Interleukin-11 Receptor Is a Candidate Target for Ligand-Directed Therapy in Lung Cancer: Analysis of Clinical Samples and BMTP-11 Preclinical Activity.

We previously isolated an IL-11-mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R-expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P < 0.0001). BMTP-11 induced in vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lung cancer is a promising feature for potential translational applications.

Personalizing lung cancer prevention through a reverse migration strategy.

Lung cancer is the deadliest cancer in the United States and worldwide. Tobacco use is the one of the primary causes of lung cancer and smoking cessation is an important step towards prevention, but patients who have quit smoking remain at risk for lung cancer. Finding pharmacologic agents to prevent lung cancer could potentially save many lives. Unfortunately, despite extensive research, there are no known effective chemoprevention agents for lung cancer. Clinical trials in the past, using agents without a clear target in an unselected population, have shown pharmacologic interventions to be ineffective or even harmful. We propose a new approach to drug development in the chemoprevention setting: reverse migration, that is, drawing on our experience in the treatment of advanced cancer to bring agents, biomarkers, and study designs into the prevention setting. By identifying molecular drivers of lung neoplasia and using matched targeted agents, we hope to personalize therapy to each individual to develop more effective, tolerable chemoprevention. Also, advances in risk modeling, using not only clinical characteristics but also biomarkers, may help us to select patients better for chemoprevention efforts, thus sparing patients at low risk for cancer the potential toxicities of treatment. Our institution has experience with biomarker-driven clinical trials, as in the recently reported Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, and we now propose to bring this trial design into the prevention setting.

MicroRNA-related genetic variations as predictors for risk of second primary tumor and/or recurrence in patients with early-stage head and neck cancer.

Second primary tumor (SPT) and/or recurrence negatively impact the prognosis of patients with curatively treated early-stage head and neck cancer. MicroRNAs (miRNAs) play important roles in cancer development. We explored whether the variations of miRNA-related pathway were associated with the risk of SPT/recurrence in patients with early-stage head and neck cancer. This study includes 150 early-stage head and neck cancer patients with SPT/recurrence and 300 patients without SPT/recurrence. Two hundred and thirty-five tagging and potentially functional single-nucleotide polymorphisms (SNPs) were genotyped from eight miRNA biogenesis pathway genes and 135 miRNA-targeted genes. Eighteen miRNA-related SNPs were significantly associated with the risk of SPT/recurrence. The most significant SNP was rs3747238, a miRNA-binding site SNP in SMC1B. The variant homozygous genotype of this SNP was associated with a 1.74-fold increased risk [95% confidence interval (CI) 1.19-2.54; P = 0.004]. Cumulative effect analysis showed joint effects for the number of unfavorable genotype in patients. Survival tree analysis further identified the high-order gene-gene interactions and categorized the study subjects into low-, medium- and high-risk groups. Patients in the high-risk group had a 4.84-fold increased risk (95% CI: 3.11-7.51; P = 2.45 × 10(-12)) and a shorter event-free median survival time of 37.9 months (log rank P = 2.28 × 10(-13)). Our results suggested that miRNA-related genetic polymorphisms may be used individually and jointly to predict the risk of SPT/recurrence of early-stage head and neck cancer patients.

Genetic variations in regulator of G-protein signaling genes as susceptibility loci for second primary tumor/recurrence in head and neck squamous cell carcinoma.

Curatively treated patients with early-stage head and neck squamous cell carcinoma (HNSCC) are at high risks for second primary tumor (SPT) and recurrence. The regulator of G-protein signaling (RGS) is important in essential signaling transduction and cellular activities. We hypothesize that genetic variations of RGS may modulate the risk of SPT/recurrence in patients with early-stage HNSCC. In a nested case-control study, we evaluated 98 single-nucleotide polymorphisms (SNPs) in 17 RGS genes for the risk of SPT/recurrence among 450 HNSCC patients. Eight SNPs showed significant associations with the risk of SPT/recurrence, with the most significant one of rs2179653, which is located in the 5'-flanking region of RGS2 gene. Under a recessive genetic model, the homozygous variant genotype of this SNP was associated with 2.95-fold [95% confidence interval (CI): 1.52-5.74] increased risk of SPT/recurrence. This association remained significant after the adjustment for multiple comparisons. Cumulative effects analysis revealed that the risk increased significantly with the increasing numbers of unfavorable genotypes. Compared with subjects carrying 0-2 unfavorable genotypes, the hazard ratios (95% CIs) for those carrying 3 or 4+ were 1.73 (1.10-2.70) and 3.05 (1.92-4.83), respectively. Furthermore, survival tree analysis revealed potential higher order gene-gene interactions and indicated different outcomes based on distinct genotype profiles. Genetic variations of RGS genes may modulate the susceptibility to SPT/recurrence in early-stage HNSCC patients individually and cumulatively. Our results stressed the importance of taking a polygenic approach to evaluate the cumulative and interaction effects of genetic variations in the prediction of cancer risk and prognosis.

Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells.

The standard treatment for most advanced cancers is multidrug therapy. Unfortunately, combinations in the clinic often do not perform as predicted. Therefore, to complement identifying rational drug combinations based on biological assumptions, we hypothesized that a functional screen of drug combinations, without limits on combination sizes, will aid the identification of effective drug cocktails. Given the myriad possible cocktails and inspired by examples of search algorithms in diverse fields outside of medicine, we developed a novel, efficient search strategy called Medicinal Algorithmic Combinatorial Screen (MACS). Such algorithms work by enriching for the fitness of cocktails, as defined by specific attributes through successive generations. Because assessment of synergy was not feasible, we developed a novel alternative fitness function based on the level of inhibition and the number of drugs. Using a WST-1 assay on the A549 cell line, through MACS, we screened 72 combinations of arbitrary size formed from a 19-drug pool across four generations. Fenretinide, suberoylanilide hydroxamic acid, and bortezomib (FSB) was the fittest. FSB performed up to 4.18 SD above the mean of a random set of cocktails or "too well" to have been found by chance, supporting the utility of the MACS strategy. Validation studies showed FSB was inhibitory in all 7 other NSCLC cell lines tested. It was also synergistic in A549, the one cell line in which this was evaluated. These results suggest that when guided by MACS, screening larger drug combinations may be feasible as a first step in combination drug discovery in a relatively small number of experiments.

Polymorphisms of FAS and FAS ligand genes involved in the death pathway and risk and progression of squamous cell carcinoma of the head and neck.

PURPOSE: Alteration of the FAS/FAS ligand (FASLG) pathway regulating cell death may lead to cancer development, but the effects of functional promoter polymorphisms of the FAS and FASLG genes on risk of squamous cell carcinoma of the head and neck (SCCHN) are unknown. DESIGN: We genotyped the FAS -1377 G>A, FAS -670 A>G, FASLG -844 C>T, and FASLG IVS2nt -124 A>G polymorphisms in 721 case patients with SCCHN and 1,234 cancer-free non-Hispanic White control subjects frequency-matched by age and sex. Multivariate logistic regression was used to assess the adjusted odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Compared with the FAS -1377 GG and -670 AA genotypes, the FAS -1377 AA and -670 (GG+AG) genotypes were associated with an increased risk of SCCHN (OR, 2.23; 95% CI, 1.07-4.64 and OR, 1.24; 95% CI, 1.01-1.52, respectively), whereas no risk of SCCHN was associated with any of the FASLG genotypes. When we used the combined FAS -1377 (GG+AG)/-670 AA genotypes as the reference, we found that the individuals carrying the FAS -1377 AA/-670 (GG+AG) had the highest risk (OR, 2.69; 95% CI, 1.24-5.83), whereas individuals carrying genotypes other than FAS -1377 (GG+AG)/-670 AA had a higher risk of SCCHN (OR, 1.24; 95% CI, 1.01-1.52). Furthermore, the elevated risk was particularly evident for pharyngeal cancer with the larger tumors without regional lymph metastasis (OR, 1.77; 95% CI, 1.07-2.94). CONCLUSIONS: The FAS (but not FASLG) polymorphisms seem to contribute to risk of developing SCCHN, particularly the pharyngeal cancer in non-Hispanic Whites. However, potential selection bias warrants future population-based studies to verify the findings.

Identification of insulin-like growth factor binding protein-3 as a farnesyl transferase inhibitor SCH66336-induced negative regulator of angiogenesis in head and neck squamous cell carcinoma.

