Five-gene Signature Research Articles

DNA replication stress response involving PLK1, CDC6, POLQ, RAD51 and CLASPIN upregulation prognoses the outcome of early/mid-stage non-small cell lung cancer patients

Lung cancer is the leading cause of cancer deaths worldwide. Clinical staging classification is generally insufficient to provide a reliable prognosis, particularly for early stages. In addition, prognostic factors are therefore needed to better forecast life expectancy and optimize adjuvant therapeutic strategy. Recent evidence indicates that alterations of the DNA replication program contribute to neoplasia from its early stages and that cancer cells are frequently exposed to endogenous replication stress. We therefore hypothesized that genes involved in the replication stress response may represent an under-explored source of biomarkers. Expressions of 77 DNA replication-associated genes implicated in different aspects of chromosomal DNA replication, including licensing, firing of origins, elongation, replication fork maintenance and recovery, lesion bypass and post-replicative repair were determined in primary tumors and adjacent normal tissues from 93 patients suffering from early- or mid-stage non-small cell lung cancer (NSCLC). We then investigated a statistically significant interaction between gene expressions and survival of early-stage NSCLC patients.The expression of five genes, that is, POLQ, PLK1, RAD51, CLASPIN and CDC6 was associated with overall, disease-free and relapse-free survival. The expression levels are independent of treatment and stage classification. Except RAD51, their prognostic role on survival persists after adjustment on age, sex, treatment, stage classification and conventional proliferation markers, with a hazard ratio of 36.3 for POLQ (95%CI 2.6–517.4, P=0.008), 23.5 for PLK1 (95%CI 1.9–288.4, P=0.01), 20.7 for CLASPIN (95%CI 1.5–275.9, P=0.02) and 18.5 for CDC6 (95%CI 1.3–267.4, P=0.03). We also show that a five-gene signature including POLQ, PLK1, RAD51, CLASPIN and CDC6 separates patients into low- and high-risk groups, with a hazard ratio of 14.3 (95% CI 5.1–40.3, P<0.001). This ‘replication stress' metamarker may be a reliable predictor of survival for NSCLC, and may also help understand the molecular mechanisms underlying tumor progression.

Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Abstract MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 463. doi:1538-7445.AM2012-463

A Five-Gene and Corresponding Protein Signature for Stage-I Lung Adenocarcinoma Prognosis

Identification of effective markers for outcome is expected to improve the clinical management of non-small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis. Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression data sets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs). The survival of all-stages (P < 0.001, HR = 2.0) or stage-I (P < 0.001, HR = 2.84) adenocarcinoma patients that expressed the five-gene in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (P < 0.001, HR = 1.95) or stage-I (P = 0.001, HR = 2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, P < 0.001, HR = 3.6; stage-I, P < 0.001, HR = 3.5; stage-IB, P < 0.001, HR = 4.6) and mortality risk (all stages, P = 0.001, HR = 4.0; stage-I, P = 0.01, HR = 3.4; stage-IB, P < 0.001, HR = 7.2) in lung adenocarcinoma patients. Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy.

Prediction of prognosis in hepatocellular carcinoma based on a gene signature from highly tumorigenic liver cancer cells.

4148 Background: Hepatocellular carcinoma (HCC) contains a minority population of cancer cells with higher tumorigenic capacity and stem cells proprieties which was called side-population (SP) cells, characterized by the ability to efflux Hoechst 33342. In this study, the clinical significance of SP cells was explored through analysis of transcript level profiles. Methods: We compared the gene expression profile of SP cells with that of non-SP cells in HCC cell lines. Differentially expressed genes were used to generate a 124-gene recurrence gene signature (RGS), which was evaluated for its association with the microarray database of 268 HCC patients according to the prognosis analysis. Based on the RGS, a refined signature containing five genes was developed and revalidated by real-time reverse-transcriptase polymerase chain reaction in an independent cohort containing 100 HCC patients. Results: The RGS was confirmed as a significant predictor of recurrence and survival for HCC patients, especially for those in early stages, with well-differentiated tumor and normal serum alpha-fetoprotein. A five-gene signature containing NEDD9, EPAS1, EPC2, JMJD1C and USP36 was an independent predictor for recurrence and survival of HCC patients in training, testing sets, and the validation cohort (p < 0.05 for all analyses). Further validation in protein level and gene function based on small-interfering RNA showed that NEDD9 expression related with tumor cell migration, invasiveness, proliferation, clonogenic abilities and recurrence of HCC. Conclusions: Gene signature from highly tumorigenic HCC cells is associated with poor prognosis of HCC. The five-gene signature can be a useful tool for clinicians to stratify HCC patients with high recurrent risk. No significant financial relationships to disclose.

