Therapeutic targeting of tumor-associated myeloid cells synergizes with radiation therapy for glioblastoma.

Tumor-associated myeloid cells (TAMCs) are key drivers of immunosuppression in the tumor microenvironment, which profoundly impedes the clinical response to immune-dependent and conventional therapeutic modalities. As a hallmark of glioblastoma (GBM), TAMCs are massively recruited to reach up to 50% of the brain tumor mass. Therefore, they have recently been recognized as an appealing therapeutic target to blunt immunosuppression in GBM with the hope of maximizing the clinical outcome of antitumor therapies. Here we report a nano-immunotherapy approach capable of actively targeting TAMCs in vivo. As we found that programmed death-ligand 1 (PD-L1) is highly expressed on glioma-associated TAMCs, we rationally designed a lipid nanoparticle (LNP) formulation surface-functionalized with an anti-PD-L1 therapeutic antibody (αPD-L1). We demonstrated that this system (αPD-L1-LNP) enabled effective and specific delivery of therapeutic payload to TAMCs. Specifically, encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into PD-L1-targeted LNPs led to a robust depletion of TAMCs and an attenuation of their immunosuppressive functions. Importantly, the delivery efficiency of PD-L1-targeted LNPs was robustly enhanced in the context of radiation therapy (RT) owing to the RT-induced up-regulation of PD-L1 on glioma-infiltrating TAMCs. Accordingly, RT combined with our nano-immunotherapy led to dramatically extended survival of mice in 2 syngeneic glioma models, GL261 and CT2A. The high targeting efficiency of αPD-L1-LNP to human TAMCs from GBM patients further validated the clinical relevance. Thus, this study establishes a therapeutic approach with immense potential to improve the clinical response in the treatment of GBM and warrants a rapid translation into clinical practice.

Tuberculosis infection and lung adenocarcinoma: Mendelian randomization and pathway analysis of genome-wide association study data from never-smoking Asian women.

We investigated whether genetic susceptibility to tuberculosis (TB) influences lung adenocarcinoma development among never-smokers using TB genome-wide association study (GWAS) results within the Female Lung Cancer Consortium in Asia. Pathway analysis with the adaptive rank truncated product method was used to assess the association between a TB-related gene-set and lung adenocarcinoma using GWAS data from 5512 lung adenocarcinoma cases and 6277 controls. The gene-set consisted of 31 genes containing known/suggestive associations with genetic variants from previous TB-GWAS. Subsequently, we followed-up with Mendelian Randomization to evaluate the association between TB and lung adenocarcinoma using three genome-wide significant variants from previous TB-GWAS in East Asians. The TB-related gene-set was associated with lung adenocarcinoma (p = 0.016). Additionally, the Mendelian Randomization showed an association between TB and lung adenocarcinoma (OR = 1.31, 95% CI: 1.03, 1.66, p = 0.027). Our findings support TB as a causal risk factor for lung cancer development among never-smoking Asian women.

Association between GWAS-identified lung adenocarcinoma susceptibility loci and EGFR mutations in never-smoking Asian women, and comparison with findings from Western populations.

To evaluate associations by EGFR mutation status for lung adenocarcinoma risk among never-smoking Asian women, we conducted a meta-analysis of 11 loci previously identified in genome-wide association studies (GWAS). Genotyping in an additional 10,780 never-smoking cases and 10,938 never-smoking controls from Asia confirmed associations with eight known single nucleotide polymorphisms (SNPs). Two new signals were observed at genome-wide significance (P < 5 × 10-8), namely, rs7216064 (17q24.3, BPTF), for overall lung adenocarcinoma risk, and rs3817963 (6p21.3, BTNL2) which is specific to cases with EGFR mutations. In further sub-analyses by EGFR status, rs9387478 (ROS1/DCBLD1) and rs2179920 (HLA-DPB1) showed stronger estimated associations in EGFR-positive compared to EGFR-negative cases. Comparison of the overall associations with published results in Western populations revealed that the majority of these findings were distinct, underscoring the importance of distinct contributing factors for smoking and non-smoking lung cancer. Our results extend the catalogue of regions associated with lung adenocarcinoma in non-smoking Asian women and highlight the importance of how the germline could inform risk for specific tumour mutation patterns, which could have important translational implications.

