Secreted recombinant P53 protein from Pichia pastoris is a useful antigen for detection of serum p53: autoantibody in patients with advanced colorectal adenocarcinoma

The detection of P53 alteration by serological method is easier to perform, does not require tumor tissues and is of interest for patients monitoring. In this study, we described the development of a home made ELISA test based on recombinant human P53 protein produced in Pichia pastoris and used as antigen for the detection of serum p53-Abs in colorectal carcinoma patients. The human P53 was secreted as a His-tagged protein by recombinant KM71 strain (Kα21) via the peptide signal α of the Saccharomyces cerevisiae mating type gene. The recombinant P53-His was able to detect p53-Abs in 23.4 % of patients. Serum p53-Abs correlated significantly with surgical treatment (P = 0.007), relapse during follow-up (P = 0.036), depth of invasion (P = 0.036) and the level of CA19-9 (P = 0.034). Survival analysis showed that patients negative for serum p53-Abs exhibited a prolonged disease free survival period (P log rank = 0.012). In conclusion, the secreted recombinant human P53-His produced in P. pastoris seems to be a useful antigen for detection of serum p53 Abs in patients with colorectal carcinoma.     

Circulating anti-p53 antibodies in esophageal cancer patients

Circulating anti-p53 protein antibodies (p53-Abs) have been detected in some cancer patients. The aim of the study was to determine the presence of circulating anti-p53 protein antibodies and their clinical significance in patients with esophageal carcinoma. Serum specimens from 75 consecutive patients with squamous cell carcinomas and 10 healthy subjects were studied. Enzyme linked immunosorbent assay (ELISA--Pharma Cell) was used to detect p53-Abs. At the time of diagnosis 20 (26.6%) of 75 analyzed patients had positive result in the p53-Abs test, but not any of the healthy subjects. The positive rate was 25% (1/4) cases in stage I, 41% (10/24) cases in stage IIA, 0% (0/8) cases in stage IIB, 28% (8/28) cases in stage III and 9% (1/11) cases in stage IV. In respect of tumour differentiation, cases graded as G1, G2 and G3 were positive in 28.5% (4/14), 25.9% (7/27) and 26.4% (9/34), respectively. There was no correlation between presence of p53-Abs and stage, rumour differentiation, lymph nodes metastases, tumour size, patient age and sex. In conclusion, the results of the present study indicate that serum p53-Abs did not correlate with cliniocopathologic feature of esophageal carcinoma.     

P21过表达和P53蛋白蓄积在判断肺癌恶性度与预后的应用研究

应用Ha-ras P21和P53单克隆抗体,采用S-P免疫组织化学方法对86例原发性肺癌进行了研究,前者总阳性率为79.07%,后者为53.48%.细支气管肺泡癌的P21强阳性(++)率明显高于腺泡状腺癌和大细胞癌(P<0.05),分化程度越高,染色越强.阴性(-)和中等阳性(+)的5年存活率明显高于强阳性(++)者.P53蛋白蓄积与肺癌的分型、分化、TNM分期无关,但P53阳性与阴性患者的术后平均存活月数间有着显著的差异(P<0.05),P53染色越强则术后存活月数越短.提示P53蛋白蓄积是判定肺癌预后的指标之一.     

早期非小细胞肺癌患者血清巨噬细胞抑制因子-1、趋化素与临床病理特征及预后的关系

目的:探讨早期非小细胞肺癌(NSCLC)患者血清巨噬细胞抑制因子-1(MIC-1)、趋化素(chemerin)水平与临床病理特征及预后的关系。方法:选择72例NSCLC患者(NSCLC组)、53例肺良性疾病患者(良性组)、50例体检健康人群(对照组),分别检测血清MIC-1、chemerin水平,分析血清MIC-1、chemerin水平与NSCLC患者临床病理参数的关系。Kaplan-Meier法分析不同血清MIC-1、chemerin水平NSCLC患者生存时间的差异,COX比例风险回归分析血清MIC-1、chemerin水平与NSCLC患者预后的关系。结果:NSCLC组患者血清MIC-1、chemerin水平高于良性组和对照组(P0.05)。血清MIC-1水平与NSCLC患者年龄、目前吸烟、肿瘤直径、TNM分期、分化程度、复发或转移、生存状态有关(P0.05),chemerin水平与NSCLC患者目前吸烟、TNM分期、复发或转移、生存状态有关(P0.05)。高MIC-1水平患者生存率低于低MIC-1水平患者(P0.05),高chemerin水平患者生存率低于低chemerin水平患者(P0.05)。COX比例风险回归分析结果显示:血清MIC-1、chemerin、TNM分期与NSCLC不良预后独立相关。结论:血清MIC-1、chemerin水平与NSCLC患者部分临床病理参数和预后相关,可作为早期NSCLC患者预后预测的潜在指标。     

