区域癌化与喉鳞癌外科手术切缘的研究
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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。
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.
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。
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.
引文
1.刘天润，杨安奎，陈福进，等。221例晚期喉鳞癌患者术后的生存和预后分析。癌症，2009，28(3)：297-302.
2. Kamangar F,Dores G. M, Anderson W. F. Patterns of cancer incidence,mortality,and prevalence across five continents：defining priorities to reducecancer disparities in different geographic regions of the world. J. Clin. Oncol.2006,24,2137–2150.
3. Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, et al. Genetically altered fieldsas origin of locally recurrent head and neck cancer：a retrospective study. Clin.Cancer Res.2004,10,3607–3613.
4. Roesch-Ely M, Nees M, Karsai S, et al. Proteomic analysis reveals successiveaberrations in protein expression from healthy mucosa to invasive head and neckcancer. Oncogene.2007,26,54–64.
5. Schaaij-Visser TB, Graveland A, Gauci S, et al. Differential proteomics identifiesprotein biomarkers that predict local relapse of head and neck squamous cellcarcinomas. Clin. Cancer Res.2009,15,7666–7675.
6. Kutler DI, Auerbach AD, Satagopan J. et al. High incidence of head and necksquamous cell carcinoma in patients with fanconi anemia. Arch. Otolaryngol.Head Neck Surg.2003,129,106–112.
7. Hopkins J, Cescon DW, Tse D, et al. Genetic polymorphisms and head and neckcancer outcomes： a review. Cancer Epidemiol. Biomark. Prev.2008,17,490–499.
8. Cloos J, Spitz MR, Schantz SP, et al. Genetic susceptibility to head and necksquamous cell carcinoma. J. Natl Cancer Inst.1996,88,530–535.
9. Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival ofpatients with oropharyngeal cancer. N. Engl. J. Med.2010,363,24–35.
10. Smith B, Sniezek C, Weed T, et al. Utilization of free tissue transfer in head andneck surgery. Otolaryngol. Head Neck Surg.2007,137,182–191.
11. Vergeer R, Doornaert A, Rietveld H, et al. Intensity-modulated radiotherapyreduces radiation-induced morbidity and improves health-related quality of life：results of a nonrandomized prospective study using a standardized follow-upprogram. Int. J. Radiat. Oncol. Biol. Phys.2009,74,1–8.
12. Boscolo-Rizzo P, Maronato F, Marchiori C, et al. Long-term quality of life aftertotal laryngectomy and postoperative radiotherapy versus concurrentchemoradiotherapy for laryngeal preservation. Laryngoscope.2008,118,300–306.
13. Slaughter P, Southwick W, Smejkal W, et al. Field cancerization in oral stratifiedsquamous epithelium； clinical implications of multicentric origin. Cancer.1953,6,963–968.
14. Braakhuis BJ, Tabor MP, Kummer JA,et al. A genetic explanation of Slaughter'sconcept of field cancerization: evidence and clinical implications. Cancer Res.2003,15；63(8)：1727-30.
15. Califano J, van P, Westra W, et al. Genetic progression model for head and neckcancer： implications for field cancerization. Cancer Res.1996,56,2488–2492.
16. van M, Tabor P, van W. et al. Mutated p53as a molecular marker for thediagnosis of head and neck cancer. J. Pathol.2002,198,476–486.
17. Jonason S, Kunala S, Price J. et al. Frequent clones of p53-mutated keratinocytesin normal human skin. Proc. Natl Acad. Sci. USA.1996.93,14025–14029.
18. Tabor P, Brakenhoff H, van M, et al. Persistence of genetically altered fields inhead and neck cancer patients：Biological and clinical implications. Clin. CancerRes.2001.7,1523–1532.
19. Tabor P, Braakhuis J, van JE, et al. Comparative molecular and histologicalgrading of epithelial dysplasia of the oral cavity and the oropharynx. J. Pathol.2003,199,354–360.
20. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
21. Slaughter P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
22. Hittelman N. Genetic instability in epithelial tissues at risk for cancer. Ann. NYAcad. Sci.2001,952,1–12.
23. Mao L,Lee J,Tockman S,et al．Microsatellite alterations as clonal markers for thedetection of human cancer. Proc Natl Acad Sci USA.1994.91(21)9871-5．
24. Mao L, Lee S, Fan H, et al. Frequent microsatellite alterations at chromosomes9p21and3p14in oral premalignant lesions and their value in cancer riskassessment. Nature Med.1996,2,682–685.
25. Abou E,Habib N,Moussa E,et al．The role of genetic susceptibility in head andneck squamous cell carcinoma． Eur Arch Otorhinolaryngol.2008.265(2)：217-222．
26. Pearlstein RP, Benninger MS, Carey TE, Zarbo RJ, Torres FX, Rybicki BA, DykeDL. Loss of18q predicts poor survival of patients with squamous cell carcinomaof the head and neck.. Genes Chromosomes Cancer.1998,21(4),333-339.
27. GuoT,Sun JW,Lv QP,Li XG. Allelicic imbalance on chromosomes3p,9p and17pin malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
28. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity incritical genome regions predict a local relapse in patients afterlaryngeetomy.Mutat Res.2006.600(1-2)：67-76．
29. Tabor P,Brakenhoff H, van Houten M, et al. Persistence of GeneticallyAltered Fields in Head and Neck Cancer Patients： Biological and ClinicalImplications Clin Cancer Res.2001,7(6)：1523-1532.
30. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head andneck cancer. Nat Rev Cancer.2011,11(1)：9-22.
1. Marx J.New colon cancer gene discovered. Science.1993May7;260(5109):751-2.
2. Sharma A, Wayne S, Nikiforova MN, Johnson JT, Walvekar RR.Two sites ofhead and neck squamous cell carcinoma: utility of loss of heterozygosity. AnnOtol Rhinol Laryngol.2008Aug;117(8):591-3.
3. Califano, J. et al. Genetic progression model for head and neckcancer:implications for field cancerization. Cancer Res.56,2488–2492(1996).
4. Yin D T,Dong M M．Micresatellite instability and loss of heterozygosity offragile histidine triad gene in laryngesl squamous cell carcinomas.Zhanghua ErBi Yan Hou Tan Jing Wai Ke Za Zhi,2005.40(1)：4548．
5. Califano J, Westra WH, Meininger G,et al.Genetic progression and clonalrelationship of recurrent premalignant head and neck lesions. Clin Cancer Res.2000Feb;6(2):347-52.
6. GuoT,Sun JW,Lv QP,Li XG. Allelicic imbalance on chromosomes3p,9p and17pin malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
7. Vander R P,Nawroz H,Hruban RH,et al. Fequent loss of chromosome9p21-22early in head and neck cancer progression.Cancer Res,1994,54(5):1156-1158.
8. Rosin M, Splieth C, Wilkens M, Meyer G..Effect of cement type on retention of atapered post with a self-cutting double thread. J Dent.2000Nov;28(8):577-82.
9. Chakraborty SB, Dasgupta S, Roy A, Sengupta A,et al..Differential deletions in3p are associated with the development of head and neck squamous CancerGenet Cytogenet.2003Oct15;146(2):130-8.
10. Ghosh S, Ghosh A, Maiti GP, Alam N, Roy A, Roychoudhury S, PandaCK.Alterations of ROBO1/DUTT1and ROBO2loci in early dysplastic lesionsof head and neck： clinical and prognostic implications.Hum Genet.2009Mar；125(2)：189-98.
11. Hogg RP, Honorio S, Martinez A, et al. Frequent3p allele loss and epigeneticinactivation of the RASSF1A tumour suppressor gene from region3p21.3in headand neck squamous cell carcinoma. Eur J Cancer.2002Aug；38(12)：1585-92.
12. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
13. Abou-Elhamd KE, Habib TN, Moussa AE, et al. The role of genetic susceptibilityin head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol.2008Feb；265(2)：217-22.
14. Koscielny S, V Eggeling F, Dahse R.Investigations to the influence of tumorsupressor gene p16inactivation on the prognosis of head and neck squamous cellcarcinoma].Laryngorhinootologie.2004Jun;83(6):374-80.
15. Smigiel R, Sasiadek M, Krecicki T,et al. Inactivation of the cyclin-dependentkinase inhibitor2A (CDKN2A) gene in squamous cell carcinoma of the larynx.Mol Carcinog.2004Mar;39(3):147-54.
16. Sun PC, Uppaluri R, Schmidt AP,et al.Transcript map of the8p23putative tumorsuppressor region. Genomics.2001Jul;75(1-3):17-25.
17. Papadimitrakopoulou VA, Oh Y, El-Naggar A, Izzo J, Clayman G, MaoL.Presence of multiple incontiguous deleted regions at the long arm ofchromosome18in head and neck cancer. Clin Cancer Res.1998Mar；4(3)：539-44.
18. Kim SK, Fan Y, Papadimitrakopoulou V, Clayman Get al. DPC4, a candidatetumor suppressor gene, is altered infrequently in head and neck squamous cellcarcinoma. Cancer Res.1996Jun1；56(11)：2519-21.
19. G tte K, Riedel F, Neubauer J, et al. The relationship between allelic imbalanceon17p, p53mutation and p53overexpression in head and neck cancer. Int JOncol.2001Aug;19(2):331-6.
20. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
21. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
22. Yuge T, Nibu K, Kondo K, et al. Loss of FHIT expression in squamous cellcarcinoma and premalignant lesions of the larynx. Ann Otol Rhinol Laryngol.2005Feb;114(2):127-31.
23. Fu W,,Wu D, Zhang X.FHIT gene is abnormal in laryngeal carcinoma. ZhonghuaZhong Liu Za Zhi.1999Nov;21(6):436-8.
24. Pavelic K,Krizanac S,Cacev T,et a1．aberration of fhit gene is associated withincreased tumour proliferation and decreased apoptosis clinical evidence in lungand head neck carcinomas.．MolMed,2001.7(7)：442-453．
25. Chan MW,Chan LW,Tang NL,et al. Freguent hypermethyiation of promoterregion of RASSF1A in tumor tissues and voided urine of urinary biadder cancerpatients. Int Cancer,2003,104(5)：611-616.
26. Yu MY,Tong JH,Chan PK,et al. Hypermethyiation of the tumor suppressor geneRASSF1A and freguent concomitant ioss of heterozygosity at3P21.3in cervicaicancers. Int Cancer,2003,105(2)：204-209.
27.许承弼;滕博;李长青;金春顺；喉鳞状细胞癌中RASSF1A基因启动子区甲基化及蛋白表达;吉林大学学报(医学版);2006年02期
28. Worsham MJ, Chen KM, Tiwari N, et al. Fine-mapping loss of gene architectureat the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genesin head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.2006Apr；132(4)：409-15.