Abstract 4817: A five-gene signature predictive of survival in lung adenocarcinoma but not in squamous cell carcinoma

Abstract Identification of novel molecular markers for lung carcinogenesis, outcome and response to therapy is expected to improve the clinical management of lung cancer such as non-small cell lung cancer (NSCLC). Previously, we have derived gene expression signatures indicative of differential gene expression among cells constituting an in vitro model of human lung carcinogenesis and relevant to survival in NSCLC. In this study we assess the prognostic efficacy of our previously described six genes by using several prediction algorithms and a leave-one-out-cross-validation (LOOCV) strategy as well as risk-score prediction models. The NCI Director's Challenge datasets (n=443) were used as a training set and gene expression data of adenocarcinomas from the Duke and Harvard cohorts (DH cohort; n=183) were pooled as a validation set. In addition, two independent published datasets comprised of 130 and 58 lung squamous cell carcinomas (SCCs) served as a SCC validation cohort. A Five-gene in vitro lung carcinogenesis model signature (FILM) classifier was derived and found to be superior in prediction as the lowest specificity or sensitivity of the six prediction algorithms was 0.943. Importantly, all six prediction algorithms showed that the overall survival of all-stage or stage-I only human lung adenocarcinoma patients in the DH validation cohort that expressed FILM was significantly poorer than that of patients predicted to lack the signature. Moreover, no differences in overall survival between lung SCCs predicted to express or lack FILM were observed demonstrating the prognostic specificity of this classifier for lung adenocarcinomas. We then developed a risk score-prediction model for lung adenocarcinoma based on the Cox regression coefficients and expression of FILM genes. Lung adenocarcinoma patients identified to be at high risk based on the FILM risk model exhibited significantly worse survival (p=5.4 × 10-7, 100 months follow-up) than patients at low risk. For validation of the FILM risk model, Cox regression coefficients and the dichotomization cut-off threshold generated from the training cohort (n=443) were directly applied to the DH validation cohort (n=183). All stages or stage-I only lung adenocarcinoma patients in the validation cohort and predicted to be at high risk displayed significantly worse survival than patients predicted to be at low risk by the FILM risk model (p=0.0006 and p=0.0005 of the log-rank test, respectively). Our findings highlight a novel five-gene signature which, although derived originally from an in vitro cell model, is highly effective in predicting survival of lung adenocarcinoma patients. Studies to validate the effectiveness of FILM in predicting, in particular, the response of lung adenocarcinoma patients to various therapies are highly warranted. Supported by DOD grant W81XWH-04-1-0142 and NCI lung cancer SPORE (P50 CA70907). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4817.

A five‐gene signature as a potential predictor of metastasis and survival in colorectal cancer

To understand the molecular mechanisms of metastasis and prognosis of colorectal cancer (CRC), we isolated single cell-derived progenies (SCPs) from SW480 cells in vitro and compared their metastatic potential in an orthotopic CRC tumour model in vivo. Two groups of SCPs with the capability of high and low metastasis, respectively, were obtained. By analysing the gene expression profiles of high (SCP51), low (SCP58) metastatic SCPs, and their parental cell line (SW480/EGFP), we demonstrated that 143 genes were differentially expressed either between SCP51 and SCP58 or between SCP58 and SW480/EGFP. Gene-annotation enrichment analysis of DAVID revealed 80 genes in the top ten clusters of the analysis (gene enrichment score > 1). Of the 80-gene set, 32 genes are potentially involved in metastasis, as revealed by Geneclip. Five putative metastatic genes (LYN, SDCBP, MAP4K4, DKK1, and MID1) were selected for further validations. Immunohistochemical analysis in a cohort of 181 CRC clinical samples showed that the individual expression of LYN, MAP4K4, and MID1, as well as the five-gene signature, was closely correlated with lymph node metastasis in CRC patients. More importantly, the individual expression of LYN, MAP4K4, SDCBP, and MID1, as well as the five-gene signature, was significantly correlated with overall survival in CRC patients. Thus, our five-gene signature may be able to predict metastasis and survival of CRC in the clinic, and opens new perspectives on the biology of CRC.

