Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Comparison of the activity of carp and rat beta-actin gene regulatory sequences in tilapia and rainbow trout embryos.

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat beta-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for beta-galactosidase expression. We provide evidence to demonstrate that the carp beta-actin promoter/ lacZ reporter gene is expressed at higher levels than the equivalent rat beta-actin construct in both species.

Identification of evolutionary conserved regulatory sequences in the 5' untranscribed region of the neural-specific ubiquitin C-terminal hydrolase (PGP9.5) gene.

The structure at the 5' end of the gene encoding neural-specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1-kb stretch of 5' untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233-bp CpG-rich minimal promoter. Truncation mutagenesis revealed the presence of essential positive cis-acting regulatory sequences within the region -182 to -123 relative to the transcription initiation site. Sequence alignment analysis of the human and Monodelphis promoter sequences showed 76% identity in this 59-bp region of the gene. A perfectly conserved 12-bp sequence (PSN) located within this region acts as a non-cell-specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16-bp sequence located at -356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negative cis-acting sequences located in the untranscribed region of the gene.

Receptor-mediated endocytosis of low-density lipoprotein by cultured human glomerular cells.

Lipoproteins might be involved in the pathogenesis of glomerular damage. Uptake of low-density lipoprotein (LDL) by cultured human glomerular cells has been studied using LDL, labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethyl-indocarbocyanine perchlorate (diI). Cells have been characterised using phase-contrast microscopy, monoclonal antibodies and lectins. Differentiated glomerular epithelial cells, epithelial-like cells and mesangial cells all took up diI-LDL. Uptake was specific for LDL, of high affinity and inhibited by excess unlabelled LDL, heparin and preloading the cells with cholesterol. Binding of diI-LDL to the cell surface was restricted to discrete areas which were arranged in linear arrays on mesangial cells. Endocytosis of surface-bound diI-LDL occurred within 3 min and breakdown of internalised diI-LDL within 30 min. These results indicate that cultured human glomerular cells take up LDL by receptor-mediated endocytosis.

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity.

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gka and Gkb, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent Km values for MgATP2- and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.

Hepatic glucokinase activity and circulating insulin concentrations in two inbred mouse strains.

Hepatic glucokinase activity in the C3H/He strain of mice is about twice that in the C58 strain. Genetic analysis of hybrids and back-crosses indicates control of activity by a single codominant gene. The adaptations of activity that occur when animals of the two strains are starved, fed a carbohydrate-free diet or made streptozotocin-diabetic are similar in the two strains. In almost all the situations tested, plasma insulin concentrations were higher in the C3H/He strain than in the C58 strain. On the assumption that insulin plays some role in the synthesis of glucokinase, the possible association between the plasma insulin concentration and hepatic glucokinase activity in mice in which plasma glucose concentrations are similar is discussed. The twofold difference in glucokinase activity between the two strains is associated with only minor differences in glucose tolerance and insulin response to a glucose load.