29. Worsham MJ, Stephen JK, Chen KM, Havard S, Shah V, Gardner G, SchweitzerVG Delineating an epigenetic continuum in head and neck cancer. Cancer Lett.2012Mar1.
30. Jares P, Nadal A, Fernández P L, et al. Disregulation of p16MTS1/CDK4I proteinand mRNA expression is associated with gene alterations in squamous‐cellcarcinoma of the larynx. International journal of cancer,1999,81(5):705-711.
31. Namazie A, Alavi S, Olopade OI, et al. Cyclin D1amplification andp16(MTS1/CDK4I) deletion correlate with poor prognosis in head and necktumors.Laryngoscope.2002Mar；112(3)：72-81.4
32. Rizos E,Sourvlnm C,Spondidm DA．Loss of heterozygosity at8p,9p and17q inlaryngeal cytological specimens. OralOncd.1998,34(6)：519．
33. Schneider-Stock R, Mawrin C, Motsch C, et al. Retention of the arginine allele incodon72of the p53gene correlates with poor apoptosis in head and neck cancer.Am J Pathol.2004Apr;164(4):1233-41.
34. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
35. Graveland AP, Golusinski PJ, Buijze M, et al. Loss of heterozygosity at9p andp53immunopositivity in surgical margins predict local relapse in head and necksquamous cell carcinoma. Int J Cancer.2011Apr15;128(8):1852-9.
36. Ma C, Quesnelle KM, Sparano A, Rao S, et al. Characterization CSMD1in alarge set of primary lung, head and neck, breast and skin cancer tissues. CancerBiol Ther.2009May；8(10)：907-16.
37. Ribeiro IP, Marques F, Caramelo F, et al.Genetic imbalances detected bymultiplex ligation-dependent probe amplification in a cohort of patients with oralsquamous cell carcinoma-the first step towards clinical personalized medicine.Tumour Biol.2014Jan31.[Epub ahead of print]
38. Urashima M, Hama T, Suda T, et al.Distinct effects of alcohol consumption andsmoking on genetic alterations in head and neck carcinoma.PLoS One.2013Nov20;8(11):e80828.
39. Scholnick SB, Richter TM.The role of CSMD1in head and neck carcinogenesis.Genes Chromosomes Cancer.2003Nov;38(3):281-3.
40. Takebayashi S, Ogawa T, Jung KY, et al. Identification of new minimally lostregions on18q in head and neck squamous cell carcinoma. Cancer Res.2000Jul1；60(13)：3397-403.
41. Leong PP, Rezai B, Koch WM, et al.Distinguish second primary tumors fromlung metastases in patients with head and neck squamous cell carcinola. J NatlCancer Inst,1998,90:972-977.
42. Scholes AG, Woolgar JA, Boyle MA, et al.Synchronous oral carcinomas:independent or common clonal origin? Cancer Res.1998May1;58(9):2003-6.
43. van Houten, V. M. M., Tabor, M. P., van den Brekel, M. W. M., Denkers, F.,Wishaupt, R. G. A., Kummer, J. A., Snow G. B., and Brakenhoff, R. H. Molecularassays for the diagnosis of minimal residual head and neck cancer: methods,reliability, pitfalls, and solutions. Clin. Cancer Res.,6:3803–3816,2000.
44. Brennan JA, Mao L, Hruban RH, et al.Molecular assessment of histopathologicalstaging in squamous-cell carcinoma of the head and neck.N Engl J Med.1995Feb16;332(7):429-35.
45. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
46. Yamamoto N,Mizoe JE,Numasawa H,et a1．Allelic loss of chmmosome2inhuman oral squamous cell carcinoma：correlation with lymphnode metastasis.0ral0ncology,2003,39(1)：64-68．
1.刘天润，杨安奎，陈福进，等。221例晚期喉鳞癌患者术后的生存和预后分析。癌症，2009，28(3)：297-302.
2. Raitiola H, Pukander J, Laippala P. Glottic and supraglottic laryngeal carcinoma:differences in epidemiology, clinical characteristics and prognosis. ActaOtolaryngol.1999;119(7):847-51.
3.孙越峰，杨蓓蓓．喉癌手术切缘的研究进展．临床耳鼻咽喉科杂志200l，15(9)428．430
4.李为民，郭睿，郭志祥．喉癌手术切缘的组织病理学研究．临床耳鼻咽喉科杂志，1998，12(11)：496—498
5. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
6. Worsham, M. Wolman, S. R., Carey, T. et al Common clonal origin ofsynchronous primary head and neck squamous cell carcinomas: analysis bytumorkaryotypes and fluorescence in situ hybridization. Hum. Pathol.,26:251–261,1995.
7. Califano, Leong, P. L., Koch, W. M., et al. Second esophageal tumors in patientswith head and neck squamous cell carcinoma: an assessment of clonalrelationships. Clin. Cancer Res.,5:1862–1867,1999.
8. Dakubo,G. D.,Jakupciak， J. P.， Birch-Machin， M. A.&Parr， R. L. Clinicalimplications and utility of field cancerization. Cancer Cell. Int.7,2(2007).
9. Califano. et al. Genetic progression model for head and neck cancer：implications for field cancerization. Cancer Res.56,2488–2492(1996).
10. Tabor,M. P. et al. Persistence of genetically altered fields in head and neck cancerpatients： Biological and clinical implications. Clin. Cancer Res.7，1523–1532(2001).
11. Tabor,M. P. et al. Comparative molecular and histological grading of epithelialdysplasia of the oral cavity and the oropharynx. J. Pathol.199，354–360(2003).
12. Hittelman,W. N. Genetic instability in epithelial tissues at risk for cancer. Ann.NY Acad. Sci.952，1–12(2001).
13. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
14. Slaughter, D. P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
15. Mao L, Lee JS, Fan YH, Ro JY, Batsakis JG, Lippman S, Hittelman W, Hong WK.Frequent microsatellite alterations at chromosomes9p21and3p14in oralpremalignant lesions and their value in cancer risk assessment. Nat Med.1996Jun；2(6)：682-5.
16. Ghosh S, Ghosh A, Maiti GP, Alam N, Roy A, Roychoudhury S, PandaCK.Alterations of ROBO1/DUTT1and ROBO2loci in early dysplastic lesionsof head and neck： clinical and prognostic implications.Hum Genet.2009Mar；125(2)：189-98.
17. Abou-Elhamd KE, Habib TN, Moussa AE, et al. The role of genetic susceptibilityin head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol.2008Feb；265(2)：217-22.
18. Chang SS, Califano J. Current status of biomarkers in head and neck cancer. JSurg Oncol.2008Jun15；97(8)：640-3.
19. Yin D T,Dong M M．Micresatellite instability and loss of heterozygosity offragile histidine triad gene in laryngesl squamous cell carcinomas.Zhanghua ErBi Yan Hou Tan Jing Wai Ke Za Zhi,2005.40(1)：4548．
20. GuoT，Sun JW，Lv QP，Li XG. Allelicic imbalance on chromosomes3p，9p and17p in malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
21. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
22. Califano J, Westra WH, Meininger G,et al.Genetic progression and clonalrelationship of recurrent premalignant head and neck lesions. Clin Cancer Res.2000Feb;6(2):347-52.
23. Worsham MJ, Chen KM, Tiwari N, et al. Fine-mapping loss of gene architectureat the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genesin head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.2006Apr；132(4)：409-15.
24. Chakraborty SB, Dasgupta S, Roy A, Sengupta A,et al..Differential deletions in3p are associated with the development of head and neck squamous CancerGenet Cytogenet.2003Oct15;146(2):130-8.
25. Jang SJ, Chiba I, Hirai A, et al.Multiple oral squamous epithelial lesions： arethey genetically related? Oncogene.2001Apr26；20(18)：2235-42.
26. Rosin M, Splieth C, Wilkens M, Meyer G..Effect of cement type on retention of atapered post with a self-cutting double thread. J Dent.2000Nov;28(8):577-82.
27. Pindborg, J. J., Daftary, D. K., and Mehta, F. S. A follow-up studyof sixty-oneoral dysplastic precancerous lesions in Indian villagers.Oral Surg. Oral Med. O.,43:383–390,1977.
1. Kamangar, F., Dores, G. M.&Anderson, W. F. Patterns of cancer incidence,mortality, and prevalence across five continents： defining priorities to reducecancer disparities in different geographic regions of the world. J. Clin. Oncol.24,2137–2150(2006).
2. Kutler, D. I. et al. High incidence of head and neck squamous cell carcinoma inpatients with fanconi anemia. Arch. Otolaryngol. Head Neck Surg.129,106–112(2003).
3. Hopkins, J. et al. Genetic polymorphisms and head and neck cancer outcomes： areview. Cancer Epidemiol. Biomark. Prev.17,490–499(2008).
4. Cloos, J. et al. Genetic susceptibility to head and neck squamous cell carcinoma.J. Natl Cancer Inst.88,530–535(1996).
5. Ang, K. K. et al. Human papillomavirus and survival of patients withoropharyngeal cancer. N. Engl. J. Med.363,24–35(2010).
6. Smith, R. B., Sniezek, J. C., Weed, D. T.&Wax, M. K. Utilization of free tissuetransfer in head and neck surgery. Otolaryngol. Head Neck Surg.137,182–191(2007).
7. Vergeer, M. R. et al. Intensity-modulated radiotherapy reduces radiation-inducedmorbidity and improves health-related quality of life： results of anonrandomized prospective study using a standardized follow-up program. Int. J.Radiat. Oncol. Biol. Phys.74,1–8(2009).
8. Boscolo-Rizzo, P., Maronato, F., Marchiori, C., Gava, A.&Da Mosto, M. C.Long-term quality of life after total laryngectomy and postoperative radiotherapyversus concurrent chemoradiotherapy for laryngeal preservation. Laryngoscope118,300–306(2008).
9. Hanahan, D.&Weinberg, R. A. The hallmarks of cancer. Cell100,57–70(2000).
10. Woolgar, J. A.&Triantafyllou, A. Pitfalls and procedures in the histopathologicaldiagnosis of oral and oropharyngeal squamous cell carcinoma and a review of therole of pathology in prognosis. Oral Oncol.45,361–385(2009).
11. Chung, C. H. et al. Molecular classification of head and neck squamous cellcarcinomas using patterns of gene expression. Cancer Cell5,489–500(2004).
12. Chung, C. H. et al. Gene expression profiles identify epithelial-to-mesenchymaltransition and activation of nuclear factor-kB signalling as characteristic of a highrisk squamous cell carcinoma. Cancer Res.66,8210–8218(2006).
13. Hermsen, M. et al. New chromosomal regions with highlevel amplifications insquamous cell carcinomas of the larynx and pharynx, identified by comparativegenomic hybridization. J. Pathol.194,177–182(2001).
