Earth's geodynamic evolution constrained by 182W in Archean seawater.

Radiogenic isotope systems are important geochemical tools to unravel geodynamic processes on Earth. Applied to ancient marine chemical sediments such as banded iron formations, the short-lived 182Hf-182W isotope system can serve as key instrument to decipher Earth's geodynamic evolution. Here we show high-precision 182W isotope data of the 2.7 Ga old banded iron formation from the Temagami Greenstone Belt, NE Canada, that reveal distinct 182W differences in alternating Si-rich (7.9 ppm enrichment) and Fe-rich (5.3 ppm enrichment) bands reflecting variable flux of W from continental and hydrothermal mantle sources into ambient seawater, respectively. Greater 182W excesses in Si-rich layers relative to associated shales (5.9 ppm enrichment), representing regional upper continental crust composition, suggest that the Si-rich bands record the global rather than the local seawater 182W signature. The distinct intra-band differences highlight the potential of 182W isotope signatures in banded iron formations to simultaneously track the evolution of crust and upper mantle through deep time.

Asymmetric multiplex-polymerase chain reaction - a high throughput method for detection and sequencing genomic fusion sites in t(4;11).

Chromosomal translocations are a characteristic feature of leukaemia and other malignant diseases. As clonal markers, they can be applied to identify and quantify the number of malignant cells by polymerase chain reaction (PCR) methods. The translocation t(4;11) is present in >60% of infant leukaemia. In order to facilitate the sequencing of chromosomal breakpoints, we developed an optimized set of 30 PCR primers and a new approach, designated as asymmetric multiplex PCR (am-PCR). Due to the high number of primers, small breakpoint-spanning DNA fragments are obtained in one nested multiplex PCR reaction. All PCR products contain an identical binding site for the initiation of direct sequencing. By using am-PCR, the translocation t(4;11) was examined in bone marrow and blood samples from children with acute leukaemia. Compared with previously described methods for the determination of genomic breakpoints, am-PCR may be advantageous with regard to its simplicity and rapidity. Breakpoint-spanning sequences were also evaluated with regard to their applicability as unique clonal markers to design primers and probes for minimal residual disease quantification by real-time PCR. This approach can easily be adapted to other chromosomal translocations in malignant diseases for the detection and analysis of clone-specific DNA markers.

Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement.

The fusion transcript AML1/ETO corresponding to translocation t(8;21)(q22;q22) can be found in approximately 7-12% of childhood de novo AML. Despite the favorable prognosis, some of these patients relapse. Most of MRD studies so far were performed on adults treated not uniformly. Therefore, we analyzed the follow-up of 15 AML1/ETO-positive children using real-time quantitative reverse transcription PCR (RQ-RT-PCR), all enrolled in the multicenter therapy trial AML-BFM 98. AML1/ETO copy numbers were normalized to the control gene ABL and the results were expressed in copy numbers AML1/ETO per 10 000 copies ABL. At diagnosis, a median of 10 789 copies AML1/ETO was found. A linear decrease to about 10 copies (2-4 log) could be seen in most of the children by the start of consolidation. In the majority of cases they remained positive at this low level during the ongoing therapy. Four children relapsed and two of them had a decrease of less than 2 log before starting consolidation. Three of the relapsed children showed, prior to relapse, an increase of the AML1/ETO fusion transcript at 6, 9, and 11 weeks, respectively. These results suggest that monitoring of minimal residual disease using RQ-RT-PCR could be helpful in detecting patients with a higher risk of relapse.

Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).

Prednisone response is the strongest predictor of treatment outcome in infant acute lymphoblastic leukemia.

To define prognostic factors in infant acute lymphoblastic leukemia (ALL), the outcome of 106 infants (age /=1,000 blasts/microL) received intensified therapy. Infant ALL was characterized by a high incidence of a white blood cell count greater than 100 x 10(3)/microL (57%), central nervous system leukemia (24%), lack of CD10 expression (59%), 11q23 rearrangement (49%) including the translocation t(4;11) (29%), and a comparatively high proportion of PPR (26%), which were all significantly associated with inferior outcome by univariate analysis. The estimated probability for an event-free survival at 6 years (pEFS) was by far better for PGR compared with PPR, who had a dismal prognosis despite intensified treatment (pEFS, 53% +/- 6% v 15% +/- 7%, P =.0001). Infant PGR, who were less than 6 months of age (n = 40), lacked CD10 expression (n = 43), and/or had an 11q23 rearrangement (n = 17) fared significantly better compared with corresponding PPR, as indicated by a pEFS of 44% +/- 8%, 49% +/- 8%, and 41% +/- 12%, respectively. In multivariate analysis, PPR was the strongest adverse prognostic factor (relative risk, 3.3; 95% confidence interval, 1.9 to 5.8; P <.0001). Infants with PGR, comprising a major subgroup (74%) among infants, might successfully be treated with conventional therapy, whereas PPR require new therapeutic strategies, including early treatment intensification or bone marrow transplantation in first remission.

Trisomy 21 is a recurrent secondary aberration in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion.

