ALBERT

Description: Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY. Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones. Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and

Description: Introduction: Alterations of TP53 (cytogenetic 17p13.1 deletions and molecular TP53 mutations) were reported to be frequent in pts with myelodysplastic syndromes (MDS) and complex abnormalities (≥3 clonal cytogenetic aberrations, CA) that represent around 15% of all MDS cases. It was suggested that pts with MDS and complex abnormalities may be further prognostically subdivided by the molecular TP53 mutation status (Bejar et al, ASH, 2014, abstract #532). In this study we investigated the frequency of different types of TP53 alterations, their cytogenetic profile and their clinical impact in the adverse cytogenetic MDS subgroup of complex abnormalities. We performed comprehensive cytogenetic and molecular genetic analysis focusing as well on the extent of cytogenetic instability. Methods: We included 105 pts (57 m/48 f; median 71 yrs, range, 47-95 yrs) with MDS (n=86) and sAML after MDS (n=19) with complex abnormalities in our study. A total of 56/89 (62.9%) pts had received azacitidine. Survival was censored at allogeneic stem cell transplantation (26/83; 31.3%). Pts were characterized by chromosome banding analysis, interphasefluorescence in situ hybridization (FISH) with a panel including a 17p13/TP53-covering probe, multicolor FISH (mFISH), Sanger sequencing of TP53 and SNP-array analysis (SNP-A). The extent of genetic imbalances was objectified by counting the number of CA, the number of cytogenetic fusions as shown by mFISH and the size of total genomic aberrations (TGA) measured by SNP-A in megabases (Mb). Results: A molecular TP53 mutation was found in 46/105 (43.8%) pts; a cytogenetic TP53 deletion in 38/105 (36.2%) pts. TP53 was not affected by a molecular mutation or a cytogenetic deletion in 44/105 (42.2%) pts, 23/105 (21.9%) pts were affected by combined TP53 alterations (molecular mutation and cytogenetic deletion), 23/105 (21.9%) pts by a molecular mutation only and 15/105 (14.3%) pts by a cytogenetic deletion only. The median number of CA was 6 (range, 3-41) in the entire cohort. Median overall survival for the entire cohort was 17 months. The degree of genomic imbalances was higher in pts with any TP53 alteration (molecular mutation and/or cytogenetic deletion) as compared to those without: The median number of CA was 8 (range, 3-41) vs. 4 (3-20) (P

