Diagnostic and prognostic significance of human kallikrein 11 (KLK11) mRNA expression levels in patients with laryngeal cancer

ObjectivesHuman kallikrein 11 gene (KLK11) encodes a secreted serine protease. In view of its diagnostic and prognostic strength in many malignancies, we investigated the mRNA expression levels of KLK11 in laryngeal tissues in order to unveil its clinical usefulness in laryngeal cancer.Design and methodsKLK11 expression was quantified in 163 tissue samples from 105 laryngeal cancer patients with the development of a highly sensitive real-time PCR methodology, using SYBR Green® chemistry.ResultsKLK11 expression in laryngeal cancer specimens of primary or recurrent nature was significantly inferior compared with their non-malignant counterparts (P < 0.001 and P = 0.026, respectively), a finding of immense diagnostic value as illustrated in the ROC curve analyses (P < 0.001). Survival analysis showed that patients harboring KLK11-positive tumors had a significantly decreased risk of death (HR = 0.26, P = 0.042).ConclusionsOur data recommend KLK11 mRNA expression as a novel and independent biomarker in laryngeal cancer for diagnostic and prognostic purposes.     

Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: comparison of real-time reverse transcription-PCR vs. relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products

The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.     

MAL基因mRNA在乳腺癌组织中表达的研究

目的 检测MAL基因mRNA在乳腺癌组织中的表达,探讨其与乳腺癌组织中雌激素受体(ER)、孕激素受体(PR)、表皮细胞生长因子受体-2(C-erbB-2)阳性表达的相关性.方法 采用RT-PCR的技术手段检测MAL基因mRNA在乳腺癌组织中的差异性表达;应用免疫组化SP法检测ER、PR、C-erbB-2在乳腺癌组织中的表达.结果 MAL基因mRNA在乳腺癌癌旁组织中表达的阳性率为100%,在乳腺癌组织中表达的阳性率为20%;≤50岁的乳腺癌患者,其MAL基因mRNA的相对表达水平为0.78±0.17,>50岁者其MAL基因mRNA的相对表达水平为0.70±0.18,两组比较差异无统计学意义(t=1.616,P>0.05);MAL基因mRNA在<2 cm组的乳腺癌组织中的表达水平为0.94±0.21,在≥2 cm组的乳腺癌组织中的表达水平为0.51±0.12,两组比较差异具有统计学意义(P<0.001);MAL基因mRNA在淋巴结阴性组乳腺癌组织中的表达水平是0.81±0.21,在淋巴结阳性组中的表达水平是0.40±0.16,两组比较差异具有统计学意义(P<0.001);经Spearman关联性分析,MAL基因mRNA在乳腺癌组织中的阳性表达与ER和PR在乳腺癌组织中的阳性表达无相关性,而与C-erbB-2在乳腺癌组织中的阳性表达呈负相关(r=-0.347,P=0.047).结论 MAL基因mRNA在乳腺癌组织中的表达水平较正常乳腺组织中明显降低,与肿瘤大小、淋巴结密切相关,而与C-erbB-2在乳腺癌组织中的阳性表达呈负相关.     

人乳腺癌细胞株KLK5 mRNA的表达及雌激素的调节作用

目的 探讨雌激素对雌激素受体阳性人乳腺癌细胞株MCF-7和B37组织激肽释放酶5（KLK 5）mRNA的表达水平,以及雌激素对KLK5 mRNA表达水平的影响。方法 分别取生长良好的MCF-7和B37细胞,在不同浓度的雌激素（17-βE2）存在下培养72小时后,收集细胞,提取总RNA,采用荧光定量逆转录-聚合酶链反应,检测KLK5 mRNA表达水平及变化。结果 在未经刺激的乙醇对照组,MCF-7细胞KLK5 mRNA的相对表达水平（KLK5 mRNA/GAPDH mR-NA）为（3.23±0.51）×10^-5,B37细胞KLK5 mRNA表达水平为（3.13±0.62）×10^-4。B37细胞KLK5 mRNA表达水平高于MCF-7细胞。当17-βE2浓度10^-11mol/L以下时,MCF-7和B37细胞中KLK5 mRNA表达水平与乙醇对照组无明显差异（P〈0.05）。随17-βE2浓度的增高,MCF-7和B37细胞中KLK5 mRNA的表达呈递增趋势,各组与乙醇对照组相比差异有显著意义（P〈0.01）。结论 雌激素对人乳腺癌细胞株MCF-7和B37细胞的KLK5 mRNA表达有剂量依赖的上调作用。     

