Ni Liu

Role of KRAS and EGFR As Biomarkers of Response to Erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21

PURPOSE To evaluate the effect of KRAS and epidermal growth factor receptor (EGFR) genotype on the response to erlotinib treatment in the BR.21, placebo-controlled trial. PATIENTS AND METHODS We analyzed 206 tumors for KRAS mutation, 204 tumors for EGFR mutation, and 159 tumors for EGFR gene copy by fluorescent in situ hybridization (FISH). We reanalyzed EGFR deletion/mutation using two highly sensitive techniques that detect abnormalities in samples with 5% to 10% tumor cellularity. KRAS mutation was analyzed by direct sequencing. RESULTS Thirty patients (15%) had KRAS mutations, 34 (17%) had EGFR exon 19 deletion or exon 21 L858R mutations, and 61 (38%) had high EGFR gene copy (FISH positive). Response rates were 10% for wild-type and 5% for mutant KRAS (P = .69), 7% for wild-type and 27% for mutant EGFR (P = .03), and 5% for EGFR FISH-negative and 21% for FISH-positive patients (P = .02). Significant survival benefit from erlotinib therapy was observed for patients with wild-type KRAS (hazard ratio [HR] = 0.69, P = .03) and EGFR FISH positivity (HR = 0.43, P = .004) but not for patients with mutant KRAS (HR = 1.67, P = .31), wild-type EGFR (HR = 0.74, P = .09), mutant EGFR (HR = 0.55, P = .12), and EGFR FISH negativity (HR = 0.80, P = .35). In multivariate analysis, only EGFR FISH-positive status was prognostic for poorer survival (P = .025) and predictive of differential survival benefit from erlotinib (P = .005). CONCLUSION EGFR mutations and high copy number are predictive of response to erlotinib. EGFR FISH is the strongest prognostic marker and a significant predictive marker of differential survival benefit from erlotinib.

Prognostic and Predictive Gene Signature for Adjuvant Chemotherapy in Resected Non–Small-Cell Lung Cancer

PURPOSE The JBR.10 trial demonstrated benefit from adjuvant cisplatin/vinorelbine (ACT) in early-stage non-small-cell lung cancer (NSCLC). We hypothesized that expression profiling may identify stage-independent subgroups who might benefit from ACT. PATIENTS AND METHODS Gene expression profiling was conducted on mRNA from 133 frozen JBR.10 tumor samples (62 observation [OBS], 71 ACT). The minimum gene set that was selected for the greatest separation of good and poor prognosis patient subgroups in OBS patients was identified. The prognostic value of this gene signature was tested in four independent published microarray data sets and by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). RESULTS A 15-gene signature separated OBS patients into high-risk and low-risk subgroups with significantly different survival (hazard ratio [HR], 15.02; 95% CI, 5.12 to 44.04; P < .001; stage I HR, 13.31; P < .001; stage II HR, 13.47; P < .001). The prognostic effect was verified in the same 62 OBS patients where gene expression was assessed by qPCR. Furthermore, it was validated consistently in four separate microarray data sets (total 356 stage IB to II patients without adjuvant treatment) and additional JBR.10 OBS patients by qPCR (n = 19). The signature was also predictive of improved survival after ACT in JBR.10 high-risk patients (HR, 0.33; 95% CI, 0.17 to 0.63; P = .0005), but not in low-risk patients (HR, 3.67; 95% CI, 1.22 to 11.06; P = .0133; interaction P < .001). Significant interaction between risk groups and ACT was verified by qPCR. CONCLUSION This 15-gene expression signature is an independent prognostic marker in early-stage, completely resected NSCLC, and to our knowledge, is the first signature that has demonstrated the potential to select patients with stage IB to II NSCLC most likely to benefit from adjuvant chemotherapy with cisplatin/vinorelbine.

