Abstract: The EUROPE phase 2 trial investigated the predictive value of biomarkers on the clinical efficacy of single agent romiplostim (ROM) treatment in patients with lower-risk myelodysplastic neoplasms (LR-MDS) and thrombocytopenia within the ‘European Myelodysplastic Neoplasms Cooperative Group‘ (EMSCO) network. A total of 77 patients with LR-MDS and a median platelet count of 25/nl were included, all patients received ROM at a starting dose of 750 μg by SC injection weekly. Thirty-two patients (42%) achieved a hematologic improvement of platelets (HI-P) with a median duration of 340 days. Neutrophil (HI-N) and erythroid (HI-E) responses were observed in three (4%) and seven (9%) patients, respectively. We could not confirm previous reports that HI-P correlated with baseline endogenous thrombopoietin levels and platelet transfusion history, but SRSF2 mutation status and hemoglobin levels at baseline were significantly linked to HI-P. Sequential analysis of variant allelic frequency of mutations like SRSF2 did not reveal an impact of ROM on clonal evolution in both responders and non-responders. In summary, our study confirms the safety and efficacy of ROM in LR-MDS patients and may allow to better define subgroups of patients with a high likelihood of response.

Abstract: The Retina.net ROP registry documents data of preterm infants developing stages of retinopathy of prematurity (ROP) that need ROP treatment. The aim of this analysis was to investigate data regarding epidemiology, therapy and changes over time (15 years) in a single participating center (Hannover Medical School, MHH). Methods Analysis of data of infants treated for ROP at a single center over time (birth 2001–2016, ROP treatment in 2002–2017). Results Overall, 65 infants were treated (23 female). In 11 infants (16.9%) ROP screening was conducted externally and infants were transferred to the MHH for ROP treatment. Between 2006 and 2016, incidence of ROP requiring treatment among infants screened for the development of ROP was 4.1%. Mean gestational age was 25.7 weeks (standard deviation, SD 1.8), mean birth weight 763 g (SD 235), postmenstrual age at treatment 38.2 weeks (SD 3.2), postnatal age 12.4 weeks (SD 3.2). There was no significant change in parameters over time. ROP zone II, stage 3+ was most frequently treated (57 eyes of 31 infants). 58 infants were treated with laser (114 eyes), 7 infants were treated with anti-VEGF (bevacizumab, bilateral, 14 eyes) from 2014 onwards. Retreatment due to recurrence of ROP was necessary in one infant after initial laser coagulation. Infants with ROP requiring treatment often presented with neonatal comorbidities, ventilation in more than 90%, bronchopulmonary dysplasia, and received transfusions. Conclusion This is the first monocentric analysis over 15 years originating from the Retina.net ROP registry. In this cohort we see a change in ROP therapy from laser coagulation to anti-VEGF (bevacizumab) from 2014 onwards, demographic data and treatment parameters remained relatively stable over time.

Abstract: *UP, LA contributed equally Introduction A significant proportion of lower risk (LR)-MDS patients present with thrombocytopenia, being associated with shortened survival and higher risk of progression to acute myeloid leukemia (AML). Treatment options for patients with LR-MDS and severe thrombocytopenia remain limited apart from transfusion support. Romiplostim (ROM), a thrombopoietin receptor agonist (TPO-RA) has shown safety and efficacy dependent on endogenous TPO levels as well as platelet transfusion history in a poorly defined subset of LR-MDS patients (Giagounidis et al. Cancer 2014, Sekeres et al. BJH 2014). Methods The multicenter phase 2 EUROPE trial investigated potential biomarkers of response (e.g. TPO levels, molecular markers) to single agent ROM in LR-MDS patients with severe thrombocytopenia. Patients were eligible if platelet counts were ≤30 G/L or ≤50 G/L in case of bleeding history. The primary efficacy endpoint was the rate of hematologic improvement of platelets (HI-P, according to IWG 2006 criteria) lasting for at least 8 weeks after 16 weeks of ROM (750µg SC qw) treatment. At the time of screening, patients were assigned into 3 different cohorts based on their previous platelet transfusion events (PTE) as well as centrally assessed TPO serum levels (A: TPO & lt;500 ng/l, PTE & lt;6 units/past year; B: TPO & lt;500 ng/l, PTE≥6 units or TPO≥500 ng/l, PTE & lt;6 units, C: TPO≥ 500 ng/l, PTE≥6 units). Bone marrows analysis were centrally reviewed. Results From 2015 to 2019, a total of 79 patients were included at 29 trial sites in Germany, France and the Czech Republic. Patients' median age was 74 years (range 42-93), median baseline platelet count was 25.5 G/L (range 3-50 G/L) and they were stratified into cohort A (n=51) or B+C (n=28), respectively. The primary endpoint was met with 34 out of 79 (43%) patients responding (HI-P), with response being markedly higher in cohort A (49%, n=25) vs. cohort B and C (32%, n=9) (p=0.145). Ten (13%) and eight (10%) patients had additional neutrophil (HI-N) and erythroid (HI-E) responses, respectively. During treatment, six patients had transient increases in peripheral blasts to more than 10% and one patient progressed to AML after one month of ROM. Although a higher number of responders was observed in group A, neither TPO level at screening (p=0.21), nor number of pretreatment PTE (p=0.12) were significantly associated with response to ROM treatment. Thus, our findings do not confirm that baseline TPO levels and number of pretreatment PTE alone allow reliable prediction of response to ROM. With the aim to identify new molecular patterns correlating with response, we performed a targeted NGS analysis for somatic variants in 54 candidate genes in 75 patients at baseline and in 44 patients after 16 weeks of ROM. Responders (R) more frequently exhibited mutations like SRSF2 (R=39%, NR=17%), RUNX1 (R=24%, NR=14%) and TET2 (R=30%, NR=29%), whereas non-responders (NR) exhibited mutations like DNMT3A (R=12%, NR=21%), U2AF1(R=9%, NR=14%) or ASXL1 (R=6%, NR 17%) more frequent. The percentages of patients with a response to ROM were similar regardless of total number of baseline somatic mutations. Comparing responders vs. non-responders, we found no significant changes of variant allelic burden of variants detected pre- and post-ROM (Fig. 1). We identified the presence of a SRSF2 mutation as a significant predictor of response to ROM treatment (p=0.031, logistic regression). Mutated SRSF2 was significantly more frequent in responders (39%) compared to non-responders (17%) (p=0.036, Fisher's exact test) (Fig. 2A,B). We used logistic regression with stepwise backward selection to assess the influence of the presence of ASXL1, DNMT3A, RUNX1, TET2 and SRSF2 mutations on response. Our final regression model excludes the non-significant ASXL1, DNMT3A, RUNX1 and TET2 mutations and includes the significant SRSF2 mutation, resulting in an overall accuracy of 64.0% for a correct ROM response prediction in this patient cohort. Conclusion: This prospective study did not confirm a significant association between response to ROM, pretreatment PTE burden and endogenous TPO levels. Instead, patients with a mutated SRSF2 displayed a significantly higher response to ROM treatment. This may allow personalized treatment approaches in patients with LR-MDS and severe thrombocytopenia. In this study, extended treatment with ROM did not lead to a significant increase in AML cases. Disclosures Kubasch: Shire: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Giagounidis:AMGEN: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Research Funding. Cony-Makhoul:Novartis: Consultancy; Pfizer: Consultancy; Incyte Biosciences: Speakers Bureau; BMS: Consultancy; BMS: Speakers Bureau. Laribi:takeda: Research Funding; novartis: Honoraria, Research Funding; amgen: Research Funding; abbvie: Honoraria, Research Funding. Park:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Other: Travel expenses. Metzeler:Astellas: Honoraria; Otsuka Pharma: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria. Thiede:AgenDix GmbH: Other: Co-owner and CEO. Schlenk:PharmaMar: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Roche: Research Funding; AstraZeneca: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Fenaux:Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Ades:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; novartis: Research Funding; Celgene/BMS: Research Funding; jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP). Limitations of Use: Romiplostim is not indicated for the treatment of thrombocytopenia due to myelodysplastic syndrome (MDS) or any cause of thrombocytopenia other than chronic ITP.

Abstract: Introduction: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and peripheral cytopenia. In about half of patients with lower-risk (LR) MDS, thrombocytopenia is present at the time of diagnosis and associated with shortened survival and an increased risk of progression to acute myeloid leukemia (AML). The thrombopoietin receptor agonist (TPO-RA) romiplostim has shown safety and marked efficacy in a still poorly-defined subset of LR-MDS patients with thrombocytopenia. Methods: The EUROPE multicenter phase 2 trial within the EMSCO network investigated the impact of biomarkers like endogenous thrombopoietin (TPO) level and platelet transfusion events (PTE) on the efficacy of romiplostim (750µg SC qw) treatment in patients with LR-MDS (IPSS low/int-1). Patients were eligible if baseline bone marrow blast count was 〈 5% as assessed by central morphology and platelet counts were ≤30 Gpt/L or ≤50 Gpt/L in case of bleeding history. According to a previously published model of response to TPO-RA (Sekeres at al. BJH 2014), patients were assigned into 3 different cohorts at the time of screening based on their previous PTE as well as centrally assessed TPO serum levels (cohort A: TPO 〈 500 ng/l, PTE 〈 6 units/past year; cohort B: TPO 〈 500 ng/l, PTE≥6 units or TPO≥500 ng/l, PTE 〈 6 units, cohort C: TPO≥500 ng/l, PTE≥6 units). Primary endpoint of the study was the rate of hematologic improvement of platelets (HI-P) according to IWG 2006 criteria after 16 weeks of romiplostim treatment. We here present the analysis for the first 16 weeks of romiplostim treatment. Results: From 2015 to 2018, a total of 68 patients were included in 20 trial sites in Germany, France and Czech Republic. Patients displayed a median age of 74 years and a median platelet count of 25 G/L (range 1-50 G/L) and were stratified into cohort A (n=47), B (n=17) or C (n=4), respectively. All patients received at least one cycle of romiplostim with a median weekly dose of 750μg and a median of 15 cycles of romiplostim until week 16. Reasons for premature study discontinuation before week 16 were investigator/patient decision (n=8), adverse events (n=5), disease progression (n=4) or death (n=1). There were 9 reported severe treatment-related adverse events in seven patients including pulmonary embolism (n=1), subacute stroke (n=1), mucocutaneous hemorrhage (n=1), asthenia (n=1), suspicion of anti-romiplostim antibodies (n=1), progression to AML (n=1) and varicella zoster infection (n=1). Two patients had transient increases in peripheral blasts to more than 10% and 1 patient progressed to AML after 1 month of treatment. HI-P was observed in 26 of 68 (38%) patients, while response was ongoing in 24 of them beyond week 16. Moreover, rate of HI-P lasting for at least 8 weeks was notably higher in cohort A (45%, n=21/47) compared to patients in cohort B and C (24%, n=5/21) (p=0.11). Median peak increase of PLT count in responding patients was 199 G/L in cohort A and 83 G/L in cohort B (p=0.25) and was observed in median after 7 weeks (range 3-16). In addition, responses occurred also in 2 patients in the neutrophil (HI-N) and in 7 patients in the erythroid (HI-E) lineage according to IWG 2006 criteria (Table 1). Explorative analysis showed a correlation between pretreatment platelet transfusion requirement and endogenous TPO-levels (spearman-test, p=0.034). Median pretreatment endogenous TPO-level was lower in responders compared to non-responders (82 vs. 103 pg/ml, p=0.15). Higher response rates occurred in patients with lower TPO-levels ( 〈 500 ng/l) and lower pre-treatment transfusion needs (PTE 〈 6 units/past year), but both variables were not significantly associated with response to romiplostim (univariable logistic regression, p= 0.13 and p=0.53, respectively). Evaluation of the mutational profile in a subgroup of 49 patients demonstrated that 67% of responders exhibited spliceosome mutations including SRSF2, SF3B1, U2AF1 and ZRSR2 compared to 35% in non-responders (p=0.06) (Table 1). Conclusion: This prospective study confirms that romiplostim treatment is highly effective in a subgroup of LR-MDS patients, but neither baseline platelet transfusion requirements nor baseline TPO levels were significantly associated with clinical response to romiplostim. Further translational analyses are ongoing to elucidate potential biomarkers of response. Disclosures Platzbecker: Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Götze:AbbVie: Membership on an entity's Board of Directors or advisory committees. Cony-Makhoul:Pfizer: Consultancy; Novartis: Consultancy; Incyte Biosciences: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Park:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Thiede:Daiichi Sankyo: Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AgenDix GmbH: Employment, Equity Ownership; Diaceutics: Membership on an entity's Board of Directors or advisory committees. Ades:Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. OffLabel Disclosure: Romiplostim is formally not licensed for the treatment of thrombocytopenia due to myelodysplastic syndrome (MDS).

Abstract: The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.

Abstract: 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.