The farnesyl transferase inhibitor (FTI) SCH66336 has been shown to have antitumor activities in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. However, its mechanism of action has not been well defined. Here, we report that the insulin-like growth factor (IGF) binding protein (IGFBP)-3 mediates antitumor activities of SCH66336 in HNSCC by inhibiting angiogenesis. SCH66336 significantly suppressed HNSCC tumor growth and angiogenesis via mechanisms that are independent of H-Ras and RhoB. By inducing IGFBP-3 secretion from HNSCC cells, this compound suppresses angiogenic activities of endothelial cells, including vessel formation in chorioallantoic membranes of chick, endothelial cell sprouting from chick aorta, and capillary tube formation of human umbilical vascular endothelial cells (HUVEC). Knockdown of IGFBP-3 expression in HNSCC cells by RNA interference or depletion of IGFBP-3 in HUVECs by neutralizing antibody effectively blocked the effects of IGFBP-3 secreted from SCH66336-treated HNSCC cells on HUVECs. These findings suggest that IGFBP-3 could be a primary target for antitumor activities of FTIs and that IGFBP-3 is an effective therapeutic approach against angiogenesis in HNSCC.

Phase II study of a liposome-entrapped cisplatin analog (L-NDDP) administered intrapleurally and pathologic response rates in patients with malignant pleural mesothelioma.

PURPOSE: To determine pathologic response rates to liposome-entrapped cis-bisneodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II) (L-NDDP) administered intrapleurally in patients with malignant pleural mesothelioma. PATIENTS AND METHODS: Thirty-three patients with malignant pleural mesothelioma and free-flowing pleural effusions received intrapleural L-NDDP once every 3 weeks at a dose of 450 mg/m2. Thoracoscopic evaluation with pleural biopsies was performed before therapy and then after every two cycles. The primary end point was pathologic response as determined by thoracoscopic biopsy. RESULTS: After at least two cycles, post-treatment pleural biopsy analysis was negative in 14 patients for a pathologic response rate of 42% (95% CI, 25% to 61%). Median survival was 11.2 months. There were three treatment-related deaths secondary to peritonitis, cellulitis at the thoracoscopy site, and empyema. Grade 3 nonhematologic toxicities included infection, fever, dyspnea, and anorexia, which occurred in five (15%), one (3%), one (3%), and one (3%) patients, respectively. There were no grade 4 nonhematologic toxicities. Grade 3 or 4 neutropenia, thrombocytopenia, and anemia occurred in five (15%), three (9%), and two (6%) patients, respectively. Two patients with pathologic responses subsequently underwent pleural decortication. Both surgical specimens revealed residual tumor in regions that were not in direct communication with the pleural space. CONCLUSION: Intrapleural L-NDDP therapy in this patient population is feasible with significant but manageable toxicity. Although pathologic responses are highly encouraging, areas of mesothelioma that are not in direct communication with the pleural space will evade drug exposure and limit efficacy in some patients. The optimal role of intrapleural L-NDDP therapy currently remains to be determined.

Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer.

PURPOSE: Bevacizumab (Avastin; Genentech, South San Francisco, CA) is a recombinant, humanized anti-vascular endothelial growth factor monoclonal antibody. Erlotinib HCl (Tarceva, OSI-774; OSI Pharmaceuticals, New York, NY) is a potent, reversible, highly selective and orally available HER-1/epidermal growth factor receptor tyrosine kinase inhibitor. Preclinical data in various xenograft models produced greater growth inhibition than with either agent alone. Additionally, both agents have demonstrated benefit in patients with previously treated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: A phase I/II study in two centers examined erlotinib and bevacizumab (A+T) in patients with nonsquamous stage IIIB/IV NSCLC with > or = one prior chemotherapy. In phase I, erlotinib 150 mg/day orally plus bevacizumab 15 mg/kg intravenously every 21 days was established as the phase II dose, although no dose-limiting toxicities were observed. Phase II assessed the efficacy and tolerability of A+T at this dose. Pharmacokinetic parameters were evaluated. ResultsForty patients were enrolled and treated in this study (34 patients at phase II dose); the median age was 59 years (range, 36 to 72 years), 21 were female, 30 had adenocarcinoma histology, nine were never-smokers, and 22 had > or = two prior regimens (three patients had > or = four prior regimens). The most common adverse events were mild to moderate rash, diarrhea, and proteinuria. Preliminary data showed no pharmacokinetic interaction between A + T. Eight patients (20.0%; 95% CI, 7.6% to 32.4%) had partial responses and 26 (65.0%; 95% CI, 50.2% to 79.8%) had stable disease as their best response. The median overall survival for the 34 patients treated at the phase II dose was 12.6 months, with progression-free survival of 6.2 months. CONCLUSION: Encouraging antitumor activity and safety of A + T support further development of this combination for patients with advanced NSCLC and other solid tumors.

