Background: The Kleihauer-Betke test (KBT) is the most widely used assay for fetomaternal hemorrhage (FMH) detection in rhesus D negative women. In the current study, we sought to evaluate the performance of a flow cytometry (FCM) kit (FMH QuikQuant) using an anti-HbF antibody.Methods: Eighty-three pregnant women, 58 umbilical cord blood (UCB) dilutions in adult blood, and 6 control samples were tested in parallel with FCM and KBT.Results

Introduction: Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) with high-risk genetics and/or measurable residual disease (MRD) are at high-risk of disease recurrence. In the previous GRAALL-2005 study, we identified KMT2A rearrangements (KMT2A-r), IKZF1 intragenic deletion (IKZF1del) and post-induction (TP1, week 6) MRD ≥ 0.01% as independent factors to predict relapse in Ph-negative B-cell precursor (BCP) ALL (Beldjord K, Blood 2014). In the GRAALL-2014 trial, high-risk (HR) patients were thus defined by the presence of at least one of these three factors. Among them, only those with higher MRD levels defined as TP1-MRD ≥ 0.1% and/or week 12 (TP2) MRD ≥ 0.01% were considered at very high risk (VHR) and proposed allogeneic hematopoietic stem cell transplant (alloSCT) in first remission (Dhedin et al., Blood 2015). Since October 2018, all these patients were eligible to be included in the GRAALL-2014-QUEST phase 2 study to receive blinatumomab as part of consolidation and maintenance phases or as a bridge to transplant. Methods: From October 2018 to December 2020, 95 patients with high-risk Ph-negative BCP-ALL without central nervous system involvement at diagnosis and in continuous complete remission after induction and consolidation 1, were prospectively included to start blinatumomab at week 12. One patient was excluded because of T-ALL phenotype (with CD19 aberrant expression). Patients with alloSCT indication and a stem cell source received blinatumomab 28 microg/d administered by continuous intravenous infusion (cIV) until transplant. A minimum of 4 weeks blinatumomab was recommended before proceeding to transplantation. All other patients received 5 cycles of blinatumomab 28 microg/day cIV (for 28 days), during consolidation 2 and 3 and at months 1/3/5 of the maintenance phase respectively. The primary objective was disease-free survival (DFS). Secondary objectives included post-blinatumomab MRD response at TP3 (after consolidation 2 or before alloSCT), overall survival (OS), and safety. Early results are reported here. Results: Median age was 35 years old (range, 18-60). Median white blood cell count (WBC) at diagnosis was 12 G/L (range, 1-449). Oncogenetic analyses allowed classifying ALL as Ph-like (18%), KMT2A-r (17%), DUX4/ERGdel (13%), ZNF384-r (11%), low hypodiploidy/near triploidy (7%), B-other (26%) or unknown (9%). An IKZF1del was found in 37/93 (40%). A TP1-MRD ≥ 0.01% was found in 46/94 patients (49%). Final risk group was HR for 45 patients and VHR for 49 patients. Last pre-blinatumomab MRD was <0.01% in 49/88 (56%) of evaluable patients. A total of 40 patients (42%) received an alloSCT. The median number of blinatumomab cycles received in patients not proceeding to alloSCT was 4 cycles (range, 1-5). Thirty-nine severe adverse events (SAEs) were reported: 1 CRS (grade 2), 8 neurotoxicities (1 grade 2, 3 grade 3, 3 grade 4, 1 grade 5), 19 infections, and 11 others. The only grade 5 SAE occurred after alloSCT (seizures). After blinatumomab, a complete MRD response (with at least 0.01% sensitivity) was achieved in 61/82 (74%) evaluable patients and in evaluable patients with pre-blinatumomab detectable MRD. MRD response to blinatumomab was lower in patients with high pre-blinatumomab MRD level, while not impacted by age, WBC, or oncogenic subgroup. With a median follow-up of 20 months, 18-month DFS and OS was 78.8% (95% CI [66.9-86.8]) and 92.1% (95% CI [83.2-96.4]) respectively (Figure 1). Patients with VHR diseases had a worse DFS (68.8%, 95% CI [51.1-81.2]) as compared to other patients (90.6%, 95% CI [72.1-97.1]); p=0.018). This difference of DFS was abrogated by censoring patients at transplant (VHR 88.1%, 95% CI [65.5-96.3] versus others 90.6%, 95% CI [72.1-97.1%], p=0.10). Other factors significantly associated with better DFS were DUX4/ERGdel subgroup, low pre-blinatumomab MRD, and complete MRD response after blinatumomab. Conclusion. In patients wih high-risk BCP-ALL, blinatumomab added to consolidation is safe and gives promising results. A comparison to similar patients treated in the same GRAALL-2014 study before October 2018 is planned with a longer follow-up. Figure 1 Figure 1. Disclosures Boissel: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; SANOFI: Honoraria; Servier: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; CELGENE: Honoraria; JAZZ Pharma: Honoraria, Research Funding; PFIZER: Consultancy, Honoraria. Huguet: Novartis: Other: Advisor; Jazz Pharmaceuticals: Other: Advisor; Celgene: Other: Advisor; BMS: Other: Advisor; Amgen: Other: Advisor; Pfizer: Other: Advisor. Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Chalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation. Delabesse: Astellas: Consultancy; Novartis: Consultancy. Dombret: Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; NOVARTIS: Research Funding; pfizer: Honoraria, Research Funding; servier: Research Funding; BMS-Celgene: Honoraria; Daiichi Sankyo: Honoraria. OffLabel Disclosure: Blinatumomab in frontline high-risk acute lymphoblastic leukemia

Tgd LGL Leukemia (Tgd LGLL) is a rare variant of T-LGLL that has been less investigated as compared with the more frequent Tab LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tgd LGLL retrospectively collected at 8 referral centers and collected from 1997 to 2020. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in around 20% of cases each. Among the various treatments, Cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 patients were available for STAT3 and STAT5b mutations analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tgd patients were also compared with a recently published Tab cohort including 129 patients. Though no differences in STAT3 and STAT5b mutational frequency were found, Tgd cases more frequently presented with neutropenia (p=0.0161), anemia (p<0.0001), severe anemia (p=0.0065) and thrombocytopenia (p=0.0187). Moreover, Vd2 negative patients displayed higher frequency of symptomatic disease. Overall, Tgd patients displayed reduced survival with respect to Tab patients (p=0.0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tgd LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared to Tab LGLL.

