Value of KRAS as prognostic or predictive marker in NSCLC: results from the TAILOR trial.

BACKGROUND: The prognostic and predictive role of KRAS mutations in advanced nonsmall-cell lung cancer (NSCLC) is still unclear. TAILOR prospectively assessed the prognostic and predictive value of KRAS mutations in NSCLC patients treated with erlotinib or docetaxel in second line. PATIENTS AND METHODS: NSCLC patients from 52 Italian hospitals were genotyped for KRAS and EGFR mutational status in two independent laboratories. Wild-type EGFR patients (N = 218) received first-line platinum-based chemotherapy and were randomly allocated at progression to erlotinib or docetaxel. Overall survival (OS) according to KRAS mutational status was the primary end point. RESULTS: KRAS mutations were present in 23% of TAILOR randomized cases. The presence of a KRAS mutation did not adversely affect progression-free (PFS) or overall (OS) survival [hazard ratio (HR) PFS = 1.01, 95% confidence interval (CI) 0.71-1.41, P = 0.977; OS = 1.24, 95% CI 0.87-1.77, P = 0.233], nor influenced treatment outcome (test for interaction: OS P = 0.965; PFS P = 0.417). Patients randomized to docetaxel treatment experienced longer survival independently from the KRAS mutational status of their tumors (HR: mutated KRAS 0.81, 95% CI 0.45-1.47; wild-type KRAS 0.79, 95% CI 0.57-1.10). CONCLUSION: In TAILOR, KRAS was neither prognostic nor predictive of benefit for either docetaxel or erlotinib. Docetaxel remains superior independently from KRAS status for second-line treatment in EGFR wild-type advanced NSCLC patients. CLINICAL TRIAL REGISTRATION: NCT00637910.

Tumor necrosis factor-alpha in airway secretions from cystic fibrosis patients upregulate endothelial adhesion molecules and induce airway epithelial cell apoptosis: implications for cystic fibrosis lung disease.

Airway inflammation plays a crucial role in lung damage in cystic fibrosis (CF) and is characterized by a persistent influx of neutrophils into the airways. We hypothesized that the high levels of inflammatory products that accumulate in the microenvironment of the CF lung contribute to induce the persistent neutrophil recruitment and the airway epithelial damage. Thus, we evaluated the in vitro effect of sputum sol phase (SSP) from CF patients on a) adhesion molecule expression by human microvascular endothelial cells (HMECs) and b) apoptosis of human bronchial epithelial cells (HBECs), both wild-type and CFTR-defective. SSP was obtained from 7 clinically stable adult CF patients and 8 patients with an acute exacerbation. HMECs and HBECs were cultured in the absence or presence of SSP. Cell adhesion molecule expression was assessed by flow cytometry and cell death by the detection of histone-associated DNA fragments, caspase activation, and cytochrome c release. SSP obtained from CF patients, especially at the time of an acute exacerbation, induced a) an upregulation of endothelial adhesion molecules on cultured HMECs that was associated with an increase of neutrophil adhesion to these cells, and was mediated at least in part by TNF-alpha and IL-1 and b) apoptosis of airway epithelial cells, mainly activated by TNF- alpha pathway. These results suggest that the high concentrations of inflammatory mediators in CF airways contribute both to the chronic neutrophil influx and the airway damage, and support the crucial role of early anti-inflammatory treatment in the disease.

Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis.

Gene therapy of cystic fibrosis (CF) lung disease needs highly efficient delivery and long-lasting complementation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene into the respiratory epithelium. The development of lentiviral vectors has been a recent advance in the field of gene transfer and therapy. These integrating vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells by virtue of their ability to infect nondividing cells and mediate long-term persistence of transgene expression. Studies in human airway tissues and animal models have highlighted the possibility of achieving gene expression by lentiviral vectors, which outlasted the normal lifespan of the respiratory epithelium, indicating targeting of a 'stem cell' compartment. Modification of the paracellular permeability and pseudotyping with heterologous envelopes are the strategies currently used to overcome the paucity of specific viral receptors on the apical surface of airway epithelial cells and to reach the basolateral surface receptors. Preclinical studies on CF mice, demonstrating complementation of the CF defect, offer hope that lentivirus gene therapy can be translated into an effective treatment of CF lung disease. Besides a direct targeting of the stem/progenitor niche(s) in the CF airways, an alternative approach may envision homing of hematopoietic stem cells engineered to express the CFTR gene by lentiviral vectors. In the context of lentivirus-mediated CFTR gene transfer to the CF airways, biosafety aspects should be of primary concern.

Differentiated and functional human airway epithelium regeneration in tracheal xenografts.

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.

A bcl-2/IgH antisense transcript deregulates bcl-2 gene expression in human follicular lymphoma t(14;18) cell lines.

