Idiopathic pulmonary fibrosis (IPF) is an untreatable lung disease with a median survival of only 3-5 years that is diagnosed using a combination of clinical, radiographic and pathologic criteria. ...Histologically, IPF is characterised by usual interstitial pneumonia (UIP), a fibrosing interstitial pneumonia with a pattern of heterogeneous, subpleural regions of fibrotic and remodelled lung. We hypothesised that gene expression profiles of lung tissue may identify molecular subtypes of disease that could classify subtypes of IPF/UIP that have clinical implications.
We collected transcriptional profiles on lung tissue from 119 patients with IPF/UIP and 50 non-diseased controls. Differential expression of individual transcripts was identified using an analysis of covariance (ANCOVA) model incorporating the clinical diagnosis of each patient as well as age, gender and smoking status. Validation was performed in an independent cohort of 111 IPF/UIP and 39 non-diseased controls. Our analysis identified two subtypes of IPF/UIP based on a strong molecular signature associated with expression of genes previously associated with fibrosis (matrix metalloproteinases, osteopontin, keratins), cilium genes and genes with unknown function. We demonstrate that elevated expression of cilium genes is associated with more extensive microscopic honeycombing and higher expression of both the airway mucin gene MUC5B and the metalloproteinase MMP7, a gene recently implicated in attenuating ciliated cell differentiation during wound repair.
Expression of cilium genes appears to identify two unique molecular phenotypes of IPF/UIP. The different molecular profiles may be relevant to therapeutic responsiveness in patients with IPF/UIP.
The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to ...27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.
The modest response of patients with head and neck squamous cell carcinoma (HNSCC) and non–small cell lung carcinoma (NSCLC)
to epithelial growth factor receptor tyrosine kinase inhibitors such as ...gefitinib and erlotinib indicates the need for the
development of biomarkers to predict response. We determined gefitinib sensitivity in a panel of HNSCC cell lines by a 5-day
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confirmed these responses with analysis of downstream
signaling by immunoblotting and cell cycle arrest. Basal gene expression profiles were then determined by microarray analysis
and correlated with gefitinib response. These data were combined with previously reported NSCLC microarray results to generate
a broader predictive index. Common markers of resistance between the two tumor types included genes associated with the epithelial
to mesenchymal transition. We confirmed that increased protein expression of vimentin combined with the loss of E-cadherin,
claudin 4, and claudin 7 by immunoblotting was associated with gefitinib resistance in both HNSCC and NSCLC cell lines. In
addition, the loss of the Ca 2+ -independent cell-cell adhesion molecules EpCAM and TROP2 in resistant lines was confirmed by immunofluorescence. Tumor xenografts
derived from the gefitinib-sensitive UM-SCC-2 were growth-delayed by gefitinib, whereas the gefitinib-resistant 1483 xenografts
were unaffected. These data support a role for epithelial to mesenchymal transition in establishing gefitinib resistance for
both HNSCC and NSCLC, and indicate that clinical trials should address whether these biomarkers will be useful for patient
selection. Mol Cancer Ther 2007;6(6):1683–91
Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell
lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as ...gefitinib exhibit limited activity in this cancer.
We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression
profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell
lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed
in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent
growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI (±)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1 H -pyrimido-4,5- d pyrimidin-2-one (RO4383596) that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation,
two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress
FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking
FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor
autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.
Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and ...emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.
Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but ...mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis. (Blood. 2004;104:3878-3885)
Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies ...have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences.
To assess whether there is larger-scale genomic instability.
We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH.
Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality.
Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.
A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment ...of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent.
Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC(50) value as sensitive (<or=1.5 micromol/L) or resistant (>5 micromol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo.
Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts.
These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials.
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority
of patients with advanced-stage non–small cell lung cancer (NSCLC), and about ...half of all treated patients progress within
6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors
of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism
of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated
with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib.
This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with
known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of
this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein
expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry.
This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations.
Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to
the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.
(Mol Cancer Res 2006;4(8):521–8)
The quality of gene expression microarray data has improved dramatically since the first arrays were introduced in the late 1990s. However, the reproducibility of data generated at multiple ...laboratory sites remains a matter of concern, especially for scientists who are attempting to combine and analyze data from public repositories. We have carried out a study in which a common set of RNA samples was assayed five times in four different laboratories using Affymetrix GeneChip arrays. We observed dramatic differences in the results across laboratories and identified batch effects in array processing as one of the primary causes for these differences. When batch processing of samples is confounded with experimental factors of interest it is not possible to separate their effects, and lists of differentially expressed genes may include many artifacts. This study demonstrates the substantial impact of sample processing on microarray analysis results and underscores the need for randomization in the laboratory as a means to avoid confounding of biological factors with procedural effects.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