Development of an immunofluorometric assay for human kallikrein 15 (KLK15) and identification of KLK15 in tissues and biological fluids

BACKGROUND: Human kallikrein 15 (KLK15) may have some utility as a prostate, ovarian, and breast cancer biomarker, based on previous studies, which examined mRNA levels of KLK15. The aim of this study was to develop analytical technology for human kallikrein 15, including recombinant protein, specific antibodies, and a sensitive and specific ELISA immunoassay. The assay was then used to examine levels of KLK15 in tissues and biological fluids. METHODS: We produced human, recombinant pro-KLK15 in HEK 293 cells. Recombinant KLK15 was purified with various chromatographic steps and used to immunize rabbits and mice for production of KLK15 polyclonal antibodies. We used these antibodies to develop a highly sensitive and specific KLK15 immunoassay and to study KLK15 expression in various tissues and biological fluids. RESULTS: Large amounts of pure, recombinant KLK15 have been produced and characterized. KLK15 mouse and rabbit polyclonal antibodies have been employed for development of a KLK15 immunoassay. This assay has a lower detection limit of 0.05 microg/L, and no cross-reactivity with any of the other fourteen kallikreins. Using this assay, KLK15 was detected in prostate, colon, and thyroid tissues, as well as in breast milk and seminal plasma. CONCLUSIONS: The KLK15 reagents developed here will allow for analysis of KLK15 protein expression levels in tissues and biological fluids, both normal and cancerous. This will expand upon previously characterized tissue KLK15 mRNA expression studies which suggested that KLK15 might be useful as a biomarker for breast, ovarian, and prostate cancer. KLK15 is another serine protease that is produced in prostate and other tissues and is secreted in seminal plasma and other fluids. Its physiological function needs to be further elucidated.     

Unfavorable prognostic value of human kallikrein 7 quantified by ELISA in ovarian cancer cytosols

BACKGROUND: Human tissue kallikrein 7 (gene, KLK7; protein, hK7) is a member of the kallikrein family of secreted serine proteases. Reports indicate that in ovarian cancer, KLK7 is significantly up-regulated at the mRNA level. The aim of this study was to determine whether hK7, measured quantitatively by ELISA in ovarian cancer cytosols, is a prognostic biomarker for ovarian cancer. METHODS: We used a newly developed ELISA with 2 monoclonal antibodies to quantify hK7 production in 260 ovarian tumor cytosols and correlated these data with various clinicopathologic variables and patient outcomes [progression-free survival (PFS) and overall survival (OS)] over a median follow-up period of 52 months. RESULTS: Median (range) hK7 concentration in ovarian tumor cytosols was 2.84 (0-32.8) ng/mg of total protein. Compared with healthy and benign ovarian tissues and nonovarian tumors that metastasized to the ovary, malignant ovarian tumor cytosols highly overproduced hK7 (P <0.001). We used the median value as the cutoff value to categorize tumors as hK7-positive and hK7-negative. Women with hK7-positive tumors most frequently had advanced-stage disease, higher tumor grade (G3), suboptimal debulking, and serous or undifferentiated histotype (P <0.001). Univariate analysis showed that hK7 positivity was associated with significantly shorter PFS (P = 0.01) but not OS. Kaplan-Meier survival curves confirmed an increased risk of relapse in women with hK7-positive tumors (P = 0.009). In multivariate analysis, hK7 was not significantly associated with either PFS or OS. CONCLUSIONS: hK7 is associated with other unfavorable characteristics of ovarian cancer, but it is not an independent prognosticator for ovarian cancer.     