9-cis-retinoic acid treatment increases serum concentrations of alpha-tocopherol in former smokers.

PURPOSE: Low serum concentrations of antioxidants may be associated with an increased risk of cancer. Based on the accumulated evidence, we hypothesized that retinoids would elevate serum alpha-tocopherol. This study was designed to determine whether 9-cis-retinoic acid (9-cis-RA), the most common chemopreventive agent, could alter serum alpha-tocopherol in former smokers. Because hyperlipidemia is a known side effect of retinoids, we also evaluated the association between serum alpha-tocopherol and lipids in the same population. EXPERIMENTAL DESIGN: Subjects who had stopped smoking at least 12 months before the study were randomly assigned to receive oral 9-cis-RA or placebo daily for 3 months. Clinical information and blood samples were obtained monthly; serum alpha-tocopherol concentrations were measured by high-performance liquid chromatography and lipid levels by enzymatic assays before treatment and every month during the treatment. RESULTS: Of the 149 subjects in the study, 113 completed 3 months of treatment and provided samples for evaluation of serum alpha-tocopherol. Serum alpha-tocopherol levels in the 9-cis-RA group (n = 52) were higher after treatment (r = 0.445, P < 0.01) than before. The incidences of grade > or =2 hypertriglyceridemia and hypercholesterolemia were higher in the 9-cis-RA group than in the placebo group (P = 0.0005 and P = 0.01, respectively), but there were no serious complications related to hyperlipidemia. CONCLUSIONS: Treatment of former smokers with 9-cis-RA significantly increased their serum alpha-tocopherol levels, and this could be a benefit. In addition, serum alpha-tocopherol could serve as a biomarker for 9-cis-RA treatment.

Results of a phase II double-blinded randomized clinical trial of difluoromethylornithine for cervical intraepithelial neoplasia grades 2 to 3.

PURPOSE: Our purpose was to conduct a double-blinded randomized trial of difluoromethylornithine (DFMO) at 0.125, 0.5 gm/m2, versus placebo in the treatment of cervical intraepithelial neoplasia (CIN) grades 2 to 3. A promising phase I study has shown histopathologic responses at these dose levels. EXPERIMENTAL DESIGN: Patients with histopathologically confirmed CIN 2-3 lesions were recruited from a colposcopy clinic and underwent Papanicolaou testing, human papillomavirus testing, and colpophotography. They took oral contraception and DFMO or placebo elixir for 28 days and filled out the National Cancer Institute common toxicity calendars. They returned for follow-up and a repeat Papanicolaou smear, colpophotograph, and loop excision of the cervix. RESULTS: There were no statistically significant differences among the arms in histopathologic response. This could no be explained by any biases in risk factors. The prominent toxicities were diarrhea, dizziness, nausea, and headaches. There were no differences in the toxicities among arms. The Papanicolaou smear was a poor biomarker of response and correlated poorly with the histopathology. CONCLUSIONS: DFMO is no active at 0.125 and 0.5 gm/m2 for 28 days when given orally in CIN 2-3. Higher oral doses or longer administration is necessary, supporting data from breast trials. Alternatively, a trial of topical DFMO might merit attention as activity has been noted in trials of actinic keratoses.

p53 Genotypes and Haplotypes Associated With Lung Cancer Susceptibility and Ethnicity.