Abstract C25: A five-gene signature is associated with tumor progression and poor prognosis in colorectal cancer

Abstract In order to develop an experimentally derived molecular signature closely associated with survival of colorectal cancer (CRC) patients from mouse tumor models, we isolated single cell-derived progenies (SCPs) from SW480 CRC cell line and compared their metastatic potential in an orthotopic implantation model of murine CRC. We compared the differences in the gene expression profiles of a high metastatic variant SCP51, a low metastatic variant SCP58 and their parent SW480/EGFP. We found that 143 genes were commonly differentially expressed between SCP51 and SCP58 cells, SCP58 and SW480/EGFP. Using Gene-annotation enrichment analysis of DAVID, 80 genes were found in top ten clusters of the analysis. Thirty-two genes of above the 80-gene set were found to be involved in metastasis by Genclip, a text mining program. We selected five genes (LYN, SDCBP, MAP4K4, DKK1 and MID1) for immunohistochemical analysis in a cohort of 181 CRC patients. Results showed those LYN, SDCBP, MAP4K4, MID1 and five-gene signatures were significantly correlated with overall survival in patients with CRC. We also found that LYN, MAP4K4 and MID1 but not SDCBP and DKK1 expression was closely correlated with lymph node metastasis and Dukes classification in patients with CRC. Our five-gene signature was closerly associated with survival of CRC patients and could be used as a potential prognostic factor for CRC. Citation Information: Cancer Res 2009;69(23 Suppl):C25.

Validation of a Reverse Transcription-Polymerase Chain Reaction-Based Five-Gene Signature in Non-small Cell Lung Cancer

Last year in the New England Journal of Medicine Chen et al.1Chen HY Yu SL Chen CH et al.A five-gene signature and clinical outcome in non–small-cell lung cancer.N Engl J Med. 2007; 356: 11-20Crossref PubMed Scopus (727) Google Scholar stated that a five-gene quantitative polymerase-chain-reaction assay stratified patients with early-stage lung cancer into low-risk and high-risk groups. Indeed, they concluded that patient with surgically resected non-small cell lung cancer that were classified as first group might be spared from adjuvant treatment. In contrast, Michiels and Hill2Michiels S Hill C Five-gene signature in non-small-cell lung cancer.N Engl J Med. 2007; 356: 1581-1582PubMed Google Scholar observed that to correctly validate such signature a different and independent group of patients needed to be used. In addition, Dobbin3Dobbin KK Five-gene signature in non-small-cell lung cancer.N Engl J Med. 2007; 356: 1582PubMed Google Scholar noted that the reuse of survival data to identify a predictor genes provide substantial bias. Taken together, these observations provide a rationale for an independent validation of such molecular signature. To do that, we analyzed a population of 38 non-small cell lung cancer patients with stage I disease and untreated after lobar resection. Using methods as described,1Chen HY Yu SL Chen CH et al.A five-gene signature and clinical outcome in non–small-cell lung cancer.N Engl J Med. 2007; 356: 11-20Crossref PubMed Scopus (727) Google Scholar we did not find difference among 19 patients who had cancer-related death within 30months after resection and 19 patients who were alive within 40 months after resection (5-gene high risk = 7/12 versus 8/11; Fisher’s exact test p = 1).

NF-kappa B genes have a major role in Inflammatory Breast Cancer

BackgroundIBC (Inflammatory Breast cancer) is a rare form of breast cancer with a particular phenotype. New molecular targets are needed to improve the treatment of this rapidly fatal disease. Given the role of NF-κB-related genes in cell proliferation, invasiveness, angiogenesis and inflammation, we postulated that they might be deregulated in IBC.MethodsWe measured the mRNA expression levels of 60 NF-κB-related genes by using real-time quantitative RT-PCR in a well-defined series of 35 IBCs, by comparison with 22 stage IIB and III non inflammatory breast cancers. Twenty-four distant metastases of breast cancer served as "poor prognosis" breast tumor controls.ResultsThirty-five (58%) of the 60 NF-κB-related genes were significantly upregulated in IBC compared with non IBC. The upregulated genes were NF-κB genes (NFKB1, RELA, IKBKG, NFKBIB, NFKB2, REL, CHUK), apoptosis genes (MCL1L, TNFAIP3/A20, GADD45B, FASLG, MCL1S, IER3L, TNFRSF10B/TRAILR2), immune response genes (CD40, CD48, TNFSF11/RANKL, TNFRSF11A/RANK, CCL2/MCP-1, CD40LG, IL15, GBP1), proliferation genes (CCND2, CCND3, CSF1R, CSF1, SOD2), tumor-promoting genes (CXCL12, SELE, TNC, VCAM1, ICAM1, PLAU/UPA) or angiogenesis genes (PTGS2/COX2, CXCL1/GRO1). Only two of these 35 genes (PTGS2/COX2 and CXCL1/GRO1)were also upregulated in breast cancer metastases. We identified a five-gene molecular signature that matched patient outcomes, consisting of IL8 and VEGF plus three NF-κB-unrelated genes that we had previously identified as prognostic markers in the same series of IBC.ConclusionThe NF-κB pathway appears to play a major role in IBC, possibly contributing to the unusual phenotype and aggressiveness of this form of breast cancer. Some upregulated NF-κB-related genes might serve as novel therapeutic targets in IBC.

