【作者】 方瑾；

【导师】 周水洪；

【作者基本信息】 浙江大学 ， 耳鼻咽喉科学， 2011， 硕士

【摘要】 背景和目的：发生在耳鼻咽喉气管部位的腺样囊性癌(adenoid cystic carcinoma, ACC)极为少见,生长位置较为隐蔽,早期症状不典型,早期确诊较为困难。与大涎腺的腺样囊性癌一样,耳鼻咽喉气管部位的腺样囊性癌生长较为缓慢但侵袭性强,常沿神经、血管生长,易复发和转移,手术与放化疗联合治疗的方法尚达不到理想的治疗效果,预后极差。不同的病变部位、病理类型、TNM分期、治疗方法、切缘等因素差异可直接影响ACC患者的预后,但影响其预后的真正原因尚不明了。因此探讨腺样囊性癌浸润与转移的发病机制及预后的相关因素,寻求新的诊断和治疗方法有良好的应用前景。随着免疫学、分子生物学技术的不断发展,从分子水平探讨ACC发生、发展的机制,对于认识ACC的发病机制、判断预后、发现潜在的治疗靶点具有重要意义。ACC与其它恶性肿瘤一样,恶性细胞所处的生理环境能量相对匮乏,呈现局部缺氧状态,并引起肿瘤细胞因子的过度表达。恶性肿瘤细胞的葡萄糖代谢率较高,这种现象已被正电子发射计算机断层(Positron emission tomography, PET)检查所证实,PET/CT也证实ACC中存在此现象。恶性肿瘤细胞对葡萄糖的代谢率增高与葡萄糖转运蛋白(Glucose transporter protein, Glut)及基因的异常表达有关,其中葡萄糖转运蛋白-1(Glut-1)在恶性肿瘤细胞的葡萄糖吸收和转运中起主导作用,Glut-1的异常表达与多种信号转导通路有关,而PI3K/Akt在Glut-1表达的调节中起重要作用。PI3K/Akt是一种对生长和增殖有重要作用的异二聚体酶,是近年来发现的生长因子信号转导途径。研究发现在人类许多肿瘤中都有PI3K-Akt的过度表达。从恶性肿瘤葡萄糖能量供应的层面已有研究发现,PI3K/Akt途径的激活与Glut-1表达的增加有关。我们的前期研究已经发现,Glut-1的异常表达与头颈部癌的生物学行为有关,与头颈部癌的预后差有关,可以作为靶向治疗的新方法之一,并且我们已通过反义寡脱氧核苷酸技术阻断喉癌Hep-2细胞Glut-1的表达,能抑制喉癌细胞的葡萄糖吸收的影响及Hep-2细胞增殖,可能是喉癌治疗的一种理想靶标。目前,Glut-1在肿瘤发生机制中的研究,多集中于头颈部较常见肿瘤类型如鳞状细胞癌,而在腺样囊性癌中Glut-1的表达情况与临床生物学特性的关系研究较为少见,尤其是腺样囊性癌中PI3K/Akt信号通路调控Glut-1表达的研究更为少见,国内尚未见有文献报道。本课题在前期研究基础上,从癌细胞能量供应的层面,采用免疫组织化学方法检测耳鼻咽喉气管腺样囊性癌组织中Glut-1及PI3K和磷酸化Akt (p-Akt)蛋白的表达,回顾性分析患者的临床病例特点,探讨Glut-1及PI3K和p-Akt蛋白表达与耳鼻咽喉腺样囊性癌生物学行为的关系。材料与方法：收集1993.1～2010.2期间浙江大学医学院附属第一医院耳鼻咽喉科收治的14例耳鼻咽喉气管腺样囊性癌病例进行回顾性分析,观察临床病理参数对患者生存状态和预后的影响。并应用免疫组织化学EliVisionTM法检测14例耳鼻喉气管腺样囊性癌病例以及对照组15例炎性病变(5例鼻息肉组织及10例声带息肉)、14例良性肿瘤(5例鼻腔良性神经鞘瘤、1例耵聍腺瘤、1例喉部良性软骨瘤、7例鼻腔内翻性乳头状瘤)的Glut-1. PI3K和p-Akt的蛋白表达情况,14例腺样囊性癌病理学分类分别为筛孔型、管状型及实体性,临床病理学分期在T1、T2、T3期；显微镜下计数阳性细胞,结果进行统计学分析。采用SPSS15.0软件,各临床病理因素的关系因素及Glut-1、PI3K和p-Akt之间的关系用χ2检验、Fisher精确概率法,以α=0.05为差异有统计学意义检验水准,相关性分析采用Kappa检验(即一致性检验),如果kappa)0.75,提示两个指标的一致性非常好,即相关性高,kappa值在0.4～0.75之间,两个指标间的一致性中等,kappa<0.4,两个指标间一致性较差。结果：1、14例腺样囊性癌(ACC)患者,男性6例,女性8例,年龄32～80岁,平均54岁。位于外耳道者4例,鼻腔3例,鼻窦3例,喉部3例,气管1例。T1 6例、T2 5例or T3 3例,所有患者均未发现淋巴结及远处转移。全部患者均行手术治疗,10例患者术后进行了放射治疗。本组资料显示,10例患者术后进行了放射治疗,随访至今仍分别存活6年9个月(病例1),4年3个月(病例3),15年(病例4),24年(病例5),9年5个月(病例6),5年7个月(病例7),1年5个月(病例9),5年8个月(病例11),6年6个月(病例12),7年(病例14),8例((病例1、3、4、6、9、11、12、14)放射剂量不清,2例放射剂量50Gy。在放射的10例中,接受手术+放疗后2年(病例6)、5年(病例5)出现局部复发,行多次手术,现1例带瘤生存(病例6),1例无瘤生存(病例5),其余8例均无复发,说明放射治疗控制局部复发有一定效果；3例无术后放射治疗,其中1例随访24个月后失访,1例随访半年后失访,‘1例随访至今存活1年8个月；1例第一次在外院手术后9个月,在我院再次手术,术后失访,有无术后放疗情况不清楚。本组鼻腔-鼻窦及外耳道ACC局部复发率高,喉及气管ACC未见复发,无颈淋巴结转移及远处转移。2、14例耳鼻喉气管腺样囊性癌组织中Glut-1阳性表达率为35.7%(5/14)PI3K阳性表达率为28.6%(4/14),p-Akt阳性表率为71.4%(10/14)；对照组：15例炎性病变Glut-1、PI3K和p-Akt均为阴性表达；14例良性肿瘤中,Glut-1均呈阴性表达,1例PI3K、2例p-Akt呈阳性表达,阳性表达率分别为7.14%(1/14)；14.3%(2/14)。耳鼻喉气管腺样囊性癌组织组Glut-1, PI3K、p-Akt阳性表达率明显高于对照组,差异有显著性(P<0.05,P<0.01)。ACC中Glut-1和PI3K表达、Glut-1p-Akt表达、p-Akt和PI3K表达,经kappa检验,kappa值分别为0.186、0.364、0.276,说明Glut-1、PI3K、p-Akt表达之间相关一致性较差。结论：临床病理特点：1、本组资料显示：本组鼻腔-鼻窦及外耳道ACC局部复发率高,喉及气管ACC未见复发,无颈淋巴结转移及远处转移。2、本组资料显示：第一次的确诊及第一次彻底手术治疗、术后放疗对控制局部复发有一定效果。3、本组资料显示：腺样囊性癌极易误诊,腺样囊性癌应作为耳鼻咽喉气管部位疾病的鉴别诊断Glut-1、PI3K/AKT信号通路的表达和意义：1、本组资料显示Glut-1表达增高与腺样囊性癌的发生可能有关。2、PI3K/Akt信号传导通路的异常活化与腺样囊性癌的发生有关。更多还原