BACKGROUND: The p53 tumor suppressor protein is important in cell-cycle control, apoptosis, and DNA repair. Mutations in p53 have been associated with inherited cancer susceptibility. Because there is a difference in the risk of lung cancer among different ethnic groups, we examined associations between ethnicity and three polymorphisms in p53 (one exonic and two intronic) and haplotypes for the three loci and risk of lung cancer. We also examined the functionality of the p53 variants in apoptosis and DNA repair. METHODS: In a case-control study, we frequency matched (by age, sex, and ethnicity) 635 lung cancer case patients and 635 control subjects. p53 genotypes and haplotypes at the three polymorphic sites were determined by restriction fragment length polymorphism analysis of lymphocyte DNA. Odds ratios (ORs) and 95% confidence intervals (CIs) for the association between genotype or haplotype and lung cancer risk were determined by logistic regression analysis. Apoptosis and DNA repair capacity were measured in 22 lymphoblastoid cell lines to determine the functional effects of the polymorphisms. All statistical tests were two-sided. RESULTS: Genotype and haplotype frequency distributions were strongly dependent on ethnicity; variant allele frequencies were highest in African-Americans (29.1%) and lowest in Mexican-Americans (12.2%). Each of the three polymorphisms was associated with an increased risk of lung cancer among all ethnic groups. Moreover, for all three polymorphisms, increased variant allele copy number was associated with increased risk of lung cancer. Similarly, the variant haplotypes were also associated with an increased risk for lung cancer. Lymphoblastoid cell lines with all wild-type alleles at the three loci had statistically significantly higher apoptotic indices (13.66%, 95% CI = 8.61% to 18.71%) and DNA repair capacity (27.63%, 95% CI = 21.72% to 33.53%) than cell lines with at least one variant allele at all three loci (3.50%, 95% CI = 1.08% to 5.91%; and 17.48%, 95% CI = 7.99% to 26.96%, respectively). CONCLUSIONS: p53 polymorphisms may be associated with increased lung cancer risk and may affect p53 function.

Retinoic acid receptor beta and telomerase catalytic subunit expression in bronchial epithelium of heavy smokers.

Expression of retinoic acid receptor beta (RAR beta) and telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) was analyzed in 285 bronchial biopsy specimens from 53 heavy cigarette smokers and four former smokers by using in situ hybridization. Of the 191 biopsy specimens that were positive for RAR beta, 69% expressed hTERT, whereas only 54% of the 94 RAR beta negative biopsy specimens expressed hTERT (P =.014). Because hTERT expression in bronchial tissue has been previously associated with increased risk of lung cancer, the association between RAR beta and hTERT expression suggests that RAR beta expression may be an indicator of increased risk of lung cancer in heavy smokers.

Cyclin D1 genotype, response to biochemoprevention, and progression rate to upper aerodigestive tract cancer.

BACKGROUND: Altered cyclin D1 expression in advanced preinvasive lesions of the upper aerodigestive tract (UADT) is associated with an increased risk of developing cancer and histologic progression during and after combination biochemopreventive therapy (13-cis-retinoic acid, alpha-interferon, and alpha-tocopherol). Both alleles of the adenine (A)/guanine (G) cyclin D1 polymorphism located at nucleotide 870 encode two alternatively spliced transcripts, but the A allele preferentially encodes a protein with an extended half-life. We investigated whether the cyclin D1 genotype at nucleotide 870 was associated with baseline levels of cyclin D1 protein, post-treatment modulation of cyclin D1 protein levels, histologic response to treatment, and the outcome for subjects with preinvasive UADT lesions after biochemopreventive therapy. METHODS: UADT tissue biopsy samples were obtained before and 6 and 12 months after biochemopreventive treatment from 31 individuals with advanced preinvasive UADT lesions. Tissues were examined for cyclin D1 genotype (by DNA single-strand conformation polymorphism analysis), for cyclin D1 protein expression (by immunohistochemistry), and for cyclin D1 gene copy number (by fluorescence in situ hybridization). Associations of cyclin D1 genotype with histologic response to therapy and time to progression to a higher degree of dysplasia or invasive cancer were investigated. All statistical tests were two-sided. RESULTS: The A allele was associated with increased baseline cyclin D1 expression in the parabasal epithelial layer (16 of 18 AA/AG subjects versus four of nine GG subjects; P =.02), decreased histologic response to biochemopreventive treatment (six of 21 AA/AG subjects versus four of 10 GG subjects; P =.70), decreased favorable modulation of cyclin D1 expression by the treatment (seven of 18 AA/AG subjects versus eight of nine GG subjects; P =.02), and shorter progression-free survival (P =.05). CONCLUSIONS: The cyclin D1 A allele was associated with a diminished modulation of normal physiologic and treatment-induced decreased expression of cyclin D1, a decreased likelihood of response to biochemopreventive intervention, and an increased rate of progression to cancer development, findings that require validation in a larger cohort.

The synthetic retinoid CD437 selectively induces apoptosis in human lung cancer cells while sparing normal human lung epithelial cells.

