ALBERT

Description: Article A range of mechanisms has been proposed for large-scale folding in polar ice sheets. Here, using new three-dimensional reconstructions of such folds in the onset region of the Greenland Petermann Glacier, the authors show that these formed due to flow convergence and the high mechanical anisotropy of ice. Nature Communications doi: 10.1038/ncomms11427 Authors: Paul D. Bons, Daniela Jansen, Felicitas Mundel, Catherine C. Bauer, Tobias Binder, Olaf Eisen, Mark W. Jessell, Maria-Gema Llorens, Florian Steinbach, Daniel Steinhage, Ilka Weikusat

Description: Abstract 3243 Objective: Previous reports indicated that short term prognosis for patients with malignant diseases and serious adverse events requiring mechanical ventilation (SAEV) is not dismal any more. The purpose of this study was to determine whether patients who survive such complications can also achieve long term cure from leukemia. It might influence end-of-live decisions on the intensive care unit if patients with an SEAV only survive intensive care to succumb to relapse. Patients and Methods: We report the outcome of children with SAEV treated in the multicenter studies ALL-BFM 95 and AML-BFM 98. Data from 1182 patients with acute lymphoblastic leukemia (ALL) and 332 patients with acute myeloid leukemia (AML) were analyzed. 88 patients (51 ALL; 37 AML) developed an SAEV. Results: The prognosis was almost identical in ALL and AML (50% survival of SAEV; 30% overall survival after 5 years). This was independent from the time between diagnosis of leukemia and SAEV. Even children who required hemodialysis (n=14) or cardiac resuscitation (n=16) achieved 20% long term survival but no patient survived (n=16) who fulfilled more than 3 out of 6 identified risk factors: age≥10 years, high risk leukemia, C-reactive Protein≥150mg/l, administration of inotropic infusion, cardiac resuscitation, and hemodialysis. Conclusions: To our knowledge, this is the first report about cure rates in a malignant disease after successful treatment of SAEV. Our results show that intensive care medicine contributes to short and long term survival of children with leukemia. Sixty percent of all children with acute leukemia who survive an SAEV achieve long term cure. However, we could also identify risk features that still indicate a devastating prognosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 344 Hemorrhagic cystitis is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of hemorrhagic cystitis is not fully understood and is influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Nothing is known about human genetic factors and the development of BK virus-associated hemorrhagic cystitis in patients after HSCT. Toll-like receptors (TLR) are essential components of the innate immune system. TLR have been discovered as the most important class of pattern recognition receptors, involved in the host defense against bacteria, viruses, fungi, and protozoa. C3H/HeJ mice that lack functional TLR4 receptors did not develop cystitis after intravesical instillation of E. coli. Individuals with the Asp299Gly polymorphism of the TLR4 gene showed a diminished inflammatory responsiveness to endotoxin. Because of the requirement of TLR4 in inflammatory response we hypothesized that TLR4 Asp299Gly reduces the risk of BK virus-associated hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT). 166 children (median age, 13 years) with acute lymphoblastic leukemia (n=72), acute myeloid leukemia (n=38), myelodysplastic syndrome (n=21), chronic myeloid leukemia (n=12), genetic disease (n=12) or severe aplastic anemia (n=11) who underwent allogeneic bone marrow (n=105) or peripheral blood stem cell transplantation (n=61; T-cell depleted: n=31) in a single center and their respective donors were genotyped of TLR4 for rs4986790 (A896G, Asp299Gly) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 57% of transplants or HLA-identical related in 33% of transplants. Conditioning regimen was myeloablative in all cases and based on total body irradiation in 48% of transplants or busulfan in 47% of transplants. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 64% of transplants and cyclosporine A alone in 25% of transplants. The Asp299Gly variant was present in 21 of the 166 patients (12.6%) and in 24 of the 166 donors (14.4%). Interestingly, we found a significantly reduced incidence of BK virus-associated hemorrhagic cystitis in patients with the Asp299Gly variant (0% versus 22.8%, p=0.009). In addition, we observed a significantly reduced incidence of BK virus-associated hemorrhagic cystitis in patients who were transplanted from a donor with this specific variant (4.2% versus 22.5%; p=0.05). In ten of the donor-patient pairs the variant was present in both individuals and no hemorrhagic cystitis was observed. The occurrence of the TLR4 Asp299Gly variant, in either recipients or donors, had no significant impact on acute and chronic GVHD, relapse rate, bacterial and fungal infectious complications, transplant related mortality, and overall survival. In conclusion, the TLR4 Asp299Gly variant in the recipient and/or the donor confers strong protection against BK virus-associated hemorrhagic cystitis after HSCT in children. This study provides the first evidence that the innate immune system through TLR4 signaling pathway seems to play an important role in the pathogenesis of BK virus-associated hemorrhagic cystitis after HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: A stepwise approach which combined genome wide expression profiling and a TaqMan realtime PCR based screening was used to identify new markers for the monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML). Leukemic cells from 52 children with AML were analyzed. Seven genes were identified which are vastly over-expressed in many patients with AML compared to healthy bone marrow: CCL23, GAGED2, MSLN, SPAG6, and ST18 as well as the previously described markers WT1 and PRAME. This set of genes was analyzed in 141 follow-up samples from 25 patients. The expression of all genes decreased to normal levels in patients who achieved a continuous complete remission. Elevated levels of MRD markers were found prior to relapse in 7 out of 10 patients who relapsed. This set of genes should allow a sensitive and specific monitoring of MRD in AML. Notably, some of these markers could also serve as therapeutic targets or might be involved in leukemogenesis. MSLN is already used as a target for immunotherapy in clinical trials in other malignancies. GAGED2 and SPAG6 belong to the family of cancer testis genes which are also studied intensively as targets for immunotherapy. ST18 is a recently discovered tumor suppressor which was not yet described in hematological malignancies. CCL23 is a chemokine that inhibits the proliferation of healthy hematological stem cells. Names, symbols, and geneID of seven MRD markers Gene Symbol Gene Name GeneID CCL23 chemokine (C–C motif) ligand 23 6368 GAGED2 G antigen, family D, 2 9503 MSLN Mesothelin 10232 SPAG6 sperm associated antigen 6 9576 ST18 suppression of tumorigenicity 18 9705 WT1 Wilms tumor 1 7490 PRAME preferentially expressed antigen in melanoma 23532