【Abstract】 Background:Adenoid cystic carcinomas(ACC) in ear, nasal cavity, paranasal sinus, larynx and trachea are uncommon. These malignant tumours often are asymptomatic or mimic inflammatory diseases, leading to a delay in diagnosis. ACC was noted for its slow growth and indolent course, like ACCs in general, patients with ACC of these sites often experienced recurrence and distant metastasis many years after diagnosis and definitive treatment. Patterns of disease included perineural and vascular invasion. The most important modalities for primary treatment of local and locoregional disease are surgery and/or irradiation. However, the outcome of these treatment is unfavorable. Several studies have identified clinicopathological factors in ACC with an unfavorable effect on survival, including old age, tumor location, advanced stage, solid histological subtype, high grade, TNM staging, major nerve involvement, the presence of perineural invasion, a positive surgical margin, and lymph node metastasis. However, real prognostic factors are still unknown. Thus, more studies are needed for pathogenesis, prognostic factors, new diagnostic and therapeutic methods, especially in the molecular level.Like other malignant tumors, cells of ACC exhibit increased glucose uptake and utilization in comparison to their nonmalignant counterparts. The phenomenon has been demonstrated by positron emission tomography (PET),using 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG).Many mechanisms of FDG uptake have been proposed for accelerated glucose use in growing tumors and in transformed and malignant cells: passive diffusion, Na+-dependent glucose transport, and via facilitative glucose transporters (GLUT). The latter is considered to be the most important mechanism for enhancing glucose influx into cells. Gluts are membrane proteins that facilitate the transport of glucose across cellular membranes. Thirteen (14) members of the facilitative sugar transporter family are now recognized (Glut-1-Glut-12 and HMIT; genename SLC2A).The human genes encoding these proteins are named Glut 1-5 and Glut 7-13; Glut-6 and Glut-14 are now known to be pseudogenes. Of the 14 isoforms, Glut-1 appears to be the most ubiquitously distributed. Increased Glutl levels and glucose uptake correlate with increased cellular growth and proliferation. This isoform is overexpressed in many human cancer cells, and its appearance is correlated with aggressive biological behavior. Therefore, control of Glutl trafficking and activity are also key elements regulating glucose uptake. Similar to the insulin-responsive glucose transporter, Glut-4, Glut-1 cell surface localization is controlled by extrinsic signals. Among signaling pathways initiated in cell activation, the PI3K/Akt pathway has been shown to promote both Glut-1 cell surface trafficking and activity.PI3K is a heterodimeric enzyme important for growth and proliferation and Akt is a downstream serine-threonine kinase that transmits survival signals from growth factors. The PI3-K/AKT pathway is frequently overactivated in a wide range of tumors and triggers a cascade of responses, from cell growth and proliferation to cell survival and motility, which drive tumor progression. It has been shown that the PI3K/Akt pathway is involved in translocating the GLUT-1 glucose transporter from the cytosol to the plasma