ALBERT

Beschreibung: Clonality assessment through Southern blot (SB) analysis ofTCRB genes or polymerase chain reaction (PCR) analysis ofTCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)–PCR analysis of Vβ-Cβ transcripts and flow cytometry with a Vβ antibody panel covering more than 65% of Vβ domains was validated using 28 SB-defined clonal T-cell receptor (TCR)αβ+ T-ALL samples and T-cell lines. Next, the diagnostic applicability of the Vβ RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vβ-Cβ RT-PCR products were detected in all 47 samples, whereas single Vβ domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vβ monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vβ gene segments not covered by the applied Vβ monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the “clonal” character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vβ-Cβ products and single Vβ domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vβ analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vβ antibody studies can be used as a powerful screening method for the detection of single Vβ domain expression, followed by molecular studies in patients with more than 20% single Vβ domain expression or large suspect T-cell populations (more than 50%-60%) without Vβ reactivity.

Beschreibung: Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a “single-cell” model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igκ gene (IGK) rearrangements, followed byIGK deletions and Igλ gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression.

Beschreibung: This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic Jh gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the Jh and Cμ regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal Jhgene rearrangements. Only 4 of the joinings were complete Vh-(D)-Jhrearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete Dh-Jh joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame Vκ-Jκgene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of Ch probes. In 5 patients the rearrangements in the Jh and Ch regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32.3). These data indicate that in many IgH− MM patients illegitimateIGH class switch rearrangement or illegitimate deletion of the functional Vh-(Dh)-Jhallele are responsible for IgH negativity. For example, the exclusive presence ofDh-Jhrearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH− MM patients.

Beschreibung: Expression of major histocompatibility complex (MHC) class II molecules in human activated T cells is under normal circumstances regulated exclusively by the CIITA-PIII subtype of the class II transactivator (CIITA). In this study, we show that the absence of MHC class II expression in leukemic T cells was due to a lack of expression of CIITA, whereas in T-lymphoma cells, expression of CIITA correlated with expression of MHC class II. Interestingly, activation of a CIITA-promoter (P)III–reporter construct was not affected in leukemic T cells. This revealed that the absence of endogenous CIITA expression was not caused by a lack of transcription factors critical for CIITA-PIII activation but suggests the involvement of an epigenetic silencing mechanism. Subsequent analysis showed that the lack of human leukocyte antigen–DR (HLA-DR) expression correlated with hypermethylation of CIITA-PIII in leukemic T-cell lines and in primary T-cell acute lymphoblastic leukemia (T-ALL) and a T-cell prolymphocytic leukemia (T-PLL). Treatment of leukemic T-cell lines with a demethylation agent showed re-expression of CIITA-PIII and HLA-DRA. Furthermore, in vitro methylation of CIITA-PIII and subsequent assessment of CIITA-PIII activity in Jurkat leukemic T cells resulted in reduction of constitutive and CREB-1 (cyclic adenosine monophosphate [cAMP]–response element binding protein 1)–induced promoter activity. Together, these results argue for an important role of DNA hyper-methylation in the control of CIITA expression in leukemic T cells.

Beschreibung: Background: Chronic Lymphocytic Leukemia (CLL) has a variable clinical course, of which IgVH mutational status of the malignant B cells is a strong independent predictor for outcome. Objective: We studied the expression of 5 different genes for their value to predict IgVH mutational status and overall survival (OS). Methods: mRNA levels were quantified by real time PCR in lymphoprep separated but unsorted blood samples from 110 morphologically and fenotypically characterised CLL patients for the following genes: ZAP70, lipoprotein lipase (LPL), ADAM29, KIAA0610 and nRIP1. Expression levels were compared wih respect to IgVH mutational status (mutation = % germline 〈 98%), using logistic regression and with respect to OS (= time from day of laboratory tests to death due to any cause), using Cox regression analysis. Results: The predictive value of the expression of these 5 genes for IgVH mutational status is shown in table 1. Each of these genes showed to be an independent marker for the prediction of IgVH mutational status. LPL as a single marker was the best predictor for mutation. These factors taken together had a high predictive value towards mutational IgVH status (area under ROC curve = 0.93). table 1 N Odds ratio P value ZAP70 no 85 1 (〉5.2) yes 25 0.098 0.002 LPL no 73 1 (〉0.04) yes 37 0.029 30) yes 37 6.61 0.012 KIAA0610 no 81 1 (〉0.25) yes 29 0.19 0.045 NRIP1 no 88 1 (〉10) yes 22 48.73 0.008 The predictive value of IgVH mutation and the expression of these 5 genes for OS is shown in table 2. From the six parameters tested, IgVH mutational status and LPL were predictors for OS at a significant level of 5%, while ZAP70 was of borderline significance. table 2 Conclusion: measured in unsorted CLL samples, all genes, and especially LPL, had predictive value towards the presence of IgVH mutation. Expression of LPL and absence of mutation were adverse prognostic factors for survival. Thus, the replacement of the assessment of both IgVH mutational status and ZAP70 by assessment of LPL levels for the prediction of prognosis in CLL patients may be considered. N survival at 24months P value mutation no 36 70% yes 44 93% 0.041 ZAP70 no 66 87% yes 18 70% 0.078 LPL no 55 94% yes 29 66% 0.001 ADAM29 no 57 84% yes 27 84% 0.486 KIAA0610 no 63 88% yes 21 71% 0.133 NRIP no 71 81% yes 13 100% 0.167