Description: The proof for the prenatal origin of childhood acute lymphoblastic leukemia (ALL) comes from the detection of concordant leukemia in monozygotic twins and the identification of translocation breakpoint genomic sequences at birth in a limited number of ALL patients with t(4;11) or t(12;21) chromosomal translocation. However, most patients with childhood ALL lack leukemia-specific fusion gene sequences. Therefore, we have used the rearranged immunoglobulin heavy chain (IgH) genes as a marker for the detection of preleukemic clones at birth. Guthrie card blood spots of 32 children with B-lineage ALL treated at our institution were available for this retrospective study. The ALL patients had a median age of 5 years (range, 15 months to 14 years) and had median presenting white blood cell (WBC) counts of 10150/μl (range, 800 to 103800/μl). In all patients a monoclonal IgH gene rearrangement was obtained from diagnostic bone marrow and sequenced. Clone-specific primers were designed using the specific D-N-J and N-D-N sequences. A two-stage polymerase chain reaction (PCR) using a semi-nested approach was developed to improve sensitivity and specificity of amplification. In all 32 patients, one leukemic cell could be detected in a background of 105 normal blood mononuclear cells. Nineteen of the 32 patients (59%) had detectable IgH gene rearrangements at birth using the sensitive semi-nested PCR. Sequencing of the PCR products obtained from Guthrie card blood spots revealed the identical sequences identified from diagnostic leukemic cells. The fetal characteristics of the leukemic cells were indicated by the small numbers of nucleotides inserted into the N region and the shortened D germ line segments. Interestingly, five of the six children (83%) with hyperdiploid ALL had detectable preleukemic clones at birth. Four of the five children (80%) with pro-B ALL, 13 of the 21 children (62%) with cALL and only two of the six children (33%) with pre-B ALL had preleukemic clones on their cards. We did not observe any differences in age at diagnosis or presenting WBC count between the 19 patients with preleukemic clones at birth and the 13 patients whose Guthrie cards were tested negative. Our results suggest that the majority of children with B-lineage ALL has preleukemic clones already at birth indicating a prenatal origin of leukemia. In addition, postnatal factors are important in leukemogenesis as well because of the long latency periods until clinical diagnosis of leukemia.