14. Jin, C. et al. Cytogenetic abnormalities in106oral squamous cell carcinomas.Cancer Genet. Cytogenet.164,44–53(2006).
15. Smeets, S. J. et al. Genetic classification of oral and oropharyngeal carcinomasidentifies subgroups with a different prognosis. Cell. Oncol.31,291–300(2009).
16. Walboomers, J. M. et al. Human papillomavirus is a necessary cause of invasivecervical cancer worldwide. J. Pathol.189,12–19(1999).
17. zur Hausen, H. Papillomaviruses and cancer： from basic studies to clinicalapplication. Nature Rev. Cancer2,342–350(2002).
18. Munoz, N. et al. Epidemiologic classification of human papillomavirus typesassociated with cervical cancer. N. Engl. J. Med.348,518–527(2003).
19. Syrjanen, S. Human papillomavirus (HPV) in head and neck cancer. J. Clin. Virol.32, S59–S66(2005).
20. Snijders, P. J. F. et al. Prevalence and expression of human papillomavirus intonsillar carcinomas, indicating a possible viral etiology. Int. J. Cancer51,845–850(1992).
21. Gillison, M. L. et al. Evidence for a causal association between humanpapillomavirus and a subset of head and neck cancers. J. Natl Cancer Inst.92,709–720(2000).
22. Meijer, C. J. et al. Guidelines for human papillomavirus DNA test requirementsfor primary cervical cancer screening in women30years and older. Int. J. Cancer124,516–520(2009).
23. Snijders, P. J., van den Brule, A. J.&Meijer, C. J. The clinical relevance ofhuman papillomavirus testing： relationship between analytical and clinicalsensitivity. J. Pathol.201,1–6(2003).
24. Van Houten, V. M. M. et al. Biological evidence that human papillomaviruses areetiologically involved in a subgroup of head and neck squamous cell carcinomas.Int. J. Cancer93,232–235(2001).
25. Wiest, T., Schwarz, E., Enders, C., Flechtenmacher, C.&Bosch, F. X.Involvement of intact HPV16E6/E7gene expression in head and neck cancerswith unaltered p53status and perturbed pRb cell cycle control. Oncogene21,1510–1517(2002).
26. Smeets, S. J. et al. Genome-wide DNA copy number alterations in head and necksquamous cell carcinomas with or without oncogene-expressing humanpapillomavirus. Oncogene25,2558–2564(2006).
27. Braakhuis, B. J. M. et al. Genetic patterns in head and neck cancers that containor lack transcriptionally active human papillomavirus. J. Natl Cancer Inst.96,998–1006(2004).
28. Slebos, R. J. C. et al. Gene expression differences associated with humanpapillomavirus status in head and neck squamous cell carcinoma. Clin. CancerRes.12,701–709(2006).
29. Smeets, S. J. et al. A novel algorithm for reliable detection of humanpapillomavirus in paraffin embedded head and neck cancer specimen. Int. J.Cancer121,2465–2472(2007).
30. Robinson, M., Sloan, P.&Shaw, R. Refining the diagnosis of oropharyngealsquamous cell carcinoma using human papillomavirus testing. Oral Oncol.46,492–496(2010).
31. D’Souza, G. et al. Case-control study of human papillomavirus andoropharyngeal cancer. N. Engl. J. Med.356,1944–1956(2007).
32. Ragin, C. C. R.&Taioli, E. Survival of squamous cell carcinoma of the head andneck in relation to human papillomavirus infection： review and meta-analysis.Int. J. Cancer121,1813–1820(2007).
33. Poeta, M. L. et al. Tp53mutations and survival in squamous-cell carcinoma ofthe head and neck. N. Engl. J. Med.357,2552–2561(2007).
34. Westra, W. H. et al. Inverse relationship between human papillomavirus-16infection and disruptive p53gene mutations in squamous cell carcinoma of thehead and neck. Clin. Cancer Res.14,366–369(2008).
35. Napier, S. S.&Speight, P. M. Natural history of potentially malignant orallesions and conditions： an overview of the literature. J. Oral Pathol. Med.37,1–10(2008).
36. van der Waal, I. Potentially malignant disorders of the oral and oropharyngealmucosa； terminology, classification and present concepts of management. OralOncol.45,317–323(2009).
37. Lodi, G., Sardella, A., Bez, C., Demarosi, F.&Carrassi, A. Interventions fortreating oral leukoplakia. Cochrane Database Syst. Rev. CD001829(2006).
38. Partridge, M. et al. A case-control study confirms that microsatellite assay canidentify patients at risk of developing oral squamous cell carcinoma within a fieldof cancerization. Cancer Res.60,3893–3898(2000).
39. Wrangle, J. M.&Khuri, F. R. Chemoprevention of squamous cell carcinoma ofthe head and neck. Curr. Opinion Oncol.19,180–187(2007).
40. Schaaij-Visser, T. B. M. et al. Evaluation of cornulin, keratin4, keratin13expression and grade of dysplasia for predicting malignant progression of oralleukoplakia. Oral Oncol.46,123–127(2010).
41. Rosin, M. P. et al. Use of allelic loss to predict malignant risk for low-grade oralepithelial dysplasia. Clin. Cancer Res.6,357–362(2000).
42. Mao, L. et al. Frequent microsatellite alterations at chromosomes9p21and3p14in oral premalignant lesions and their value in cancer risk assessment. NatureMed.2,682–685(1996).
43. Torres-Rendon, A., Stewart, R., Craig, G. T., Wells, M.&Speight, P. M. DNAploidy analysis by image cytometry helps to identify oral epithelial dysplasiaswith a high risk of malignant progression. Oral Oncol.45,468–473(2009).
44. Shpitzer, T. et al. Salivary analysis of oral cancer biomarkers. Brit. J. Cancer101,1194–1198(2009).
45. Bremmer, J. F. et al. A noninvasive genetic screening test to detect oralpreneoplastic lesions. Lab. Invest.85,1481–1488(2005).
46. Bremmer, J. F. et al. Screening for oral precancer with noninvasive geneticcytology. Cancer Prev. Res.2,128–133(2009).
47. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
48. Califano, J. et al. Genetic progression model for head and neck cancer：implications for field cancerization. Cancer Res.56,2488–2492(1996).
49. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
50. Tabor, M. P. et al. Comparative molecular and histological grading of epithelialdysplasia of the oral cavity and the oropharynx. J. Pathol.199,354–360(2003).
51. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
52. Slaughter, D. P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
53. Hittelman, W. N. Genetic instability in epithelial tissues at risk for cancer. Ann.NY Acad. Sci.952,1–12(2001).
54. Tabor, M. P. et al. Genetically altered fields as origin of locally recurrent headand neck cancer： a retrospective study. Clin. Cancer Res.10,3607–3613(2004).
55. Roesch-Ely, M. et al. Proteomic analysis reveals successive aberrations in proteinexpression from healthy mucosa to invasive head and neck cancer. Oncogene26,54–64(2007).
56. Schaaij-Visser, T. B. M. et al. Differential proteomics identifies proteinbiomarkers that predict local relapse of head and neck squamous cell carcinomas.Clin. Cancer Res.15,7666–7675(2009).
57. Braakhuis, B. J. M., Tabor, M. P., Kummer, J. A., Leemans, C. R.&Brakenhoff,R. H. A genetic explanation of Slaughter’s concept of field cancerization：evidence and clinical implications. Cancer Res.63,1727–1730(2003).
58. van Houten, V. M. et al. Mutated p53as a molecular marker for the diagnosis ofhead and neck cancer. J. Pathol.198,476–486(2002).
59. Jonason, A. S. et al. Frequent clones of p53-mutated keratinocytes in normalhuman skin. Proc. Natl Acad. Sci. USA93,14025–14029(1996).
60. Dakubo, G. D., Jakupciak, J. P., Birch-Machin, M. A.&Parr, R. L. Clinicalimplications and utility of field cancerization. Cancer Cell. Int.7,2(2007).
61. Negrini, S., Gorgoulis, V. G.&Halazonetis, T. D. Genomic instability—anevolving hallmark of cancer. Nature Rev. Mol. Cell Biol.11,220–228(2010).
62. Patmore, H. S., Cawkwell, L., Stafford, N. D.&Greenman, J. Unraveling thechromosomal aberrations of head and neck squamous cell carcinoma： a review.Ann. Surg. Oncol.12,831–842(2005).
63. Wreesmann, V. B.&Singh, B. Chromosomal aberrations in squamous cellcarcinomas of the upper aerodigestive tract： biologic insights and clinicalopportunities. J. Oral Pathol. Med.34,449–459(2005).
64. Ha, P. K., Chang, S. S., Glazer, C. A., Califano, J. A.&Sidransky, D. Moleculartechniques and genetic alterations in head and neck cancer. Oral Oncol.45,335–339(2009).
65. Carbone, M., Klein, G., Gruber, J.&Wong, M. Modern criteria to establishhuman cancer etiology. Cancer Res.64,5518–5524(2004).
66. Kastan, M. B.&Bartek, J. Cell-cycle checkpoints and cancer. Nature432,316–323(2004).
67. Balz, V. et al. Is the p53inactivation frequency in squamous cell carcinomas ofthe head and neck underestimated? Analysis of p53exons2–11and humanpapillomavirus16/18E6transcripts in123unselected tumor specimens. CancerRes.63,1188–1191(2003).
68. Opitz, O. G. et al. Cyclin D1overexpression and p53inactivation immortalizeprimary oral keratinocytes by a telomerase-independent mechanism. J. Clin.Invest.108,725–732(2001).
69. Rheinwald, J. G. et al. A two-stage, p16INK4A-and p53-dependent keratinocytesenescence mechanism that limits replicative potential independent of telomerestatus. Mol. Cell. Biol.22,5157–5172(2002).
70. Smeets, S. J. et al. Immortalization of oral keratinocytes by functionalinactivation of the p53and pRb pathways. Int. J. Cancer (in the press).
71. Reed, A. L. et al. High frequency of p16(CDKN2/MTS-1/INK4A) inactivationin head and neck squamous cell carcinoma. Cancer Res.56,3630–3633(1996).
72. Gibcus, J. H. et al. Amplicon mapping and expression profiling identify theFas-associated death domain gene as a new driver in the11q13.3amplicon inlaryngeal/pharyngeal cancer. Clin. Cancer Res.13,6257–6266(2007).
73. Berns, K. et al. A large-scale RNAi screen in human cells identifies newcomponents of the p53pathway. Nature428,431–437(2004).
74. Dickson, M. A. et al. Human keratinocytes that express hTERT and also bypass ap16INK4A-enforced mechanism that limits life span become immortal yet retainnormal growth and differentiation characteristics. Mol. Cell. Biol.20,1436–1447(2000).
75. Snijders, P. J. F. et al. Telomerase activity exclusively in cervical carcinomas anda subset of cervical intraepithelial neoplasia grade III lesions： strong associationwith elevated messenger RNA levels of its catalytic subunit and high-risk humanpapillomavirus DNA. Cancer Res.58,3812–3818(1998).
