ALBERT

Description: Abstract 4830 Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myeloproliferative disorder characterized by the clonal hyperproliferation of myelomonocytic cells. The pathogenesis of JMML involves disruption of signal transduction through the RAS pathway, which may be an early event during leukemogenesis. Additional genetic lesions may be necessary for full malignant transformation. We applied microarray BAC/PAC- and high resolution 244k oligo-arrayCGH to bone marrow samples from 21 JMML patients in order to identify subtle genomic alterations. In 8 of13 JMML patients with normal karyotype and 2 of 8 patients with monosomy 7, additional copy number alterations were identified. A recurrent deletion of around 1.4 Mb at 17q11.2 targeting the NF1 gene was identified in 2 patients with clinical diagnosis of neurofibromatosis due to germline mutations and LOH in the tumor as described previously. In addition to the monosomy 7, one patient (D600) with a somatic mutation in PTPN11 showed an additional marker chromosome. The origin of this marker chromosome was resolved by arrayCGH. The BAC/PAC array indicated a gain of the centromeric region: arr8p12-q11.21(37,204,000-49,686,000)×3. Breakpoints were refined to 8p12-q11.21(36,673,794- 50,142,678) by high resolution oligo-aCGH. Interestingly, in patient D703 with a heterozygous CBL germline mutation and homozygosity in hematopoietic cells, we detected a nearly identical gain arr8p12-q11.21(40,076,917-49,622,319)×3 not accompanied by -7 and with slightly different breakpoints. The gain was confirmed by means of FISH using the centromere-specific probe for chromosome 8 CEP8 showing three signals. This patient was also found to have a small additional marker chromosome. In order to rule out that the marker chromosome was inherited, we performed chromosome analyses in the parents of patient D703. Both had a normal karyotype and did not carry an extra copy of the region 8p12-q11.21. However, FISH on buccal epithelial mucosa cells of the patient D703 revealed a mosaic constellation with 34% cells positive for trisomy 8. Likewise, in the second patient, the dup 8p12-q11.21 (D600) was constitutional as 38% of fibroblasts showed 3 CEP8 FISH signals. There has been a longstanding discussion as to whether mosaic trisomy 8 may be constitutional. Our findings provide evidence that at least partial trisomy 8 may indeed be present as a germline mutation. Future work is needed to determine how constitutional partial trisomy 8 might contribute to leukemogenesis in JMML with different mutational subtypes. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic diseases characterized by impaired hematopoiesis and progression to acute myeloid leukemia (AML). Although rare, several monogenic causes of familial MDS/AML have recently been molecularly defined. We studied two families with variable manifestation of cytopenia, MDS with cytogenetic aberrations involving chromosome 7, immunodeficiency, and neurologic disease consistent with ataxia-pancytopenia syndrome. Genetic studies uncovered heterozygous missense variants (p.Arg986Cys and p.Ile891Thr) in SAMD9L, a tumor suppressor gene located on chromosome 7. Consistent with a gain-of-function effect, transfection and over-expression of both SAMD9L variants decreased cell proliferation relative to wild-type protein. In the two families, a total of 10 individuals heterozygous for either SAMD9L mutation were identified. Three individuals developed MDS, with monosomy 7 or der(1;7)(q10;p10) as cytogenetic aberrations that encompassed the mutant SAMD9L locus. In an additional five individuals, three of which experienced a spontaneously resolved cytopenic episodes in infancy, we detected mosaic copy-neutral loss of heterozygosity of 7q by uniparental disomy, with loss of the mutated allele, or mosaic cis SAMD9L mutations. By digital PCR, we identified these events in hematopoietic progenitor cell populations, which were further enriched in B and NK cell lineages. Absent in non hematological tissues, these mutations thus represented somatic revertant mosaicism. Clinically, revertant mosaicism was associated with reduced disease severity, although in two individuals neurological manifestations persisted. Of note, two unaffected carriers without revertant mosaicism harbored an additional rare in trans germline SAMD9L p.Thr233Asn missense variant. In cellular assays, the SAMD9L p.Thr233Asn variant increased proliferation, indicating a loss-of-function effect that potentially compensates for the SAMD9L p.Arg986Cys mutation. Together, our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause a disease with cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with chromosome 7 aberrations, where hematopoietic revertant mosaicism is common and ameliorates the clinical presentation. Disclosures Fioretos: Cantargia: Equity Ownership.