Description: Monitoring of minimal residual disease (MRD) has become one of the strongest diagnostic tools in the treatment of acute leukemia. MRD data after induction therapy can be used for risk group stratification. Long-term monitoring of MRD may allow predicting pending relapses. Suitable MRD markers are available for almost all ALL patients. In AML, many patients can not be monitored because there are no specific markers available that distinguish between leukemic cells and healthy blood (hB) or healthy bone marrow (hBM). It was recently shown that MRD in AML can be monitored by measuring the expression of WT1 (Wilms tumor gene) or PRAME (Preferentially expressed antigen in melanoma). Both genes are strongly overexpressed in the leukemic cells of many AML patients. The aim of the present study was to identify additional genes which are strongly overexpressed in AML. In a first step, five microarrays (Affymetrix U133 chip) were performed. 1st: pooled RNA from 10 samples of healthy CD34+ stem cells (hSC); 2nd: pooled RNA from 10 samples of hBM; 3rd: pooled RNA from 10 children with AML M1 or M2; 4th: pooled RNA from 10 children with AML M4; 5th: pooled RNA from 10 children with AML M5. Twenty-eight genes were found to be absent in hBM and hSC but present in all three subtypes of AML. About 200 genes were absent in hBM and hSC but present in at least two subtypes of AML. In a second step, 35 genes were selected from these lists and analyzed by real time PCR in the pools of RNA from hBM and from AML patients. Using real time PCR none of the genes was found completely negative in hBM. However, for eight genes the expression in the AML pools was more than 100 times higher than in the normal control (PRAME, WT1, TRH, CTGF, POU4F1, CCL23, ST18, and CCNA1). These eight genes were analyzed by real time PCR in 51 samples from children with previously untreated AML, 10 samples of hBM and 10 samples of hB. The most promising candidates for MRD markers were PRAME and ST18. The expression of both genes was up to 10000 times higher in AML patients than the highest levels that were found in hBM or hB. WT1 was completely negative in all hB samples but positive in some samples of hBM. The expression in the patient samples was up to 100 times higher than the highest expression in hBM. This is less than previously described for adult patients. TRH and CTGF could be very useful markers for the analysis of MRD in peripheral blood but the expression in hBM is probably too high to allow a reliable detection of MRD. CCL23 and POU4F1 could be useful marker for the detection of MRD in blood and bone marrow but very high levels were only found in a few patients. CCNA1 was expressed at relatively high levels in some hBM and some hB samples and therefore does not seem to be a useful MRD marker. It is not yet clear what level of expression is needed to use a gene as MRD marker. It was recently shown that WT1 can be used to predict pending relapses in some children with AML. Our results suggest that PRAME and ST18 are at least as useful. We are currently analyzing the expression of these genes retrospectively in follow up samples of 35 children with AML who were treated in our hospital. We hope to show that the combined analysis of these markers allows a more specific and sensitive monitoring of MRD.

Description: The family of multidrug resistance-associated proteins (MRPs) belongs to the superfamily of adenosine triphosphate-binding-cassette (ABC) transporters, which have the ability to function as outward pumps for chemotherapeutic drugs and therefore might be involved in drug resistance. In this study the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time polymerase chain reaction (PCR) in 103 children with previously untreated acute lymphoblastic leukemia (ALL) (precursor B-cell ALL [B-ALL], n = 71; T-cell ALL [T-ALL], n = 32). All 5 genes were expressed with a great variability. Only MRP3 expression was associated with a significantly worse prognosis (P = .008). The median expression of MRP3 was 10-fold higher in T-ALL than in precursor B-ALL (P 〈 .001) and 4-fold higher in male patients than in female patients (P 〈 .001). The prognostic impact of MRP3 was independent of immunophenotype or sex. Higher levels of MRP3 were found in patients with a poor in vivo response to prednisone, but this could not be confirmed in an independent case-control study (40 patients) for prednisone response. In healthy donors, the median expression of MRP4 was 4-fold higher in bone marrow and 8-fold higher in CD34+ stem cells compared with peripheral blood (P = .002). Our results suggest that MRP3 is involved in drug resistance in childhood ALL. It therefore represents an interesting target to overcome multidrug resistance. High levels of MRP3 could possibly be the reason for the poorer prognosis of male patients or patients who have T-ALL. Similar to other members of the family of ABC transporters, MRP4 seems to be a marker for immature stem cells. (Blood. 2003;102:4493-4498)