The 14;18 chromosome translocation, characteristic of most human follicular B-cell lymphomas, juxtaposes the bcl-2 gene with the IgH locus, creating a bcl-2/IgH hybrid gene. By mechanisms that are still under investigation, this event increases the cellular levels of the bcl-2 mRNA and thereby induces an overproduction of the antiapoptotic BCL-2 protein which is likely responsible for neoplastic transformation. In an effort to identify potential upregulators of bcl-2 activity in t(14;18) cells, we found, by strand-specific RT-PCR, a bcl-2 antisense transcript that is present in the t(14;18) DOHH2 and SU-DHL-4 but not in the t(14;18)-negative Raji and Jurkat lymphoid cell lines, and thus appears to be dependent on the bcl-2/IgH fusion. This antisense transcript is a hybrid bc1-2/IgH RNA, that originates in the IgH locus, encompasses the t(14;18) fusion site and spans at least the complete 3' UTR region of the bcl-2 mRNA. To achieve some insight into its biological function, we treated the t(14;18) DOHH2 cell line with oligonucleotides (ODNs) by specifically targeting the bc1-2/IgH antisense strand. These ODNs lowered bcl-2 gene expression, inhibited neoplastic cell growth by inducing apoptosis. We would like to propose the hypothesis that the bc1-2/IgH antisense transcript may contribute, by an unknown mechanism, to upregulation of bcl-2 gene expression in t(14;18) cells. The possibility has been considered that the hybrid antisense transcript mask AU-rich motifs present in the 3' UTR of the bcl-2 mRNA characterized in other genes as mRNA destabilizing elements.

Terminally L-modified oligonucleotides: pairing, stability and biological properties.

An oligodeoxynucleotide (ODN), capable of reducing the growth of human B lymphocytes carrying the t(14;18) chromosome translocation, was prepared in different 'chemical versions': unmodified phosphodiester, phosphorothioate and phosphodiester capped with L-2'-deoxycytidine. Their binding affinity to the complementary synthetic target was studied by the melting point assay. The ODNs, administered to DOHH2 cells, were compared for stability in the culture medium, cellular uptake, time course of the intact sequence concentration within the cell and ability to inhibit cell growth. The 5', 3'-L-capped derivative and the phosphorothioate had comparable potency, superior to that of the unmodified ODN, in agreement with the concentration of undegraded ODNs within the cell.

Oligonucleotides induce apoptosis restricted to the t(14;18) DHL-4 cell line.

Most human follicular B-cell lymphomas are associated with t(14;18) chromosome translocation that joins the bcl-2 gene with the IgH locus. This hybrid gene causes upregulation of BCL-2 protein expression, endowing cells with survival advantage. Although early BCL-2 overexpression is definitely responsible for immortalization/transformation, its exact role in the overt transformation as well as in the maintenance of the tumor phenotype is not known. The capacity of oligodeoxynucleotides (ODN) to modulate gene expression specifically has been exploited to downregulate the overexpression of BCL-2 protein in the SU-DHL-4 human follicular B-cell lymphoma line by the use of sense ODN or antisense ODN or antisense ODN designed to encompass the unique nucleotide sequence in the fusion region of the hybrid transcript. The specific downregulation of the bcl-2 transcript and of the relevant BCL-2 protein in the treated cells activated programed cell death and inhibited growing cells. The antitumor activity was restricted to the DHL-4 cell line carrying the specific nucleotide sequence at the bcl-2/IgH joining region. Thus, DHL-4 lymphoma cells derived from the acute phase of human follicular B-cell lymphoma, although endowed with additional activated oncogenes, were growth inhibited by bcl-2 downregulation with additional activated oncogenes, were growth inhibited by bcl-2 downregulation in a genetically restricted fashion. The biological activity was exerted exclusively by ODNs synthesized in the sense orientation. The sense ODNs have been proposed to anneal the hybrid bcl-2/IgH antisense RNA as identified in this study.

Quantitation of bcl-2 oncogene in cultured lymphoma/leukemia cell lines and in primary leukemia B-cells by a highly sensitive RT-PCR method.

BACKGROUND: The bcl-2 gene, isolated from the t(14;18) chromosomal translocation breakpoint, is able to prevent apoptotic death induced by various stimuli in different tissues. Therefore bcl-2 oncogene expression could be a key parameter for investigating the molecular mechanisms involved in the apoptosis of normal and neoplastic hematopoietic cells. METHODS: In order to evaluate bcl-2 expression in both follicular B-lymphomas carrying or not carrying the 14;18 translocation and in lymphatic leukemias, we optimized an internal standard-based method of reverse transcriptase-polymerase chain reaction (RT-PCR) for the rapid quantitation of bcl-2 mRNA cellular levels. A simple purification of the reverse transcription products resulted in very high PCR efficiency, so that radioactive labelling of the amplification products was avoided. RESULTS: bcl-2 mRNA levels proved to be higher in t(14;18) than in t(14;18) negative cell lines, and higher in primary leukemia pre-B cells than in early-B cells. Tested for sensitivity by identifying minimal residual t(14;18) B cells expressing the bcl-2/IgH gene, this RT-PCR method was able to detect bcl-2/IgH mRNA from just one t(14;18) positive cell out of ten million t(14;18) negative cells. CONCLUSIONS: The RT-PCR method we optimized appears to be suitable for clinical use in both leukemia/lymphoma characterization and in lymphomatous disease follow-up.