Description: Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow (BM) characterized by a dysfunction of hematopoiesis commonly resulting in cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML) development. In up to 90% of MDS cases acquired somatic mutations can be identified. Additionally, these aberrations constitute important markers for diagnosis and risk stratification. Currently, the use of BM aspirates is the gold standard for cytogenetic and molecular genetic analysis. However, frequent analyses of peripheral blood (PB) samples are of special interest for monitoring the natural course and therapy response in MDS since this might not be feasible with BM specimens due to ethical and other reasons. The aim of this study was to investigate whether the high sensitivity of targeted deep sequencing (TDS) allows reliable detection of somatic variants also from PB samples and whether the molecular profiles are comparable between these different sources of material. Additionally robustness, feasibility and comparability of the method were verified by inter-laboratory comparisons. This study included 31 patients from two centers, in Barcelona (Spain) and Göttingen (Germany) (12x RCMD, 4x RAEB-2, 4x sAML, 4x CMML-1, 2x MDS-RA, 1x MDS-U, 1x RAEB-1, 1x RCMD-SA, 1x RARS, 1x CCUS). For all patients, genomic DNA was extracted from concurrent mononuclear or total bone marrow cells (BMC), mononuclear peripheral blood cells (PBMC) and immunomagnetically (MACS) enriched circulating CD34+ cells (CD34+). Library preparation was performed using a custom hybridization-probe based panel (Nimblegen SeqCap EZ, Roche) including 83 myeloid-related genes (Barcelona) or the TruSight Myeloid Sequencing-Panel (Illumina) including 53 target genes (Göttingen). Sequencing was performed on MiSeq instruments. Reads were analyzed using local bioinformatic pipelines. Variants were filtered according to read depth (〉 100x), population frequency (〈 1%), their impact on protein integrity or function and evaluated by visualization on the Integrative Genome Viewer Software. Somatic origin of the variants was confirmed by sequencing of control samples from MACS enriched circulating T-lymphocytes (CD3+). Somatic mutations were discovered in 29 of the 31 analyzed patients. Overall we identified 76 aberrations in 22 genes (12x TET2, 7x SF3B1, 7x SRSF2, 5x EZH2, 5x ASXL1, 5x U2AF1, 5x RUNX1, 4x ZRSR2, 4x TP53, 3x NRAS, 3x STAG2, 2x DNMT3A, 2x IDH1, 2x BCOR, 2x KRAS, 2x PTPN11, 1x PDGFRB, 1x IKZF1, 1x IDH2, 1x ATRX, 1x CSNK1A1, 1x ETV6). Literally all variants were found in BMC (n=74) as well as in circulating CD34+ cells (n=72) and PBMC (n=73). The discordance between the three sample types was random (5x RCMD, 2x sAML, 1x MDS-RA, 1x RARS). However, for five variants the allele frequency (VAF) values, which correlate with the clone size of the malignant cell population, were below 5% in PBMC samples. Therefore these variants were likely to be overlooked in case only PBMC would have been tested. There was no significant difference (p=0.774) between the VAF values measured in BMC (average: 40.0%) and enriched CD34+ cells (average: 41.3%). In contrast VAF values of PBMC (average: 30.1%) deviate significantly from both, BMC (p=0.007) as well as circulating CD34+ cells (p=0.027) (Figure 1). Our findings indicate that TDS enables the adequate detection of somatic mutations from BM, circulating CD34+ cells and for the most part also in PBMC preparations. However, the malignant cell population seems to be less abundant in the PBMC fraction and therefore the detection and especially the quantification of clonal somatic variations is more challenging in this sample type. In the present study we demonstrate that enrichment of circulating CD34+ cells from PB can overcome this problem and provide data that are equivalent to the results obtained from the analysis of BMC, especially for follow up or minimal residual disease analyses. We conclude that enrichment of CD34+ cells from PB constitutes an appropriate alternative for the reliable detection and quantification of somatic aberrations in MDS patients. Usage of circulating CD34+ cells in routine diagnostic next generation sequencing applications would significantly reduce invasive clinical interventions and could therefore improve diagnostics and disease monitoring. Support: PI 11/02010, PI/14/00013, RD12/0036/0044, AR 14/34, R14/03, SGR225 Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Complex (≥3) abnormalities (cA) are associated with an inferior outcome in myelodysplastic syndromes (MDS). About 50% of MDS with cA show mutations in TP53 that might contribute to the formation of the cA and worsen prognosis (Haase et al., Leukemia, 2019). In former single nucleotid polymorphism (SNP) analysis we found chromosome 17q being affected in several patients with cA with a higher incidence as by chance. In just this region is a gene called PPM1D located which already has been observed as one of the most frequently mutated genes in pts./individuals with clonal hematopoiesis with indetermined significance (CHIP). PPM1D is encoding for a protein named Wip1. This protein acts as an inhibitor of p53. About 5% of MDS with 5q deletions show mutations in PPM1D (Panagiota et al., ASH 2017). Mutations in PPM1D are even more common among pts with therapy-related MDS (15%, Lindsley et al., 2017). The aim of our study was to determine the frequency of PPM1D mutations in MDS with cA and to shed light upon their possible contribution to the formation of cA. Methods and patients: We included 100 patients characterized by conventional cytogenetics in our analysis (67x MDS; 30x secondary acute myeloid leukemia, AML; 3x chronic myelomonocytic leukemia, CMML). 20 pts had a therapy-related MDS. All the included pts had cA with a median number of aberrations of 8 (range: 3-50). The median age at first diagnosis of MDS with cA was 72 (range 29-95). A deletion of 5q was found in 71 patients (71%). The TP53 status was known for all pts by fluorescence in situ hybridization (FISH) and/or molecular karyotyping (TP53 deletion status) and sequencing (TP53 mutation status). 68 of 100 pts had an alteration on TP53 (68%, 4 deletions, 34 mutations, 30 biallelic changes). All pts were subjected to next generation sequencing of PPM1D. Amplicons for exons 1 to 6 were generated by multiplex polymerase chain reaction (PCR). The pooled amplicons were processed using the Nextera XT2 sample preparation kit (Illumina, San Diego, Ca, USA) followed by sequencing on a MiniSeq platform (Illumina, San Diego, Ca, USA). We used our local bioinformatics pipeline to identify single-nucleotide variants (SNVs) and indels. Results: In ten pts (10%) we found single-nucleotide variants of PPM1D. The median number of aberrations was 8 (range: 5-15). Six of those PPM1D variants have already been described as very rare SNPs. Three of them were located in the 3'UTR (untranslated region), the other three seem to be silent mutations. The other four are not listed in common databases. Three of those four are potential missense mutations, one is a potential nonsense mutation. Two variants are located at the same -previously undescribed- position (c.230A〉C, p.D77A). Two of those four patients showed an additional TP53 mutation, one of them biallelic. A deletion of 5q was identified in two of them. One pt had therapy related MDS. At a clone size of the complex karyotype of 94% and 90%, the VAF of three of the recurrent mutation was just 7% and 8%, indicating that the PPM1D mutation arised in a subclone in these pts. In one pt the VAF was 33,6%. The VAF of 30-38% in the other cases implies PPM1D being an ancestral or co-dominant mutation. Conclusion: We were able to show that PPM1D is mutated in MDS with cA in a relevant fraction of pts. In our cohort, 10% of MDS pts with cA are affected. 4% may have a deleterious mutation of PPM1D. Although PPM1D mutations were described to preferentially occur in therapy related diseases (Lindsley et al., 2017), in our cohort three of four patients with potential PPM1D mutation had no known prior chemo-/radiation therapy. Mutations in PPM1D might contribute to the formation or toleration of cA alternatively to TP53 mutations as two of four patients with PPM1D mutations did not show TP53 mutations and the PPM1D mutations could be the ancestral or co-dominant mutation in two of four cases. Our data imply that also mutations in PPM1D may be important for prognosis and therapy decisions in MDS patients with cA. We will continue observing our patients in order to enlarge the database and to find out which impact mutations in PPM1D may have on overall survival and whether they can affect the prognosis of patients with cA. Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria. Hertenstein:RS Media: Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.