76. Hynes, N. E.&Lane, H. A. ERBB receptors and cancer： the complexity oftargeted inhibitors. Nature Rev. Cancer5,341–354(2005).
77. Lin, S. Y. et al. Nuclear localization of EGF receptor and its potential new role asa transcription factor. Nature Cell Biol.3,802–808(2001).
78. Lo, H. W. et al. Nuclear interaction of EGFR and STAT3in the activation of theiNOS/NO pathway. Cancer Cell7,575–589(2005).
79. Goessel, G. et al. Creating oral squamous cancer cells： a cellular model oforal–esophageal carcinogenesis. Proc. Natl Acad. Sci. USA102,15599–15604(2005).
80. Ozanne, B., Richards, C. S., Hendler, F., Burns, D.&Gusterson, B.Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas.J. Pathol.149,9–14(1986).
81. Grandis, J. R.&Tweardy, D. J. Elevated levels of transforming growth factor αand epidermal growth factor receptor messenger RNA are early markers ofcarcinogenesis in head and neck cancer. Cancer Res.53,3579–3584(1993).
82. Hama, T. et al. Prognostic significance of epidermal growth factor receptorphosphorylation and mutation in head and neck squamous cell carcinoma.Oncologist14,900–908(2009).
83. Bonner, J. A. et al. Radiotherapy plus cetuximab for squamous-cell carcinoma ofthe head and neck. N. Engl. J. Med.354,567–578(2006).
84. Morandell, S. et al. Phosphoproteomics strategies for the functional analysis ofsignal transduction. Proteomics6,4047–4056(2006).
85. Lee, J. W. et al. Somatic mutations of EGFR gene in squamous cell carcinoma ofthe head and neck. Clin. Cancer Res.11,2879–2882(2005).
86. Loeffler-Ragg, J. et al. Low incidence of mutations in EGFR kinase domain inCaucasian patients with head and neck squamous cell carcinoma. Eur. J. Cancer42,109–111(2006).
87. Ekstrand, A. J., Sugawa, N., James, C. D.&Collins, V. P. Amplified andrearranged epidermal growth factor receptor genes in human glioblastomas revealdeletions of sequences encoding portions of the Nand/or C-terminal tails. Proc.Natl Acad. Sci. USA89,4309–4313(1992).
88. Sok, J. C. et al. Mutant epidermal growth factor receptor (EGFRvIII) contributesto head and neck cancer growth and resistance to EGFR targeting. Clin. CancerRes.12,5064–5073(2006).
89. Ishitoya, J. et al. Gene amplification and overexpression of EGF receptor insquamous cell carcinomas of the head and neck. Brit. J. Cancer59,559–562(1989).
90. Temam, S. et al. Epidermal growth factor receptor copy number alterationscorrelate with poor clinical outcome in patients with head and neck squamouscancer. J. Clin. Oncol.25,2164–2170(2007).
91. Sheu, J. J. C. et al. Functional genomic analysis identified epidermal growthfactor receptor activation as the most common genetic event in oral squamouscell carcinoma. Cancer Res.69,2568–2576(2009).
92. Knudsen, B. S.&Vande Woude, G. Showering c-MET-dependent cancers withdrugs. Curr. Opin. Genet. Dev.18,87–96(2008).
93. Seiwert, T. Y. et al. The MET receptor tyrosine kinase is a potential noveltherapeutic target for head and neck squamous cell carcinoma. Cancer Res.69,3021–3031(2009).
94. Knowles, L. M. et al. HGF and c-Met participate in paracrine tumorigenicpathways in head and neck squamous cell cancer. Clin. Cancer Res.15,3740–3750(2009).
95. Wang, D. et al. Mutation and downregulation of the transforming growth factorbeta type II receptor gene in primary squamous cell carcinomas of the head andneck. Carcinogenesis18,2285–2290(1997).
96. Huntley, S. P. et al. Attenuated type II TGF-B receptor signalling in humanmalignant oral keratinocytes induces a less differentiated and more aggressivephenotype that is associated with metastatic dissemination. Int. J. Cancer110,170–176(2004).
97. Qiu, W., Schonleben, F., Li, X.&Su, G. H. Disruption of transforming growthfactor β–Smad signaling pathway in head and neck squamous cell carcinoma asevidenced by mutations of SMAD2and SMAD4. Cancer Lett.245,163–170(2007).
98. Bornstein, S. et al. Smad4loss in mice causes spontaneous head and neck cancerwith increased genomic instability and inflammation. J. Clin. Invest.119,3408–3419(2009).
99. Mishra, A., Bharti, A. C., Varghese, P., Saluja, D.&Das, B. C. Differentialexpression and activation of NF-κB family proteins during oral carcinogenesis：role of high risk human papillomavirus infection. Int. J. Cancer119,2840–2850(2006).
100.Karin, M. Nuclear factor-κB in cancer development and progression. Nature441,431–436(2006).
101.Perkins, N. D. Integrating cell-signalling pathways with NF-κB and IKK function.Nature Rev. Mol. Cell Biol.8,49–62(2007).
102.Cohen, J. et al. Attenuated transforming growth factor β signaling promotesnuclear factor-κB activation in head and neck cancer. Cancer Res.69,3415–3424(2009).
103.Engelman, J. A. Targeting PI3K signalling in cancer： opportunities, challengesand limitations. Nature Rev. Cancer9,550–562(2009).
104.Kozaki, K. et al. PIK3CA mutation is an oncogenic aberration at advanced stagesof oral squamous cell carcinoma. Cancer Sci.97,1351–1358(2006).
105.Qiu, W. L. et al. PIK3CA mutations in head and neck squamous cell carcinoma.Clin. Cancer Res.12,1441–1446(2006).
106.Murugan, A. K., Hong, N. T., Fukui, Y., Munirajan, A. K.&Tsuchida, N.Oncogenic mutations of the PIK3CA gene in head and neck squamous cellcarcinomas. Int. J. Oncol.32,101–111(2008).
107.Okami, K. et al. Analysis of PTEN/MMAC1alterations in aerodigestive tracttumors. Cancer Res.58,509–511(1998).
108.Redon, R. et al. A simple specific pattern of chromosomal aberrations at earlystages of head and neck squamous cell carcinomas：PIK3CA but not p63gene asa likely target of3q26-qter gains. Cancer Res.61,4122–4129(2001).
109.Woenckhaus, J. et al. Genomic gain of PIK3CA and increased expression ofp110alpha are associated with progression of dysplasia into invasive squamouscell carcinoma. J. Pathol.198,335–342(2002).
110.Pantel, K.&Brakenhoff, R. H. Dissecting the metastatic cascade. Nature Rev.Cancer4,448–456(2004).
111.Rosenthal, E. L.&Matrisian, L. M. Matrix metalloproteases in head and neckcancer. Head Neck28,639–648(2006).
112.Sun, P. C. et al. Transcript map of the8p23putative tumor suppressor region.Genomics75,17–25(2001).
113.Scholnick, S. B. et al. Chromosome8allelic loss and the outcome of patientswith squamous cell carcinoma of the supraglottic larynx. J. Natl Cancer Inst.88,1676–1682(1996).
114.Sunwoo, J. B. et al. Localization of a putative tumor suppressor gene in thesub-telomeric region of chromosome8p. Oncogene18,2651–2655(1999).
115.Kraus, D. M. et al. CSMD1is a novel multiple domain complement-regulatoryprotein highly expressed in the central nervous system and epithelial tissues. J.Immunol.176,4419–4430(2006).
116.Roepman, P. et al. An expression profile for diagnosis of lymph node metastasesfrom primary head and neck squamous cell carcinomas. Nature Genet.37,182–186(2005).
117.Thiery, J. P. Epithelial–mesenchymal transitions in tumour progression. NatureRev. Cancer2,442–454(2002).
118.Ikushima, H.&Miyazono, K. TGFβ signalling： a complex web in cancerprogression. Nature Rev. Cancer10,415–424(2010).
119.Folkman, J. Role of angiogenesis in tumor growth and metastasis. Semin. Oncol.29,15–18(2002).
120.Kerbel, R. S. Tumor angiogenesis. N. Engl. J. Med.358,2039–2049(2008).
121.Kyzas, P. A., Cunha, I. W.&Ioannidis, J. P. A. Prognostic significance ofvascular endothelial growth factor immunohistochemical expression in head andneck squamous cell carcinoma： a meta-analysis. Clin. Cancer Res.11,1434–1440(2005).
122.Fei, J. et al. Prognostic significance of vascular endothelial growth factor insquamous cell carcinomas of the tonsil in relation to human papillomavirus statusand epidermal growth factor receptor. Ann. Surg. Oncol.16,2908–2917(2009).
123.Virgilio, L. et al. FHIT gene alterations in head and neck squamous cellcarcinomas. Proc. Natl Acad. Sci. USA93,9770–9775(1996).
124.Hibi, K. et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc.Natl Acad. Sci. USA97,5462–5467(2000).
125.Rocco, J. W., Leong, C. O., Kuperwasser, N., DeYoung, M. P.&Ellisen, L. W.p63mediates survival in squamous cell carcinoma by suppression ofp73-dependent apoptosis. Cancer Cell9,45–56(2006).
126.Rodrigo, J. P., Lazo, P. S., Ramos, S., Alvarez, I.&Suarez, C. MYCamplification in squamous cell carcinomas of the head and neck. Arch.Otolaryngol. Head Neck Surg.122,504–507(1996).
127.Carvalho, A. L. et al. Deleted in colorectal cancer is a putative conditionaltumor-suppressor gene inactivated by promoter hypermethylation in head andneck squamous cell carcinoma. Cancer Res.66,9401–9407(2006).
128.Avissar, M., Christensen, B. C., Kelsey, K. T.&Marsit, C. J. MicroRNAexpression ratio is predictive of head and neck squamous cell carcinoma. Clin.Cancer Res.15,2850–2855(2009).
129.Childs, G. et al. Low-level expression of microRNAs let-7d and miR-205areprognostic markers of head and neck squamous cell carcinoma. Am. J. Pathol.174,736–745(2009).
130.Cervigne, N. K. et al. Identification of a microRNA signature associated withprogression of leukoplakia to oral carcinoma. Hum. Mol. Genet.18,4818–4829(2009).
131.Kies, M. S. et al. Induction chemotherapy and cetuximab for locally advancedsquamous cell carcinoma of the head and neck： results from a phase IIprospective trial. J. Clin. Oncol.28,8–14(2010).
2. Kamangar F,Dores G. M, Anderson W. F. Patterns of cancer incidence,mortality,and prevalence across five continents：defining priorities to reducecancer disparities in different geographic regions of the world. J. Clin. Oncol.2006,24,2137–2150.
3. Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, et al. Genetically altered fieldsas origin of locally recurrent head and neck cancer：a retrospective study. Clin.Cancer Res.2004,10,3607–3613.
4. Roesch-Ely M, Nees M, Karsai S, et al. Proteomic analysis reveals successiveaberrations in protein expression from healthy mucosa to invasive head and neckcancer. Oncogene.2007,26,54–64.
5. Schaaij-Visser TB, Graveland A, Gauci S, et al. Differential proteomics identifiesprotein biomarkers that predict local relapse of head and neck squamous cellcarcinomas. Clin. Cancer Res.2009,15,7666–7675.
6. Kutler DI, Auerbach AD, Satagopan J. et al. High incidence of head and necksquamous cell carcinoma in patients with fanconi anemia. Arch. Otolaryngol.Head Neck Surg.2003,129,106–112.
7. Hopkins J, Cescon DW, Tse D, et al. Genetic polymorphisms and head and neckcancer outcomes： a review. Cancer Epidemiol. Biomark. Prev.2008,17,490–499.
8. Cloos J, Spitz MR, Schantz SP, et al. Genetic susceptibility to head and necksquamous cell carcinoma. J. Natl Cancer Inst.1996,88,530–535.
9. Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival ofpatients with oropharyngeal cancer. N. Engl. J. Med.2010,363,24–35.
10. Smith B, Sniezek C, Weed T, et al. Utilization of free tissue transfer in head andneck surgery. Otolaryngol. Head Neck Surg.2007,137,182–191.
11. Vergeer R, Doornaert A, Rietveld H, et al. Intensity-modulated radiotherapyreduces radiation-induced morbidity and improves health-related quality of life：results of a nonrandomized prospective study using a standardized follow-upprogram. Int. J. Radiat. Oncol. Biol. Phys.2009,74,1–8.
12. Boscolo-Rizzo P, Maronato F, Marchiori C, et al. Long-term quality of life aftertotal laryngectomy and postoperative radiotherapy versus concurrentchemoradiotherapy for laryngeal preservation. Laryngoscope.2008,118,300–306.
13. Slaughter P, Southwick W, Smejkal W, et al. Field cancerization in oral stratifiedsquamous epithelium； clinical implications of multicentric origin. Cancer.1953,6,963–968.
14. Braakhuis BJ, Tabor MP, Kummer JA,et al. A genetic explanation of Slaughter'sconcept of field cancerization: evidence and clinical implications. Cancer Res.2003,15；63(8)：1727-30.
15. Califano J, van P, Westra W, et al. Genetic progression model for head and neckcancer： implications for field cancerization. Cancer Res.1996,56,2488–2492.
16. van M, Tabor P, van W. et al. Mutated p53as a molecular marker for thediagnosis of head and neck cancer. J. Pathol.2002,198,476–486.
17. Jonason S, Kunala S, Price J. et al. Frequent clones of p53-mutated keratinocytesin normal human skin. Proc. Natl Acad. Sci. USA.1996.93,14025–14029.
18. Tabor P, Brakenhoff H, van M, et al. Persistence of genetically altered fields inhead and neck cancer patients：Biological and clinical implications. Clin. CancerRes.2001.7,1523–1532.
19. Tabor P, Braakhuis J, van JE, et al. Comparative molecular and histologicalgrading of epithelial dysplasia of the oral cavity and the oropharynx. J. Pathol.2003,199,354–360.
20. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
21. Slaughter P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
22. Hittelman N. Genetic instability in epithelial tissues at risk for cancer. Ann. NYAcad. Sci.2001,952,1–12.
23. Mao L,Lee J,Tockman S,et al．Microsatellite alterations as clonal markers for thedetection of human cancer. Proc Natl Acad Sci USA.1994.91(21)9871-5．
24. Mao L, Lee S, Fan H, et al. Frequent microsatellite alterations at chromosomes9p21and3p14in oral premalignant lesions and their value in cancer riskassessment. Nature Med.1996,2,682–685.
25. Abou E,Habib N,Moussa E,et al．The role of genetic susceptibility in head andneck squamous cell carcinoma． Eur Arch Otorhinolaryngol.2008.265(2)：217-222．
26. Pearlstein RP, Benninger MS, Carey TE, Zarbo RJ, Torres FX, Rybicki BA, DykeDL. Loss of18q predicts poor survival of patients with squamous cell carcinomaof the head and neck.. Genes Chromosomes Cancer.1998,21(4),333-339.
27. GuoT,Sun JW,Lv QP,Li XG. Allelicic imbalance on chromosomes3p,9p and17pin malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
28. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity incritical genome regions predict a local relapse in patients afterlaryngeetomy.Mutat Res.2006.600(1-2)：67-76．
29. Tabor P,Brakenhoff H, van Houten M, et al. Persistence of GeneticallyAltered Fields in Head and Neck Cancer Patients： Biological and ClinicalImplications Clin Cancer Res.2001,7(6)：1523-1532.
30. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head andneck cancer. Nat Rev Cancer.2011,11(1)：9-22.
1. Marx J.New colon cancer gene discovered. Science.1993May7;260(5109):751-2.
2. Sharma A, Wayne S, Nikiforova MN, Johnson JT, Walvekar RR.Two sites ofhead and neck squamous cell carcinoma: utility of loss of heterozygosity. AnnOtol Rhinol Laryngol.2008Aug;117(8):591-3.
3. Califano, J. et al. Genetic progression model for head and neckcancer:implications for field cancerization. Cancer Res.56,2488–2492(1996).
4. Yin D T,Dong M M．Micresatellite instability and loss of heterozygosity offragile histidine triad gene in laryngesl squamous cell carcinomas.Zhanghua ErBi Yan Hou Tan Jing Wai Ke Za Zhi,2005.40(1)：4548．
5. Califano J, Westra WH, Meininger G,et al.Genetic progression and clonalrelationship of recurrent premalignant head and neck lesions. Clin Cancer Res.2000Feb;6(2):347-52.
6. GuoT,Sun JW,Lv QP,Li XG. Allelicic imbalance on chromosomes3p,9p and17pin malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
7. Vander R P,Nawroz H,Hruban RH,et al. Fequent loss of chromosome9p21-22early in head and neck cancer progression.Cancer Res,1994,54(5):1156-1158.
8. Rosin M, Splieth C, Wilkens M, Meyer G..Effect of cement type on retention of atapered post with a self-cutting double thread. J Dent.2000Nov;28(8):577-82.
9. Chakraborty SB, Dasgupta S, Roy A, Sengupta A,et al..Differential deletions in3p are associated with the development of head and neck squamous CancerGenet Cytogenet.2003Oct15;146(2):130-8.
10. Ghosh S, Ghosh A, Maiti GP, Alam N, Roy A, Roychoudhury S, PandaCK.Alterations of ROBO1/DUTT1and ROBO2loci in early dysplastic lesionsof head and neck： clinical and prognostic implications.Hum Genet.2009Mar；125(2)：189-98.
11. Hogg RP, Honorio S, Martinez A, et al. Frequent3p allele loss and epigeneticinactivation of the RASSF1A tumour suppressor gene from region3p21.3in headand neck squamous cell carcinoma. Eur J Cancer.2002Aug；38(12)：1585-92.
12. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
13. Abou-Elhamd KE, Habib TN, Moussa AE, et al. The role of genetic susceptibilityin head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol.2008Feb；265(2)：217-22.
14. Koscielny S, V Eggeling F, Dahse R.Investigations to the influence of tumorsupressor gene p16inactivation on the prognosis of head and neck squamous cellcarcinoma].Laryngorhinootologie.2004Jun;83(6):374-80.
15. Smigiel R, Sasiadek M, Krecicki T,et al. Inactivation of the cyclin-dependentkinase inhibitor2A (CDKN2A) gene in squamous cell carcinoma of the larynx.Mol Carcinog.2004Mar;39(3):147-54.
16. Sun PC, Uppaluri R, Schmidt AP,et al.Transcript map of the8p23putative tumorsuppressor region. Genomics.2001Jul;75(1-3):17-25.
17. Papadimitrakopoulou VA, Oh Y, El-Naggar A, Izzo J, Clayman G, MaoL.Presence of multiple incontiguous deleted regions at the long arm ofchromosome18in head and neck cancer. Clin Cancer Res.1998Mar；4(3)：539-44.
18. Kim SK, Fan Y, Papadimitrakopoulou V, Clayman Get al. DPC4, a candidatetumor suppressor gene, is altered infrequently in head and neck squamous cellcarcinoma. Cancer Res.1996Jun1；56(11)：2519-21.
19. G tte K, Riedel F, Neubauer J, et al. The relationship between allelic imbalanceon17p, p53mutation and p53overexpression in head and neck cancer. Int JOncol.2001Aug;19(2):331-6.
20. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
21. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
22. Yuge T, Nibu K, Kondo K, et al. Loss of FHIT expression in squamous cellcarcinoma and premalignant lesions of the larynx. Ann Otol Rhinol Laryngol.2005Feb;114(2):127-31.
23. Fu W,,Wu D, Zhang X.FHIT gene is abnormal in laryngeal carcinoma. ZhonghuaZhong Liu Za Zhi.1999Nov;21(6):436-8.
24. Pavelic K,Krizanac S,Cacev T,et a1．aberration of fhit gene is associated withincreased tumour proliferation and decreased apoptosis clinical evidence in lungand head neck carcinomas.．MolMed,2001.7(7)：442-453．
25. Chan MW,Chan LW,Tang NL,et al. Freguent hypermethyiation of promoterregion of RASSF1A in tumor tissues and voided urine of urinary biadder cancerpatients. Int Cancer,2003,104(5)：611-616.
26. Yu MY,Tong JH,Chan PK,et al. Hypermethyiation of the tumor suppressor geneRASSF1A and freguent concomitant ioss of heterozygosity at3P21.3in cervicaicancers. Int Cancer,2003,105(2)：204-209.
27.许承弼;滕博;李长青;金春顺；喉鳞状细胞癌中RASSF1A基因启动子区甲基化及蛋白表达;吉林大学学报(医学版);2006年02期
28. Worsham MJ, Chen KM, Tiwari N, et al. Fine-mapping loss of gene architectureat the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genesin head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.2006Apr；132(4)：409-15.
29. Worsham MJ, Stephen JK, Chen KM, Havard S, Shah V, Gardner G, SchweitzerVG Delineating an epigenetic continuum in head and neck cancer. Cancer Lett.2012Mar1.
30. Jares P, Nadal A, Fernández P L, et al. Disregulation of p16MTS1/CDK4I proteinand mRNA expression is associated with gene alterations in squamous‐cellcarcinoma of the larynx. International journal of cancer,1999,81(5):705-711.