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the western world and is characterized by the accumulation in blood, bone marrow and secondary lymphoid organs of long-lived resting clonal B lymphocytes with a very low proliferation index and/or that proliferate abnormally. The disease has a notable clinical course diversity, with different prognosis and distinct treatment needs. However, the exact etiology is still unknown. Since cancer cells have a global DNA hypomethylation and local hypermethylation, there are evidences that epigenetic modifications have an important role in CLL pathogenesis. DNA methyltransferases (DNMT), including DNMT1, DNMT3A and DNMT3B, are responsible for DNA methylation and changes in the expression of these enzymes were observed in other hematological malignancies. Aims: The aim of this study was to evaluate the expression levels of DNMT1, DNMT3A and DNMT3A genes in chronic lymphocytic leukemia patients and analyze its correlation with clinical and laboratory data, in order to identify potential biomarkers of diagnosis and/or prognosis. Methods: We evaluated the DNMTs in peripheral blood of 68 CLL patients and 23 controls (CTL). Informed consent was obtained in accordance with the Helsinki Declarations. qPCR was used to evaluate the gene expression levels of DNMT1, DNMT3A, DNMT3B and GUSB (endogenous control). MannWhitney U and KruskalWallis tests were used to assess the statistical significance between groups. Receiver operating characteristic (ROC) curves analysis was performed to analyze variables accuracy as diagnostic biomarker. Survival analysis was performed by Kaplan Meier method. Patients were dichotomized according to the cut off points obtained from the ROC curves constructed to predict death. All statistical analysis were two sided and a p < 0.05 was considered statistically significant. Results: This study enrolled 68 CLL patients, 37 males and 31 females with a median age of 65 years (48-101), and 23 CTL, 8 males and 15 females with a median age of 72 years (58-88). According with Binet staging system 54 patients (89%) were low risk (A), 2 (3%) intermediate (B) and 5 (8%) high risk (C). Cytogenetic abnormalities usually associated with good prognosis were found in 28 patients (62%) and with bad prognosis in 17 patients (38%), including 12 with del(17p) (27%). We observe that CLL patients had a higher DNMT1 expression [median: 0691; interquartile range (IR): 0.990] and a significant lower DNMT3B gene expression levels [median: 0.023, IR: 0.040; p = 0.020] compared with controls [DNMT1 median: 0.603, IR: 0.995; DNMT3B median: 0.038; IR: 0.267] and in 12 patients (18%) no expression of DNMT3B was detected. On the other hand, DNMT3A expression levels are similar in patients and controls (CLL median: 0.281, IR: 0.283; CTL median: 0.283, IR: 0.268). Using ROC analysis, DNMT3B expression levels shown to be a possible diagnosis biomarker (AUC: 0.720; IC95%: 0.596-0,843; cut-off<0.026; sensitivity: 63%; specificity: 78%; p = 0.002). The ...

for 10 days. NK-cell cytotoxicity was assessed using Calcein AM + labeled K562 target cells. Additionally, the cytotoxic capacity of T and NK cells was evaluated by MPFC, utilizing the degranulation marker CD107a and the effector molecules Perforin and Granzyme B. Results: Idelalisib induced a dose-dependent inhibition of T-cell proliferation from healthy donors. However, significant effects on the number of CD4+ and CD8+ dividing T cells were only seen at concentrations higher than 5 µM, indicating that T-cell proliferation is not affected at therapeutic concentrations of the agent. CD4+ T cells, as well as CD8+ T cells, showed reduced expression of CTLA-4 and PD-1 after treatment with 0.5 µM Idelalisib upon CD3/CD28 stimulation. Similar effects were detected for the respective Treg subsets. Interestingly, cytokine secretion of T cells was significantly impaired for IL-10, TNF and IFNγ for all tested Idelalisib concentrations (p = 0.0008; p = 0.03; p = 0.03; n = 11-23). We also found Idelalisib to significantly decrease upregulation of Granzyme B and Perforin in CD8+ and CD4+ T cells (CD8+: p = 0.0002; p = 0.0215; CD4+: p = 0.0002; p = 0.015, n = 12-13) upon CD3/CD28 stimulation. NK cells treated with 0.5 µM Idelalisib showed decreased overall proliferation (p = 0.02; n = 12). NK cell cytotoxicity against K562 target cells was significantly reduced in a concentration dependent manner (0.5 µM, p = 0.0031, n = 12). Summary/Conclusion: Idelalisib impaired the cytotoxicity of T and NK cells at therapeutic concentrations in our in vitro studies. In addition, secretion of pro-inflammatory and immune-mediating cytokines like IL-10,TNF and IFNγ was significantly reduced for T cells by Idelalisib. These observations support the hypothesis that the T-and NK-cell function is directly impaired by Idelalisib, and not by B-cell malignancy-inflicted immunosuppression.

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