ALBERT

Description: Background: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by somatic mutations in peripheral blood mononuclear cells (PBMC) of otherwise healthy adults that has been associated with increased risk of developing hematological malignancies. Clonal hematopoiesis has been shown to be present in patients with therapy-related myeloid neoplasms (therapy-related acute myeloid leukemia, t-AML) / therapy-related myelodysplastic syndrome, t-MDS) at the time of their primary cancer diagnosis and before exposure to treatment. Such clones expand under selective pressure from cytotoxic treatment for the primary cancer and can subsequently give rise to overt myeloid neoplasms. Somatic mutations in the gene encoding the TP53-inducible protein phosphatase Mg2+/Mn2+ 1D (PPM1D) were initially reported in PBMC of patients with solid tumors (breast, ovary, lung) and lymphoma. They are associated with older age and seem to reflect prior exposure to cytotoxic treatment. Moreover, the mutations, all of which are nonsense or frameshift mutations in exon 6, have been described as one of the most recurrent mutations in CHIP and to be frequent in t-MDS. Methods and results: To resolve this issue and to determine whether clones harboring PPM1D mutations that expand into CHIP after cytotoxic therapy for solid tumors drive leukemogenesis and might be useful as markers to identify patients at risk for t-MDS/t-AML development, we performed PPM1D mutational analysis in 87 patients with de novo AML and in 49 patients with t-AML. As mutations in TP53 as a representative DNA damage response gene are rare in de novo AML, we enriched our de novo AML patient cohort towards poor risk cases with complex karyotypes in order to increase the chance of identifying PPM1D mutations. Patients with core-binding factor AML were excluded from the analysis. Using the published frequency of 15% PPM1D mutations in t-MDS (Lindsley RC et al., N Engl J Med 2017;376(6):536-547) as surrogate for the expected frequency in t-AML, a minimum of 44 t-AML patients was determined to be required to allow for the detection of mutations of PPM1D in t-AML (Chi square with Fisher's exact test for independent groups, α-error 0.05, power 0.8). We performed focused mutational analysis by targeted Sanger sequencing of PPM1D exon 6 on DNA from bone marrow mononuclear cells or PBMC at diagnosis of de novo or t-AML samples taken prior to treatment initiation. Overall, only one patient with de novo AML (1/87, 1.2%) proved mutation positive. He was diagnosed with AML, FAB M4, at the age of 57 years and harbored a complex karyotype with marker chromosomes in the absence of a TP53 mutation. Unexpectedly, none of the 49 patients with t-AML harbored a mutation in PPM1D. Conclusion: In this study, we found that PPM1D mutations, which frequently occur in CHIP especially following prior cytotoxic therapy, are uncommon in AML, whether de novo or after prior cytotoxic therapy. These data are in contrast to previous observations on a high frequency of PPM1D mutations in t-MDS samples relative to primary MDS (15% vs. 3%, Lindsley RC et al., N Engl J Med 2017;376(6):536-547). Our findings suggest that while cytotoxic therapy favors the expansion of PPM1D-mutant CHIP clones, possibly even up to the development of t-MDS, mutations in PPM1D seem to be irrelevant for progression to t-AML. Disclosures Stoelzel: Neovii: Speakers Bureau. Rollig:Bayer: Research Funding; Janssen: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership.

Description: Abstract 855 The cAMP signaling pathway has emerged as a key regulator of hematopoietic cell proliferation, differentiation, and apoptosis. Signal specificity is achieved through local activation of signaling enzymes that are anchored to subcellular organelles and membranes. In particular, A-kinase anchoring proteins (AKAPs) coordinate and control cAMP responsive events. AKAPs were originally classified based on their ability to bind cAMP-dependent protein kinase (protein kinase A; PKA). The activity of PKA is regulated by its two regulatory subunits, which from a dimer that binds to the two catalytic subunits. Binding of cAMP to the regulatory dimer dissociates the catalytic subunits and activates PKA. Anchoring of PKA by AKAPs constrains PKA activity to a relevant subset of potential substrates. Thus, AKAPs contribute to the precision of intracellular signaling events by directing anchored enzyme pools to a subset of their physiological substrates at specific subcellular localizations. Using an in vitro short hairpin RNA (shRNA) screen against potentially druggable targets, we have uncovered a requirement for AKAP12 in the proliferation of a cultured pre-B cell leukemia cell line. In the hematopoietic system of mice and humans, expression of AKAP12 is tightly restricted to the pro/pre/immature stages of B lymphopoiesis, suggesting a potential role in pre-B cell receptor (pre-BCR) or BCR signaling. We find that retroviral knockdown or germline knockout of AKAP12 in mice leads to an increase in pre B and immature B cells in the bone marrow. In contrast, B cell numbers in the spleen are significantly reduced, as are recirculating B cells in the bone marrow. Transplantation of AKAP12 null hematopoietic stem and progenitor cells from fetal liver into wildtype recipients demonstrates an autonomous defect in the development of AKAP12−/− B cells. Competitive bone marrow transplantations confirm that this defect is cell autonomous and not due to a defective bone marrow environment or secretion of a B cell inhibitory factor. To identify AKAP12 interaction partners, we overexpressed FLAG-epitope tagged AKAP12 in a pre-B cell leukemia cell line. Affinity purification of AKAP12 showed a repeated co-immunoprecipitation of poorly characterized RIO kinase 1 (RIOK1). Our current efforts are focused on investigating the interaction between RIOK1 and AKAP12 and their role in the control of B cell development and cell cycle progression. Further, we are focusing on a likely role for AKAP12 in the scaffolding of PKA, PKC and phosphodiesterases by analyzing the activation of signaling cascades in cultured primary wildtype and AKAP12−/− pre B cells. Additionally, we are investigating the role of the BCR in vivo by testing if enforced expression of BCR components rescue B cell development in a AKAP12−/− BCR transgenic mouse model (SWHEL mouse). In summary, we have confirmed a novel role for AKAP12 in B lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome. Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p