Genetic variants associated with longer telomere length are associated with increased lung cancer risk among never-smoking women in Asia: a report from the female lung cancer consortium in Asia.

Recent evidence from several relatively small nested case-control studies in prospective cohorts shows an association between longer telomere length measured phenotypically in peripheral white blood cell (WBC) DNA and increased lung cancer risk. We sought to further explore this relationship by examining a panel of seven telomere-length associated genetic variants in a large study of 5,457 never-smoking female Asian lung cancer cases and 4,493 never-smoking female Asian controls using data from a previously reported genome-wide association study. Using a group of 1,536 individuals with phenotypically measured telomere length in WBCs in the prospective Shanghai Women's Health study, we demonstrated the utility of a genetic risk score (GRS) of seven telomere-length associated variants to predict telomere length in an Asian population. We then found that GRSs used as instrumental variables to predict longer telomere length were associated with increased lung cancer risk (OR = 1.51 (95% CI = 1.34-1.69) for upper vs. lower quartile of the weighted GRS, p value = 4.54 × 10(-14) ) even after removing rs2736100 (p value = 4.81 × 10(-3) ), a SNP in the TERT locus robustly associated with lung cancer risk in prior association studies. Stratified analyses suggested the effect of the telomere-associated GRS is strongest among younger individuals. We found no difference in GRS effect between adenocarcinoma and squamous cell subtypes. Our results indicate that a genetic background that favors longer telomere length may increase lung cancer risk, which is consistent with earlier prospective studies relating longer telomere length with increased lung cancer risk.

Single nucleotide polymorphisms of MAGE-A3 gene and its clinical implications in Chinese patients with non-small cell lung cancer (NSCLC).

BACKGROUND: Available study revealed advanced tumors have a higher expression rate of MAGE-A3 gene which has a lot of single nucleotide polymorphism (SNP) loci with polymorphisms. This study aimed to analyze the allele frequency of SNP loci in MAGE-A3 gene and investigate the relationship between MAGE-A3 gene polymorphisms and clinical factors. METHODS: Tumor samples of a cohort of 191 NSCLC patients were collected. EGFR mRNA expression were detected by qRT-PCR. SNPs in whole length of MAGE-A3 gene were detected by direct sequencing. Frequencies of the SNPs were correlated to gene expression, mutation status of EGFR and clinical factors. RESULTS: Sequencing analysis confirmed that allele frequencies of genotypes on SNP loci rs5970360, rs5925210, rs5970361, rs5925211 and rs35123853 were CC (0.681)/CT (0.319), CC (0.660)/CG (0.340), CC (0.681)/CA (0.319), AA (0.984)/AT (0.016) and GG (1.000)/GA (0.000), respectively, which were different from the frequencies and genotypes of MAGE-A3 in SNP database. Chi-square tests showed the EGFR mRNA expression level had significant correlation with the genotypes of SNP loci rs5970360 and rs5925210. But all frequencies of each MAGE-A3 SNPs were not found significantly different between EGFR mutant and wild type patients. MAGE-A3 gene polymorphisms had no significant effects on survival of NSCLC patients. CONCLUSIONS: Chinese patients with NSCLC had different SNP patterns of MAGE-A3 in comparison with those in international SNP database. These MAGE-A3 SNP loci might have not prognostic significance. MAGE-A3 SNP loci rs5970360 and rs5925210 might be predictive for EGFR mRNA expression levels and helpful to the selection of patients for epidermal growth factor receptor (EGFR) targeted immunotherapy.

miR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment. METHODS: Formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays. RESULTS: The expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression. CONCLUSIONS: Our findings suggest that miR-638 plays an important role in TNBC progression via BRCA1 deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer.

