Plasma Of Non-small Cell Lung Cancer Patients Research Articles

Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer

Background:Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals.Methods:Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals.Results:The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan–Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS.Conclusions:Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.

Abstract 4157: Identification and validation of galectin-7 as a novel blood biomarker for NSCLC diagnosis

Abstract Lung cancer is the leading cause of cancer-related moralities in the US and worldwide. Non small cell lung cancer (NSCLC) accounts for 80 % of all diagnosed lung cancer cases. The lack of reliable, non-invasive diagnostic tests prompted the search for viable diagnostic protein markers. We report here the identification and validation of galectin7 as a novel blood-based biomarker for diagnosis of NSCLC. To identify potential blood protein markers that could be used for diagnosis of NSCLC, we implemented a multiphase validation scheme exploiting a large pool of novel protein markers shown to be over expressed in NSCLC tissues by our group by shotgun- mass spectrometry. Top candidates were first selected using bioinformatics tools including Ingenuity Pathway Analysis (IPA), Web gestalt and DAVID by applying a combination of statistical and biological filters including expression levels in cancer tissues, reported presence in plasma and other biofluids, and statistical significance. The resulting list of candidates was further refined by cross examination against other publically available data bases such as Plasma Proteome project of the Human Proteome Organization (PPP-HUPO) and Oncomine. We decided to focus our efforts on galectin 7, one of our top candidates that has not been reported previously as a biomarker for lung cancer. A case-control pilot study was designed to evaluate the relative levels of galectin 7 in plasma samples of peripheral blood from a small cohort of 30 NSCLC and 15 control nonmalignant lung disease patients using ELISAs. The results of this study were subjected to a second round of validation in an independent set of plasma samples from controls and NSCLC patients. Finally, we verified the identity of our protein candidates by means of immunoprecipitation (IP) and Western blot analyses. Statistical analysis of our ELISA data on galectin7 using the Wilcoxon cox nonparametric test revealed that galectin7 was detected at significantly higher levels in NSCLC patient plasma compared to controls. To further validate our findings, a second round of interrogation was performed using a separate sample set of 30 plasma samples and their matched tissues from nonmalignant and NSCLC patients. To verify the identity of our protein markers detected by ELISA, we successfully detected galectin 7 from both tissue lysates and plasma from NSCLC patients by Western blot analysis. Based on our preliminary results, we believe that we present the first evidence that galcetin 7 is a potential diagnostic blood-based biomarker for NSCLC. Retrospective clinical studies are currently underway to test the utility of galectin7 for diagnosis of early stage NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2011-4157

ENO1 protein levels in the tumor tissues and circulating plasma samples of non-small cell lung cancer patients

背景与目的肺癌严重威胁人类生存健康，有效的肿瘤标志物可以辅助诊断、判断预后和指导治疗。本研究旨在检测非小细胞肺癌患者肿瘤组织和血浆中烯醇化酶1（alpha-enolase, ENO1）蛋白水平，初步探讨ENO1作为肺癌相关蛋白标志物的可能性。方法采用Western blot方法检测16例肺鳞癌患者的肿瘤组织及其配对正常肺组织中ENO1蛋白水平。采用双抗体夹心酶联免疫吸附分析（enzyme-linked immunosorbent assay, ELISA）测定42例健康体检者、34例肺部良性疾病患者和84例非小细胞肺癌患者三组人群血浆中ENO1蛋白水平。结果在87.5%（14/16）的肺鳞癌患者肿瘤组织中ENO1蛋白表达量高于其配对正常肺组织；非小细胞肺癌患者血浆中ENO1蛋白水平高于健康体检者（P=0.031）和肺部良性疾病者（P=0.019），且ENO1蛋白在肺腺癌患者血浆中的水平高于肺鳞癌患者（P=0.023）。结论非小细胞肺癌肿瘤组织和血浆中ENO1蛋白水平升高，提示ENO1可作为潜在的非小细胞肺癌相关血浆蛋白标志物。

Abstract B180: A novel mutant-enriched liquidchip technology (MEL) for detecting circulating EGFR somatic mutations in non small cell lung cancer