Serum anti-p53 autoantibodies in primary resected non-small-cell lung carcinoma

 Mutated p53 proteins accumulate in the nuclei of tumor cells, and anti-p53 autoantibodies are found in the sera of patients with non-small-cell lung carcinoma (NSCLC). We analyzed the correlation among serum anti-p53 autoantibodies, immunohistochemical staining for p53, and clinical features (age, gender, smoking history, histological type, differentiation, stage, T factor, tumor size, and N factor) in resected non-small-cell lung carcinomas. A total of 62 cases of resected NSCLC were studied (43 men and 19 women; 33 adenocarcinomas, 21 squamous cell carcinomas, 8 large-cell carcinomas). Preoperative serum titers of anti-p53 autoantibodies were detected in 13/62 cases (21.0%). A correlation between histological type and positive titers of serum p53 autoantibodies was seen (large-cell carcinoma versus squamous cell carcinoma and adenocarcinoma, P = 0.031, χ²-test). Out of 25 cases, 10 (40%) with positive immunohistochemical staining for p53 had positive titers, whereas 3 positive titers were found in 37 patients with negative immunohistochemical staining for p53 (P = 0.0025, χ²-test). Serum titers of anti-p53 autoantibodies were present in approximately 20% of the cases of NSCLC, and overexpression of p53 protein in tumor cells was detectable in approximately 40%. Serum anti-p53 autoantibodies may be a clinical parameter for the presence of p53 mutations and p53 overexpression in NSCLC patients. Received: 22 October 1997 / Accepted: 22 April 1998     

Serum Levels of the Cancer-Testis Antigen POTEE and Its Clinical Significance in Non-Small-Cell Lung Cancer

Background

POTEE (POTE ankyrin domain family, member E) is a newly identified cancer-testis antigen that has been found to be expressed in a wide variety of human cancers including cancers of the colon, prostate, lung, breast, ovary, and pancreas.

Aim

To measure the serum levels of POTEE in patients with non-small-cell lung cancer (NSCLC) and to explore the clinical significance of POTEE in NSCLC.

Patients and Methods

104 NSCLC patients, 66 benign lung disease patients and 80 healthy volunteers were enrolled in this study from May 2013 to February 2014. Serum POTEE levels were measured using enzyme-linked immunosorbent assay (ELISA). Numerical variables were recorded as means ± standard deviation (SD) and analyzed by independent t tests. Categorical variables were calculated as rates and were analyzed using a χ² test or Fisher’s exact test. Survival curves were estimated and compared using the Kaplan-Meier method and log-rank tests.

Results

Serum POTEE levels were significantly higher in NSCLC patients than in benign lung disease patients and healthy controls (mean ± SD [pg/ml], 324.38± 13.84 vs. 156.93 ± 17.38 and 139.09 ± 15.80, P<0.001) and were significantly correlated with TNM stage. Survival analysis revealed that patients with low serum POTEE had longer progression-free survival (PFS) than those with high serum POTEE (P=0.021). Cox multivariate analysis indicated that POTEE was an independent prognostic factor of progression-free survival (P =0.009, hazard ratio, 2.440).

Conclusions

Serum POTEE level in NSCLC patients is associated with TNM stage and is a potential prognostic factor.     

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry

In this study the entirep53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations inp53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation inp53. No conclusions or trends could be drawn from the immunohistochemical studies ofBcl-2 andBax.     

