Cross-lineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease.

A large series of 202 childhood precursor-B cell acute lymphoblastic leukemia (ALL) patients was analyzed by Southern blotting (SB) for cross-lineage rearrangements and/or deletions in the T cell receptor TCRB, TCRG and TCRD loci. In 93% (187/201) of the precursor-B-ALL patients one or more genes were rearranged and/or deleted. TCRB gene rearrangements were found in 35% (69/196), TCRG gene rearrangements in 59% (113/192), TCRD gene rearrangements in 55% (112/202), and isolated monoallelic or biallelic deletions of TCRD loci in 34% (68/202) of the cases. TCRB gene rearrangements involved exclusively the Jbeta2 locus with complete V(D)Jbeta2 joinings in 53% of gene rearrangements and incomplete Dbeta-Jbeta2 gene rearrangements in 33%. TCRG gene rearrangements frequently occurred on both alleles (65% of cases) and in approximately 70% concerned rearrangements to Jgamma1 gene segments. Most rearranged TCRD alleles (80%) represented incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 gene rearrangements, while the remaining TCRD gene rearrangements remained unidentified. Subsequently, we evaluated, whether heteroduplex PCR analysis of rearranged TCRG and TCRD genes can be used for reliable identification of PCR targets for detection of minimal residual disease (MRD). The concordance between SB and heteroduplex PCR analysis for detection of the various types of clonal TCRG and TCRD gene rearrangements ranged between 78% and 87%. The discrepancies could be assigned to the presence of 'atypical' TCRD gene rearrangements or translocations only detectable by SB, but also to efficient PCR-based detection of rearrangements derived from small subclones, which are difficult to detect with SB. Indications for oligoclonality were observed in 38% and 30% of patients with TCRG and TCRD gene rearrangements, respectively, which is comparable to the frequency of oligoclonality in IGH locus. Based on the combined data it was possible to reduce the broad panel of six TCRD and 12 TCRG primer combinations for MRD studies to two TCRD combinations (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and six TCRG combinations (VgammaI, VgammaII, VgammaIV family-specific primers with Jgamma1.1/2.1 and Jgamma1.3/2.3 primers) resulting in the detection of 80% and 97% of all TCRD and TCRG gene rearrangements, respectively. Finally, the heteroduplex PCR data indicate that MRD monitoring with TCRG and/or TCRD targets is possible in approximately 80% of childhood precursor-B-ALL patients; approximately 55% of patients even have two TCRG and/or TCRD targets.

Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood.

BACKGROUND: Sensitive techniques for detection of minimal residual disease (MRD) at degrees of one leukaemic cell per 10(3)-10(6) cells (10(-3)-10(-6)) during follow-up of children with acute lymphoblastic leukaemia (ALL) can provide insight into the effectiveness of cytotoxic treatment. However, it is not yet clear how information on MRD can be applied to treatment protocols. METHODS: We monitored 240 patients with childhood ALL who were treated according to national protocols of the International BFM Study Group. 60 patients relapsed and the patients in continuous complete remission (CCR) had a median event-free follow-up of 48 months. Bone-marrow samples were collected at up to nine time points during and after treatment. Standardised PCR analysis of patient-specific immunoglobulin and T-cell receptor gene rearrangements and TAL1 deletions were used as targets for semiquantitative estimation of MRD. Amount of MRD was classed as 10(-2) or more, 10(-3), and 10(-4) or less. FINDINGS: MRD negativity at the various follow-up times was associated with low relapse rates (3-15% at 3 years), but five-fold to ten-fold higher relapse rates (39-86% at 3 years) were found in MRD-positive patients. The distinct degrees of MRD appeared to have independent prognostic value (p [trend]<0.001) at all separate time points, especially at the first two time points (at the end of induction treatment and before consolidation treatment). At these two time points a high degree of MRD (> or = 10(-2)) was associated with a three-fold higher relapse rate when compared with patients with a low degree of MRD (< or = 10(-4)). At later time points (including the end of treatment) even a low degree of MRD was associated with a poor outcome. Positivity in patients in CCR after treatment was rare (< 1%). With the combined MRD information from the first two follow-up time points, it was possible to recognise three different risk groups--55 (43%) were in a low-risk group and had a 3-year relapse rate of only 2% (95% CI 0.05-12%); 19 (15%) were in a high-risk group and had a relapse rate of 75% (55-95%); and 55 (43%) were in an intermediate-risk group and had a 3-year relapse rate of 23% (13-36%). INTERPRETATION: Our collaborative MRD study shows that monitoring patients with childhood ALL at consecutive time points gives clinically relevant insight into the effectiveness of treatment. Combined information on MRD from the first 3 months of treatment distinguishes patients with good prognoses from those with poor prognoses, and this helps in decisions whether and how to modify treatment.