妊娠中肾细胞因子mRNA在人食管癌组织中的表达

目的 研究妊娠中肾细胞因子（midkine, MK)mRNA在食管癌组织中的表达。方法 用Trizol提取食管癌组织和相应癌旁正常组织RNA，经RT－PCR获得扩增的MK cDNA，溴化乙锭琼脂糖凝胶电泳检测PCR产物。结果 6例食管癌组织在450bp处均出现一条MK的特征条带，其中1例低分化食管癌MK条带最深。6例癌旁正常组织均检测不出MK mRNA。此外，2例食管癌组织除显示MK的特征条带外，在280bp处出现一条截短型MK（tMK)区带。结论 MK在食管癌组织特异表达，表达程度可能与细胞分化程度相关。     

两种定量聚合酶链反应模式检测乳腺癌组织中缓激肽释放酶表达的比较

目的比较两种荧光定量聚合酶链反应（FQ-PCR）检测乳腺癌组织中激肽释放酶6（KLK6）基因的表达。方法分别用TaqMan探针和SYBR Green I荧光染料作为荧光指示剂,实时荧光定量PCR检测KLK6在乳腺癌中的相对表达水平,应用统计软件分析两种检测模式的差异。结果两种检测模式的检测结果均显示乳腺癌中KLK6的表达均高于正常组织,KLK6的相对表达水平无显著性差异。结论在扩增产物特异的条件下应用SYBR Green Ⅰ检测的效果同Taq Man探针,SYBR Green Ⅰ染料应用范围广泛,成拳低。     

乳腺癌组织KLK5mRNA的转录及其与临床病理特征的关系

目的对正常乳腺组织和癌组织KLK5 mRNA进行定量,分析其表达与临床病理特征的关系,探讨其临床意义。方法用实时荧光定量PCR检测48例乳腺癌切除的组织中KLK5基因mRNA的表达量,分析其表达量与年龄、TNM分期、组织学类型、淋巴结转移、雌激素受体(ER)、孕激素受体(PR)等临床病理特征的关系。结果79%(38/48)的乳腺癌组织KLK5 mRNA表达下降。KLK5 mRNA在正常乳腺组织的相对表达水平为0.338±0.324,在乳腺癌组织为0.088±0.128,明显低于正常组织(P<0.01)。KLK5 mRNA在有淋巴结转移的乳腺癌患者组织中的表达水平明显高于未转移组(P<0.01);在Ⅲ~Ⅳ期患者乳腺癌组织中高于Ⅰ~Ⅱ期患者(P<0.01);ER( )阳性乳腺癌组织明显低于ER(-)乳腺癌组织(P<0.01)。KLK5 mR-NA表达水平与患者年龄、组织分类以及PR状态无相关性(P>0.05)。结论乳腺癌组织KLK5 mRNA表达水平降低,并与TNM分期、ER状态及淋巴结转移与否相关。     

Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.     

Prognostic value of quantitatively assessed KLK7 expression in ovarian cancer

BACKGROUND: Among females, ovarian cancer is the sixth most common malignancy. Women with ovarian cancer have poor overall survival rates, largely because the disease is often diagnosed at an advanced, less curable stage. Several lines of evidence suggest that members of the kallikrein family are involved in various malignancies such as prostate (PSA, KLK2, KLK15), ovarian (KLK4, KLK5, KLK6, KLK8, KLK10), and breast cancer (KLK10, KLK13, KLK14). Recent evidence has indicated that expression of KLK7 appears to be increased in ovarian cancer. We hypothesized that overexpression of the KLK7 gene in ovarian cancer may serve as a prognostic marker of the disease. METHODS: Using the LightCycler technology we quantified the level of KLK7 mRNA expression in 125 ovarian tumors. Different disease stages and tumor grades were analyzed. Univariate and multivariate analyses were performed to establish the associations between clinicopathological parameters and KLK7 expression. RESULTS: We here report that patients with KLK7-negative tumors have a significantly higher disease-free survival than patients with KLK7-positive tumors. KLK7 expression levels were significantly higher in patients with grade 3 than in patients with grade 1 to 2 tumors (p = 0.030). KLK7 status also correlated with size of residual tumor postsurgery. KLK7 expression is an independent predictor of both disease-free and overall survival for patients with low grade tumors. In this subgroup of patients the hazard ratios for disease-free and overall survival were 3.28 and 3.09, respectively. Similarly, patients who had undergone optimal debulking but harbored KLK7-positive tumors had a high hazard ratio (HR) for relapse (HR = 8.2) and death (HR = 4.6). CONCLUSIONS: We conclude that higher KLK7 expression in ovarian cancer tissue is associated with poorer prognosis of ovarian cancer patients, especially those with lower grade disease and those who have been optimally debulked.