Abstract Epidermal growth factor receptor (EGFR) is a key target for developing targeted therapeutics to treat cancer. The US FDA has approved some EGFR targeted drugs that include monoclonal antibody drugs Erbitux, Panitumumab, as well as the EGFR tyrosine kinase inhibitors Iressa and Tarceva. However, clinical data indicated that only a small percentage of patients were responsive to these targeted drugs. Further studies have revealed that up to 20% of non small cell lung cancer (NSCLC) tumors that harbor the EGFR Exon 19 or 21 somatic mutations are more likely responsive, while patients without these mutations are not responsive. Therefore, physicians have a responsibility to test whether or not the patient's EGFR is mutated before prescribing these targeted drugs. In general, a very small amount of free DNA fragments can be found in the blood, especially in patients with malignant tumors. However, it is technically very difficult to detect a very small amount of DNA somatic mutations that are usually masked under a huge amount of the wild-type (WT) sequence. To cater to the clinical need, we have successfully developed a novel technology that creatively combines a mutant-enriched method to a liquidchip platform. The principle of MEL is to create a unique restriction enzyme site at the WT gene sequence in order to remove the WT gene by the restriction enzyme digestion before the mutated gene sequence could be selectively amplified by the PCR. The mutated PCR product will then be hybridized to a specific probe on polystyrene microspheres and analyzed by the Luminex. This technology is extremely sensitive and therefore allows us to use the serum or plasma sample to detect somatic gene mutations. It is also high-throughput; which allows us to assay up to 96 samples at once and read up to 100 different mutations from one sample. MEL technology has been successfully used in the clinic for detecting other somatic mutations from serum and plasma samples as well, such as KRAS, BRAF, PIK3CA, EGFR exon 20, etc. The detection sensitivity is one mutant DNA in the presence of 1×103 wild-type copies. As low as 10 copies of mutant DNA in a sample could be detected. To evaluate the MEL, the EGFR mutation status of NSCLC was tested on tissue samples using both sequencing and MEL methods. The positive samples of EGFR mutation by sequencing method were in agreement with the results by the MEL. However, 31.8% (7/22) of the negative samples by sequencing were positive by MEL. To date, our results have shown that 32.1% (146/455) serum samples of NSCLC patients showed EGFR mutant positive, from which 9.2% (42/455) were exon 19 deletion mutations; 19.8% (90/455), exon 21 single nucleotide polymorphism mutation; and 3.1% (14/455), both. Our study also showed that EGFR mutations were detected more frequently in patients with partial response following the targeted therapy than those with stable disease or progressive disease (p=0.027). In conclusion, MEL is a valuable method for detecting circulating EGFR mutations in serum or plasma of NSCLC patients for pharmacogenomic diagnostics. This method could be used on tissue samples as well. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B180.

Serum Proteomic Classifier for Predicting Response to Epidermal Growth Factor Receptor Inhibitor Therapy: Have We Built a Better Mousetrap?

The ability to predict treatment response and clinical (survival) outcomes is one of the two “holy grails” of cancer biomarker development, the other being early detection or risk stratification by population screening ( 1 ). In advanced non – small-cell lung cancer (NSCLC), small-molecule tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) represent a breakthrough for targeted therapy, yet only a small proportion of patients (especially among non – East Asians) appear to benefit from these expensive agents ( 2 , 3 ). Previous studies have shown that EGFR immunohistochemistry, tyrosine kinase domain mutations, and/or EGFR gene copy number by fluorescent in situ hybridization (FISH) are biomarkers that are potentially useful to select patients more likely to benefit from EGFR TKIs ( 4 – 6 ). Aside from the incomplete validation of these markers for routine clinical implementation, mutation and FISH analyses are limited by tissue availability and, at times, technical feasibility ( 5 ). Therefore, a strongly predictive but simple blood-based test has great potential for becoming the ultimate biomarker for selecting patients who are likely to benefit from EGFR TKI therapy. In this issue of the Journal, Taguchi et al. ( 7 ) report a proteomic algorithm (signature) that classifi es patients according to their likely outcomes after EGFR TKI therapy. This algorithm was generated by matrix-assisted laser desorption ionization (MALDI) mass spectroscopy (MS) analysis of pretreatment sera or plasma of patients. The authors used a training set of 139 patients treated with second- or greater-line gefi tinib who were recruited from three separate institutions in Europe or Asia to discover eight MALDI signature peaks that form the predictive algorithm. This classifi er was then validated in two independent cohorts of gefi tinibor erlotinib-treated patients from Italy and the United States. In fact, this study marks the fi rst large-scale multi-institutional evaluation of an MS-based protein signature in the serum or plasma of NSCLC patients for predicting EGFR TKI – related clinical outcomes. The study showed that, despite variability in the source or nature of samples studied and in the spectra generated by different operators, the data preprocessing and normalization procedures were able to generate MALDI MS features that were reproducible in two independent laboratories. Furthermore, the clinical outcome classifi er (which classifi ed patients into good, poor, or undefi ned classes) was validated in two independent cohorts of patients receiving fi rst- or greater-line gefi tinib or erlotinib therapy. More important, the authors claim that the algorithm is predictive and not prognostic, in that its classifi cation capability failed in three separate cohorts of patients who were not treated with EGFR TKIs. The authors conclude that their eight-peak MALDI MS algorithm might assist in the pretreatment selection of appropriate subgroups of NSCLC pa tients for