31. Namazie A, Alavi S, Olopade OI, et al. Cyclin D1amplification andp16(MTS1/CDK4I) deletion correlate with poor prognosis in head and necktumors.Laryngoscope.2002Mar；112(3)：72-81.4
32. Rizos E,Sourvlnm C,Spondidm DA．Loss of heterozygosity at8p,9p and17q inlaryngeal cytological specimens. OralOncd.1998,34(6)：519．
33. Schneider-Stock R, Mawrin C, Motsch C, et al. Retention of the arginine allele incodon72of the p53gene correlates with poor apoptosis in head and neck cancer.Am J Pathol.2004Apr;164(4):1233-41.
34. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
35. Graveland AP, Golusinski PJ, Buijze M, et al. Loss of heterozygosity at9p andp53immunopositivity in surgical margins predict local relapse in head and necksquamous cell carcinoma. Int J Cancer.2011Apr15;128(8):1852-9.
36. Ma C, Quesnelle KM, Sparano A, Rao S, et al. Characterization CSMD1in alarge set of primary lung, head and neck, breast and skin cancer tissues. CancerBiol Ther.2009May；8(10)：907-16.
37. Ribeiro IP, Marques F, Caramelo F, et al.Genetic imbalances detected bymultiplex ligation-dependent probe amplification in a cohort of patients with oralsquamous cell carcinoma-the first step towards clinical personalized medicine.Tumour Biol.2014Jan31.[Epub ahead of print]
38. Urashima M, Hama T, Suda T, et al.Distinct effects of alcohol consumption andsmoking on genetic alterations in head and neck carcinoma.PLoS One.2013Nov20;8(11):e80828.
39. Scholnick SB, Richter TM.The role of CSMD1in head and neck carcinogenesis.Genes Chromosomes Cancer.2003Nov;38(3):281-3.
40. Takebayashi S, Ogawa T, Jung KY, et al. Identification of new minimally lostregions on18q in head and neck squamous cell carcinoma. Cancer Res.2000Jul1；60(13)：3397-403.
41. Leong PP, Rezai B, Koch WM, et al.Distinguish second primary tumors fromlung metastases in patients with head and neck squamous cell carcinola. J NatlCancer Inst,1998,90:972-977.
42. Scholes AG, Woolgar JA, Boyle MA, et al.Synchronous oral carcinomas:independent or common clonal origin? Cancer Res.1998May1;58(9):2003-6.
43. van Houten, V. M. M., Tabor, M. P., van den Brekel, M. W. M., Denkers, F.,Wishaupt, R. G. A., Kummer, J. A., Snow G. B., and Brakenhoff, R. H. Molecularassays for the diagnosis of minimal residual head and neck cancer: methods,reliability, pitfalls, and solutions. Clin. Cancer Res.,6:3803–3816,2000.
44. Brennan JA, Mao L, Hruban RH, et al.Molecular assessment of histopathologicalstaging in squamous-cell carcinoma of the head and neck.N Engl J Med.1995Feb16;332(7):429-35.
45. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
46. Yamamoto N,Mizoe JE,Numasawa H,et a1．Allelic loss of chmmosome2inhuman oral squamous cell carcinoma：correlation with lymphnode metastasis.0ral0ncology,2003,39(1)：64-68．
1.刘天润，杨安奎，陈福进，等。221例晚期喉鳞癌患者术后的生存和预后分析。癌症，2009，28(3)：297-302.
2. Raitiola H, Pukander J, Laippala P. Glottic and supraglottic laryngeal carcinoma:differences in epidemiology, clinical characteristics and prognosis. ActaOtolaryngol.1999;119(7):847-51.
3.孙越峰，杨蓓蓓．喉癌手术切缘的研究进展．临床耳鼻咽喉科杂志200l，15(9)428．430
4.李为民，郭睿，郭志祥．喉癌手术切缘的组织病理学研究．临床耳鼻咽喉科杂志，1998，12(11)：496—498
5. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
6. Worsham, M. Wolman, S. R., Carey, T. et al Common clonal origin ofsynchronous primary head and neck squamous cell carcinomas: analysis bytumorkaryotypes and fluorescence in situ hybridization. Hum. Pathol.,26:251–261,1995.
7. Califano, Leong, P. L., Koch, W. M., et al. Second esophageal tumors in patientswith head and neck squamous cell carcinoma: an assessment of clonalrelationships. Clin. Cancer Res.,5:1862–1867,1999.
8. Dakubo,G. D.,Jakupciak， J. P.， Birch-Machin， M. A.&Parr， R. L. Clinicalimplications and utility of field cancerization. Cancer Cell. Int.7,2(2007).
9. Califano. et al. Genetic progression model for head and neck cancer：implications for field cancerization. Cancer Res.56,2488–2492(1996).
10. Tabor,M. P. et al. Persistence of genetically altered fields in head and neck cancerpatients： Biological and clinical implications. Clin. Cancer Res.7，1523–1532(2001).
11. Tabor,M. P. et al. Comparative molecular and histological grading of epithelialdysplasia of the oral cavity and the oropharynx. J. Pathol.199，354–360(2003).
12. Hittelman,W. N. Genetic instability in epithelial tissues at risk for cancer. Ann.NY Acad. Sci.952，1–12(2001).
13. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
14. Slaughter, D. P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
15. Mao L, Lee JS, Fan YH, Ro JY, Batsakis JG, Lippman S, Hittelman W, Hong WK.Frequent microsatellite alterations at chromosomes9p21and3p14in oralpremalignant lesions and their value in cancer risk assessment. Nat Med.1996Jun；2(6)：682-5.
16. Ghosh S, Ghosh A, Maiti GP, Alam N, Roy A, Roychoudhury S, PandaCK.Alterations of ROBO1/DUTT1and ROBO2loci in early dysplastic lesionsof head and neck： clinical and prognostic implications.Hum Genet.2009Mar；125(2)：189-98.
17. Abou-Elhamd KE, Habib TN, Moussa AE, et al. The role of genetic susceptibilityin head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol.2008Feb；265(2)：217-22.
18. Chang SS, Califano J. Current status of biomarkers in head and neck cancer. JSurg Oncol.2008Jun15；97(8)：640-3.
19. Yin D T,Dong M M．Micresatellite instability and loss of heterozygosity offragile histidine triad gene in laryngesl squamous cell carcinomas.Zhanghua ErBi Yan Hou Tan Jing Wai Ke Za Zhi,2005.40(1)：4548．
20. GuoT，Sun JW，Lv QP，Li XG. Allelicic imbalance on chromosomes3p，9p and17p in malignant progression of IaryngeaI mueosa.JLaryngoIOtoI.2008.122(l):72一77.
21. Szukala K,Sowinska A,Wierzbicka M,et al. Does loss of heterozygosity in criticalgenome regions predict a local relapse in patients after laryngeetomy.MutatRes.2006.600(1—2)：67-76．
22. Califano J, Westra WH, Meininger G,et al.Genetic progression and clonalrelationship of recurrent premalignant head and neck lesions. Clin Cancer Res.2000Feb;6(2):347-52.
23. Worsham MJ, Chen KM, Tiwari N, et al. Fine-mapping loss of gene architectureat the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genesin head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.2006Apr；132(4)：409-15.
24. Chakraborty SB, Dasgupta S, Roy A, Sengupta A,et al..Differential deletions in3p are associated with the development of head and neck squamous CancerGenet Cytogenet.2003Oct15;146(2):130-8.
25. Jang SJ, Chiba I, Hirai A, et al.Multiple oral squamous epithelial lesions： arethey genetically related? Oncogene.2001Apr26；20(18)：2235-42.
26. Rosin M, Splieth C, Wilkens M, Meyer G..Effect of cement type on retention of atapered post with a self-cutting double thread. J Dent.2000Nov;28(8):577-82.
27. Pindborg, J. J., Daftary, D. K., and Mehta, F. S. A follow-up studyof sixty-oneoral dysplastic precancerous lesions in Indian villagers.Oral Surg. Oral Med. O.,43:383–390,1977.
1. Kamangar, F., Dores, G. M.&Anderson, W. F. Patterns of cancer incidence,mortality, and prevalence across five continents： defining priorities to reducecancer disparities in different geographic regions of the world. J. Clin. Oncol.24,2137–2150(2006).
2. Kutler, D. I. et al. High incidence of head and neck squamous cell carcinoma inpatients with fanconi anemia. Arch. Otolaryngol. Head Neck Surg.129,106–112(2003).
3. Hopkins, J. et al. Genetic polymorphisms and head and neck cancer outcomes： areview. Cancer Epidemiol. Biomark. Prev.17,490–499(2008).
4. Cloos, J. et al. Genetic susceptibility to head and neck squamous cell carcinoma.J. Natl Cancer Inst.88,530–535(1996).
5. Ang, K. K. et al. Human papillomavirus and survival of patients withoropharyngeal cancer. N. Engl. J. Med.363,24–35(2010).
6. Smith, R. B., Sniezek, J. C., Weed, D. T.&Wax, M. K. Utilization of free tissuetransfer in head and neck surgery. Otolaryngol. Head Neck Surg.137,182–191(2007).
7. Vergeer, M. R. et al. Intensity-modulated radiotherapy reduces radiation-inducedmorbidity and improves health-related quality of life： results of anonrandomized prospective study using a standardized follow-up program. Int. J.Radiat. Oncol. Biol. Phys.74,1–8(2009).
8. Boscolo-Rizzo, P., Maronato, F., Marchiori, C., Gava, A.&Da Mosto, M. C.Long-term quality of life after total laryngectomy and postoperative radiotherapyversus concurrent chemoradiotherapy for laryngeal preservation. Laryngoscope118,300–306(2008).
9. Hanahan, D.&Weinberg, R. A. The hallmarks of cancer. Cell100,57–70(2000).
10. Woolgar, J. A.&Triantafyllou, A. Pitfalls and procedures in the histopathologicaldiagnosis of oral and oropharyngeal squamous cell carcinoma and a review of therole of pathology in prognosis. Oral Oncol.45,361–385(2009).
11. Chung, C. H. et al. Molecular classification of head and neck squamous cellcarcinomas using patterns of gene expression. Cancer Cell5,489–500(2004).
12. Chung, C. H. et al. Gene expression profiles identify epithelial-to-mesenchymaltransition and activation of nuclear factor-kB signalling as characteristic of a highrisk squamous cell carcinoma. Cancer Res.66,8210–8218(2006).
13. Hermsen, M. et al. New chromosomal regions with highlevel amplifications insquamous cell carcinomas of the larynx and pharynx, identified by comparativegenomic hybridization. J. Pathol.194,177–182(2001).
14. Jin, C. et al. Cytogenetic abnormalities in106oral squamous cell carcinomas.Cancer Genet. Cytogenet.164,44–53(2006).
15. Smeets, S. J. et al. Genetic classification of oral and oropharyngeal carcinomasidentifies subgroups with a different prognosis. Cell. Oncol.31,291–300(2009).
