ALBERT

Description: Noonan syndrome is characterized by short stature, facial dysmorphism, and cardiac defects. We and our colleagues discovered novel de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with NS and a P34R alteration in an individual with cardio-facio-cutaneous (CFC) syndrome, which has overlapping phenotypic features with NS. Recombinant V14I and T58I K-Ras proteins display defective intrinsic GTP hydrolysis and impaired responsiveness to the GTPase activating proteins (GAPs) p120 GAP and neurofibromin. We also found that V14I and T58I K-Ras render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage specific manner (Nature Genetics38, 331, 2006). We recently began interrogating the P34R and D153V K-Ras mutant proteins and a novel CFC-associated F156L K-Ras mutant protein. Both P34R and D153V K-Ras display normal levels of intrinsic GTP hydrolysis. In contrast, F156L K-Ras displays defective intrinsic GTP hydrolysis that resembles oncogenic G12D K-Ras. P34R K-Ras is completely resistant to both GAPs, which is intriguing as these data suggest that the ability of GAPs to down-regulate Ras-GTP levels is dispensible for development. In contrast, D153V K-Ras is responsive to both GAPs. We expressed mutant Ras proteins in COS-7 monkey kidney cells to investigate activation of Ras and downstream effectors. Cells expressing P34R, D153V, F156L, and G12D K-Ras demonstrate elevated levels of Ras-GTP and phospho-MEK in basal and serum-starved conditions. We conclude that germline KRAS mutations that cause human disease encode proteins with distinct mechanisms of biochemical activation. Further investigation of this allele series will provide insights about the relative importance of the intrinsic Ras GTPase, GAPs, and other biochemical mechanisms in controlling the growth of different cell lineages.

Description: Neonatal thrombocytopenia occurs in about 1% of all newborns. Inherited forms like 11q- or Jacobsen syndrome are rare. However, they may remain undetected with karyotyping because the deleted regions in 11q often involve small subtelomeric regions. Here we report on the detection of deletions in 11q in two newborns with normal routine karyotypes who were shown to carry subtelomeric deletions in 11q by means of fluorescence in situ hybridization (FISH) using a subtelomeric 11q probe (Abbott, Diagnostics, Wiesbaden, Germany). Both children showed thrombocytopenia (18.000/μl and 26.000/μl, respectively) and dysmegakaryopoiesis (absence of normal megakaryocytes and presence of micromegakaryocytes) associated with facial dysmorphism, cardiac defects and psychomotoric retardation. In the second case, the mother and the grandmother also showed mild thrombocytopenia. In both patients, FISH analyses on peripheral blood and bone marrow showed the loss of the telomere-associated region of 11q distal of the MLL gene. In the first patient, the deletion of 11q resulted from an unbalanced complex rearrangement with duplication of 11p. As the source of this chromosomal aberration, a paternal pericentric inversion of chromosome 11 was identified. The partial monosomy 11q and the partial trisomy 11p in the first patient were confirmed by comparative genomic hybridization (CGH) analysis. Array/matrix CGH assisted in determining the breakpoints at 11p15.1 and 11q24.1. No structural aberrations of 11q were found in the mother of the second patient, but further investigations are under way. These findings give further evidence that small subtelomeric deletions of 11q and probably mutations of genes located therein cause thrombocytopenia. Since it can be very difficult to detect these deletions by karyotyping, FISH using a subtelomeric 11q probe seems to be an extremely useful new diagnostic tool. This new method should be applied in children with congenital thrombocytopenia, in particular if they have additional complex dysmorphic features.