Lung cancer treatment disparities in China: a question in need of an answer.

BACKGROUND: Substantial progress has been made in the treatment of malignancies in the People's Republic of China in recent years. The goal of this study was to identify the extent to which national treatment guidelines are being used to customize patient care in lung cancer and to analyze the reasons for treatment disparities. METHODS: Patient characteristics and treatments were investigated retrospectively for the period from October 2004 to January 2013 using the outpatient database of the Guangdong Lung Cancer Institute (GLCI) in China. RESULTS: A total of 2,535 outpatients with lung cancer were studied in this retrospective analysis. The treatment disparity was 45.3%. Overall, 20.6% of patients with stage I non-small cell lung cancer (NSCLC) were overtreated, and 20.1% of stage II patients were undertreated. Only 19.6% of stage IIIA patients and 30.7% of stage IIIB patients underwent the recommended combination of chemotherapy and radiotherapy, respectively. For advanced NSCLC, the greatest treatment disparity appeared in the second-line setting and beyond. Patients who were positive for epidermal growth factor receptor (EGFR) and receiving EGFR tyrosine kinase inhibitors experienced significant prolongation of survival compared with patients who were EGFR negative or whose EGFR mutation status was unknown (hazard ratio: 0.79; p = .037). The treatment disparities were significantly larger among patients aged younger than 65 years and in patients from developing regions compared with patients aged 65 years and older and from developed regions, respectively (p < .001, p = .046). The difference in treatment disparity was statistically significant between GLCI and other hospitals (p < .001). CONCLUSION: This retrospective study of a large number of patients from an outpatient oncology database demonstrated large disparities in the treatment of lung cancer in China. It is important to develop a new guideline for recommendations that are based on resource classification.

Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay.

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

KRAS mutation in patients with lung cancer: a predictor for poor prognosis but not for EGFR-TKIs or chemotherapy.

BACKGROUND: The prognostic and predictive value of KRAS mutations in patients with lung cancer is controversial. Biases in disease stage, treatment regimen, small-scale patient studies, and biomarker status have led to inconsistent results in previous reports. METHODS: The KRAS and EGFR genes were examined in 1935 consecutive patients with non-small cell lung cancer. All patients were divided into KRAS mutation (KRAS group), EGFR mutation (EGFR group), and KRAS/EGFR wild type (WT group) groups. Randomly selected cases were paired with patients with the KRAS mutation, the EGFR mutation, and KRAS/EGFR wild type patients according to tumor, node, metastasis stage, time of first visit within 1 year, and pathology. Progression-free survival (PFS) and overall survival were evaluated by Kaplan-Meier and Cox models. RESULTS: The KRAS mutation rate for lung adenocarcinoma was 5.90 %. The overall survival was 14.47, 20.57, and 42.73 months for the KRAS group, WT group, and EGFR group, respectively (P < 0.001). Multivariate analysis indicated that KRAS mutation status was an independent prognostic factor (hazard ratio 2.69, 95 % confidence interval 1.91-3.80, P < 0.001). No difference was found in PFS and tumor responsiveness between patients with a KRAS mutation and those with wild type KRAS/EGFR for chemotherapy and EGFR tyrosine kinase inhibitors (TKI). PFS did not significantly differ for chemotherapy among the three groups (P = 0.270). CONCLUSIONS: KRAS mutation is a poor prognosis factor, but it is not an independent predictor of response to EGFR-TKI or chemotherapy in patients with lung cancer.

LIN28B and Let-7 in Diffuse Midline Glioma: A Review.

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

Docetaxel targets aggressive methylation profiles and serves as a radiosensitizer in high-risk meningiomas.