16. Walboomers, J. M. et al. Human papillomavirus is a necessary cause of invasivecervical cancer worldwide. J. Pathol.189,12–19(1999).
17. zur Hausen, H. Papillomaviruses and cancer： from basic studies to clinicalapplication. Nature Rev. Cancer2,342–350(2002).
18. Munoz, N. et al. Epidemiologic classification of human papillomavirus typesassociated with cervical cancer. N. Engl. J. Med.348,518–527(2003).
19. Syrjanen, S. Human papillomavirus (HPV) in head and neck cancer. J. Clin. Virol.32, S59–S66(2005).
20. Snijders, P. J. F. et al. Prevalence and expression of human papillomavirus intonsillar carcinomas, indicating a possible viral etiology. Int. J. Cancer51,845–850(1992).
21. Gillison, M. L. et al. Evidence for a causal association between humanpapillomavirus and a subset of head and neck cancers. J. Natl Cancer Inst.92,709–720(2000).
22. Meijer, C. J. et al. Guidelines for human papillomavirus DNA test requirementsfor primary cervical cancer screening in women30years and older. Int. J. Cancer124,516–520(2009).
23. Snijders, P. J., van den Brule, A. J.&Meijer, C. J. The clinical relevance ofhuman papillomavirus testing： relationship between analytical and clinicalsensitivity. J. Pathol.201,1–6(2003).
24. Van Houten, V. M. M. et al. Biological evidence that human papillomaviruses areetiologically involved in a subgroup of head and neck squamous cell carcinomas.Int. J. Cancer93,232–235(2001).
25. Wiest, T., Schwarz, E., Enders, C., Flechtenmacher, C.&Bosch, F. X.Involvement of intact HPV16E6/E7gene expression in head and neck cancerswith unaltered p53status and perturbed pRb cell cycle control. Oncogene21,1510–1517(2002).
26. Smeets, S. J. et al. Genome-wide DNA copy number alterations in head and necksquamous cell carcinomas with or without oncogene-expressing humanpapillomavirus. Oncogene25,2558–2564(2006).
27. Braakhuis, B. J. M. et al. Genetic patterns in head and neck cancers that containor lack transcriptionally active human papillomavirus. J. Natl Cancer Inst.96,998–1006(2004).
28. Slebos, R. J. C. et al. Gene expression differences associated with humanpapillomavirus status in head and neck squamous cell carcinoma. Clin. CancerRes.12,701–709(2006).
29. Smeets, S. J. et al. A novel algorithm for reliable detection of humanpapillomavirus in paraffin embedded head and neck cancer specimen. Int. J.Cancer121,2465–2472(2007).
30. Robinson, M., Sloan, P.&Shaw, R. Refining the diagnosis of oropharyngealsquamous cell carcinoma using human papillomavirus testing. Oral Oncol.46,492–496(2010).
31. D’Souza, G. et al. Case-control study of human papillomavirus andoropharyngeal cancer. N. Engl. J. Med.356,1944–1956(2007).
32. Ragin, C. C. R.&Taioli, E. Survival of squamous cell carcinoma of the head andneck in relation to human papillomavirus infection： review and meta-analysis.Int. J. Cancer121,1813–1820(2007).
33. Poeta, M. L. et al. Tp53mutations and survival in squamous-cell carcinoma ofthe head and neck. N. Engl. J. Med.357,2552–2561(2007).
34. Westra, W. H. et al. Inverse relationship between human papillomavirus-16infection and disruptive p53gene mutations in squamous cell carcinoma of thehead and neck. Clin. Cancer Res.14,366–369(2008).
35. Napier, S. S.&Speight, P. M. Natural history of potentially malignant orallesions and conditions： an overview of the literature. J. Oral Pathol. Med.37,1–10(2008).
36. van der Waal, I. Potentially malignant disorders of the oral and oropharyngealmucosa； terminology, classification and present concepts of management. OralOncol.45,317–323(2009).
37. Lodi, G., Sardella, A., Bez, C., Demarosi, F.&Carrassi, A. Interventions fortreating oral leukoplakia. Cochrane Database Syst. Rev. CD001829(2006).
38. Partridge, M. et al. A case-control study confirms that microsatellite assay canidentify patients at risk of developing oral squamous cell carcinoma within a fieldof cancerization. Cancer Res.60,3893–3898(2000).
39. Wrangle, J. M.&Khuri, F. R. Chemoprevention of squamous cell carcinoma ofthe head and neck. Curr. Opinion Oncol.19,180–187(2007).
40. Schaaij-Visser, T. B. M. et al. Evaluation of cornulin, keratin4, keratin13expression and grade of dysplasia for predicting malignant progression of oralleukoplakia. Oral Oncol.46,123–127(2010).
41. Rosin, M. P. et al. Use of allelic loss to predict malignant risk for low-grade oralepithelial dysplasia. Clin. Cancer Res.6,357–362(2000).
42. Mao, L. et al. Frequent microsatellite alterations at chromosomes9p21and3p14in oral premalignant lesions and their value in cancer risk assessment. NatureMed.2,682–685(1996).
43. Torres-Rendon, A., Stewart, R., Craig, G. T., Wells, M.&Speight, P. M. DNAploidy analysis by image cytometry helps to identify oral epithelial dysplasiaswith a high risk of malignant progression. Oral Oncol.45,468–473(2009).
44. Shpitzer, T. et al. Salivary analysis of oral cancer biomarkers. Brit. J. Cancer101,1194–1198(2009).
45. Bremmer, J. F. et al. A noninvasive genetic screening test to detect oralpreneoplastic lesions. Lab. Invest.85,1481–1488(2005).
46. Bremmer, J. F. et al. Screening for oral precancer with noninvasive geneticcytology. Cancer Prev. Res.2,128–133(2009).
47. Slaughter, D. P., Southwick, H. W.&Smejkal, W. Field cancerization in oralstratified squamous epithelium； clinical implications of multicentric origin.Cancer6,963–968(1953).
48. Califano, J. et al. Genetic progression model for head and neck cancer：implications for field cancerization. Cancer Res.56,2488–2492(1996).
49. Tabor, M. P. et al. Persistence of genetically altered fields in head and neckcancer patients： Biological and clinical implications. Clin. Cancer Res.7,1523–1532(2001).
50. Tabor, M. P. et al. Comparative molecular and histological grading of epithelialdysplasia of the oral cavity and the oropharynx. J. Pathol.199,354–360(2003).
51. Willis, R. A. The mode of origin of tumors. Solitary localized squamous cellgrowths of the skin. Cancer Res.4,469–479(1944).
52. Slaughter, D. P. Multicentric origin of intraoral carcinoma. Surgery133–146(1946).
53. Hittelman, W. N. Genetic instability in epithelial tissues at risk for cancer. Ann.NY Acad. Sci.952,1–12(2001).
54. Tabor, M. P. et al. Genetically altered fields as origin of locally recurrent headand neck cancer： a retrospective study. Clin. Cancer Res.10,3607–3613(2004).
55. Roesch-Ely, M. et al. Proteomic analysis reveals successive aberrations in proteinexpression from healthy mucosa to invasive head and neck cancer. Oncogene26,54–64(2007).
56. Schaaij-Visser, T. B. M. et al. Differential proteomics identifies proteinbiomarkers that predict local relapse of head and neck squamous cell carcinomas.Clin. Cancer Res.15,7666–7675(2009).
57. Braakhuis, B. J. M., Tabor, M. P., Kummer, J. A., Leemans, C. R.&Brakenhoff,R. H. A genetic explanation of Slaughter’s concept of field cancerization：evidence and clinical implications. Cancer Res.63,1727–1730(2003).
58. van Houten, V. M. et al. Mutated p53as a molecular marker for the diagnosis ofhead and neck cancer. J. Pathol.198,476–486(2002).
59. Jonason, A. S. et al. Frequent clones of p53-mutated keratinocytes in normalhuman skin. Proc. Natl Acad. Sci. USA93,14025–14029(1996).
60. Dakubo, G. D., Jakupciak, J. P., Birch-Machin, M. A.&Parr, R. L. Clinicalimplications and utility of field cancerization. Cancer Cell. Int.7,2(2007).
61. Negrini, S., Gorgoulis, V. G.&Halazonetis, T. D. Genomic instability—anevolving hallmark of cancer. Nature Rev. Mol. Cell Biol.11,220–228(2010).
62. Patmore, H. S., Cawkwell, L., Stafford, N. D.&Greenman, J. Unraveling thechromosomal aberrations of head and neck squamous cell carcinoma： a review.Ann. Surg. Oncol.12,831–842(2005).
63. Wreesmann, V. B.&Singh, B. Chromosomal aberrations in squamous cellcarcinomas of the upper aerodigestive tract： biologic insights and clinicalopportunities. J. Oral Pathol. Med.34,449–459(2005).
64. Ha, P. K., Chang, S. S., Glazer, C. A., Califano, J. A.&Sidransky, D. Moleculartechniques and genetic alterations in head and neck cancer. Oral Oncol.45,335–339(2009).
65. Carbone, M., Klein, G., Gruber, J.&Wong, M. Modern criteria to establishhuman cancer etiology. Cancer Res.64,5518–5524(2004).
66. Kastan, M. B.&Bartek, J. Cell-cycle checkpoints and cancer. Nature432,316–323(2004).
67. Balz, V. et al. Is the p53inactivation frequency in squamous cell carcinomas ofthe head and neck underestimated? Analysis of p53exons2–11and humanpapillomavirus16/18E6transcripts in123unselected tumor specimens. CancerRes.63,1188–1191(2003).
68. Opitz, O. G. et al. Cyclin D1overexpression and p53inactivation immortalizeprimary oral keratinocytes by a telomerase-independent mechanism. J. Clin.Invest.108,725–732(2001).
69. Rheinwald, J. G. et al. A two-stage, p16INK4A-and p53-dependent keratinocytesenescence mechanism that limits replicative potential independent of telomerestatus. Mol. Cell. Biol.22,5157–5172(2002).
70. Smeets, S. J. et al. Immortalization of oral keratinocytes by functionalinactivation of the p53and pRb pathways. Int. J. Cancer (in the press).
71. Reed, A. L. et al. High frequency of p16(CDKN2/MTS-1/INK4A) inactivationin head and neck squamous cell carcinoma. Cancer Res.56,3630–3633(1996).
72. Gibcus, J. H. et al. Amplicon mapping and expression profiling identify theFas-associated death domain gene as a new driver in the11q13.3amplicon inlaryngeal/pharyngeal cancer. Clin. Cancer Res.13,6257–6266(2007).
73. Berns, K. et al. A large-scale RNAi screen in human cells identifies newcomponents of the p53pathway. Nature428,431–437(2004).
74. Dickson, M. A. et al. Human keratinocytes that express hTERT and also bypass ap16INK4A-enforced mechanism that limits life span become immortal yet retainnormal growth and differentiation characteristics. Mol. Cell. Biol.20,1436–1447(2000).