Prognostic and Predictive Importance of p53 and RAS for Adjuvant Chemotherapy in Non–Small-Cell Lung Cancer

PURPOSE p53 and RAS are multifunctional proteins that are critical to cell cycle regulation, apoptosis, cell survival, gene transcription, response to stress, and DNA repair. We have evaluated the prognostic and predictive value of p53 gene/protein aberrations using tumor samples from JBR.10, a North American phase III intergroup trial that randomly assigned 482 patients with completely resected stage IB and II non-small-cell lung cancer (NSCLC) to receive four cycles of adjuvant cisplatin plus vinorelbine or observation alone. METHODS p53 protein expression was evaluated by immunohistochemistry. Mutations in exons 5 to 9 of the p53 gene were determined by denaturing high-performance liquid chromatography and confirmed by sequencing. RAS mutations were identified by allelic specific oligonucleotide hybridization. RESULTS Of 253 patients, 132 (52%) were positive for p53 protein overexpression. Untreated p53-positive patients had significantly shorter overall survival than did patients with p53-negative tumors (hazard ratio [HR] = 1.89; 95% CI, 1.07 to 3.34; P = .03). However, these p53-positive patients also had a significantly greater survival benefit from adjuvant chemotherapy (HR = 0.54; P = .02) compared with patients with p53-negative tumors (HR = 1.40; P = .26; interaction P = .02). Mutations of p53 and RAS genes were found in 124 (31%) of 397 and 117 (26%) of 450 patients, respectively. Mutations in these genes were neither prognostic for survival nor predictive of a differential benefit from adjuvant chemotherapy. CONCLUSION p53 protein overexpression is a significant prognostic marker of shortened survival, and also a significant predictive marker for a differentially greater benefit from adjuvant chemotherapy in completely resected NSCLC patients.

Immortal Human Pancreatic Duct Epithelial Cell Lines with Near Normal Genotype and Phenotype

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Three-gene prognostic classifier for early-stage non small-cell lung cancer.

PURPOSE Several microarray studies have reported gene expression signatures that classify non-small-cell lung carcinoma (NSCLC) patients into different prognostic groups. However, the prognostic gene lists reported to date overlap poorly across studies, and few have been validated independently using more quantitative assay methods. PATIENTS AND METHODS The expression of 158 putative prognostic genes identified in previous microarray studies was analyzed by reverse transcription quantitative polymerase chain reaction in the tumors of 147 NSCLC patients. Concordance indices and risk scores were used to identify a stage-independent set of genes that could classify patients with significantly different prognoses. RESULTS We have identified a three-gene classifier (STX1A, HIF1A, and CCR7) for overall survival (hazard ratio = 3.8; 95% CI, 1.7 to 8.2; P < .001). The classifier was also able to stratify stage I and II patients and further improved the predictive ability of clinical factors such as histology and tumor stage. The predictive value of this three-gene classifier was validated in two large independent microarray data sets from Harvard and Duke Universities. CONCLUSION We have identified a new three-gene classifier that is independent of and improves on stage to stratify early-stage NSCLC patients with significantly different prognoses. This classifier may be tested further for its potential value to improve the selection of resected NSCLC patients in adjuvant therapy.

Comparative Phenotypic Studies of Duct Epithelial Cell Lines Derived from Normal Human Pancreas and Pancreatic Carcinoma

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-alpha, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-alpha, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.

Novel candidate tumor marker genes for lung adenocarcinoma

Using the representational difference analysis (RDA) technique on pooled mRNA of five primary lung adenocarcinomas and their corresponding non-neoplastic lung tissues, we identified six genes that were putatively overexpressed in this type of lung cancer. Five corresponded to previously isolated genes, while one (Lc19) matched with the sequence of an unannotated EST. Real-time RT–PCR analyses of expression levels in a panel of 34 paired primary non-small cell lung cancer (NSCLC) and corresponding grossly normal appearing lung tissues confirmed the common overexpression of these genes in non-small cell lung cancer. Among these genes, overexpression of Lc19, hyaluronan binding protein 2 (HABP2) and crystalline-mu appeared more specific to adenocarcinoma, whereas ceruloplasmin, integrin α-11 and collagen type XI alpha 1 were overexpressed at high frequency among both adenocarcinoma and squamous cell carcinoma. These genes represent novel candidate tumor biomarker genes for NSCLC and its histological subtypes.

Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma

Glypican-3 is a membrane-bound proteoglycan whose expression has been linked to malignancies through the existence of both mutations and aberrant protein expression. Reports on glypican-3 expression in lung cancer were limited, with some evidence for loss of expression, which suggested a tumor-suppressor role. We sought to evaluate glypican-3 expression in lung cancer at the protein and mRNA levels and correlate it with clinical, histological and genomic characteristics such as RAS mutation status. We used immunohistochemistry on tissue microarray to study glypican-3 expression in 97 patients, evaluated glypican-3 mRNA levels by quantitative polymerase chain reaction in 143 patients and identified RAS mutations by allele-specific oligonucleotide hybridization. We correlated the results with clinical and histological data. Glypican-3 immunostaining was negative in all normal lung tissues, but positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology (positive stain in 55% of squamous cell carcinoma vs 8% of adenocarcinoma, P<0.0001 for both immunostaining and mRNA). RAS mutations were highly associated with adenocarcinoma and low glypican-3 mRNA expression (P<0.0001 for both). Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients (P=0.013), and was elevated in those with squamous cell carcinoma (P=0.03, interaction P=0.0009). These opposing associations also correlated with the smoking burden. Patients with tumors staining positively for glypican-3 smoked significantly more than patients with tumors staining negatively (P=0.013). No association was found between glypican-3 expression and patient outcome. In conclusion, glypican-3 was overexpressed in cancerous compared with normal lung tissue. Adenocarcinoma and squamous cell carcinoma had differential expression of glypican-3, with predilection to squamous cell carcinoma patients who smoked. Glypican-3 expression in squamous cell carcinoma as an oncofetal protein renders it a potential candidate marker for early detection of lung squamous cell carcinoma.

Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer.

Telomerase reactivation is a hallmark of human carcinogenesis. Increased telomerase activity may result from gene amplification and/or overexpression. This study evaluates the prognostic value of hTERT gene amplification and mRNA overexpression in 144 resectable non-small-cell lung cancer (NSCLC) specimens. The hTERT gene copy number was assessed by quantitative polymerase chain reaction (qPCR) on laser-capture microdissected tumour cells of 81 tumours, and by fluorescence in situ hybridisation (FISH) on a subset of 59 tumours. hTERT mRNA level was determined by reverse transcription (RT)–qPCR in 130 tumours. In total, 57% of (46 out of 81) primary NSCLC specimens demonstrated hTERT amplification, which was significantly more common (P<0.001) in adenocarcinoma (30 out of 40) than in squamous cell carcinoma (13 out of 37). The hTERT mRNA overexpression was noted in 74% (94 out of 130) of tumours; it was more frequent in squamous cell than in adenocarcinoma (87 vs 68%, P=0.03). Overexpression was significantly associated with amplification (P=0.03), especially in adenocarcinoma. The hTERT gene amplification was prognostic for shorter recurrence-free survival (hazard ratio=2.16, P=0.03). These data indicate that gene amplification is an important mechanism for hTERT overexpression in lung adenocarcinoma and is an independent poor prognostic marker for disease-free survival in NSCLC.

Skp2 Gene Copy Number Aberrations Are Common in Non-Small Cell Lung Carcinoma, and Its Overexpression in Tumors with ras Mutation Is a Poor Prognostic Marker

Purpose: Skp2 plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins. The Skp2 gene is located on a region of chromosome 5p that is commonly overrepresented in lung cancer. The present study aimed to evaluate Skp2 abnormalities and their prognostic value in non-small cell lung cancer (NSCLC). Experimental Design: In total 16 NSCLC cell lines and 163 primary tumors were included in studies to measure Skp2 relative gene copy number, mRNA abundance, and protein level. The tumors were also evaluated for p27 protein expression level and ras mutation. These values were correlated with the clinical and pathological features of the patients. Results: Skp2 relative gene copy number aberrations were found in 88 and 65% of NSCLC cell lines and primary tumors, respectively. Overrepresentation was especially common among squamous cell carcinoma (74%). Both gene copy overrepresentation (13%) and loss (35%) were found in adenocarcinoma. Skp2 relative gene copy number was significantly correlated with mRNA and protein levels, but none of these were correlated with p27 protein levels. Neither high Skp2 protein expression nor ras mutation was prognostically significant. In NSCLCs with ras mutation, however, high Skp2 protein expression was a significant independent poor prognostic marker. Conclusion: There appears to be a synergistic interaction between high Skp2 protein expression and ras mutation with negative impact on the survival of NSCLC patients.