Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5–10% of AML and 2–3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5–9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse. © 1999 Cancer Research Campaig

Enhanced Transduction and Replication of RGD-Fiber Modified Adenovirus in Primary T Cells

Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replicationcompetent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45 % of splenic T cells were transduced by Ad-RGD. Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary

Absence of a specific radiation signature in post-Chernobyl thyroid cancers

Thyroid cancers have been the main medical consequence of the Chernobyl accident. On the basis of their pathological features and of the fact that a large proportion of them demonstrate RET-PTC translocations, these cancers are considered as similar to classical sporadic papillary carcinomas, although molecular alterations differ between both tumours. We analysed gene expression in post-Chernobyl cancers, sporadic papillary carcinomas and compared to autonomous adenomas used as controls. Unsupervised clustering of these data did not distinguish between the cancers, but separates both cancers from adenomas. No gene signature separating sporadic from post-Chernobyl PTC (chPTC) could be found using supervised and unsupervised classification methods although such a signature is demonstrated for cancers and adenomas. Furthermore, we demonstrate that pooled RNA from sporadic and chPTC are as strongly correlated as two independent sporadic PTC pools, one from Europe, one from the US involving patients not exposed to Chernobyl radiations. This result relies on cDNA and Affymetrix microarrays. Thus, platform-specific artifacts are controlled for. Our findings suggest the absence of a radiation fingerprint in the chPTC and support the concept that post-Chernobyl cancer data, for which the cancer-causing event and its date are known, are a unique source of information to study naturally occurring papillary carcinomas

Increasing the Number of Thyroid Lesions Classes in Microarray Analysis Improves the Relevance of Diagnostic Markers

BackgroundGenetic markers for thyroid cancers identified by microarray analysis have offered limited predictive accuracy so far because of the few classes of thyroid lesions usually taken into account. To improve diagnostic relevance, we have simultaneously analyzed microarray data from six public datasets covering a total of 347 thyroid tissue samples representing 12 histological classes of follicular lesions and normal thyroid tissue. Our own dataset, containing about half the thyroid tissue samples, included all categories of thyroid lesions. Methodology/Principal Findings Classifier predictions were strongly affected by similarities between classes and by the number of classes in the training sets. In each dataset, sample prediction was improved by separating the samples into three groups according to class similarities. The cross-validation of differential genes revealed four clusters with functional enrichments. The analysis of six of these genes (APOD, APOE, CLGN, CRABP1, SDHA and TIMP1) in 49 new samples showed consistent gene and protein profiles with the class similarities observed. Focusing on four subclasses of follicular tumor, we explored the diagnostic potential of 12 selected markers (CASP10, CDH16, CLGN, CRABP1, HMGB2, ALPL2, ADAMTS2, CABIN1, ALDH1A3, USP13, NR2F2, KRTHB5) by real-time quantitative RT-PCR on 32 other new samples. The gene expression profiles of follicular tumors were examined with reference to the mutational status of the Pax8-PPARγ, TSHR, GNAS and NRAS genes. Conclusion/Significance We show that diagnostic tools defined on the basis of microarray data are more relevant when a large number of samples and tissue classes are used. Taking into account the relationships between the thyroid tumor pathologies, together with the main biological functions and pathways involved, improved the diagnostic accuracy of the samples. Our approach was particularly relevant for the classification of microfollicular adenomas

Flavopiridol Pharmacogenetics: Clinical and Functional Evidence for the Role of SLCO1B1/OATP1B1 in Flavopiridol Disposition

Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy

Carbon inputs from Miscanthus displace older soil organic carbon without inducing priming

The carbon (C) dynamics of a bioenergy system are key to correctly defining its viability as a sustainable alternative to conventional fossil fuel energy sources. Recent studies have quantified the greenhouse gas mitigation potential of these bioenergy crops, often concluding that C sequestration in soils plays a primary role in offsetting emissions through energy generation. Miscanthus is a particularly promising bioenergy crop and research has shown that soil C stocks can increase by more than 2 t C ha−1 yr−1. In this study, we use a stable isotope (13C) technique to trace the inputs and outputs from soils below a commercial Miscanthus plantation in Lincolnshire, UK, over the first 7 years of growth after conversion from a conventional arable crop. Results suggest that an unchanging total topsoil (0–30 cm) C stock is caused by Miscanthus additions displacing older soil organic matter. Further, using a comparison between bare soil plots (no new Miscanthus inputs) and undisturbed Miscanthus controls, soil respiration was seen to be unaffected through priming by fresh inputs or rhizosphere. The temperature sensitivity of old soil C was also seen to be very similar with and without the presence of live root biomass. Total soil respiration from control plots was dominated by Miscanthus-derived emissions with autotrophic respiration alone accounting for ∼50 % of CO2. Although total soil C stocks did not change significantly over time, the Miscanthus-derived soil C accumulated at a rate of 860 kg C ha−1 yr−1 over the top 30 cm. Ultimately, the results from this study indicate that soil C stocks below Miscanthus plantations do not necessarily increase during the first 7 years

HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of a neoplastic disease of CD4+ T cells, adult T-cell leukemia (ATL), and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis, dermatitis, and inflammatory lung diseases. ATL cells, which constitutively express CD25, resemble CD25+CD4+ regulatory T cells (Treg). Approximately 60% of ATL cases indeed harbor leukemic cells that express FoxP3, a key transcription factor for Treg cells. HTLV-1 encodes an antisense transcript, HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases. In this study, we show that transgenic expression of HBZ in CD4+ T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals. In HBZ-transgenic mice, CD4+Foxp3+ Treg cells and effector/memory CD4+ T cells increased in vivo. As a mechanism of increased Treg cells, HBZ expression directly induced Foxp3 gene transcription in T cells. The increased CD4+Foxp3+ Treg cells in HBZ transgenic mice were functionally impaired while their proliferation was enhanced. HBZ could physically interact with Foxp3 and NFAT, thereby impairing the suppressive function of Treg cells. Thus, the expression of HBZ in CD4+ T cells is a key mechanism of HTLV-1-induced neoplastic and inflammatory diseases

Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses

Ancient hepadnavirus sequences found in bird genomes reveal that this family of viruses, which includes the human hepatitis B virus, is much older than previously thought