BACKGROUND: Meningioma is the most common primary intracranial tumor in adults. A subset of these tumors recur and invade the brain, even after surgery and radiation, resulting in significant disability. There is currently no standard-of-care chemotherapy for meningiomas. As genomic DNA methylation profiling can prognostically stratify these lesions, we sought to determine whether any existing chemotherapies might be effective against meningiomas with high-risk methylation profiles. METHODS: A previously published dataset of meningioma methylation profiles was used to screen for clinically significant CpG methylation events and associated cellular pathways. Based on these results, patient-derived meningioma cell lines were used to test candidate drugs in vitro and in vivo, including efficacy in conjunction with radiotherapy. RESULTS: We identified 981 genes for which methylation of mapped CpG sites was related to progression-free survival in meningiomas. Associated molecular pathways were cross-referenced with FDA-approved cancer drugs, which nominated Docetaxel as a promising candidate for further preclinical analyses. Docetaxel arrested growth in 17 meningioma cell sources, representing all tumor grades, with a clinically favorable IC50 values ranging from 0.3 nM to 10.7 mM. The inhibitory effects of this medication scaled with tumor doubling time, with maximal benefit in fast-growing lesions. The combination of Docetaxel and radiation therapy increased markers of apoptosis and double-stranded DNA breaks, and extended the survival of mice engrafted with meningioma cells relative to either modality alone. CONCLUSIONS: Global patterns of DNA methylation may be informative for the selection of chemotherapies against meningiomas, and existing drugs may enhance radiation sensitivity in high-risk cases.

Genome-wide association study of lung adenocarcinoma in East Asia and comparison with a European population.

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (Pinteraction = 0.0058). These findings provide new insights into the etiology of lung adenocarcinoma in individuals from East Asian populations, which could be important in developing translational applications.

EGFR mutation heterogeneity and the mixed response to EGFR tyrosine kinase inhibitors of lung adenocarcinomas.

BACKGROUND: Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutations have mixed responses to tyrosine kinase inhibitors (TKIs). Intertumor heterogeneity in EGFR mutations is one potential explanation for this phenomenon. METHODS: We performed direct sequencing to identify EGFR mutations in 180 pairs of lung adenocarcinoma samples (from 3,071 patients). The high-resolution melting method was used in discordant cases to confirm EGFR mutation status. Matching samples were divided into four groups: primary lesions detected at different times, primary tumors with matched metastatic lymph nodes, multiple pulmonary nodules, and primary tumors with matched distant metastases. Multivariate analyses were performed to evaluate correlations between heterogeneity and patient characteristics. RESULTS: In the study population, the discordance rate was 13.9% (25 of 180). The multiple pulmonary nodules group had the highest discordance rate of 24.4% (10 of 41; odds ratio for heterogeneity in primary lesions detected at different times, 6.37; 95% confidence interval, 1.71-23.72; p = .006). Discordance rates in the metachronous and synchronous settings were 15.7% (22 of 140) and 7.5% (three of 40), respectively. In the 34 patients who developed EGFR TKI resistance, 10 (29.4%) cases exhibited heterogeneity and five (14.7%) patients exhibited a mixed response to the drug. Three (8.8%) of the patients with a mixed response also exhibited discordant EGFR mutations. CONCLUSIONS: The overall discordance rate of EGFR mutation heterogeneity in Asian patients with pulmonary adenocarcinoma is relatively low, but the rate in patients with multiple pulmonary nodules is significantly higher. This observation may explain the mixed tumor response to EGFR TKIs.

Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression.

BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer. RESULTS: RACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups. CONCLUSIONS: The EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

The -271 G>A polymorphism of kinase insert domain-containing receptor gene regulates its transcription level in patients with non-small cell lung cancer.