Interim results of two prospective trials measuring plasma total DNA and methylated genes before and after surgery, or chemotherapy, in patients with non-small cell lung cancer (NSCLC)

7123 Background: Measuring DNA in plasma using quantitative RT-PCR is a promising method for surveillance of NSCLC. This strategy has the potential to assess response more accurately or quickly than radiologic tests, or to confirm the success of surgical resection and select patients for adjuvant therapy. Methylated genes may be more specific than total DNA for the presence of cancer. Methods: We are conducting two prospective trials measuring total DNA, and 10 commonly hypermethylated genes in the plasma of NSCLC patients before and after therapy. For patients undergoing surgery, plasma samples are collected before, and at three timepoints after surgery (3–8 d, 2–5 w, 2–4 m). For patients on chemotherapy, collections are before, and coincident with up to 3 radiologic response assessments. DNA is extracted from a fixed volume of plasma, and measured using quantitative PCR of the β-Actin gene. After treating DNA with sodium metabisulfite, methylation-specific RT-PCR is performed to amplify the promoter region of APC, p16, MGMT, GSTP1, DAPK, CDH1, RASSF1A, WIF-1, SOCS-3, METH-2, assuming the promoter is fully methylated. Gene levels are normalized to β-Actin in modified DNA. Results: To date, plasma analysis has been completed on 50 patients with advanced NSCLC undergoing chemotherapy (320 planned), and 60 patients with early-stage NSCLC undergoing surgery (400 planned). Pretreatment levels of total DNA are higher in metastatic NSCLC (median 9.2, range 2.2–1020 ng/ml) compared to early-stage (median 3.6, range 0–1280 ng/ml), p &lt; 0.001 by Wilcoxon rank sum test. At least one methylated gene is detectable in pretreatment plasma in 36% of patients with metastatic NSCLC, and 20% of patients with early-stage NSCLC. Conclusions: Methylated genes are rarely found in the pre-treatment plasma of NSCLC patients in this experiment, despite testing a panel of 10 genes. Plasma total DNA levels correlate with NSCLC stage. Correlation of baseline levels of total DNA, and temporal trends during treatment, to overall survival and radiologic response are planned. Supported by R01-CA092315. [Table: see text]

Increased plasma levels of urokinase plasminogen activator and matrix metalloproteinase-9 in nonsmall cell lung cancer patients

Background The involvements of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) in the pathogenesis of various types of lung cancers have been established. The aim of the present study was to determine the concentrations of MMP-9 and uPA in the plasma of nonsmall cell lung cancer (NSCLC) patients. Methods Gelatin zymography and ELISA were used to measure MMP-9 and uPA concentrations, respectively, in 90 NSCLC patients and 50 control subjects. Results Compared with that of control subjects, MMP-9 and uPA levels were significantly higher in the plasma of NSCLC patients ( P<0.001 and P<0.01, respectively). Further statistical analysis for clinic pathological parameters revealed that MMP-9 and uPA levels were significantly increased in patients with metastasis ( P<0.01 and P<0.05, respectively). Furthermore, the MMP-9 level was significantly different between smokers and nonsmokers ( P<0.05) while uPA levels varied between histological types ( P<0.05). Conclusions Plasma MMP-9 and uPA might play an important role in the metastasis of NSCLC by involvement in the processes of invasion and metastasis.