Immunoglobulin and T cell receptor gene rearrangement patterns in acute lymphoblastic leukemia are less mature in adults than in children: implications for selection of PCR targets for detection of minimal residual disease.

In order to gain insight into immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements in adult acute lymphoblastic leukemia (ALL), we studied 48 adult patients: 26 with precursor-B-ALL and 22 with T-ALL. Southern blotting (SB) with multiple DNA probes for the IGH, IGK, TCRB, TCRG, TCRD and TAL1 loci revealed rearrangement patterns largely comparable to pediatric ALL, but several differences were found for precursor-B-ALL patients. Firstly, adult patients showed a lower level of oligoclonality in the IGH gene locus (five out of 26 patients; 19%) despite a comparable incidence of IGH gene rearrangements (24 out of 26 patients; 92%). Secondly, all detected IGK gene deletions (n = 12) concerned rearrangements of the kappa deleting element (Kde) to Vkappa gene segments, which represent two-thirds of the Kde rearrangements in pediatric precursor-B-ALL and only half of the Kde rearrangements in mature B cell leukemias. Thirdly, a striking predominance of immature Ddelta2-Ddelta3 cross-lineage recombinations was observed (seven out of 16 TCRD rearrangements; 44%), whereas more mature Vdelta2-Ddelta3 gene rearrangements occurred less frequently (six out of 16 TCRD rearrangements; 38% vs >70% in pediatric precursor-B-ALL). Together these data suggest that the Ig/TCR genotype of precursor-B-ALL is more immature and more stable in adults than in children. We also evaluated whether heteroduplex analysis of polymerase chain reaction (PCR) products of rearranged Ig and TCR genes can be used for identification of molecular targets for minimal residual disease (MRD) detection. Using five of the major gene targets (IGH, IGK, TCRG, TCRD and TAL1 deletion), we compared the SB data and heteroduplex PCR results. High concordance between the two methods ranging from 96 to 100% was found for IGK, TCRG and TAL1 genes. The concordance was lower for IGH (70%) and TCRD genes (90%), which may be explained by incomplete or 'atypical' rearrangements or by translocations detectable only by SB. Finally, the heteroduplex PCR data indicate, that MRD monitoring is possible in almost 90% of adult precursor-B-ALL and >95% of adult T-ALL patients.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vkappa gene family usage in the Vkappa-Kde rearrangements in our series of B-lineage leukemias was comparable to Vkappa gene family usage in functional Vkappa-Jkappa rearrangements in normal and malignant mature B cells, except for a higher frequency of VkappaII family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10(-4) to 10(-5). Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be 'end-stage' rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

Interactions between organic anions, micelles and vesicles in model bile systems.

Biliary lipid secretion probably involves both 'micellization' and 'vesiculization' of bile-canalicular membrane lipids. Several hydrophilic organic anions inhibit the secretion of lipids into the bile without altering bile salt secretion [Verkade, Vonk and Kuipers (1995) Hepatology 21, 1174-1189]. Hydrophobic organic anions do not interfere with biliary lipid secretion. We investigated whether the organic-anion-induced inhibition of biliary lipid secretion in vivo could be attributed to inhibition of micellization, by the application of in vitro models of micellization. Carboxyfluorescein was entrapped in a self-quenching concentration in small unilamellar vesicles (SUV) composed of cholesterol/egg phosphatidylcholine (molar ratios 0, 0.2 and 0.5). Certain organic anions clearly affected the bile-salt-induced release of fluorescence from these SUV, reflecting interference with micellization. However, the effects of hydrophilic and hydrophobic organic anions did not correspond with their effects on biliary lipid secretion in vivo, irrespective of the bile salt species used (taurocholate, taurodeoxycholate or tauroursodeoxycholate) and of the lipid composition of the SUV. Ultracentrifugation and dynamic light-scattering studies indicated that organic anions do interact with bile salt/ phosphatidylcholine/cholesterol mixed micelles, but that they do not inhibit micellization, for example by competing with phosphatidylcholine and/or cholesterol for incorporation into mixed micelles. In conclusion, the present in vitro data indicate that the in vivo mechanism of organic-anion-induced inhibition of biliary lipid secretion is not mediated by inhibition of micellization.

Characterization of the cholesterol crystallization-promoting low-density particle isolated from human bile.