Description: Key Points Marker chromosomes are frequently found in AML, particularly among aneuploid adverse-risk karyotypes and confer a poor prognosis. About one-third of marker and ring chromosome karyotypes arise from chromothripsis.

Description: This study compares the macrofaunal communities along two bathymetric transects (1000 – 2500 m water depth) in predominantly ice-covered western (offshore Greenland) and generally ice-free eastern (offshore Svalbard) regions of the Fram Strait. Material was collected using an USNEL 0.25 m2 box corer and all sediment samples were processed through a 500-μm sieve. A total of 1671 organisms from 169 species were found. Densities off Greenland were generally lower than those observed off Svalbard. On both sides of the Fram Strait, density, biomass and biodiversity generally decreased with increasing water depth. An exception was observed at the deepest station off Greenland (2500 m water depth), which was located within the Marginal Ice Zone. At this station, macrofaunal density was elevated (992 ± 281 ind. m−2) compared to the adjacent shallower sampling areas off Greenland (272 ± 208 ind. m−2 to 787 ± 172 ind. m−2) and the deeper stations (2000 and 2500 m water depth) off Svalbard (552 ± 155 ind. m−2 and 756 ± 182 ind. m−2). The most abundant species along both transects was the polychaete Galathowenia fragilis (off Greenland: 288 ind. m−2, off Svalbard: 740 ind. m−2). Sea ice coverage and water depth, as well as the associated food availability at the seafloor, seem to be crucial factors driving the macrofaunal community patterns. A strong pelago-benthic coupling is observed to be typical in Arctic deep-sea ecosystems, and is also confirmed by our study.

Description: While numerous regional studies of bathymetric zonation of benthic fauna globally have been done, few large-scale analyses exist, and no ocean-scale studies have focused on the Arctic Ocean to date. In the present work we, hence, examined bathymetric zonation of macro- and megabenthos over a depth range spanning from the shelf to the abyssal plain (14 – 5416 m) and regionally extending from the Fram Strait to the Beaufort Sea (as a whole hereafter called the Central Arctic). Based on 104 quantitative (box-corers and grabs) and 37 semi- quantitative (trawls) samples compiled from different studies we evaluated bathymetric zonation patterns in abundance, biomass and diversity, and also compared species composition among samples. Abundance and biomass decreased with depth from 〉3000 ind. m-2 and 〉40 g ww m-2 to ∼130 ind. m-2 and 〈1 g ww m-2 corroborating previous studies. Diversity showed a parabolic pattern, peaking at ∼100-600 m. Cluster analysis revealed four (macrofauna) and five (megafauna) groups of benthic assemblages, including three that covered the upper and lower continental slope and the abyssal plains with relatively little overlap (named the Lower Shelf – Upper Slope 1, the Lower Slope and the Abyss). Substantial changes in benthic community composition were observed at depths 650-950 m (between the Lower Shelf – Upper Slope 1 and the Lower Slope) and 2600-3000 m (between the Lower Slope and the Abyss), so we interpreted these two depth horizons as major bathymetric boundaries. The first boundary (650-950 m) corresponds to the transition from sublittoral to bathyal fauna consistent with previous studies. The second boundary (2600-3000 m) reflects a decrease in benthic abundance, biomass and diversity within the Central Arctic abyssal plain. Bathymetric patterns and species overturn of benthos were relatively uniform throughout the entire Central Arctic continental slope and abyssal plain. For some regions of the Arctic Ocean, foremost for the area north from Greenland and Canadian Archipelago, benthic data are still unavailable and further research is needed.