Description: Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22) leading to a constitutively active ABL kinase as disease driver. For tailored treatment, specific tyrosine kinase inhibitors (TKI) were developed. Still, drug resistance is frequently observed and mostly caused by point mutations in the ABL kinase domain. However, further mechanisms promoting disease progression and TKI resistance are discussed. The GA-binding protein (GABP) consists of two distinct subunits, GABPα and GABPβ. GABPα belongs to the E26 transformation-specific (ETS) transcription factor family and bears the DNA-binding domain (DBD). GABPβ contains the transcriptional activation (TAD) and the nuclear localization signal (NLS) domains. In mice, GABP is known as an important regulator in myeloid and lymphoid differentiation. Recently, its influence on murine CML-like myeloproliferative disease (MPD) was shown in independent studies (Yu et al., 2012, Cell Stem Cell; Yang et al., 2013, PNAS). Knockout of GABPα or GABPβ in BCR-ABL-transfected hematopoietic stem cells leads to prolonged survival of recipient mice. Focusing on these findings in murine models, we aimed to investigate GABP’s role in human CML. Initially, GABPA transcript expression was quantified in 70 untreated CML patients at time of diagnosis and displayed a positive and significant correlation to the BCR-ABL/ABL ratio, which is a rough estimate for the tumor cell burden in peripheral blood. In subsequent in vitro experiments, effects on imatinib response were studied following stable knockdown and ectopic overexpression of GABPA as well as overexpression of a dominant-negative GABPβ TAD deletion mutant in the BCR-ABL+ cell lines K-562 and NALM-1. As observed by enhanced apoptosis and reduced proliferation, knockdown of GABPA in K-562 cells resulted in significantly elevated imatinib sensitivity at concentrations below the determined IC50 value. In accordance with this, ectopic overexpression of GABPA led to enhanced proliferation and clonogenic capacity in comparison to empty vector controls, thus indicating a protective effect against imatinib. To study GABP’s capability to affect imatinib resistance, the GABPβ TAD deletion mutant was overexpressed in the TKI-resistant cell line NALM-1. This mutant construct lacks the transcriptional activation domain but is still capable of interacting with GABPα as confirmed by co-immunoprecipitation. Consequently, the result is a functionally impaired GABP transcription factor complex. Remarkably, overexpression of the deletion construct sensitizes NALM-1 cells to imatinib as observed in subsequent viability assays. Furthermore, we analyzed expression of two putative GABP targets with known impact on murine MPD, i.e. protein kinase D2 (PRKD2) and Rho-GTPase RAS-related C3 botulinum substrate 2 (RAC2), in K-562 cells as well as in primary CML samples. Knockdown and overexpression of GABPA in K-562 cells led to accordingly reduced or enhanced expression of PRKD2 and RAC2, respectively. In line with this, PRKD2 expression is significantly elevated in CML patients with prominent GABPA expression. In summary, our findings demonstrate that GABP plays a role in human CML and highlight the potential clinical relevance of high GABP expression possibly acting in TKI resistance mechanisms. It remains to be shown at which stage of BCR-ABL driven transformation GABP is involved and how GABP and its direct target PRKD2 are functionally embedded in BCR-ABL-triggered pro-proliferative and anti-apoptotic pathways. In this context, it is of interest whether GABP or PRKD2 may serve as alternative treatment targets for TKI-resistant CML. Disclosures No relevant conflicts of interest to declare.