Description: A major issue in the treatment of acute myeloid leukemia (AML) is the development of resistance to chemotherapeutic drugs. While more than 80% of children with AML may initially achieve complete remission with current therapeutic regimens, a large number of these patients relapse with resistant disease; even with aggressive therapy, the event free survival rate is only about 50%. Several mechanisms of drug resistance have been identified. One of these is the overexpression of ATP-binding-cassette (ABC)-transporters that function as drug efflux pumps. The best characterized member of this family is the P-glycoprotein (P-gp or MDR1 or ABCB1), which is an important cause of drug resistance in adult AML but not in children with AML. We could recently show that the multidrug resistance-associated protein 3 (MRP3 or ABCC3) and the breast cancer resistance protein (BCRP or ABCG2) are associated with a poor response to therapy in childhood AML. The aim of the present study was to identify other members of this family of proteins which are involved in drug resistance. We were particularly interested in ABC-transporters which were overexpressed in AML samples compared to healthy bone marrow because this might be an important prerequisite for using such proteins as therapeutic targets. A new microarray (DualChip human ABC) was developed for the simultaneous detection and quantification of 38 ABC transporter genes. Samples from 30 children with AML were analyzed with this microarray and the results were compared with normal bone marrow. The expression of five genes (ABCA2, ABCA3, ABCB2, ABCC1, and ABCC10) which were overexpressed in most patient samples was then analyzed by real time PCR in 40 selected samples from children with previously untreated AML. Half of the patients had achieved complete remission after the first course of chemotherapy. The other 20 patients had also undergone the full induction therapy but had failed to achieve remission at this stage. Only the expression of ABCA3 was significantly higher (p=0.015) in patients with a poor response to therapy. There was a 4-fold difference between the median expressions in both groups. The expression of ABCA3 was not significantly associated with the expression of P-gp, MRP3 or BCRP. Our results show that ABCA3 is overexpressed in childhood AML compared to healthy bone marrow and that the expression of ABCA3 is associated with a poor response to therapy. Interestingly, there are two ways in which ABCA3 might influence the prognosis of AML. Like other ABC-transporters, it could be involved in drug efflux. On the other hand, it was recently shown that ABCA3 plays a role in apoptosis and that many cancer cell lines show amplifications of the ABCA3 gene. Further studies are warranted to analyze the clinical and biological relevance of ABCA3 in leukemia.

Description: Insulin-like growth factor (IGF) system plays an important role in regulating cellular proliferation. Alterations to the Insulin-like growth factor system have been reported for different tumors. They are of particular interest in the search for new prognostic and therapeutic approaches to cancer treatment. This study aimed to investigate the expression of IGFBP-2, IGFBP-3, IGF-I and IGF-II genes in leukemic cells from children with previously untreated acute myeloblastic leukemia (AML).The expression of IGF-I and IGF-II genes as well as IGFBP-2 and IGFBP-3 genes were measured using TaqMan real-time PCR in 50 children (mean age 10.8±4.8 years). All four genes were expressed with a great variability. RNA samples were extracted from leukemic cells enriched by Ficoll-Hypaque density gradient separation of bone marrow or peripheral blood mononuclear cells (MNC). MNC samples from peripheral blood and bone marrow of healthy children were used as controls. The median expression of IGFBP-2 was 25 times higher in AML cells than in peripheral MNC (p

Description: We could recently show that the expression of the multidrug resistance-associated protein 3 (MRP3; ABCC3) is an important prognostic factor in ALL and in AML (Blood2003; 102:4493–8 and Clin Cancer Res2003; 9:1083–6). Lang et al. showed that the single nucleotide polymorphism −211C〉T (NCBI SNP ID: rs4793665) in the promoter region of the MRP3 gene is associated with the expression of the gene in liver cells (Pharmacogenetics2004;14:155–64). They found that individuals who were homozygous for cytosine in position 211 showed higher levels of MRP3 mRNA and protein. Lang et al. also provided evidence that the polymorphism is located at a binding site for transcription factors and that the polymorphism affected the binding of such factors. This could explain the influence of the genotype on the transcription level of the gene. Because of these findings, we hypothesized that the SNP −211C〉T might also determine the expression of MRP3 in acute leukemia and therefore be an inborn determinant of drug resistance. In order to prove this hypothesis we analyzed this SNP in our previously described groups of ALL and AML patients. The genotype was determined using a ready to use TaqMan® SNP Genotyping Assay and the ABI PrismTM 7700 Sequence Detector (Assay ID: C_27829307_10; Applied Biosystems, Foster City, CA, USA). Samples from 139 patients were analyzed. The frequency of the genotypes was: TT = 32% (n=44), CC = 22% (n=31), and CT = 46% (n=64). We did not find any correlation between the genotype and the expression of MRP3 (p=0.44). The result was the same, when patients with AML, T-lineage ALL, and precursor B-lineage ALL were analyzed separately. We also did not find a correlation between the genotype and response to therapy or the chance of survival. In conclusion, the SNP −211C〉T in the promoter region of MRP3 does not have a major impact on the level of MRP3 gene expression in acute leukemia and it does not determine the response to therapy. Possibly, the putative transcription regulator which binds at this part of the MRP3 gene is not active in acute leukemia.