membrane in some endocrine organs such as the thyroid gland, pancrea.Our previous study revealed that FDG uptake was higher in malignant lymphoma than in other head and neck malignant tumors by SPECT/CT, and a significant correlation was found between FDG levels and glucose transporter-1 (Glut-1) mRNA or Glut-1 protein levels. We also showed that the increased expression of Glut-1 in head and neck carcinomas was correlated with lymph node metastasis, poor survival and clinical stage.Then,we revealed that Glut-1 overexpression in vitro is associated with cell proliferation and increased glucose uptake by laryngeal carcinoma Hep-2 cells. Conversely, the suppression of Glut-1 expression by antisense oligodeoxynucleotides (AS-ODNs) may decrease glucose uptake and inhibit the proliferation of Hep-2 cells. Therefore, we propose the suppression of Glut-1 expression as a new therapeutic target for laryngeal carcinoma.To date, only a few studies have investigated the expression of Glut-1 in ACC,especilly in the effect of regulation of expression of Glut-1 by PI3K/Akt signal pathway.In present study, we retrospectively investigated the clincopathigical features of external auditory canal, sinonasal, laryngeal and tracheal ACC and we also assessed expression of Glut-1, PI3K and p-Akt protein in these ACCs by immunohistochemistry. Additionally, we examined possible correlations between Glut-1, PI3K and p-Akt protein and clinicopathological parameters in this cohort of patients. Materials and methods:14 patients with ACC from Otolaryngology Department, the First Affiliated Hospital, College of Medicine, Zhejiang University were collected between January 1993 and February 2010. The institutional review board approved this study and written informed consent was obtained from the patients before inclusion. The clinicopathological findings (including age, sex, site, TNM stage, pathological type, recurrence, metastasis, follow-up) were analysed. The expression of Glut-1, PI3K and p-Akt proteins were examined in ACC tissue samples by an EliVisionTM plus IHC Kit (Maixin Biological, Fuzhou, China) immunohistochemical analysis.5 paraffin-embedded archival tissue blocks from patients with nasal polyps,10 paraffin-embedded archival tissue blocks from patients with vocal cord polyps,5 tissue blocks from patients with nasal benign schwannomas,1 tissue block from patient with laryngeal benign chondroma and 7 tissue blocks from patients with nasal inverted papilloma were also obtained. Formalin fixed, paraffin-embedded archival tissues were obtained from institutional and consultation files.Statistical analyses:Analyses were performed using SPSS 15.0 software package. Associations between Glut-1, PI3K and p-Akt protein expression and the other pretreatment parameters were analyzed using the chi-squared test. A P-value of<0.05 was deemed to indicate statistical significance.The correlation analysis using Kappa test. A value of 1 indicates perfect agreement. We calculated weighted k-values to describe concordance in reporting as slight ( 0.4), moderate(0.4-0.75), or almost perfect (>0.75). Results:Patient Characteristics:Of