凋亡抑制蛋白Livin和抑癌基因p53在非小细胞肺癌中的表达及临床病理学意义

目的检测livin和P53蛋白在非小细胞肺癌(NSCLC)组织中的表达并分析其临床病理学意义。方法采用免疫组织化学(SABC)的方法检测livin和P53蛋白在40例NSCLC患者肺癌组织切片中的表达,分析其与临床病理学参数之间的关系。结果40例NSCLC中livin和P53蛋白阳性表达率分别为47.5%和52.5%,在淋巴结转移的NSCLC组织中livin的阳性率明显高于无淋巴结转移的肺癌组织(P<0.05),且与肺癌不同分期有关(P<0.05)。P53蛋白表达亦与肿瘤分期有关,P53在鳞癌中的表达率显著高于腺癌(P<0.05)。P53和livin均与年龄、性别、肿瘤分化程度等临床病理学参数无关(P>0.05)。Livin与P53蛋白的表达无明显相关性(P>0.05)。结论livin与P53异常表达与NSCLC的恶性生物学行为有关,其表达对临床上判断NSCLC的预后有重要意义。     

53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer

53BP1 is a conserved nuclear protein that is implicated in the DNA damage response. After irradiation, 53BP1 localizes rapidly to nuclear foci, which represent sites of DNA double strand breaks, but its precise function is unclear. Using small interference RNA (siRNA), we demonstrate that 53BP1 functions as a DNA damage checkpoint protein. 53BP1 is required for at least a subset of ataxia telangiectasia-mutated (ATM)-dependent phosphorylation events at sites of DNA breaks and for cell cycle arrest at the G2-M interphase after exposure to irradiation. Interestingly, in cancer cell lines expressing mutant p53, 53BP1 was localized to distinct nuclear foci and ATM-dependent phosphorylation of Chk2 at Thr 68 was detected, even in the absence of irradiation. In addition, Chk2 was phosphorylated at Thr 68 in more than 50% of surgically resected lung and breast tumour specimens from otherwise untreated patients [corrected]. We conclude that the constitutive activation of the DNA damage checkpoint pathway may be linked to the high frequency of p53 mutations in human cancer, as p53 is a downstream target of Chk2 and ATM.     

P53抗体联合CYFRA21-1和CEA在非小细胞肺癌中的应用

目的：研究血清P53抗体在非小细胞肺癌临床病理特征之间的关系,并结合血清中的癌胚抗原、角质蛋白21-1以指导对临床上肺癌复发和转移的分析,用来选择合理的治疗方案。方法：正常组30例,肺良性疾病组10例,肺癌组45例,肺癌全组分别于手术前1天、术后10、30、60和90天时抽取清晨空腹静脉血2ml,23例肺癌病例于手术后120天,15例病例于手术后180天抽取清晨空腹静脉血2ml,肺良性疾病组分别于手术前1天、抽取清晨空腹静脉血2ml。正常组清晨空腹采集静脉血2ml。采用酶联免疫吸附法（ELISA）检测血清P53抗体和角质蛋白21-1,采用荧光酶标免疫法检测血清癌胚抗原。结果：血清P53抗体、CYFRA21-1和CEA在正常人组、良性疾病组、肺癌组术前阳性率的比较三种肿瘤标志物阳性率经X2检验,在肺癌组分别与正常人组和良性病例组有显著性差异（P〈0.05）,良性病例组和正常人组之间无显著行差异（P〉0.05）。并与手术后复发与转移相关。结论：联合检测癌胚抗原、角质蛋白21-1及血清P53抗体水平有助于肺部良恶性疾病的诊断;手术前后动态测定肺癌患者血清P53抗体和角质蛋白21-1的变化规律,有助于判断疗效,监测预后和指导肺癌术后的综合治疗。     

Peroxiredoxin-I is an autoimmunogenic tumor antigen in non-small cell lung cancer

In eukaryotic cells, peroxiredoxins are both antioxidants and regulators of H(2)O(2)-mediated signaling. We previously found that peroxiredoxin-I (Prx-I) was overexpressed in non-small cell lung cancer (NSCLC) tissue. Since overexpressed protein can induce a humoral immune response, we examined whether serum from NSCLC patients exhibited immunoreactivity against Prx-I using Western blotting. We found that 25 (47%) of 53 NSCLC patients tested had autoantibodies against Prx-I in their sera, whereas such activity was detected in 4 (8%) sera from 50 healthy subjects. Prx-I itself was detected in the sera from 18 (34%) of 53 NSCLC patients but in only 1 (2%) serum from 50 controls. Moreover, 17% of NSCLC sera were positive to both Prx-I antibody and antigen but none in control sera. The data indicate both Prx-I autoantibody and circulating antigen are potential biomarkers for use in serological diagnosis of NSCLC. Interestingly enough, we found that Prx-I was secreted by lung adenocarcinoma cells (A549) but not by non-cancer lung cells (BEAS 2B) or breast cancer cells (MCF7). This cell culture study suggests the possibility of Prx-I secretion from NSCLC tumor tissues.     

