Role of CD81 and CD58 in minimal residual disease detection in pediatric B lymphoblastic leukemia.

INTRODUCTION: Minimal residual disease (MRD) in B lymphoblastic leukemia has been demonstrated to be a powerful predictor of clinical outcome in numerous studies in both children and adults. In this study, we evaluated 86 pediatric patients with both diagnostic and remission flow cytometry studies and compared expression of CD81, CD58, CD19, CD34, CD20, and CD38 in the detection of MRD. METHODS: We evaluated 86 patients with B lymphoblastic leukemia who had both diagnostic studies and remission studies for the presence of MRD using multicolor flow cytometry. We established our detection limit for identifying abnormal lymphoblasts using serial dilutions. We also compared flow cytometry findings with molecular MRD detection in a subset of patients. RESULTS: We found that we can resolve differences between hematogones and lymphoblasts in 85 of 86 cases using a combination of CD45, CD19, CD34, CD10, CD20, CD38, CD58, and CD81. Our detection limit using flow cytometry is 0.002% for detecting a population of abnormal B lymphoblasts. Comparison with MRD assessment by molecular methods showed a high concordance rate with flow cytometry findings. CONCLUSIONS: Our study highlights importance of using multiple markers to detect MRD in B lymphoblastic leukemia. Our findings indicate that including both CD58 and CD81 markers in addition to CD19, CD34, CD20, CD38, and CD10 are helpful in MRD detection by flow cytometry.

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA.

Increasing numbers of patients with chronic myeloid leukaemia (CML) treated with tyrosine kinase inhibitors achieve undetectable levels of BCR-ABL mRNA using sensitive quantitative real-time reverse transcriptase PCR (RT-qPCR) methods and a method to measure minimal residual disease (MRD) in patients with low levels could be of value. Following isolation and sequencing of the patient-specific BCR-ABL breakpoint, a DNA-based nested qPCR assay was established, and MRD was measured by this method and one-round RT-qPCR in 38 samples from 24 patients with CML. Mixing experiments using patient DNA in normal DNA indicated that DNA qPCR could detect BCR-ABL sequences at a limit of approximately 10⁻6. In 22 samples in which MRD was detectable by both methods, comparison of the results of DNA qPCR with the results obtained on the same sample by RT-qPCR showed good correlation. In another 16 samples, BCR-ABL mRNA was not detectable by RT-qPCR. In 8 of the 16 samples, BCR-ABL DNA was detected at levels ranging from 1.1 × 10⁻5 up to 2.8 × 10⁻4 and in the remaining eight samples BCR-ABL was not detected by either method. In one patient, who had stopped imatinib, an almost 1000-fold rise in MRD, to 5.2 × 10⁻4 was observed in sequential samples. Nested DNA qPCR was more sensitive than one-round RT-qPCR and could be used for the monitoring of patients with CML with very low levels of MRD.

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis.

During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.

In the absence of extrinsic signals, nutrient utilization by lymphocytes is insufficient to maintain either cell size or viability.

Without receptor stimulation, cells from multicellular organisms die by apoptosis. Here we show that lymphocytes deprived of receptor stimulation undergo progressive atrophy before commitment to apoptosis. Following loss of receptor engagement, lymphocytes rapidly downregulated the glucose transporter, glut1. This was accompanied by reduction in mitochondrial potential and cellular ATP, suggesting that atrophy resulted from depletion of glucose-derived metabolic substrates. Expression of the antiapoptotic protein, Bcl-X(L), prevented death but not atrophy following either growth factor or glucose withdrawal. In Bcl-X(L) transgenic animals, size and metabolic activity of naive T cells were regulated through the TCR and correlated with TCR-dependent glut1 expression. These data suggest that ligands for cell-specific receptors promote cell survival by regulating nutrient uptake and utilization.

Role of oxidative phosphorylation in Bax toxicity.

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability.

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191

The BH3 domain of Bcl-x(S) is required for inhibition of the antiapoptotic function of Bcl-x(L).

bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-x(L), is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-x gene codes for the protein Bcl-x(S), which lacks 63 amino acids present in Bcl-x(L) and has proapoptotic activity. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-x(S) does not seem to induce cell death in the absence of an additional death signal. However, Bcl-x(S) does interfere with the ability of Bcl-x(L) to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-x(S) was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-x(S) which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-x(L). Bcl-x(S) was able to form heterodimers with Bcl-x(L) in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-x(L). Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-x(L). The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-x(L) survival function.

Apicoectomy and retrograde amalgam in mandibular molar teeth.

The apicoectomy with retrograde amalgam is an excellent and accepted surgical technique for salvaging teeth. Anatomic considerations in molar teeth are not a contraindication to this technique.