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces apoptosis in a variety of cancer cells including lung cancer cells. Our previous studies have demonstrated that cancer cells with wild-type p53 are more sensitive to CD437 than those having mutant p53, although CD437 can induce both p53-dependent and -independent apoptosis. Because normal human lung epithelial cells have wild-type p53, the question arose as to whether they are also sensitive to CD437-induced apoptosis. To address this question, we compared and contrasted the effects of CD437 on apoptosis induction and the expression of several p53-regulated apoptosis-related genes between normal human lung epithelial cells and human lung cancer cells containing wild-type p53. CD437 induced apoptosis as evidenced by apoptotic morphological changes, increased DNA fragmentation, and activation of caspase cascades in two lung cancer cell lines but not in two normal human lung epithelial cells. CD437 selectively increased the p53 protein level and concomitantly induced the expression of several p53-regulated apoptosis-related genes including Bax, Fas, DR4, and DR5 only in the two lung cancer cell lines. Furthermore, the normal lung epithelial cells, which expressed constitutively higher levels of two antiapoptotic decoy receptors DcR1 and DcR2 than lung cancer cells, exhibited an increase in the expression of these receptors after CD437 treatment, whereas no increase was detected in lung cancer cells. These results predict a differential effect of CD437 on tumor and normal cells in vivo and strongly suggest that CD437 may be a useful agent for chemoprevention and/or treatment of human cancer, especially lung cancer.

A novel polymorphism in human cytosine DNA-methyltransferase-3B promoter is associated with an increased risk of lung cancer.

DNA repair is central to genomic integrity. Reduced expression of several nucleotide excision repair genes has been demonstrated to be associated with increased risk of lung cancer. Because methylation of gene promoters is one of the major regulatory mechanisms of gene expression and most nucleotide excision repair gene promoters have not been fully characterized, we hypothesized that genetic variants of the genes that are responsible for regulating genomic methylation are associated with increased risk of lung cancer. Recently, we identified a C-->T transition at a novel promoter region of cytosine DNA-methyltransferase-3B (DNMT3B) and found that this polymorphic transition significantly increases the promoter activity. In this hospital-based case-control study of 319 patients with incident lung cancer and 340 healthy controls frequency matched on age (+/-5 years), sex, ethnicity, and smoking status, we genotyped subjects for this DNMT3B promoter polymorphism to determine the association between this genetic variant and risk of lung cancer. Compared with CC homozygotes, CT heterozygotes had a >2-fold increased risk of lung cancer [adjusted odds ratio (OR), 2.13; 95% confidence interval (CI), 1.47-3.08] and TT homozygotes an OR of 1.42 (95% CI, 0.91-2.21). The combined variant genotype (CT + TT) was associated with a nearly 2-fold increased risk (adjusted OR, 1.88; 95% CI, 1.32-2.66). These results suggest that this novel variant of DNMT3B is associated with increased risk of lung cancer and may contribute to identifying individuals genetically susceptible to tobacco-induced cancers. Additional studies on the underlying molecular mechanism of this polymorphism are warranted.

Cyclin B1 overexpression and resistance to radiotherapy in head and neck squamous cell carcinoma.

Radiotherapy (RT) and surgery are the cornerstones in treating head and neck squamous cell carcinoma (HNSCC). RT is effective in the initial treatment of early to intermediate stage HNSCC but less effective for locally advanced disease, some cases of which are best managed using the combination of surgery and RT. Although various clinical/pathologic parameters have been used to classify patients according to their likelihood of responding to RT, they have generally low predictive value. We have shown previously that cyclin B1 is associated with a poor clinical outcome in HNSCC patients. In this study, we investigate the potential role of cyclin B1 in assessing RT response in patients with HNSCC. Tumor specimens obtained from 80 patients participated in a prospective Phase III clinical trial addressing the dose and fractionation regimen of postoperative RT were analyzed for cyclin B1 expression by immunohistochemistry. Patients were classified according to currently accepted clinical/pathologic parameters into three risk groups, i.e., low, intermediate, and high risk, and received surgery alone, surgery plus intermediate-dose RT, and surgery plus high-dose RT, respectively. The median follow-up duration was 4.9 years. Cyclin B1 overexpression was noted in 38 of the 80 (47%) HNSCC tumors. Interestingly, 11 of the 38 patients (29%) with cyclin B1-overexpressing tumors experienced local or nodal recurrence compared with only 3 of 42 patients (7%) having carcinomas with no or weak cyclin B1 expression (P = 0.01). When locoregional control was used as the end point for the high-risk group, patients whose tumors showed cyclin B1 overexpression had a statistically significant higher tumor recurrence and metastasis compared with patients whose tumors showed no cyclin B1 overexpression (P = 0.01). Our results indicate that tumors overexpressing cyclin B1 may be resistant to RT, and cyclin B1 may be an indicator of the risk of locoregional recurrence and metastasis in patients having HNSCC receiving RT.