非小细胞肺癌患者血清CYFRA21-1、VEGF及CEA的表达及与临床病理特征的相关性研究

目的:研究非小细胞肺癌(NSCLC)患者血清细胞角蛋白19片段(CYFRA21-1)、血管内皮生长因子(VEGF)、癌胚抗原(CEA)的表达及与临床病理特征的相关性。方法:选取2015年12月至2016年4月在我院接受治疗的NSCLC患者120例作为观察组,另选取同期在我院接受治疗的肺部良性病变患者50例作为良性对照组,比较两组患者血清中CYFRA21-1、VEGF及CEA的表达,分析观察组患者血清中CYFRA21-1、VEGF及CEA的表达与临床病理特征之间的关系,采用Pearson相关系数分析观察组患者血清中CYFRA21-1、VEGF、CEA的相关性。结果:观察组患者血清中的CYFRA21-1、VEGF及CEA水平均高于良性对照组(P0.05)。鳞状细胞癌患者血清中CYFRA21-1水平高于腺癌患者,CEA水平低于腺癌患者(P0.05),鳞状细胞癌患者和腺癌患者血清中VEGF水平比较无统计学差异(P0.05)。TNM分期为III-IV期的观察组患者血清中CYFRA21-1、VEGF及CEA水平均明显高于I-II期患者,有统计学差异(P0.05)。经Pearson相关系数分析显示,观察组患者血清中CYFRA21-1与VEGF、CEA呈正相关(r=0.512,0.423,P=0.000,0.000),VEGF与CEA呈正相关(r=0.452,P=0.000)。结论:NSCLC患者血清中CYFRA21-1、VEGF及CEA呈高表达,且CYFRA21-1、CEA与病理类型和TNM分期有关,VEGF与TNM分期有关,且三种指标存在一定的相关性。     

Livin蛋白在非小细胞肺癌中的表达及其临床意义

目的：研究livin蛋白在非小细胞肺癌中的表达及其与非小细胞肺癌的生物学特性及临床预后的关系。方法：通过免疫组化的方法检测和比较88例非小细胞肺癌组织和20例癌旁正常肺组织中livin蛋白的表达,并分析其与非小细胞肺癌的临床病理特征和预后的相关性。结果：非小细胞肺癌组织及癌旁正常肺组织中livin蛋白的阳性表达率分别为54．55％和5％,差异有显著统计学差异（P〈0．05）。非小细胞肺癌组织中livin蛋白的表达水平与淋巴结转移、TNM分期显著相关（P〈O．05）,但与患者的性别、年龄、分化程度及病理学类型无关（P〉0．05）。Livin高表达的非小细胞肺癌患者生存时间显著短于livin低表达的患者（P〈0．05）。结论：Livin蛋白在非小细胞肺癌的发生及发展中起重要作用并与患者的预后相关,可能作为非小细胞肺癌新的防治靶点。     

血清Lipocalin-2、Periostin及AGAP2-AS1在非小细胞肺癌中的表达及与病理特征的相关性

摘要 目的：探究血清脂质运载蛋白-2（Lipocalin-2）、成骨细胞特异性因子2（periostin）及长链非编码RNA NR_027032（AGAP2-AS1）表达与非小细胞肺癌（NSCLC）患者临床特征及预后的相关性。方法：选取2015年12月-2017年12月到我院确诊的84例NSCLC患者为研究组,选取同时期在我院健康体检的健康人群为健康对照组,采用ELISA法检测血清Lipocalin-2、periostin水平、采用荧光定量PCR定量检测血清外泌体AGAP2-AS1的表达水平,并分析其表达差异性;分析血清Lipocalin-2、Periostin及AGAP2-AS1水平与NSCLC患者各临床病理特征及预后的相关性。结果：NSCLC组患者血清Lipocalin-2、Periostin及AGAP2-AS1表达水平显著高于健康对照组（P<0.05）,且与淋巴结转移、TNM分期及分化程度具有相关性（P<0.05）,血清Lipocalin-2与Periostin及AGAP2-AS1在NSCLC血液中呈正相关（P<0.01或<0.05）,血清Lipocalin-2、Periostin及AGAP2-AS1高表达组NSCLC患者中位OS分别显著低于低表达（P<0.05）。结论：血清Lipocalin-2、Periostin及AGAP2-AS1在NSCLC患者血液中的表达升高,NSCLC患者血清Lipocalin-2、Periostin及AGAP2-AS1表达水平与分化程度、TNM分期、淋巴结转移及预后具有相关性;有望成为评估NSCLC患者预后的生物标志物。     