BACKGROUND: Kinase insert domain-containing receptor (KDR) plays a critical role in the metastasis of cancer and is used as a molecular target in cancer therapy. We investigated the characteristics of the -271 G>A polymorphism of the KDR gene to gain information that may benefit the development of individualized therapies for patients with non-small cell lung cancer (NSCLC). METHODS: The -271 G>A polymorphism of the KDR gene in 106 lung cancer patients and 203 healthy control individuals was analyzed by polymerase chain reaction (PCR) and DNA sequencing methods. Real-time quantitative PCR and immunohistochemical methods were used to evaluate KDR mRNA and protein expression levels, respectively, in frozen tumor specimens. RESULTS: The -271 G>A polymorphism was associated with the mRNA expression level of the KDR gene in tumor tissues (t = 2.178, P = 0.032, independent samples t-test). Compared with the AG/GG genotype, the AA genotype was associated with higher KDR mRNA expression in tumor tissues. We found no relationship between the genotype and the KDR protein expression level and no significant difference in the distribution of the KDR gene polymorphism genotypes between lung cancer patients and the control group (chi2 = 1.269, P = 0.264, Fisher's exact test). CONCLUSION: This study is the first to show that the -271 G>A polymorphism of the KDR gene may be a functional polymorphism related to the regulation of gene transcription. These findings may have important implications for therapies targeting KDR in patients with NSCLC.

An autologous therapeutic dendritic cell vaccine transfected with total lung carcinoma RNA stimulates cytotoxic T lymphocyte responses against non-small cell lung cancer.

OBJECTIVE: The development of immunotherapy for malignancy is greatly limited by the characteristic weak antigenicity of tumors. The primary goal of this study was to circumvent the isolation and purification of tumor-specific antigen determinants by producing a vaccine using lung tumor RNA-loaded dendritic cells (DCs), and to test the response against lung cancer. METHODS: Total RNA was isolated from 18 lung carcinomas with positive carcinoembryonic antigen (CEA) and mucin-1 (MUC1) staining, as identified by immunohistochemistry. DCs and T-cells from peripheral blood mononuclear cells were generated in vitro, and then DCs in different stages were transfected with RNA using several different methods. The expression of CEA and MUC1 in RNA-transfected DCs was measured using flow cytometry. T-cells stimulated by DCs were harvested as effectors, and primary tumor cells cultured in vitro were used as targets. Cytotoxicity was determined by lactic dehydrogenase detection assay. RESULTS: Immature RNA-transfected DCs significantly increased the expression of CEA and MUC1, compared to mature transfected DCs. RNA transfection via electroporation resulted in significantly greater CEA and MUC1 expression than did transfection via lipofection or passive pulsing. Lymphocytes stimulated by DCs transfected with lung tumor RNA initiated a cytotoxic T lymphocyte (CTL) tumor-specific response. CONCLUSION: Immature DCs transfected with total lung carcinoma RNA by electroporation in vitro effectively stimulate antigen-specific CTL responses against tumor cells.

Better survival with EGFR exon 19 than exon 21 mutations in gefitinib-treated non-small cell lung cancer patients is due to differential inhibition of downstream signals.

Somatic mutations in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Our clinical data showed NSCLC patients with exon 19 deletions survived longer following gefitinib treatment than those with exon 21 point mutations. We aimed to investigate whether these two mutations produced differences in phosphorylation of EGFR and downstream signals. Two stable cell lines expressing these mutations were obtained by transfection. Inhibition of phosphorylation of EGFR, Akt, and Erk by gefitinib was detected using Western blotting, and cell inhibition tests were conducted to evaluate the bio-behavior. Gefitinib inhibited the phosphorylation of EGFR, Akt, and Erk to a greater degree in exon 19 deletion cells than in L858R cells. Gefitinib produced G1 arrest in more of the cells with exon 19 deletion than with L858R. This might be attributable to patient selection in TKIs therapy.

Impact of menopausal status and HER-2/neu protein on efficacy of EGFR-TKI in EGFR mutant patients with non-small cell lung cancer.