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

MicroRNA Signature Predicts Survival and Relapse in Lung Cancer

We investigated whether microRNA expression profiles can predict clinical outcome of NSCLC patients. Using real-time RT-PCR, we obtained microRNA expressions in 112 NSCLC patients, which were divided into the training and testing sets. Using Cox regression and risk-score analysis, we identified a five-microRNA signature for the prediction of treatment outcome of NSCLC in the training set. This microRNA signature was validated by the testing set and an independent cohort. Patients with high-risk scores in their microRNA signatures had poor overall and disease-free survivals compared to the low-risk-score patients. This microRNA signature is an independent predictor of the cancer relapse and survival of NSCLC patients.

Five-Gene Signature in Non–Small-Cell Lung Cancer

Five-Gene Signature in Non–Small-Cell Lung Cancer

Gene signature for predicting NSCLC outcome

Researchers have identifi ed fi ve genes that can predict clinical outcome in patients with non-small-cell lung cancer (NSCLC; N Engl J Med 2007; 356: 11–20). Pan-Chyr Yang and colleagues (National Taiwan University Hospital, Taipei, Taiwan) analysed gene expression in lung-cancer specimens from 125 patients who had undergone surgical resection for NSCLC, and identifi ed 16 genes that correlated with survival. Five genes were selected for decision tree analysis: dual-specifi city phosphatase 6 (DUSP6), monocyte-tomacrophage diff erentiation-associated protein (MMD), signal transducer and activator of transcription 1 (STAT1), v-erb-b2 avian erythroblastic leukaemia viral oncogene homolog3 (ERBB3), and lymphocyte-specifi c protein tyrosine kinase (LCK). The fi ve-gene signature was found to be an independent predictor of overall survival (sensitivity 98%; specifi city 93%; positive predictive value 95%; negative predictive value 98%). Median overall survival for patients with the high-risk gene signature was shorter than for those with the low-risk gene signature (20 months vs 40 months; p<0·001). The highrisk signature was also associated with recurrence (hazard ratio 1·92 [95% CI 1·06–3·46]; p=0·03). The researchers propose that patients with lung cancer who have a high-risk gene signature might benefi t from cisplatin-based adjuvant chemo therapy, unlike those with a low-risk gene signature. John Nemunaitis (Mary Crowley Medical Research Center, Dallas, TX, USA) says, “it can guide chemo therapy to the higher risk population based on molecular criteria and poten tially not expose patients at low risk of recurrence to the toxic eff ects of chemotherapy”. Yang explains the fi ndings could be a “useful tool for risk prediction and designation of adjuvant treatment for high risk patitents after validation in a prospective clinical trial”.

A Five-Gene Signature and Clinical Outcome in Non–Small-Cell Lung Cancer

Current staging methods are inadequate for predicting the outcome of treatment of non-small-cell lung cancer (NSCLC). We developed a five-gene signature that is closely associated with survival of patients with NSCLC. We used computer-generated random numbers to assign 185 frozen specimens for microarray analysis, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, or both. We studied gene expression in frozen specimens of lung-cancer tissue from 125 randomly selected patients who had undergone surgical resection of NSCLC and evaluated the association between the level of expression and survival. We used risk scores and decision-tree analysis to develop a gene-expression model for the prediction of the outcome of treatment of NSCLC. For validation, we used randomly assigned specimens from 60 other patients. Sixteen genes that correlated with survival among patients with NSCLC were identified by analyzing microarray data and risk scores. We selected five genes (DUSP6, MMD, STAT1, ERBB3, and LCK) for RT-PCR and decision-tree analysis. The five-gene signature was an independent predictor of relapse-free and overall survival. We validated the model with data from an independent cohort of 60 patients with NSCLC and with a set of published microarray data from 86 patients with NSCLC. Our five-gene signature is closely associated with relapse-free and overall survival among patients with NSCLC.