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Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1␣ (HIF-1␣)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity. (Blood. 2011; 117(2):459-469) IntroductionHuman multipotent stromal cells or mesenchymal stem cells (MSCs), capable of self-renewal and differentiating into various mesenchymal tissues, 1 have emerged as a promising tool for clinical applications in, for example, cell-based therapy for osteogenesis imperfecta 2 and tissue engineering in cartilage and bone. 3 MSCs are also applied in cardiac therapeutics because they prevent deleterious remodeling and improve recovery. 4 However, variations in the isolation techniques, growth media, and culture conditions used cause a remarkable difference in their proliferation and differentiation capacity. 5 Furthermore, many studies have consistently noticed a senescent tendency of MSCs upon expansion. 6,7 Thus, the difference in stem cell properties and the senescence encountered during expansion hinder the clinical applications of MSCs.Hypoxia has been known to regulate several cellular processes and signal transductions via the expression of hypoxia inducible factor-1 (HIF-1), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response factor HIF-1␣. HIF-1␣ is regulated by the cellular O 2 concentration and determines the transcriptional activity of HIF-1. 8 Most of the effects of HIF-1␣ were investigated on cancer cells. HIF-1␣, induced during ischemia that occurred in the course of tumor progression or after treatment, stimulates proliferation 9 and induces vascular endothelial growth factor expression and angiogenesis. 9 Hypoxia has also been reported to enhance proliferation, survival, and dopaminergic differentiation of central nervous system (CNS) precursors. 10 In parallel, hypoxia also determines the cell fate of neural crest stem cells. 11 These findings suggest neural stem cells may exhibit a conserved response ...

Purpose: Mutations in epidermal growth factor receptor (EGFR) can be used to predict the tumor response of patients receiving gefitinib for non^small cell lung cancer (NSCLC).We investigated the association between mutations in EGFR tyrosine kinase domain and tumor response and survival in gefitinib-treated NSCLC patients. Experimental Design: EGFR mutations in exons 18 to 21were analyzed by DNA sequencing of paraffin-embedded tumor tissues from gefitinib-treated NSCLC patients. The results were correlated with clinical variables. Results: EGFR mutations were found in 61.1% (33 of 54) of cases; response rate and disease control rate were 56.8% and 68.5%, respectively. There was no significant difference in mutation rates between adenocarcinoma (29 of 43) and nonadenocarcinoma (4 of 11; P = 0.085). However, all four nonadenocarcinomas with EGFR mutations had no response to gefitinib. Presence of EGFR mutations was the only independent predictor for disease control (P = 0.003) and tumor response (P = 0.017) in multivariate analysis; positive predictive values were 87.9% and 70.8% and negative predictive values were 61.9% and 69.2%, respectively. In comparison with patients whose tumor was negative for EGFR mutations, patients with EGFR mutations had better progression-free survival (median, 7.6 versus 1.7 months; P = 0.011) and overall survival (median, 14.7 versus 4.7 months; P = 0.046). Conclusions: Mutations in EGFR tyrosine kinase correlate with treatment response and survival in gefitinib-treated NSCLC patients and can be used as a predictive and prognostic factor. Thus, analysis of EGFR tyrosine kinase mutations in lung adenocarcinoma is of clinical significance, as it can permit the customization of treatment with EGFR tyrosine kinase inhibitors.

Introduction: Osimertinib has shown promising activity in patients with leptomeningeal metastases (LMs) of EGFRpositive NSCLC at 160 mg once daily (qd) (BLOOM; NCT02228369). We report LM activity with osimertinib (80 mg qd) in a retrospective analysis of studies across the AURA program (AURA extension, AURA2, AURA17, and AURA3). Methods: Patients with EGFR T790M-positive advanced NSCLC and progression after previous EGFR-tyrosine kinase inhibitor therapy received osimertinib (80 mg qd). Patients with central nervous system (CNS) metastases (including LMs) were eligible if the lesions were neurologically asymptomatic and stable. Patients with evidence of LMs at the study entry were retrospectively included for the analysis; brain scans were assessed for radiologic LM response by neuroradiologically blinded, independent central review per the modified Response Assessment in Neuro-Oncology LM criteria. LM objective response rate, duration of response, progression-free survival, and overall survival were assessed. A longitudinal analysis was performed to investigate the relationship between changes from the baseline in non-CNS tumor sizes and LM responses at each visit of patients in AURA LM and BLOOM studies. Results: For the 22 patients included in the analysis, LM objective response rate was 55% (95% confidence interval [CI]: 32-76). Median LM duration of response was not reached (95% CI: 2.8-not calculable [NC]). Median LM progression-free survival and overall survival were 11.1 months (95% CI: 4.6-NC) and 18.8 months (95% CI: 6.3-NC), respectively. The longitudinal analysis revealed similar non-CNS and LM responses between the patients in AURA LM and BLOOM programs. Conclusions: Patients with EGFR T790M-positive NSCLC and radiologically detected LM obtained clinical benefit from osimertinib (80 mg qd).

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