Beschreibung: The t(10;11) translocation is recurrent in T-ALL and AML. The AF10 gene on chromosome 10 is rearranged either with MLL or CALM located on chromosome 11. CALM-AF10 fusion gene is found in T-ALLs in immature (IM) and TCRγδ-expressing (TCRGD+) T-ALLs. We compared 6 CALM-AF10+ T-ALLs cases (4 IM, 2 TCRGD+) to 17 CALM-AF10 negative T-ALLs cases (14 IM, 3 TCRGD+) using Affymetrix U133A microarrays. 44 genes were significantly overexpressed in CALM-AF10+ T-ALLs, the most significant being HOXA9, a homeobox gene overexpressed in MLL-translocated acute leukemias (MLL-t AL), BMI1, a polycomb family member whose function in regulation of HOX genes expression is opposite to Trithorax genes (whose MLL belongs), SOX4, a frequent insertion site in retroviral-induced leukemogenesis, SFRS6 and COMMD3 (p≤0.001). Only two other HOX genes, HOXA5 and HOXA10, were significantly increased. 89 genes were significantly underexpressed in CALM-AF10+ T-ALLs, the most significant being GGH, ARL6IP4, NBS1, OGFR and TUBB (p≤0.001). An independent analysis of the expression of HOXA5, HOXA9, HOXA10 and BMI1 genes was done by quantitative RT-PCR in 10 CALM-AF10+ T-ALLs and 27 CALM-AF10 negative T-ALLs. These were compared to 19 MLL-translocated acute leukemias (2 MLL-AF10, 5 MLL-AF4, 3 MLL-AF6, 5 MLL-AF9, 3 MLL-ELL and 1 MLL-ENL), since HOXA9 overexpression had been previously associated with MLL-t AL. HOXA5, HOXA9 and HOXA10 expression were higher in CALM-AF10+ T-ALLs than in CALM-AF10 negative T-ALLs (p

Beschreibung: This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic Jh gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the Jh and Cμ regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal Jhgene rearrangements. Only 4 of the joinings were complete Vh-(D)-Jhrearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete Dh-Jh joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame Vκ-Jκgene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of Ch probes. In 5 patients the rearrangements in the Jh and Ch regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32.3). These data indicate that in many IgH− MM patients illegitimateIGH class switch rearrangement or illegitimate deletion of the functional Vh-(Dh)-Jhallele are responsible for IgH negativity. For example, the exclusive presence ofDh-Jhrearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH− MM patients.

Beschreibung: The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2−/−γc−/− mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2Rβ, (IL-2Rβ) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1−TCRαβ+ CD4+ or CD8+single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA+) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO+ cells. These data suggest that all elements of a functional immune system were present in these animals.

Beschreibung: T-cell receptor (TCR) gene rearrangements are mediated via V(D)J recombination, which is strictly regulated during lymphoid differentiation, most probably through the action of specific transcription factors. Investigated was whether cotransfection ofRAG1 and RAG2 genes in combination with lymphoid transcription factors can induce TCR gene rearrangements in nonlymphoid human cells. Transfection experiments showed that basic helix-loop-helix transcription factors E2A and HEB induce rearrangements in the TCRD locus (Dδ2-Dδ3 and Vδ2-Dδ3) and TCRG locus (ψ Vγ7-Jγ2.3 and Vγ8-Jγ2.3). Analysis of these rearrangements and their circular excision products revealed some peculiar characteristics. The Vδ2-Dδ3 rearrangements were formed by direct coupling without intermediate Dδ2 gene segment usage, and most Dδ2-Dδ3 recombinations occurred via direct coupling of the respective upstream and downstream recombination signal sequences (RSSs) with deletion of the Dδ2 and Dδ3 coding sequences. Subsequently, the E2A/HEB–induced TCR gene recombination patterns were compared with those in early thymocytes and acute lymphoblastic leukemias of T- and B-lineage origin, and it was found that the TCR rearrangements in the transfectants were early (immature) and not necessarily T-lineage specific. Apparently, some parts of theTCRD (Vδ2-Dδ region) and TCRG genes are accessible for recombination not only in T cells, but also in early B-cells and even in nonlymphoid cells if the appropriate transcription factors are present. The transfection system described here appeared to be useful for studying the accessibility of immunoglobulin and TCR genes for V(D)J recombination, but might also be applied to study the induction of RSS-mediated chromosome aberrations.