75. Snijders, P. J. F. et al. Telomerase activity exclusively in cervical carcinomas anda subset of cervical intraepithelial neoplasia grade III lesions： strong associationwith elevated messenger RNA levels of its catalytic subunit and high-risk humanpapillomavirus DNA. Cancer Res.58,3812–3818(1998).
76. Hynes, N. E.&Lane, H. A. ERBB receptors and cancer： the complexity oftargeted inhibitors. Nature Rev. Cancer5,341–354(2005).
77. Lin, S. Y. et al. Nuclear localization of EGF receptor and its potential new role asa transcription factor. Nature Cell Biol.3,802–808(2001).
78. Lo, H. W. et al. Nuclear interaction of EGFR and STAT3in the activation of theiNOS/NO pathway. Cancer Cell7,575–589(2005).
79. Goessel, G. et al. Creating oral squamous cancer cells： a cellular model oforal–esophageal carcinogenesis. Proc. Natl Acad. Sci. USA102,15599–15604(2005).
80. Ozanne, B., Richards, C. S., Hendler, F., Burns, D.&Gusterson, B.Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas.J. Pathol.149,9–14(1986).
81. Grandis, J. R.&Tweardy, D. J. Elevated levels of transforming growth factor αand epidermal growth factor receptor messenger RNA are early markers ofcarcinogenesis in head and neck cancer. Cancer Res.53,3579–3584(1993).
82. Hama, T. et al. Prognostic significance of epidermal growth factor receptorphosphorylation and mutation in head and neck squamous cell carcinoma.Oncologist14,900–908(2009).
83. Bonner, J. A. et al. Radiotherapy plus cetuximab for squamous-cell carcinoma ofthe head and neck. N. Engl. J. Med.354,567–578(2006).
84. Morandell, S. et al. Phosphoproteomics strategies for the functional analysis ofsignal transduction. Proteomics6,4047–4056(2006).
85. Lee, J. W. et al. Somatic mutations of EGFR gene in squamous cell carcinoma ofthe head and neck. Clin. Cancer Res.11,2879–2882(2005).
86. Loeffler-Ragg, J. et al. Low incidence of mutations in EGFR kinase domain inCaucasian patients with head and neck squamous cell carcinoma. Eur. J. Cancer42,109–111(2006).
87. Ekstrand, A. J., Sugawa, N., James, C. D.&Collins, V. P. Amplified andrearranged epidermal growth factor receptor genes in human glioblastomas revealdeletions of sequences encoding portions of the Nand/or C-terminal tails. Proc.Natl Acad. Sci. USA89,4309–4313(1992).
88. Sok, J. C. et al. Mutant epidermal growth factor receptor (EGFRvIII) contributesto head and neck cancer growth and resistance to EGFR targeting. Clin. CancerRes.12,5064–5073(2006).
89. Ishitoya, J. et al. Gene amplification and overexpression of EGF receptor insquamous cell carcinomas of the head and neck. Brit. J. Cancer59,559–562(1989).
90. Temam, S. et al. Epidermal growth factor receptor copy number alterationscorrelate with poor clinical outcome in patients with head and neck squamouscancer. J. Clin. Oncol.25,2164–2170(2007).
91. Sheu, J. J. C. et al. Functional genomic analysis identified epidermal growthfactor receptor activation as the most common genetic event in oral squamouscell carcinoma. Cancer Res.69,2568–2576(2009).
92. Knudsen, B. S.&Vande Woude, G. Showering c-MET-dependent cancers withdrugs. Curr. Opin. Genet. Dev.18,87–96(2008).
93. Seiwert, T. Y. et al. The MET receptor tyrosine kinase is a potential noveltherapeutic target for head and neck squamous cell carcinoma. Cancer Res.69,3021–3031(2009).
94. Knowles, L. M. et al. HGF and c-Met participate in paracrine tumorigenicpathways in head and neck squamous cell cancer. Clin. Cancer Res.15,3740–3750(2009).
95. Wang, D. et al. Mutation and downregulation of the transforming growth factorbeta type II receptor gene in primary squamous cell carcinomas of the head andneck. Carcinogenesis18,2285–2290(1997).
96. Huntley, S. P. et al. Attenuated type II TGF-B receptor signalling in humanmalignant oral keratinocytes induces a less differentiated and more aggressivephenotype that is associated with metastatic dissemination. Int. J. Cancer110,170–176(2004).
97. Qiu, W., Schonleben, F., Li, X.&Su, G. H. Disruption of transforming growthfactor β–Smad signaling pathway in head and neck squamous cell carcinoma asevidenced by mutations of SMAD2and SMAD4. Cancer Lett.245,163–170(2007).
98. Bornstein, S. et al. Smad4loss in mice causes spontaneous head and neck cancerwith increased genomic instability and inflammation. J. Clin. Invest.119,3408–3419(2009).
99. Mishra, A., Bharti, A. C., Varghese, P., Saluja, D.&Das, B. C. Differentialexpression and activation of NF-κB family proteins during oral carcinogenesis：role of high risk human papillomavirus infection. Int. J. Cancer119,2840–2850(2006).
100.Karin, M. Nuclear factor-κB in cancer development and progression. Nature441,431–436(2006).
101.Perkins, N. D. Integrating cell-signalling pathways with NF-κB and IKK function.Nature Rev. Mol. Cell Biol.8,49–62(2007).
102.Cohen, J. et al. Attenuated transforming growth factor β signaling promotesnuclear factor-κB activation in head and neck cancer. Cancer Res.69,3415–3424(2009).
103.Engelman, J. A. Targeting PI3K signalling in cancer： opportunities, challengesand limitations. Nature Rev. Cancer9,550–562(2009).
104.Kozaki, K. et al. PIK3CA mutation is an oncogenic aberration at advanced stagesof oral squamous cell carcinoma. Cancer Sci.97,1351–1358(2006).
105.Qiu, W. L. et al. PIK3CA mutations in head and neck squamous cell carcinoma.Clin. Cancer Res.12,1441–1446(2006).
106.Murugan, A. K., Hong, N. T., Fukui, Y., Munirajan, A. K.&Tsuchida, N.Oncogenic mutations of the PIK3CA gene in head and neck squamous cellcarcinomas. Int. J. Oncol.32,101–111(2008).
107.Okami, K. et al. Analysis of PTEN/MMAC1alterations in aerodigestive tracttumors. Cancer Res.58,509–511(1998).
108.Redon, R. et al. A simple specific pattern of chromosomal aberrations at earlystages of head and neck squamous cell carcinomas：PIK3CA but not p63gene asa likely target of3q26-qter gains. Cancer Res.61,4122–4129(2001).
109.Woenckhaus, J. et al. Genomic gain of PIK3CA and increased expression ofp110alpha are associated with progression of dysplasia into invasive squamouscell carcinoma. J. Pathol.198,335–342(2002).
110.Pantel, K.&Brakenhoff, R. H. Dissecting the metastatic cascade. Nature Rev.Cancer4,448–456(2004).
111.Rosenthal, E. L.&Matrisian, L. M. Matrix metalloproteases in head and neckcancer. Head Neck28,639–648(2006).
112.Sun, P. C. et al. Transcript map of the8p23putative tumor suppressor region.Genomics75,17–25(2001).
113.Scholnick, S. B. et al. Chromosome8allelic loss and the outcome of patientswith squamous cell carcinoma of the supraglottic larynx. J. Natl Cancer Inst.88,1676–1682(1996).
114.Sunwoo, J. B. et al. Localization of a putative tumor suppressor gene in thesub-telomeric region of chromosome8p. Oncogene18,2651–2655(1999).
115.Kraus, D. M. et al. CSMD1is a novel multiple domain complement-regulatoryprotein highly expressed in the central nervous system and epithelial tissues. J.Immunol.176,4419–4430(2006).
116.Roepman, P. et al. An expression profile for diagnosis of lymph node metastasesfrom primary head and neck squamous cell carcinomas. Nature Genet.37,182–186(2005).
117.Thiery, J. P. Epithelial–mesenchymal transitions in tumour progression. NatureRev. Cancer2,442–454(2002).
118.Ikushima, H.&Miyazono, K. TGFβ signalling： a complex web in cancerprogression. Nature Rev. Cancer10,415–424(2010).
119.Folkman, J. Role of angiogenesis in tumor growth and metastasis. Semin. Oncol.29,15–18(2002).
120.Kerbel, R. S. Tumor angiogenesis. N. Engl. J. Med.358,2039–2049(2008).
121.Kyzas, P. A., Cunha, I. W.&Ioannidis, J. P. A. Prognostic significance ofvascular endothelial growth factor immunohistochemical expression in head andneck squamous cell carcinoma： a meta-analysis. Clin. Cancer Res.11,1434–1440(2005).
122.Fei, J. et al. Prognostic significance of vascular endothelial growth factor insquamous cell carcinomas of the tonsil in relation to human papillomavirus statusand epidermal growth factor receptor. Ann. Surg. Oncol.16,2908–2917(2009).
123.Virgilio, L. et al. FHIT gene alterations in head and neck squamous cellcarcinomas. Proc. Natl Acad. Sci. USA93,9770–9775(1996).
124.Hibi, K. et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc.Natl Acad. Sci. USA97,5462–5467(2000).
125.Rocco, J. W., Leong, C. O., Kuperwasser, N., DeYoung, M. P.&Ellisen, L. W.p63mediates survival in squamous cell carcinoma by suppression ofp73-dependent apoptosis. Cancer Cell9,45–56(2006).
126.Rodrigo, J. P., Lazo, P. S., Ramos, S., Alvarez, I.&Suarez, C. MYCamplification in squamous cell carcinomas of the head and neck. Arch.Otolaryngol. Head Neck Surg.122,504–507(1996).
127.Carvalho, A. L. et al. Deleted in colorectal cancer is a putative conditionaltumor-suppressor gene inactivated by promoter hypermethylation in head andneck squamous cell carcinoma. Cancer Res.66,9401–9407(2006).
128.Avissar, M., Christensen, B. C., Kelsey, K. T.&Marsit, C. J. MicroRNAexpression ratio is predictive of head and neck squamous cell carcinoma. Clin.Cancer Res.15,2850–2855(2009).
129.Childs, G. et al. Low-level expression of microRNAs let-7d and miR-205areprognostic markers of head and neck squamous cell carcinoma. Am. J. Pathol.174,736–745(2009).
130.Cervigne, N. K. et al. Identification of a microRNA signature associated withprogression of leukoplakia to oral carcinoma. Hum. Mol. Genet.18,4818–4829(2009).
131.Kies, M. S. et al. Induction chemotherapy and cetuximab for locally advancedsquamous cell carcinoma of the head and neck： results from a phase IIprospective trial. J. Clin. Oncol.28,8–14(2010).