Description: (Very) Severe acquired aplastic anemia ((v)SAA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. (V)SAA is a bone marrow failure syndrome characterized by immune mediated destruction of hematopoietic progenitors. MDS is a malignant clonal stem cell disorder, in which the hypoplastic variant is, in case of absence of a cytogenetic clone, difficult to separate from (v)SAA. Recently, studies provided a “molecular signature” of autoimmunity in adult (v)SAA, by showing oligoclonality of TCR Vß CDR3 region, which is refered to as TCR Vß skewing. We investigated the value of TCR Vß repertoire analysis in pediatric MDS-RC and (v)SAA patients. Peripheral blood and/or bone marrow mononuclear samples of patients with (v)SAA (n=38), MDS-RC (n=28) and 18 controls were analysed with Vß heteroduplex analysis of extracted RNA. Skewing was found in 21/38 (55%) of the (v)SAA patients and in 17/28 (61%) of the RC patients. Seventeen patients with clinical (v)SAA showed no oligoclonality. A significant difference in TCR skewing was found between the (v)SAA + MDS-RC patients as compared to the controls (χ2 analysis, p=0.001), but not between MDS-RC and (v)SAA (χ2 analysis, p=0.8). In this study paired samples (PB and BM) of 25 cases showed a high correlation between the skewing results in both compartments (Pearson correlation, rr 0.98). In this study TCR Vß repertoire analysis did not discriminate between MDS-RC and (v)SAA. Prospective studies will be necessary to investigate whether there is a role for this molecular tool in pediatric MDS-RC for the identification of a subset of patients that is associated with auto-immunity and therfore could be treated with IST up-front, and whether it can be used for molecular response monitoring.

Description: Introduction JMML is a rare leukemia characterized by aberrant myeloid proliferation and hypersensitivity to GM-CSF. Mutually exclusive mutations in PTPN11, N/K-RAS, CBL, or NF1 are found in ~90% of patients. These mutations cause the disease at least in part by activating STAT5 through phosphorylation and promoting cell growth. MicroRNAs (miRs) are small (~22 nucleotides) noncoding RNAs, regulating gene expression and often deregulated in leukemia. We investigated whether miRs are deregulated in JMML and whether miRs target critical proteins involved in the disease pathogenesis. Patients and Methods MiRs expression profile of 40 bone marrow (BM) samples from untreated JMML patients and 8 BM samples from healthy controls was performed using the Ncounter Human v2 miRNA Expression Assay (nanostring). MiR150-5p and STAT5b mRNA expression were assessed using quantitative (q)RT-PCR. Selected miRs and target expression were measured in BM and spleen from a JMML (PTPN11) murine model (D61Y mutation). We overexpressed miR-150-5p in AML cell lines (K562, OCI-AML-3, KG1a) using a miR-150-5p precursor plasmid and STAT5b mRNA was assessed by qRT-PCR. STAT5 and phospho-STAT5 protein levels were assessed by Western Blot in miR-150-5p transfected AML cell lines, human JMML and mice BM cells. STAT5b 3'UTR sequence was cloned in a Firefly/Renilla Luc construct and co-transfected with miR-150-5p miRNA mimic into 293T cell line. Results We found that 25 miRs were differentially expressed in whole BM cells from JMML patients respect to healthy controls (Table 1). MiR-150-5p was the most downregulated miR in JMML. We focused on miR-150-5p, since it has been described to be downregulated in AML cases and is predicted to target STAT5b, a critical gene in JMML biology. We validated that miR-150-5p was down-regulated in JMML cases respect to controls performing qRT-PCR on 38 BM samples from JMML patients. Likewise, miR-150-5p was downregulated in BM and spleen samples from PTPN11 mutated mice respect to controls (0.35 and 0.27 Average Fold Change decrease respectively). STAT5 protein, a predicted target for miR-150-5p, was highly expressed in the JMML patients and mice BM samples respect to their controls (4.3 and 1.3 Average Fold Change increase respectively). MiR-150-5p overexpression in K562, OCI-AML-3 and KG1a cell lines led to decrease of STAT5 protein levels and phosphorilation at 48 hours. Direct interaction of STAT5b 3'UTR with miR-150-5p was demonstrated by luciferase assay (~50% Luc activity inhibition, P

