、病理分级、T分期均无关（P＞0.05），与淋巴转移相关（P=0.049<0.05）。其余指标在癌旁黏膜（F1-F4，F5）检出与喉鳞癌患者的年龄、肿瘤发生的部位、淋巴转移、病理分级、T分期均无关（P＞0.05）。所有分指标在切缘（Ma-Md）检出与喉鳞癌患者的年龄、肿瘤发生的部位、淋巴转移、病理分级、T分期均无关（P＞0.05）。结论：1.微卫星位点D3S1300、D3S1234、D3S1568、D9S171、D9S1870、TP53在肿瘤中检测超过40%，说明在喉鳞癌中这6个位点易发生改变，可考虑作为检测喉鳞癌LOH的位点；2.复发的喉鳞癌与原发肿瘤存在克隆相关性，是属于喉鳞癌的局部原位复发，说明组织学阴性的切缘作为手术切除范围的判定存在一定风险；3.研究中只有TP53的LOH与病理分级相关(p<0.05)，但该结果仍需进一步深入研究。其他指标与年龄、性别、肿瘤部位、T分期以及淋巴结转移均无相关性；4.喉鳞癌是多因素、多阶段、多步骤参与的渐进的病变过程；5.本研究中大多数的喉鳞癌患者癌旁黏膜存在区域癌化，而且癌化区域的范围随患者不同而发生变化，有相当大的一部分的患者区域癌化扩展到手术的切缘；6. D8S518和D18S1110位点在癌前区域向喉鳞癌发展的过程中可能起关键作用，目前还需进一步的研究；7.癌旁黏膜的TP53的LOH可以考虑用来评估喉鳞癌是否发生淋巴结转移。更多还原

【Abstract】 Objective：Laryngeal cancer is frequently occurring in head and neck cancers. Most laryngealcancers are squamous cell carcinomas. Radiotherapy, chemotherapy, surgery aloneand combination of therapy have been used in the treatment of laryngocarcinoma.However, the survival rate of patients with laryngocarcinoma is low due to its latemetastases and resistance to chemotherapy and radiotherapy post surgery.“Fieldcancerization” is a biological process in which one or groups of cells in specificorgans changes into the pre-cancerous cells under stimulation of the externalcarcinogenic factors. In the chornic carcinogenic factors stimulation, further geneprecancerous occurs to induce malignant tumor, even in peripheral tissues of normalmucosa pathology. Loss of heterozygosity (LOH) reflects the characteristic geneticchanges during tumorigenesis, which is commonly used in study of fieldcancerization. There was less report on field concretization in laryngeal cancers. Inthis study, we attempt to, verify the field cancerization in laryngeal cancer patients byLOH analysis. Through analysis and comparison of gene alternation, possible sitesplay a key role in the process of tumor progression would be investigated. Moreover,the security of sugical margin in current laryngeal cancer treatment was alsoevaluated.Methods：The paraffin blocks from35cases of patients with laryngeal cancer was collected,including five cases of recurrent patients post-surgery. The patients were divided intodifferent groups according to TNM staging, histological type and other circumstances.The paraffin blocks were used to make7μm serial sections. Then HE staining wasperformed, different positions of tumor were distinguished under a microscope.Sugical margins, center area and paracancerous were observed, the appropriatesections were selected to perform laser capture microdissection (LCM). The center ofthe tumor (T), paracancerous (F1-F4, paraneoplastic distance <5mm; F5, adjacentdistance>5mm) and sugical margins (Ma-Md) position microdissection were cut off via LCM, respectively. The DNA was extracted from the cut cells. Then PCRtechniques were used to detect LOH in different region of tumor tissues. Incombination with medical record data, the LOH analysis was compared in eachpartition. To test whether field cancerization was occurred in laryngeal carcinoma, themicrosatellite located on chromosome9p,3p,17p,18q and8p, which were identifiedas potential or known tumor suppressor genes, were studied in this study. Accordingto the difference LOH patterns, to determine the recurrent tumor clone was analyzed.Meanwhile, the differences in adjacent mucosa and surgical margin were alsoanalyzed with above microsatellites.Results：1. There are25cases of laryngeal cancer patients at the cancer center position (T)detected in one or more sites of LOH, the detection rate was83.3%(25/30);2. Nine microsatellites in the center of the tumor detection rate were: D3S1284（31.6%）、D3S1300（44.4%）、D3S1234（55%）、D3S1568（47.1%）、D9S171（44.4%）、D9S1870（42.1%）、TP53（58.8%）、D18S1110（33.3%）and以及D8S518（35.3%）respectively;3. In the five patients with recurrent tumor, LOH patterns were consistent with theprimary tumor;4. It was found that the age of patient, regions of tumor, lymphatic metastasis and Tstage were not related to TP53LOH (P>0.05). Except tumor grade (P=0.021<0.05), the rest microsatellites in LOH detection were also not related to the age ofpatients, regions of tumor, lymphatic metastasis, histological grade and T stage(P>0.05);5. There were some microsatellites loci detected in mucosa of tumor-adjacenttissues (F1-F4, F5), the detection rate of each index is: D3S1284（83.3%）、D3S1300（75%）、D3S1234（72.7%）、D3S1568（75%）、D9S171（87.5%）、D9S1870（75%）、TP53（80%）、 D8S518（33.3%） and D18S1110（33.3%）,respectively;6. Microsatellites loci in the sugical margins (Ma-Md) were also detected with different degrees of LOH, the detection rate of each index are:D3S1284（83.3%）、D3S1300（37.5%）、D3S1234（54.5%）、D3S1568（50%）、D9S171（62.5%）、D9S1870（62.5%）、TP53（70%）、D8S518（16.7%）and D18S1110（16.7%）, respectively;7. There were at