Clinical studies have confirmed epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) used in lung cancer patients with EGFR mutations can obtain a better result, but still part of the patients with poor efficacy. EGFR mutation is highly related to female, nonsmoking and adenocarcinoma. Thus, we hypothesize that estrogen and circulating HER-2/neu protein might influence the efficacy of EGFR-TKIs in EGFR mutant patients with non-small cell lung cancer. HER-2/neu expression level of 357 eligible patients in its peripheral serum was determined using ELISA. The median progression-free survival (PFS) in five groups (premenopausal group, perimenopause group, peri to postmenopausal group, postmenopausal group and control group) was statistically difference (P = 0.025). Premenopausal group could predict the efficacy of EGFR-TKI (HR = 2.45, 95% CI = 1.42-4.23, P = 0.001). No statistical significance was found in median overall survival (OS) among five groups. Optimal diagnostic cut off value of HER-2/neu was set at 47.5 ng/ml, with P = 0.0607. As the cutoff value to 47.5 ng/ml division, concentrations and menopausal status was of no significant difference (P = 0.874). PFS of the group below 47.5 ng/ml was significantly longer than that of the group over 47.5 ng/ml (P = 0.000). HER-2/neu concentration was positively correlated with optimal efficacy (P = 0.042). HER-2/neu concentration over than 47.5 ng/ml was a risk factor of EGFR-TKI prognosis. Premenopausal status is an independent predictor of EGFR-TKI curative effect and circulating HER-2/neu protein is an independent prognostic factor in patients with advanced NSCLC.

KDR expression is associated with the stage and cigarette smoking of the patients with lung cancer.

PURPOSE: Kinase insert domain-containing receptor (KDR) is one of the molecular targets used in cancer therapy. We studied the KDR expression characteristics and the relationship with the clinical parameters of the patients with lung cancer, to give the basic evidence and clue for tailoring therapy. METHODS: Reverse transcriptase and real-time PCR were used to evaluate the KDR mRNA expression levels in 222 tissue samples (106 tumor tissues, 106 matched normal tissues obtained from the same patients with lung cancer, and 10 normal lung specimens from individuals without lung cancer). The KDR mRNA expression level and clinical parameters were analyzed by paired-sample t test, ANOVA and linear regression, respectively. The Kaplan-Meier method and the log-rank test were used for survival analysis. Expression of KDR protein was also examined immunohistochemically in 15 tumor samples and 15 matched normal lung specimens. RESULTS: The KDR mRNA expression levels were significantly higher in normal tissues (mean 4.50 +/- 0.51) than that in the carcinoma tissues (mean 4.12 +/- 0.50, P < 0.0005). KDR expression in tumor tissues is associated with the histological status, tumor stage, cigarette smoking, and N stage of the patients with lung cancer (P < 0.05) analyzed by using ANOVA methods. Multivariate analysis showed that tumor stages and cigarette smoking status were the two most important independent predictors for the KDR expression levels in tumor tissues (R = 0.415, R (2) = 0.172, F = 10.694, P < 0.0005). Tumors with KDR mRNA expression levels above the mean had a shorter survival (466 +/- 313 days) than did patients with KDR expression levels below the mean (671 +/- 264 days), whereas Kaplan-Meier analysis and log-rank test showed no significant difference in the overall survival between the patients (P = 0.2055). All the 15 normal lung tissues detected showed scale 2 KDR immunostaining. The intensity of immunostaining for KDR in tumor specimens varied from negative (scale 0) to strongest (scale 3) staining. CONCLUSIONS: Locally advanced and non-cigarette smoking patients with lung cancer may be the two valuable surrogate markers for KDR mRNA higher levels. Non-squamous lung cancer, N 2 stage may be the secondary markers for that. The KDR expression level in normal lung tissue is stable, but varied in tumor tissues. Targeting KDR therapy in lung cancer might considerate these clinical and KDR expression information. Further confirmation study must be needed.