Description: MDS in children is a rare disorder characterized by dysplasia and defined genetic abnormalities. In most patients (pts) MDS arises without known predisposing conditions (primary MDS). Here, we report the results of 55 males and 30 females with advanced primary MDS enrolled in the prospective EWOG-MDS trial 97. Data were analysed according to the most advanced FAB-type prior to SCT: 32 pts were classified as RAEB, 40 as RAEB-t and 13 as myelodysplasia-related AML (MDR-AML). Median age at diagnosis was 9.5 yrs (0.1–17.6) and median time from diagnosis of advanced MDS to SCT 4 mo (0.5–31). Cytogenetics revealed monosomy 7 in 32 pts, trisomy 8 in 7, a complex karyotype in 9 and other abnormalities in 9; karyotype was normal in 26 pts and unknown in 2. 31 pts had received AML-like therapy prior to SCT. All pts were given an unmanipulated graft after condititioning with busulfan 16 mg/kg, cyclophosphamide 120 mg/kg and melphalan 140 mg/m. Source of stem cells was bone marrow in 56 pts, peripheral blood in 25, cord blood in 2 and unknown in 2. 36 pts were transplanted from an HLA-identical relative (MFD), 49 pts from an HLA-identical or 1-antigen disparate unrelated donor (UD). GVHD prophylaxis consisted of CSA alone for MFD, whereas recipients of a UD graft generally received CSA, methotrexate and anti-lymphocyte globulin. Two pts suffered graft failure. The cumulative incidence of grade II-IV acute GVHD and chronic GVHD was 40% (SE 5%) and 25% (SE 5%), respectively. 18 pts suffered transplant-related mortality (TRM), the cumulative incidence of TRM in pts grafted from a MFD or UD being 14 and 25%, respectively (p=n.s.). Presence of acute GVHD II-IV (p

Description: Abstract 533 Juvenile myelomonocytic leukemia (JMML) is a rare and lethal myeloproliferative disease of young childhood. Currently, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment option. DFS at 5 years after HLA identical and unrelated HSCT was 55% (n=48) and 49% (n=52) in a largest series of patients published so far and mainly transplanted with bone marrow cells. Unrelated Cord Blood Transplantation (UCBT) is considered an alternative option for patients who lack an HLA-matched donor. We retrospectively analyzed 110 children, given a first single unmanipulated UCBT, from 1995 to 2010, and reported to Eurocord-EBMT and CIBMTR. Median age was 1 year (range 0.08–6.4) at diagnosis and 2 years (0.5-7.5) at transplantation, respectively. Median time interval between diagnosis and UCBT was 6 months (1-58); before transplantation, 88 patients were treated with low- or high-dose chemotherapy and splenectomy was performed in 24 children. Among 100 patients with available cytogenetic data, monosomy of chromosome 7 was the most frequent abnormality (24%). All but 8 patients received a myeloablative conditioning, Busulfan-Cyclophosphamide-Melphalan (BuCyMel) was used in 48 patients, total body irradiation (TBI) and Cyclophosphamide in 19 patients and combination of Busulfan-Cyclophosphamide with other drugs in 21 patients. Cyclosporin+steroid was the most common graft-versus-host disease (GvHD) prophylaxis (80%) and ATG was added in 86% of patients. Nineteen percent of units were HLA-identical (antigen level for HLA-A and B, allelic for DRB1), while 43% and 38% had 1 or 2–3 mismatches, respectively. Median TNC infused was 7.1×10e7/kg (1.7-27.6). Median follow-up was 44 months (3-169). At 60 days, cumulative incidence (CI) of neutrophil (PMN) recovery was 80±4%, with a median time to PMN recovery of 25 days. Grades II-IV acute GvHD developed in 45 patients, 100 days-CI of grade II-IV aGvHD was 40±5%. Among 90 patients at risk, 17 developed chronic GvHD and 4 years-CI was 16±4%. At 4 years CI of relapse was 37±5% (n=38); age older than 1 year at diagnosis was the only independent factor associated with increased risk of relapse (HR 2.3, p=0.038). Of note, among 58 patients with available data for level of fetal hemoglobin (HbF), a higher level of HbF (〉35%) seemed to be associated with increased relapse incidence (57% versus 31% for remainders; p=0.05). At 4 years, DFS was 43±5%, in multivariate analysis independent factors associated with better DFS were: age younger than 1 year at diagnosis (53% vs 30%, HR 2.4, p=0.001), graft with 0 or 1 HLA mismatched cord blood unit (48% vs 34%, HR=2.1, p=0.006) and cytogenetic without monosomy 7 (48% vs 26%; HR=1.95, p=0.027). At 4 years, CI of transplant related mortality (TRM) was 20±4%; in multivariate analysis, cytogenetic with monosomy 7 (HR=2.7, p=0.036) and transplantation performed before 2003 (HR=3.7, p=0.015) were factors associated with increased TRM. In fact, CI of TRM was 14% after 2003 compared to 30% before 2003. Estimated overall survival (OS) at 4 years was 51±5%, and in multivariate analysis factors associated with decreased OS were: age older than 1 year at diagnosis (42% vs 60%; HR=2.03, p=0.032), and cytogenetic with monosomy 7, (30% vs 57%; HR=2.6, p=0.004). Fifty-one patients died after transplant, 53% for relapse and 47% for transplant related causes. In conclusion, UCBT may cure approximately 50% of patients with JMML who lack a matched related donor. Presence of monosomy 7 is associated with decreased DFS and increased TRM, independent of other factors. Other patient- (age at diagnosis) and transplantation-related factors (HLA and year of transplantation) were also associated with outcomes. Disease recurrence remains the major cause of treatment failure, and strategies to reduce the risk of relapse are warranted. Disclosures: Wagner: CORD:USE: Membership on an entity's Board of Directors or advisory committees; VidaCord: Membership on an entity's Board of Directors or advisory committees.