least one microsatellite loci LOH in tumor-adjacent mucosa in18patients (60%). In which10patients (No:1,4,6,9,10,12,14,23,26,27)mucosal paraneoplastic manifestations in different regions had the same geneticchanges, six patients (No:11,17,19,20,22,25) adjacent mucosa showed loss ofdifferent loci. Meanwhile, there are also two patients with only one regionoccurred LOH (No:3,8);8. In adjacent mucosa, the D8S518LOH was detected in patients No.17and23,especially the No.17patient, the changing expanded to the sugical margins.D18S1110LOH was detected in patients No.3and20patients in adjacentmucosa, No.20patients8months after surgery recurrence of laryngeal cancer(No.33) occurred;9. The sugical margins of15patients (50%) were detected with at least one locusLOH. In which six patients (NO:4,8,9,10,12,22) mucosal resection marginperformance in different regions had the same genetic changes, one patient (No:3) mucosal resection margin performance for different loci lost. There was onlyone sugical margins region of the remaining eight patients (No:6,11,14,17,19,23,26,27) had been detected with LOH;10. Cancer center (T) and adjacent regions in8patients (No:1,6,10,11,14,19,26,27) showed the same genetic alterations. There were additional genetic changesbetween tumor center (T) and paracancerous mucosa in10patients (No:3,4,8,9,11,12,17,20,23,25). In addition, cancer Center (T) and the sugical marginsperformance had the same genetic changes in6patients (No:3,6,10,11,14,19).However, the genetic changes in mucosal resection margin was unlike cancercenter (T) in the other10patients (No:3,4,8,9,12,17,22,23,26,27);11.12patients (No:3,4,6,8,9,10,11,12,14,17,19,26) surgical margin and adjacentmucosa with the same LOH model, these two regions have a common genetic alterations process. While patients No:3patients margin (Mb), No:22(Mb, Md),No:27(Ma) showed differences in adjacent mucosa LOH patterns;12. The LOH was found in five cancer patients with recurrent tumors. The LOH ofadjacent mucosa was similar to tumor tissues in3patients (No:31,32,34).However, there were genetic changes between cancer center (T) and mucosaadditional in No.33and No.35patients. The sugical margins of4patients withrecurrent tumors (No:32,33,34,35) had been found LOH,and the LOH was lessin sugical margins than cancer center (T) with recurrent tumors. In comparisonwith primary tumor, there was difference of LOH between sugical margins andadjacent mucosa in patients with recurrent tumors. There was less LOH in thesugical margins of No.31patients, and the LOH was also different to primarytumor in No.32,33,34,35patients;13. The LOH of adjacent mucosa (F1-F4, F5) was unrelated with the age of patients,location of tumor, T stage, histological grade (P>0.05). However, the lymphaticwas significantly related (P=0.008<0.05);14. The LOH of sugical margins (Ma-Md) was unrelated with age, location of tumor,T stage, histological grade (P>0.05), but correlated with lymphatic (P=0.003<0.05);15. TP53in adjacent mucosa (F1-F4, F5) was unrelated with age, location of tumor,tumor grade, T stage (P>0.05), but correlated with lymphatic (P=0.049<0.05).The remaining indicators in adjacent mucosa (F1-F4, F5) was unrelated with age,location of tumor, lymph node metastasis, histological grade, T stage (P>0.05).All indexes in the sugical margins (Ma-Md) detected in patients were not relatedto age, location of tumor, lymph node metastasis, histological grade, T stage (P>0.05).Conclusions：1. Microsatellites D3S1300, D3S1234, D3S1568, D9S171, D9S1870, TP53tumorwas detected with over40%, which indicated that in laryngeal cancers, thesespecific sites can be considered for the detection of LOH in laryngeal cancer;2. There was clonality correlation exists between recurrent laryngeal cancer and primary tumor, which could be local recurrence. It suggested that negativemargin determination in histology could be imperfection as surgical resection;3. The relationship of other indicators between LOH and the age of laryngeal cancerpatients, location of tumor, lymph node metastasis, histological grade, T stage werenot certain yet,but TP53associated with pathological grading;4. Laryngeal cancer is multifactorial, multi-stage, multi-step process during itsdevelopment;5. There was field cancerization in most mucosa in laryngeal patients. The area wasdifferent in each patient. Importantly, a significant portion of patients had fieldcancerization in the surgical margins;6. D18S1110and D8S518may play a key role in the development of precancerouscell to tumor cells, further study should be performed;7. The LOH of TP53in adjacent mucosa can be used to assess whether the lymphnode metastasis in laryngeal cancer occurs.更多还原