Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure.

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

Microclustering of TEL-AML1 translocation breakpoints in childhood acute lymphoblastic leukemia.

TEL-AML1 fusions are the most common chromosome translocations in childhood leukemia and often, if not always, occur in utero. We previously reported the genomic sequencing of nine TEL-AML1 translocations and showed unique structural features of a breakpoint cluster region in TEL intron 5. We now report data on sequencing and mapping of TEL-AML1 from an additional 11 patients and, using Monte Carlo statistical methods, have analyzed the intronic distribution of the 24 TEL-AML1 fusion junctions sequenced to date. Compared to a null hypothesis of random breakpoint allocation within TEL intron 5 and AML1 introns 1 and 2, significant microclustering was evident on both TEL and AML1. In contrast to previous reports, the two strongest microclusters on TEL were 3' to an unstable repeat region. AML1 demonstrated four highly significant microclusters, two of which were proximal to exons. We note the necessity of sequencing multiple breakpoints before the description of putative microcluster regions. TEL-AML1 breakpoints may be distributed into microclusters because of specific DNA sequence or chromatin features in susceptible cells. We also report on additional features of breakpoints, including a complex t(12;3;21) in one patient and an inverted sequence in another.

Cytogenetic abnormalities during clinical, immunophenotypic, and molecular remission in pediatric acute lymphoblastic leukemia.

The significance of random cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL) during remission is unknown. We studied 10 of 72 consecutive ALL patients who developed cytogenetic abnormalities during clinical remission to determine their effect on remission status. The cytogenetic abnormalities occurred at a median of 14.5 months (range 5-72) from the initial diagnosis. Five abnormalities were designated as clonal (monosomy 21 in three metaphases and 64 approximately 69,XXY in three metaphases from one patient at different times, and del(20)(q12q13) in three metaphases, add(13)(q34) in two metaphases, and ?del(19)(p11) in two metaphases from three different patients). Seven abnormalities were previously described: del(5)(q12), del(5)(q33), -7, del(11)(q23), +12, and +13 each in one metaphase, and del(20)(q12q13) in three metaphases). The remaining cytogenetic abnormalities were nonclonal and random. Flow cytometry and analysis of IgH and TcR gene rearrangements showed that all evaluable patients were in immunophenotypic and molecular remission, respectively. Eight patients remain in clinical and molecular remission a median of 9 months (range 7-18) after detecting the cytogenetic abnormality, and the leukemia in two patients has relapsed. During remission, cytogenetic abnormalities may not be a harbinger of leukemia relapse in pediatric ALL; therefore, a wait-and-see approach is prudent.

Trisomy 5 as a sole cytogenetic abnormality in pediatric acute lymphoblastic leukemia.

We describe a case of pediatric acute lymphoblastic leukemia (ALL) with trisomy 5 as a sole cytogenetic abnormality. A comparison is made with the two cases of adult acute lymphoblastic leukemia with trisomy 5 in the literature. This rare cytogenetic abnormality may portend an especially poor prognosis in patients with ALL.

Quantitation of leukemia clone-specific antigen gene rearrangements by a single-step PCR and fluorescence-based detection method.

PCR-based detection of minimal residual disease (MRD) in children with precursor B cell acute lymphoblastic leukemia (pre-B ALL) by amplification of clone-specific antigen gene rearrangements has been labor-intensive. In this study we present a simpler, yet accurate, method to assess and quantitate MRD in pediatric pre-B ALL that utilizes these markers. From the sequence of the immunoglobulin heavy chain (IgH) and T cell receptor (TcR) gene rearrangements characterized at the time of diagnosis or relapse, primers were designed and tested in a clone-specific, single-round PCR assay, then analyzed by fluorescence staining of the PCR products. The most critical step involved an adherence to a new set of guidelines for the design of clone-specific primers. Application of this method to 54 IgH and 13 TcR (nine Vdelta2Ddelta3 and four Ddelta2-Ddelta3) gene rearrangements in 47 patients resulted in an intense band within the region of the predicted molecular weight, confirming the reproducibility of the assay. Quantitative applications of the approach were examined by performing a 10-replicate limiting dilution clone-specific PCR on six diagnostic samples and an asymptotic response model of the Von Krogh form was found to fit the data well. From this model, estimation of leukemic cells of remission bone marrow samples was achieved at a detection sensitivity of 2 x 10(-6). The method is demonstrated on 18 patients whose marrows were prospectively analyzed during therapy. We conclude this methodology is useful in the quick and accurate assessment of MRD in children with pre-B ALL, and could be applied to other DNA quantitation assays.

Detectable molecular residual disease at the beginning of maintenance therapy indicates poor outcome in children with T-cell acute lymphoblastic leukemia.

The aims of this study were twofold: (1) to assess the marrow of patients with T-lineage acute lymphoblastic leukemia (T-ALL) for the presence of molecular residual disease (MRD) at different times after diagnosis and determine its value as a prognostic indicator; and (2) to compare the sensitivity, rapidity, and reliability of two methods for routine clinical detection of rearranged T-cell receptor (TCR). Marrow aspirates from 23 patients with T-ALL diagnosed consecutively from 1982 to 1994 at the Division of Pediatric Hematology and Oncology, University of Catania, Italy, were obtained at diagnosis, at the end of induction therapy (6 to 7 weeks after diagnosis), at consolidation and/or reinforced reinduction (12 to 15 weeks after diagnosis), at the beginning of maintenance therapy (34 to 40 weeks after diagnosis), and at the end of therapy (96 to 104 weeks after diagnosis). DNA from the patients' marrow was screened using the polymerase chain reaction (PCR) for the four most common TCR delta rearrangements in T-ALL (Vdelta1 Jdelta1, Vdelta2 Jdelta1, Vdelta3 Jdelta1, and Ddelta2 Jdelta1) and, when negative, further tested for the presence of other possible TCR delta and TCR gamma rearrangements. After identification of junctional rearrangements involving V, D, and J segments by DNA sequencing, clone-specific oligonucleotide probes 5' end-labeled either with fluorescein or with [gamma-32P]ATP were used for heminested PCR or dot hybridization of PCR products of marrows from patients in clinical remission. For 17 patients with samples that were informative at the molecular level, the estimated relapse-free survival (RFS) at 5 years was 48.6% (+/-12%). The sensitivity and specificity for detection of MRD relating to the outcome were 100% and 88.9% for the heminested fluorescence PCR and 71.4% and 88.9% for Southern/dot blot hybridization, respectively. Predictive negative and positive values were 100% and 90.7% for heminested fluorescence PCR, respectively. The probability of RFS based on evidence of MRD as detected by heminested fluorescence PCR at the time of initiation of maintenance therapy was 100% and 0% for MRD-negative and MRD-positive patients, respectively. Thus, the presence of MRD at the beginning of maintenance therapy is a strong predictor of poor outcome, and the molecular detection of MRD at that time might represent the basis for a therapeutic decision about such patients. By contrast, the absence of MRD at any time after initiation of treatment strongly correlates with a favorable outcome. The heminested fluorescence PCR appears to be more accurate and more rapid than other previously used methods for the detection of residual leukemia.