Description: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of childhood characterized by a high frequency of somatic mutations in NRAS, KRAS2, or PTPN11 genes as well as LOH of the NF1 allele (mainly in patients with type 1 neurofibromatosis). Activation of the Ras pathway is the biochemical consequence of these lesions. Hematopoietic stem cell transplantation (HSCT) is the only known curative treatment, with event-free survival (EFS) rates approaching 50–60%. Leukemia relapse still remains the main cause of treatment failure. Monitoring minimal residual disease (MRD) before and after HSCT is challenging due to the lack of tractable molecular markers. We designed and validated a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA that can detect point mutations in eight of the most common RAS or PTPN11 mutations with a sensitivity of 1/1000 copies of the wildtype allele. We analyzed the peripheral blood (PB) and/or bone marrow (BM) in 22 patients for the levels of these mutant alleles at diagnosis and pre-HSCT. Eighteen of these patients had weekly-monthly PB and/or BM samples available after 21 HSCTs (3 children had second transplants). We compared the percentage of mutant DNA with rising levels of autologous cells measured by chimerism analysis after HSCT, which is the current surrogate marker used to detect relapse. Analysis of patients at initial diagnosis and pre-HSCT revealed a broad distribution of the frequency of the mutant alleles, regardless of the use or intensity of pre-HSCT chemotherapy. As shown in the table, levels of disease burden prior to HSCT were not predictive of relapse. After HSCT, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with analysis of chimerism (r=0.86, p

Description: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C〉T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p

Description: Approximately 75% of patients with juvenile myelomonocytic leukemia (JMML) harbour mutations in PTPN11, NF1 and RAS genes. The remaining cases presumably carry somatic mutations in other genes in the RAS pathway. BRAF plays a central role in this pathway between RAS and downstream molecules including MEK and ERK. BRAF mutations frequently occur in cancer. Recently, BRAF mutations were found in leukemia. Besides that, germline BRAF mutations cause cardio-facio-cutaneous syndrome, which shares many features with Noonan syndrome (NS). NS predisposes to a myeloproliferative disease resembling JMML. In 65 JMML patients screening for V600E mutations in exon 15 of the BRAF gene was performed from mononuclear cells. In a subset of patients, without RAS or PTPN11 mutations, and no clinical signs of NF1, the entire coding sequence of BRAF was analyzed. Sequence analysis was performed by direct, bidirectional sequencing of purified polymerase chain reaction products. In none of the 65 cases a V600E mutation of the BRAF gene was found. In a subset of patients in which the entire coding sequence of BRAF was analyzed, no mutations were identified either. Mutant proteins of the RAS-RAF-MEK-ERK pathway play an important role in the pathogenesis of JMML, resulting in GM-CSF hypersensitivity. In about 75% of the JMML cases these mutations affect RAS, NF1 or PTPN11 genes. The hypothesis for this study was that BRAF might play an important role in JMML as it is an important downstream effector of RAS. Our data show that apparently BRAF mutations do not play a role in JMML. Therefore, additional analysis of genes of the RAS pathway will be necessary to identify genetic aberrations in cases without known mutations.