TEL/AML1 gene fusion is the most frequent genetic lesion in pediatric acute lymphoblastic leukemia (ALL). It occurs as a consequence of the cryptic chromosomal translocation t(12;21)(p13;q22). In a cohort of 50 RT-PCR-positive TEL/AML1 patients, karyotype examination by GTG banding and fluorescence in situ hybridization (FISH) allowed us to identify chromosome anomalies in addition to the already existing t(12;21). Secondary aberrations were found in 29 out of 41 patients (71%) at initial diagnosis and in all 9 patients with relapse. Structural rearrangements affected chromosome arms 2p, 2q, 5q, 9p, 12p (n = 2), 6q, 11p (n = 3), and 21q (n = 4). An extra chromosome 21 was found to be the most frequent anomaly. It was detected in 6 out of 41 patients at initial diagnosis (15%) and in 7 out of the 9 patients at relapse. No karyotype with trisomy 21 exceeded 47 chromosomes. Gain of chromosome 21 was the sole anomaly in GTG-banding analysis in 2/41 patients at initial diagnosis and in 4/9 at relapse. Notably, chromosome painting analysis performed in 11 out of the 13 patients with an extra chromosome 21 revealed duplication of the normal chromosome 21 in 8, and duplication of der(21)t(12;21) in 3 patients. Furthermore, gain of der(21)t(12;21) chromosome was confined exclusively to the relapse patients.

Multiplex PCR--a rapid screening method for detection of gene rearrangements in childhood acute lymphoblastic leukemia.

Chromosomal rearrangements in childhood acute lymphoblastic leukemia (ALL) play an important role in the identification of clinical relevant subgroups. For rapid and easy detection of the clinically most important gene rearrangements, a nested multiplex reverse transcriptase polymerase chain reaction (multiplex PCR) was developed. This multiplex PCR enables the detection of M-BCR/ABL, m-BCR/ABL, TEL/ AML1, and MLL/AF4 fusion transcripts in one PCR reaction. However, the existence of splicing variants and different breakpoints on the DNA level hampers the discrimination of the rearrangements by their fragment size on an agarose gel. Therefore, one of the internal primers of each translocation (ABL-2, TEL-2, AF4-2) was labeled with a characteristic fluorescent dye, and an automatic fluorescence-based DNA fragment analysis was performed. The sensitivity of this multiplex PCR is in the same range as that of the corresponding single PCR reaction and allows a fast screening for the detection of therapy-relevant rearrangements, with a high turnover of samples.

Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials. Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Study Group.

The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9;22) and t(4;11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12;21)(p13;q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12;21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12;21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre-B immunophenotype. Among the B-lineage subgroup, the t(12;21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (> or = 1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index > or = 1.16 < 1.6). Our findings reinforce the need to include the molecular screening of the t(12;21) translocation within ongoing prospective ALL trials to prove definitively its prognostic impact.

Origin and evolution of mitochondria: what have we learnt from red algae?

The purpose of this review is to present an account of our current understanding of the structure, organization and evolution of mitochondrial genomes, and to discuss the origin and evolution of mitochondria from the perspective recently provided by the extensive sequencing of various mitochondrial genomes. Mitochondrial-en-coded protein phylogenies are congruent with nuclear phylogenies and strongly support a monophyletic origin of mitochondria. The newly available data from red-algal mitochondrial genomes, in particular, show that the structural and functional diversity of mitochondrial genomes can be accounted for by paralogous evolution. We also discuss the influence of other constraints, such as uniparental inheritance, on the evolution of genome organization in mitochondria.

Incidence of TEL/AML1 fusion gene analyzed consecutively in children with acute lymphoblastic leukemia in relapse.

The translocation t(12; 21)(p13; q22) is difficult to detect by classic cytogenetics. However, using fluorescence in situ hybridization (FISH) and by screening for the TEL/AML1 rearrangement by the polymerase chain reaction (PCR), it has been demonstrated to be the most frequent known structural chromosomal abnormality in childhood acute lymphoblastic leukemia (ALL). It is closely correlated with a B-cell precursor (BCP) phenotype and is considered a favorable prognostic factor. However, little is known about the incidence of the translocation in relapsed patients and the duration of complete remission (CR) in children expressing the TEL/AML1 fusion gene. We therefore examined 49 bone marrow samples from children with ALL at first or second relapse that were consecutively mailed to our laboratory to test for the presence of t(12; 21) using reverse transcriptase (RT)-PCR. The TEL/AML1 rearrangement could be identified in nine of 44 (20%) of the patients, a result similar to the reported incidence at diagnosis. Most of the TEL/AML1-positive children showed no adverse clinical features at diagnosis (eg, white blood cell [WBC] count <100 x 10(9)/L or age <10 years), and regarding these data, there were no differences versus children who were negative for the fusion gene. However, the period of remission was about 1 year longer in children expressing TEL/AML1 (P = .046), and the majority of relapses in this group appeared late (<2 years after diagnosis). Our findings therefore reinforce the urgent need for further prospective studies with a long follow-up period to determine the true prognostic significance of t(12; 21) and to avoid premature changes of treatment strategies.

Genes for two subunits of succinate dehydrogenase form a cluster on the mitochondrial genome of Rhodophyta.

Mitochondrial DNA from the unicellular rhodophyte Cyanidium caldarium RK-1 and the multicellular Chondrus crispus were isolated, cloned, and sequenced. Two genes, sdhB and sdhC, that encode subunits of the succinate dehydrogenase, were identified by similarity. These genes form a cluster (sdhCB) in both red algae.