the 14 ACC tumor tissues,4 cases (28.6%) were located in the external auditory canal,3 (21.40%) in the nasal cavity,3 (21.4%) in the paranasal sinus,3 (21.4%) in larynx and 1(7.2%) in the trachea. The median age of the 14 patients was 54 (range, 32-80).6 patients were males and 8 patients were females. All patients had no lymph node and distant metastasis. Six of the patients (42.9%) had cancer classified as T1 stage, 5 (35.7%) with T2 stage and 3(21.4%) with T3 stage. Surgery was performed in all patients. Ten patients were received radiotherapy postoperative. The patients’average follow-up period was 74 months (range,6-288 months). Three patients were lost to follow-up.Of 10 patients received radiotherapy, two patients suffered disease recurrence(one patient had local recurrence 2 years after initial surgery and another had local recurrence 5 years after initial surgery) and they received several surgeries. The two patients were alive over 24 years,9 years, respectively, one was living without evidence disease and one was living with recurrence. Other eight patients were living without evidence disease at a mean follow-up time of 8.6 years after diagnosis (range,1.4-24 years). Three patients were not received radiotherapy after surgery. Of these patients, one was lost to follow-up 24 months after surgery, one was lost to follow-up six months after surgery, and one was living without evidence disease over twenty months.One was performed operation at another hospital 9 months ago and lost to follow-up after surgery in our hospital.Glut-1, PI3K and Aktprotein expression in ACCThe positive rate of Glut-1,PI3K and Akt protein in ACC was 35.7%(5/14), 28.6%,71.4%, respectively. The expression of Glut-1 or PI3K or Akt protein in ACC was higher than that in inflammatory lesions or benign tumor (p<0.05).The correlation between Glut-1, PI3K and Akt protein expressionKappa test showed that correlation between Glut-1 and PI3K expression, between Glut-1 and p-Akt expression, between p-Akt and PI3K expression was slight correlation(K=0.186,0.364,0.276, respectively).Association of Glut-1, PI3K and Akt with Clinicopathological ParametersThere was no statistically significant association between Glut-1,PI3K and Akt protein in ACC and all clinicopathological variables examined (P>0.05).Conclusions:Clinicopathological findings1、The presnt study showed that there were high recurrent rate in the sinonasal and external auditory canal ACC. had no lymph node and distant metastasis.2、This study also revealed that it was very important for the initial treatment and radiotherapy may be effective on control local recurrenc.3、Additionally, this study suggested that adenoid cystic carcinoma shoule be as differentiatial diagnosis in diseases of the external auditory canal, nasal cavity, paranasal sinus,larynx and trachea. The implication of Glut-1 expression and PI3K/AKT signal pathway in ACC1. Our results showed that over-expression of Glut-1 may be associated with the occurrence of ACC.2、Our data also suggested that abnormal activation of PI3K/Akt signaling pathways may play a role in the process of occurrence of ACC.更多还原