Description: Background Germline mutations in the N-terminal of CCAAT/enhancer binding protein α (CEBPA) are a feature of autosomal dominant AML. Despite the strong penetrance of these mutations, the age of disease onset varies considerably and is usually precipitated by acquiring a C-terminal mutation. Although biallelic CEBPA-mutations in sporadic AML are associated with favorable clinical outcomes, little is known about long-term survival or the secondary molecular events linked with familial cases. Aims We sought to establish the long term clinical outcomes in familial CEBPA-mutated AML and to examine the patterns of secondary mutations associated with leukemic transformation. Methods and Results Disease specific and follow up information was collected in 16 affected patients, from 7 pedigrees, published between 2004 and 2011. In 94% of patients (n=15), at least 3 years follow up was achieved. All pedigrees had a germline N-terminal CEBPA mutation and 17 of 18 documented disease episodes had an acquired C-terminal mutation. The age at AML diagnosis varied from 2-39 years (median 24.5 yrs) with a single asymptomatic carrier detected (now 23 yrs). With the exception of 1 patient diagnosed in 1963, all cases received combination chemotherapy at diagnosis. Additional consolidation comprised autologous stem cell transplantation (SCT, n=3) and allogeneic SCT in 1 patient failing to achieve CR post induction therapy. Ten patients had at least 1 further disease episode, the first recurrence presenting after a median of 2.1 years (range 0.5-14 yrs), 5 continued in CR and 1 patient was lost to follow up. In 3 out of 4 patients, where CEBPA was screened at recurrence, the acquired C-terminal mutations differed from diagnosis, signifying new episodes of AML. The median overall survival (OS) for the entire cohort was 20 years (1.1-46 yrs, n=16) and 17.3 years for patients with multiple disease episodes, reflecting durable responses to second line therapies. To identify potential co-operating mutations in CEBPA pedigrees, whole exome sequencing (WES) was performed in 7 tumor samples from 5 patients across 3 pedigrees, all with the germline mutation p.P23fs (Figure 1). All tumor DNA samples were sequenced with matched remission or skin DNA as a germline control. The number of acquired mutations in familial tumors was similar to sporadic disease, with 10-22 (median=14) non-synonymous tier 1 mutations per sample. In addition to the acquired C-terminal CEBPA mutation, these included established AML loci such as EZH2, TET2, WT1, GATA2, NRAS, CSF3R and the recently identified cohesin complex gene, SMC3. Of note, FLT3-ITD, NPM1 and DNMT3A mutations were absent in all tumors. A minimum of 2 established mutations were identified in each tumor and, at present, we can only speculate on which additional mutations are ‘driver' or ‘passenger' events. Reflecting findings in sporadic AML, biallelic CEBPA and GATA2 mutations co-occurred in both siblings from Pedigree 1 and were subsequently identified by Sanger sequencing in the child III.2 (Figure 1). All 3 patients continue in long term remission following chemotherapy. We were able to trace the clonal evolution in patient I.2 (Pedigree 3) by WES of 3 consecutive tumor samples which arose over a 17 year period. At diagnosis (Dx) the patient received induction and consolidation chemotherapy and remained disease free for 14 years. The second disease episode (R1) was treated with chemotherapy followed by autologous SCT and the third presentation (R2) was chemo-refractory. Tumor DNA from R2 was clonally related to Dx, sharing 7 identical mutations, including the original C-terminal CEBPA deletion. In contrast, R1 appeared molecularly distinct from Dx and R2, most likely representing a new clone which was subsequently eradicated with treatment. Conclusion This is the first report of long term clinical outcomes in familial CEBPA-mutated AML. Although many patients experienced disease recurrence, our extended follow up showed that OS remained favorable despite multiple episodes of disease. Assessment of C-terminal CEBPA mutations provided a unique insight into the recurrence of AML, with some patients appearing to develop completely new leukemic episodes. Although the penetrance of germline mutations is high, healthy carriers and late onset disease are noted, emphasizing the need for clinical vigilance and screening of all related potential SCT donors. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Autosomal-dominant SAMD9L gain-of-function mutations predispose to myeloid malignancies involving chromosome 7 aberrations. Hematopoietic reversions frequently occur postnatally and are associated with milder disease manifestations.