BACKGROUND & AIMS: Biliary concanavalin A-binding glycoprotein (CABG) contains cholesterol crystallization-promoting activity that is not accounted for by the pronucleators that have been characterized in this fraction. The aim of this study was to isolate and characterize the missing activity. METHODS: Biliary glycoprotein was isolated using concanavalin A-Sepharose. Promoting activity in CABG was purified using density gradient ultracentrifugation. RESULTS: Activity in CABG separated into two fractions at low (1.08) and high (1.29) density, which showed different crystallization kinetics in a model bile. The high-density fraction had a late onset time (49.2 +/- 17.8 hours) but a high crystal growth rate (13.4 +/- 5.2 micrograms. mL-1.h-1). The low-density fraction had a rapid onset time (33.9 +/- 20.9 hours) but a slower growth rate (6.5 +/- 3.8 micrograms.mL-1 .h-1). The high-density fraction was not further characterized in this study. The low-density fraction contained solid particles consisting of lipid and very little protein, and the activity was fully pronase resistant. Delipidation of the low-density fraction removed all activity. CONCLUSIONS: A potent pronase-resistant nucleation-promoting activity was activated from human bile and characterized. The low-density fraction may be responsible for the rapid nucleation in bile from typical patients with fast-nucleating gallstones.

Detection of minimal residual disease in acute leukemia patients.

Diagnostic techniques, routinely used in clinical practice for monitoring acute leukemia patients, are able to detect only 1-5% of malignant cells. At present, two main techniques are being introduced for detection of minimal residual disease (MRD) in leukemia, namely immunological marker analysis and the polymerase chain reaction (PCR) technique with general sensitivity of 10(-4)-10(-5). Immunological marker analysis allows detection of unusual and aberrant immunophenotypes, and is usually performed by flow cytometry. PCR analysis allows detection of leukemia-specific DNA sequences, such as fusion regions of chromosome aberrations and junctional regions of rearranged immunoglogulin (Ig) genes and T-cell receptor (TcR) genes. The applicability of the immunophenotyping and PCR-mediated MRD techniques is dependent on the type of leukemia. In virtually all acute lymphoblastic leukemias, PCR analysis of Ig and TcR genes can be used, and immunophenotypic MRD detection is also possible in 70-80% of cases. In AML, immunophenotypic MRD detection can be applied in approximately 80% of cases and PCR analysis of chromosome aberrations in 25-40%. Each MRD technique has its advantages and limitations, which have to be weighed carefully to make an appropriate choice. Furthermore, standardization of the MRD techniques is needed before they are used for stratification or adaptation of treatment protocols. Finally, the clinical impact of MRD detection for the various subtypes of acute leukemias has to be established.

Interaction of cholesterol-crystallization-promoting proteins with vesicles.

In this study, the interaction of mucin and concanavalin A-binding proteins isolated from human bile with cholesterol/phospholipid vesicles was investigated. Using resonance energy transfer assays originally developed by Struck, Hoekstra and Pagano [(1981) Biochemistry 20, 4093-4099], no significant protein-induced fusion or aggregation of vesicles was demonstrated. Instead of fusion, these proteins induced destabilization of cholesterol/phospholipid vesicles, as monitored by release of entrapped carboxyfluorescein. A good correlation (rho = 0.81) was obtained between the extent of leakage and the nucleation-promoting activity of the concanavalin A-binding proteins. We conclude that aggregation or fusion of cholesterol/phospholipid vesicles is not an obligatory step in cholesterol crystallization. Biliary protein-induced crystallization seems to be preceded by vesicle disruption.

Immunoglobulins and alpha 1-acid glycoprotein do not contribute to the cholesterol crystallization-promoting effect of concanavalin A-binding biliary protein.

Human bile contains cholesterol crystallization-stimulating proteins that can be isolated by concanavalin A-Sepharose chromatography. In the past few years an increasing number of different pronucleating proteins have been identified in the concanavalin A-binding fraction. In this study we attempted to estimate the relative contribution of a number of these proteins to total concanavalin A-binding pronucleating activity. For this purpose, concanavalin A-binding glycoproteins were isolated from gallbladder bile samples from 12 patients with gallstones. The role of IgA, IgG and IgM and alpha 1-acid glycoprotein was investigated by means of immunoextraction. No decrease in crystallization-promoting activity was observed after precipitation of more than 98% of the different immunoglobulins. In addition, removal of more than 95% of alpha 1-acid glycoprotein from different concanavalin A-binding fractions had no significant effect on cholesterol crystallization-promoting activity. The influence of fibronectin was estimated by addition of physiological concentrations to a model bile system. At these concentrations fibronectin did not promote crystallization. From these data we conclude that immunoglobulins, alpha 1-acid glycoprotein and probably also fibronectin do not significantly contribute to total concanavalin A-binding activity.

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.

Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts.

We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.