p27~(Kip1)和CyclinD1在非小细胞肺癌中表达及意义

为了探讨p27Kip1蛋白和CyclinD1蛋白在非小细胞肺癌(NSCLC)中的表达及意义,收集临床手术切除的非小细胞肺癌组织蜡块64例及正常肺组织10例,应用免疫组化(S-P法)检测组织中p27Kip1蛋白和CyclinD1蛋白的表达,结合临床病理资料和随访资料进行回顾性研究。实验发现NSCLC组织中p27Kip1蛋白表达和CyclinD1蛋白表达均明显不同于正常肺组织(P<0.01)。p27Kip1蛋白表达降低与NSCLC肿瘤大小、病理分级、分期增加、淋巴结转移之间有相关性(P<0.05),但与肿瘤组织学分型无相关性(P>0.05)。CyclinD1蛋白过表达与组织学分型、肿瘤大小、病理分级、临床分期、淋巴结转移无相关性(P>0.05)。p27Kip1蛋白表达与CyclinD1蛋白表达之间呈显著负相关(P<0.01)。cox单因素及多因素分析,p27Kip1蛋白低表达及CyclinD1过表达是影响NSCLC患者预后的主要因素。实验结果显示,NSCLC组织中,p27Kip1蛋白表达降低,而CyclinD1过表达,二者与NSCLC的发生发展机制有关,可作为预后指标,有利于NSCLC患者预后判断及个体化治疗。     

Ornithine aminotransferase promoted the proliferation and metastasis of non-small cell lung cancer via upregulation of miR-21

The incidence and mortality of lung cancer ranked the first among all types of cancer in China, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for 85% of all lung cancers. Given that the survival rate of patients with advanced NSCLC is still poor nowadays, identification of novel therapeutic targets and the development of effective therapies are desired for the treatment of NSCLC in clinics. In this study, we reported the upregulation of ornithine aminotransferase (OAT) in NSCLC cells and clinical tumor samples as well as its association with the advanced TNM stage, metastasis, and poor tumor differentiation of lung cancer. Using different NSCLC cell lines, we demonstrated that OAT promoted the proliferation, invasion, and migration, inhibited the apoptosis, and altered cell cycle of NSCLC cells; besides, the involvement of OAT-miR-21-glycogen synthase kinase-3β signaling in the functional role of OAT in NSCLC was also revealed. Importantly, in the absence of OAT, the growth and metastasis of tumor lung cancer xenograft was significantly suppressed in the nude mice. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.     

Fn14 在 EGFR 外显子 19 缺失突变的非小细胞肺癌中的表达及其临床意义

目的:探讨成纤维细胞生长因子诱导14(Fn14)在表皮生长因子受体(EGFR)外显子19缺失的非小细胞肺癌(NSCLC)组织中的表达及其临床意义。方法:选择2010年9月至2013年11月第四军医大学唐都医院收治的125例原发性EGFR外显子19缺失的NSCLC组织及与之相对应的30例正常肺组织为研究对象,应用免疫组化法检测和比较其Fn14的表达,分析Fn14的表达与原发性EGFR外显子19缺失的的NSCLC临床病理特征的关系。结果:Fn14阳性表达主要定位于胞膜和胞质中。在125例原发性EGFR外显子19缺失的NSCLC组织中,Fn14的阳性表达率为100%,显著高于正常肺组织(P<0.001)。鳞癌和腺癌NSCLC组织中Fn14的阳性表达率比较无统计学差异(P=0.106);不同病理分化程度、不同TNM分期的NSCLC组织中Fn14的阳性表达率比较均具有显著性差异(P=0.000、P=0.000)。Fn14蛋白的表达与EGFR外显子19缺失的NSCLC的淋巴结转移状态、肿瘤大小以及肿瘤解剖学分类均显著相关(P=0.000、P=0.029、P=0.000、P=0.026),而与患者的性别、年龄、吸烟史均无关(P=0.816、P=0.122、P=0.816)。结论:Fn14在原发性EGFR外显子19缺失的NSCLC中呈异常高表达,EGFR外显子19缺失突变很可能通过上调Fn14通路,促进NSCLC的发生和发展。