Induction of TIG3, a putative class II tumor suppressor gene, by retinoic acid in head and neck and lung carcinoma cells and its association with suppression of the transformed phenotype.

Retinoids can regulate the proliferation and differentiation of various tumor cells. It is thought that nuclear retinoid receptors mediate these effects by regulating gene transcription. The identity of specific retinoid target genes is only beginning to be unraveled. One candidate for mediating retinoid-induced growth suppression is the novel class II tumor suppressor gene tazarotene-induced gene 3 (TIG3). We examined the constitutive and all-trans retinoic acid (ATRA)-inducible expression of TIG3 mRNA in five head and neck squamous cell carcinoma (HNSCC) and five nonsmall cell lung carcinoma (NSCLC) cell lines to determine whether it is associated with their responsiveness to ATRA. The expression patterns of retinoic acid receptor beta (RARbeta), another putative retinoid-inducible tumor suppressor gene, were also examined. The constitutive TIG3 expression was high in one HNSCC cell line and two NSCLC cell lines, and moderate to very low in the other cells. Some RARbeta-expressing cells had either low or undetectable TIG3 levels and vice versa. ATRA (1 microM; 48 h) increased TIG3 mRNA in 4/5 HNSCCs and 3/5 NSCLCs and RARbeta mRNA in some of the same cell lines, but also in cells that did not show TIG3 induction. TIG3 mRNA was induced by ATRA between 6 and 12 h in most of the responsive cells. ATRA concentrations required for TIG3 induction ranged from 1 to 500 nM depending on the cell line. The pan-RAR antagonists AGN193109 and the RARalpha antagonist Ro 41-5253 blocked TIG3 induction by ATRA. ATRA suppressed anchorage-independent colony formation in most cells that had a high or moderate constitutive or induced TIG3 expression level. In contrast, RARbeta mRNA expression pattern was not correlated with sensitivity to ATRA. These results suggest that TIG3 is regulated by ATRA via retinoid receptors in certain aerodigestive tract cancer cells, and its induction by ATRA is associated with the suppression of anchorage-independent growth.

Prognostic factors in resected stage I non-small-cell lung cancer: a multivariate analysis of six molecular markers.

PURPOSE: To analyze the prognostic significance of six molecular biomarkers (death-associated protein kinase [DAPK] promoter methylation, interleukin-10 [IL-10] protein expression, cyclooxygenase-2 [COX-2] mRNA expression, human telomerase reverse transcriptase catalytic subunit [hTERT] mRNA expression, retinoic acid receptor-beta [RAR-beta] mRNA expression, and K-ras mutational status) in stage I non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: Biomarker analyses were performed on tumors from 94 patients with stage I NSCLC who underwent surgical resection at our institution. A minimum follow-up period of 5 years was required. DAPK methylation was assessed by methylation-specific polymerase chain reaction (PCR). RAR-beta, COX-2, and hTERT mRNA levels were determined by in situ hybridization with digoxigenin-labeled antisense riboprobes. K-ras mutation status was determined by the PCR-primer introduced restriction with enrichment for mutant alleles method. IL-10 protein expression was analyzed by immunohistochemistry using a polyclonal antihuman IL-10 antibody. Cancer-specific survival was analyzed with a Cox proportional hazards model. To identify independent prognostic factors, a stepwise selection method was used. RESULTS: DAPK methylation, IL-10 lack of expression, COX-2 expression, hTERT expression, RAR-beta expression, and K-ras mutations were observed in 46.8%, 29.8%, 59.6%, 34.0%, 23.4%, and 34.0% of patients, respectively. In the final model, DAPK methylation and IL-10 lack of expression were significant negative prognostic factors for cancer-specific survival, whereas COX-2 expression was of borderline significance. CONCLUSION: In this cohort of resected stage I NSCLC patients, molecular markers that independently predict cancer-specific survival have been identified. The prognostic roles of DAPK methylation, IL-10, and other biomarkers in NSCLC merit further investigation.