Citations

Abstract We evaluated somatic genetic alterations in the kinase domain of the EGFR gene in the tumors of 219 non–small cell lung cancer patients of primarily Caucasian and African American origins. We identified 26 patients (12%) whose tumors had a mutation in the EGFR gene, and 11 (5%) patients carried novel genomic variations consistent with germ-line polymorphisms. All but one mutation were identified in Caucasian patients affected with adenocarcinoma. EGFR mutations were more frequent in women and in nonsmokers, but a significant portion of the affected patients were men (12 of 26) and current or past smokers accounted for half of the patients affected (13 of 26). Screening subjects with EGFR mutations may identify patients whose tumors could respond to targeted therapy using tyrosine kinase inhibitors.

Abstract Much has been learned regarding the biology and clinical implications of genetic abnormalities in multiple myeloma. Because of recent advances in the field, an International Workshop was held in Paris in February of 2003. This summary describes the consensus recommendations arising from that meeting with special emphasis on novel genetic observations. For instance, it is increasingly clear that translocations involving the immunoglobulin heavy-chain locus are important for the pathogenesis of one-half of patients. As a corollary, it also clear that the remaining patients, lacking IgH translocations, have hyperdiploidy as the hallmark of their disease. Several important genetic markers are associated with a shortened survival such as chromosome 13 monosomy, hypodiploidy, and others. The events leading the transformation of the monoclonal gammopathy of undetermined significance (MGUS) to myeloma are still unclear. One of the few differential genetic lesions between myeloma and MGUS is the presence of ras mutations in the latter. Gene expression platforms are capable of detecting many of the genetic aberrations found in the clonal cells of myeloma. Areas in need of further study were identified. The study of the genetic aberrations will likely form the platform for targeted therapy for the disease.

AbstractWe have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.

Background Temsirolimus (CCI-779) is a small-molecule inhibitor of the mammalian target of rapamycin (mTOR) and represents a rational therapeutic target against glioblastoma multiforme (GBM). Methods Recurrent GBM patients with ≤ 1 chemotherapy regimen for progressive disease were eligible. Temsirolimus was administered in a 250-mg intravenous dose weekly. Results Sixty-five patients were treated. The incidence of grade 3 or higher nonhematologic toxicity was 51%, and consisted mostly of hypercholesterolemia (11%), hypertriglyceridemia (8%), and hyperglycemia (8%). Grade 3 hematologic toxicity was observed in 11% of patients. Temsirolimus peak concentration (Cmax), and sirolimus Cmax and area under the concentration-time curve were decreased in patients receiving p450 enzyme–inducing anticonvulsants (EIACs) by 73%, 47%, and 50%, respectively, but were still within the therapeutic range of preclinical models. Twenty patients (36%) had evidence of improvement in neuroimaging, consisting of decrease in T2 signal abnormality +/− decrease in T1 gadolinium enhancement, on stable or reduced steroid doses. Progression-free survival at 6 months was 7.8% and median overall survival was 4.4 months. Median time to progression (TTP) for all patients was 2.3 months and was significantly longer for responders (5.4 months) versus nonresponders (1.9 months). Development of grade 2 or higher hyperlipidemia in the first two treatment cycles was associated with a higher percentage of radiographic response (71% v 31%; P = .04). Significant correlation was observed between radiographic improvement and high levels of phosphorylated p70s6 kinase in baseline tumor samples (P = .04). Conclusion Temsirolimus is well tolerated in recurrent GBM patients. Despite the effect of EIACs on temsirolimus metabolism, therapeutic levels were achieved. Radiographic improvement was observed in 36% of temsirolimus–treated patients, and was associated with significantly longer TTP. High levels of phosphorylated p70s6 kinase in baseline tumor samples appear to predict a patient population more likely to derive benefit from treatment. These findings should be validated in other studies of mTOR inhibitors.

ABSTRACT We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(–) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region (146RRGRKRKAEKQ156) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/–) and IBD(+/–) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFβ family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFβ proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.