Description: RUNX1-associated familial leukemia is a rare hematologic malignancy. It is inherited in an autosomal dominant manner and mainly causes myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Since constitutional mutations in RUNX1 can lead to thrombocytopenia or dysfunctional platelets, the disease is termed familial platelet disorder with associated myeloid malignancies (FPDMM, MIM 601399). Here, we report two novel families with early onset MDS illustrating the variability of the disorder. The index patient (P1) of family 1 (Fig. 1A) was known to have asymptomatic thrombocytopenia (90 G/l) since his neonatal period. Platelet assays revealed impaired in vitro aggregation. Asymptomatic thrombocytopenia was also known in his younger sister, his mother and his maternal grandfather. A maternal uncle had died from leukemia at age 4. While P1 was treated with antibiotics for a middle ear infection, he developed petechiae and hematoma. He was found to have a platelet count of 11 G/l with a normal white blood count (WBC), hemoglobin concentration (Hb) and MCV. Bone marrow investigations led to the diagnosis of MDS with excess blasts (MDS-EB) with normal karyotype. Sequencing of RUNX1 identified a heterozygous truncating RUNX1 mutation in P1 that was also present in his mother: c.90_117dup; p.(Leu40AlafsX80). P1 underwent hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD) following a preparative regimen consisting of busulfan, cyclophosphamide and melphalan (BuCyMel). He is alive and well 4 years post HSCT. Two years after his diagnosis, a 17-year-old maternal uncle of P1 (P2) was also diagnosed with MDS-EB with normal karyotype. Successively, the disorder progressed to AML and karyotyping showed a clonal derivative chromosome 7 due to an unbalanced translocation der(7)t(2;7)(p12;q21), and later on, a clonal evolution with trisomy 12. RUNX1 sequencing indicated that P2 also carries the familial RUNX1 mutation. Finally, the causative RUNX1 mutation was also identified in the younger sister and two further maternal uncles, all showing mild, asymptomatic thrombocytopenia without evidence of malignant transformation. In the second family (Fig. 1B), the male index patient (P3) had congenital, asymptomatic thrombocytopenia with subsequent platelet counts of 60-80 G/l. At age 3.5, platelet count declined over several months reaching values of 3-20 G/l. WBC, Hb and MCV were normal. The patient had recurrent nose bleeds and prolonged bleeding after cuts and blood drawing. Diagnostic bone marrow examination showed MDS-EB with normal karyotype. Since, thrombocytopenia was also known in P3's mother, RUNX1 was sequenced and showed a typical heterozygous truncating mutation in P3 and his mother: c.415C〉T; p.(Arg139X). The mutation was absent in P3's brother. At age 16, a maternal aunt of P3 had died due to a cervical cancer. Her DNA was not available for testing. P3 received a MUD HSCT following a BuCyMel preparative regimen and is alive and well 2 years post HSCT. These two new pedigrees with germline RUNX1 mutation emphasize a few interesting points. For both index patients the history of familial thrombocytopenia had been known since birth, but genetic work-up was only initiated after the platelet count had rapidly decreased and bleeding had become obvious. At the time of genetic confirmation, MDS-EB had to be diagnosed despite normal values and blood morphology for white cells and red cells. Thus, decreasing platelet counts in patients with known RUNX1 germline mutations or long-standing thrombocytopenias of unknown cause should result in prompt bone marrow examination, even in the absence of other signs or symptoms suspicious of hematopoietic neoplasia. Furthermore, the platelet count might be a suitable screening instrument in surveillance of patients known to carry germline RUNX1 mutations. Although both index patients had been successfully transplanted following a standard myeloablative regimen, earlier diagnosis of MDS might have saved toxicity and late effects. With our current knowledge of myeloid neoplasms with germline predisposition and pre-existing platelet disorders, children with thrombocytopenia and a family history should have complete genetic work-up for currently known alterations. Consensus guidelines need to be developed by specialists in hematology/oncology, coagulation, general pediatrics and human genetics. Figure 1 Pedigrees Figure 1. Pedigrees Disclosures No relevant conflicts of interest to declare.