Description: Pediatric myeloid neoplasms are a clinically and genetically heterogeneous group of clonal diseases. Unlike in adults inherited predisposition plays a major role in leukemogenesis. A number of germline mutations are known to increase the risk of somatically occurring myeloid transformation. These include the genes associated with the classical inherited bone marrow failure syndromes, but also transcriptopathies resulting from RUNX1, CEBPA or GATA2 deficiency. Herein we review the most significant findings from recent years and our present experience on the clonal landscape of myeloid malignancies in children with a focus on pediatric MDS and JMML. In both disease entities, monosomy 7 is the most frequent chromosomal aberration. It is likely that haploinsufficiency of genes located on chromosome 7 contributes to clonal evolution. Several of the more than 1000 lost genes were postulated as mechanistic drivers. However, in most cases additional somatic mutations not related to chromosome 7 are found at diagnosis, making it difficult to pinpoint the initial oncogenic hit. Driver somatic hits frequently encountered in adults with MDS, affecting TET2, DNMT3A or the spliceosome genes, do not play a major role in the pathogenesis of childhood disease. Also, considering the young age, environmental effects and age-related pre-malignant clonal mutations can be disregarded. In fact, GATA2 germline mutations can be considered the most common "initial hit" in pediatric MDS. In our experience, GATA2 deficiency accounts for 37% of primary pediatric MDS with monosomy 7. The prevalence of GATA2 mutations in MDS with monosomy 7 increases dramatically in adolescence where they are found in up to 72%. In comparison, 16% of pediatric patients with MDS and trisomy 8 have an underlying GATA2deficiency. The somatic mutational landscape in childhood MDS is associated with cytogenetic subgroups, i.e. SETBP1 and ASXL1 mutations are overrepresented in cases with monosomy 7. Interestingly, longitudinal NGS analysis and single colony sequencing indicate that SETBP1 lesions precede the development of ASXL1 mutations. We identified the genes SETBP1, ASXL1, NRAS, KRAS, RUNX1, PTPN11 and BCOR/ BCORL as the most frequent targets of recurrent mutations in a large cohort of pediatric patients with primary MDS. The biological significance of these genomic changes is currently not understood and warrants further studies. In contrast to pediatric MDS, signaling pathways involved in JMML have been better defined during recent years. Somatic aberrations in PTPN11, NRAS, KRAS, CBL, and NF1 genes lead to a constitutively overactive RAS pathway in leukemic cells. Additional activating mutations were identified in some JMML patients, including the RAS pathway gene RRAS, and concomitant mutations in SETBP1 and JAK3. The latter were postulated to develop as subclonal events during disease progression and to confer poor prognosis. However most recently, very rare clones with SETBP1 mutation were reported to be present at diagnosis in as much as 30% of JMML patients. This might indicate that multiple clones with different clonal composition arise during initial stages of disease, and therapy-driven selection pressure results in the expansion of these subclones. It is not clear how the somatic mutations interact with the epigenetic landscape in JMML clones, especially with regard to promoter hypermethylation, and what are the definitive biological consequences. In summary, a number of germline and somatic mutations have been identified which can initiate the development of abnormal hematopoiesis in children. Premalignant stem cells undergo evolutionary selection and produce somatically altered malignant clones resulting in myeloid transformation. Disclosures No relevant conflicts of interest to declare.