ALBERT

Description: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecular studies in 96 childhood precursor-B–ALL at diagnosis and at relapse (n = 91) or at presumably secondary acute myeloid leukemia (n = 5). Clonal Ig and TCR targets for MRD detection were identified in 94 patients, with 71% of these targets being preserved at relapse. The best stability was found for IGK-Kde rearrangements (90%), followed byTCRG (75%), IGH (64%), and incompleteTCRD rearrangements (63%). Combined Southern blot and PCR data for IGH, IGK-Kde, and TCRDgenes showed significant differences in stability at relapse between monoclonal and oligoclonal rearrangements: 89% versus 40%, respectively. In 38% of patients all MRD-PCR targets were preserved at relapse, and in 40% most of the targets (≥ 50%) were preserved. In 22% of patients most targets (10 cases) or all targets (10 cases) were lost at relapse. The latter 10 cases included 4 patients with secondary acute myeloid leukemia with germline Ig/TCR genes. In 5 other patients additional analyses proved the clonal relationship between both disease stages. Finally, in 1 patient all Ig/TCR gene rearrangements were completely different between diagnosis and relapse, which is suggestive of secondary ALL. Based on the presented data, we propose stepwise strategies for selection of stable PCR targets for MRD monitoring, which should enable successful detection of relapse in most (95%) of childhood precursor-B–ALL.

Description: In vivo and in vitro response to glucocorticoids (GC) are important prognostic factors in childhood ALL. To induce apoptosis, GC bind to the intracellular GC receptor (GR), which subsequently triggers transactivation and repression of downstream genes. As somatic mutations and polymorphisms have been linked to both increased and decreased GC sensitivity in healthy individuals, we studied the relationship between these gene alterations and GC sensitivity in childhood ALL. Methods: We analysed leukemic cells taken at diagnosis and normal lymphocytes at time of complete remission (CR) from 57 children. DNA mutations were screened for using a PCR - SSCP technique. PCR products with abnormal SSCP patterns were sequenced. In vivo response to prednisone was determined according to BFM criteria as prednisone good response (PGR) or prednisone poor response (PPR). The in vitro prednisolone sensitivity was determined by MTT assay. Results: The polymorphism ER22/23EK was found in 4% of cases, N363S in 12%, Bcl1 in 55%, intron mutation 16 basepairs upstream of exon 5 in 40%, H588H in 2% and N766N in 21%. They were proven to be polymorphisms as they were found in normal lymphocytes of the same patients at CR as well. The incidence of the polymorphisms was comparable to reports in the literature in the healthy population, except for N363S, which was found at a 2–4 fold higher incidence in ALL patients. Using the Chi-square test and the Mann-Whitney U test, no relation could be found between the occurrence of polymorphisms and in vivo prednisone response or in vitro sensitivity to prednisolone. Conclusion: GC resistance in childhood ALL can not be attributed to polymorphisms of the hGR. The association between a higher incidence of the N363S polymorphism in childhood ALL as compared to the normal population needs further investigation.

Description: In vitro resistance to Glucocorticoids (GC) is an important adverse risk factor in the treatment of ALL. To induce apoptosis in ALL cells, GC have to bind to the GC receptor (hGR), which is tightly regulated by various (co)chaperone molecules. HSP70, ST13 and HSP40 facilitate hGR binding to HSP90 and HOP in an energy dependent fashion. This complex is stabilised by immunophillins FKBP 51, FKBP59, CYP40 and P23 and is necessary for the hGR to be able to bind GC. The ATPase BAG1 can function as negative regulator of HSP70, and may downregulate hGR activity. After the GC-hGR complex is formed, it is transported to the nucleus to regulate GC responsive genes. In this study, we tested the hypothesis that RNA expression levels of these (co) chaperone molecules are important determinants for in vitro prednisolone sensitivity in childhood ALL. Methods: 20 children with leukemic cells in vitro sensitive to prednisolone (LC50 150 μg/ml). RNA expression levels of the different (co)chaperone molecules were measured by a quantitative real-time RT-PCR (Taqman) assay and standardised to endogenous GAPDH and RNAseP mRNA levels. In vitro resistance to prednisolone was measured by the MTT assay. Results. The highest median expression levels, indicated as percentage of GAPDH levels were found for HSP90 (29%) and p23 (10%), whereas FKBP51 and ST13 were expressed at 2% and 1.6% respectively. HSP70 (0.1%), HSP40 (0.44%), HOP (0.2%), FKBP59 (0.4%), PPID (0.3%) and BAG-1 (0.4%) were expressed at relatively low levels. Using matched pair analysis, no significant differences were found for the expression levels of the various (co)chaperone molecules between in vitro sensitive and resistant patients. The ratio of different (co)chaperone molecules with opposing effect (HIP versus BAG-1, FKBP-51 versus FKBP-52, HSP-90 versus P23, and HSP-90 versus the functional GR-alpha receptor, positive and negative effect respectively) was not related to GC resistance as well. Conclusion: Glucocorticoid resistance in childhood ALL can not be attributed to basal expression levels of the diverse (co)chaperone molecules involved in GC binding and transport of the GC-GR complex to the nucleus of the GR.

Description: Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoptosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC exposure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure).