ALBERT

Description: The role of thrombophilia and LMWH use in pregnancy loss (PL) and pregnancy complications (PC) is debated. In this retrospective study from a single center we analyzed the clinical outcome of pregnancies in relation to thrombophilic factors and the use of LMWH, aspirin and folic acid in 143 women followed up for a total of 173 pregnancies referred to our center from 2003 to 2016. Methods: Women were referred to our unit for: more than 2 unexplained PL (n=96, 78 experienced only early PL, 11 had only late PL, 7 had both early and late), one pregnancy loss(n=45) or one pregnancy complication (placenta abruption, intrauterine growth restriction, eclampsia, n=2). Mutations in Factor V-Leiden (FVL, G1691A), Prothrombin (PTG, G20210A) and MTHFR (C677T, A 1206C) were checked by DNA hybridization Kit. Plasma levels of antithrombin-III, protein-C, free Protein-S, APCR, FVIII, FXII, PT aPTT, fibrinogen, homocysteine and La-test were measured by photometry (DACO). Anticardiolopin and anti-β2GPI antibodies (IGG and IGM) were measured by ELISA in serum (APLA). End points were live birth and pregnancy complications. The prevalence of thrombophilia in our cohort was similar with previous studies and 34/143 (23,4%) women were negative for all thrombophilic factors. We observed mutations in FVL(11,6%), PTG (9,6%), MTHFR (homozygous or double heterozygous, 33,3%) and deficiencies of AT-III (3,3%), Prot-C (1,6%), Prot-S (8,8%), APS (8,7%). Combined severe trombophilic factors were found in 31 women (21,5%) (FVL+PTG 4/31, Natural Anticoagulants one out 3 Def + MTHFR 3/31, APS + MTHFR 2/31, FVL+MTHFR 16/31, PTG + MTHFR 6/31). We then separated our cohort into women with 2 complications. The second group had significantly higher incidence of FVL mutation (12,5 vs 8,3%, p=0.05) and deficiencies of AT-III and Free Prot-S ( 6,5 vs 0 %, p=0.01) compared to the first one. By contrast, women in the first group had higher incidence of La-test (12,5 vs 4,5%, p=0,03), APLA ( 12 vs 6,6%, p=0.03) and Prot.C deficiency (4,5 vs 0%, p=0.01). In univariate analysis both hereditary and acquired thrombophilic factors did not correlated with pregnancy outcome (live birth or pregnancy complications). Only age as a continuous parameter correlated negatively with live birth and positively with pregnancy complications (p=0.01 and p=0.025, respectively), whereas high BMI as a continuous parameter also negatively affected live births (p= 0.049). Logistic regression analysis reveals that the age of 35 is the cut off for unfortunate pregnancy outcome. Pregnancies were proceeded with (n=143, 81,7%) or without (n=32, 18,3%,) LMWH. The decision to use LMWH were based in a positive thrombophilia screening test (n=84) or to prior history 〉2 pregnancy complications with negative trombophilia testing (n=59). Concomitant use of ASA was prescribed in 78 pregnancies (dose less than 100 mg/day) and concomitant follic acid in 143 pregnancies. The percentage of live births were identical in women treated with LMWH (87,4%) or not (87,5%, p=0.9). In multivariate analysis, the only factor that was strongly correlated to live birth was the duration of LMWH treatment (odds ratio, OR =3,567, 95% CI (1.845, 6,894), p= 0,01) and the titration of the dose with anti-Xa (OR=5,138, 95% CI (1,717, 15,376), p = 0,01, fig.1a). By ROC analysis the duration of LMWH which correlated to live birth was ≥ 5.5 months(fig. 1b). The addition of ASA was insignificant for live birth (p=0.7), while the duration (〉6months) of follic acid also appeared to add a benefit in combination with LMWH (p=0.01). Moreover, pregnancies proceeded without LMWH exhibited higher rates of pregnancy complications (18,75 vs 11,2%, p=0.08) and prematurity (14,3 vs 8,8%, p=0.05). In summary, our findings argue against hereditary thrombophilia screening in the cases of previous pregnancy loss or pregnancy complications. On the contrary, testing for APS even after the first event might be of value as this population often has laboratory evidence of APS and may benefit from proper anticoagulation. The use of LMWH and folic acid but not of ASA was related to less pregnancy complications or prematurity, whereas proper titration of LMWH by using anti-Xa and long duration of therapy were the only important factors for successful pregnancy outcome. Disclosures No relevant conflicts of interest to declare.

Description: Hypomethylating agents (HMA) such as azacytidine and decitabine are the mainstay of treatment for higher risk myelodysplastic syndromes (MDS) and are also used to treat older, unfit patients with acute myeloid leukemia (AML). Being cytidine analogues, both azacytidine and decitabine are incorporated into DNA of highly proliferating cells leading to genome-wide decrease of methylation levels (Stresemann & Lyko., 2008; Gnyszka et al., 2013), whereas azacytidine is additionally incorporated into RNA molecules. Although several putative modes of action have been suggested for HMA, the precise mechanism underlying treatment success or failure remains incompletely understood. One possible mechanism of HMA action is through 'viral mimicry' of transcriptionally repressed endogenous retroelements (EREs), which is thought to trigger innate immune pathways. EREs comprise nearly half of the human genome and their transcriptional activity is repressed by diverse mechanisms including DNA methylation. According to the 'viral mimicry' hypothesis, HMA induce unphysiological levels of ERE transcription in transformed cells, which in turn generated nucleic acid species, such as double-stranded RNAs from complementary ERE transcripts, activating innate immune sensors. Although support and a mechanistic basis for this hypothesis is provided from a number of in vitro studies, in vivo evidence from the clinical use of HMA is currently lacking. To explore the possible involvement of EREs in the HMA mode of action, we have compared the transcriptional profiles of CD34+ HSCs isolated from bone marrow samples of healthy donors (n=9) and patients diagnosed with AML (n=9), chronic myelomonocytic leukemia - II (CMML-II, n=9) or high-risk MDS (n=11). For MDS and CMML, samples were obtained before, 15 days (D15) after the initiation of azacytidine and/or after cycle 6. Our analysis revealed that ERE transcription, measured as a proportion of the total polyA-selected transcriptome, is globally repressed in untreated MDS and CMML, in line with the proposed epigenetic repression that characterizes these conditions. Treatment with azacytidine had a measureable effect in overall ERE transcription in HSCs from MDS and CMML patients, which by the 6th cycle was raised to levels equivalent to those seen in HSCs healthy controls. Comparable results were also obtained following analysis of a publicly available dataset from CD34+ HSCs isolated from MDS and CMML patients prior to and after the 6th cycle of azacytidine treatment (GSE76203). However, despite noticeable upregulation of overall ERE transcription relative to gene transcription by azacytidine, the therapeutic response was not correlated with or predicted by ERE activity. Indeed, ERE transcriptional activation was frequently observed in azacytidine-treated patients who failed to respond to treatment, whereas it was frequently low or absent in patients who attained complete remission (figures 1a & b). It remained theoretically possible that a therapeutic response to azacytidine depended on the transcriptional activation of a select few ERE loci with innate immune stimulatory properties, which might have been masked by the analysis of global ERE activity. However, few individual ERE loci differed in their activity between patients who responded or not to azacytidine treatment. Moreover, our analysis failed to detect induction of either interferon-inducible genes or interferon-inducible EREs, irrespective of treatment outcome(figures 2a & b). Together, our current results do not support a role for transcriptional activation of EREs or for innate sensing of their nucleic acid products in the therapeutic response of MDS and CMML patients to azacytidine. Investigation of alternative potential mechanisms of azacytidine is therefore warranted. Disclosures No relevant conflicts of interest to declare.