ALBERT

Description: Introduction The presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube. Objectives: improvement of PNH cells detection technique. Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group. The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed. Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1). Figure 1 Figure 1. In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations. Table 1. Average value of PNH clone on leukocytes in groups of patients both techniques PNH clone Gr, % (mean) Mon, % (mean) CD24-/FLAER- CD157-/FLAER- CD14-/FLAER- CD157-/FLAER- 0%-0,99% 0,178 0,349 0,323 0,378 1%-9,99% 3,98 3,76 9,54 9,98 〉10% 71,52 71,52 71,85 71,34 Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone. Disclosures No relevant conflicts of interest to declare.

Description: Pregnancy and childbirth associated with a high risk of severe maternal and fetal complications in women with paroxysmal nocturnal hemoglobinuria (PNH). Recently the management of PNH during pregnancy has been challenging and childbearing was practically contraindicated in these patients. Eculizumab treatment improved the prognosis in PNH and made it possible to minimize complications during pregnancy. Establishment of effective and safe algorithms for the management of pregnancy, delivery and postpartum period in PNH patients is crucial to their lives. Since 1999, we have analyzed 15 pregnancies in six women with PNH. The median age at PNH diagnosis was 22 years (18-27), the median age at the start of pregnancy - 25 years (21-34). All of them were diagnosed with PNH following treatment for aplastic anemia (AA) with antithymocyte globulin, cyclosporine A and splenectomy in two cases before pregnancy. The median of PNH granulocyte clone at the start of pregnancy was 74,7% (17,8-94,1). Pregnancy occurred during complete remission of AA with PNH clinical signs in 5 (33,3%) cases. Most patients were in partial remission at the time of pregnancy-7 (46,7%) or continued to receive immunosuppressive therapy with minimal effect-3 (20%). Progression of aplasia observed during 4 (26,7%) pregnancies, but it was not severe and special treatment delayed until the completion of pregnancy. Two patients exposed to eculizumab before conceiving and remained on the treatment during pregnancy. Other women received only symptomatic therapy. Anticoagulation with low molecular weight heparin was used in 5 (33,3%) pregnancies. No thrombotic events during pregnancy and postpartum have been observed. Skin hemorrhages were revealed in 2 (13,3%) patients. During 4 (26,7%) gestations patients underwent erythrocytes and/or platelets transfusion. Pregnancies resulted in the birth of healthy infants in 7 (46,7%) cases - two girls and five boys. There were no adverse effects in the newborns from PNH patients both on eculizumab and without it. Successful outcomes were in 2/2 pregnancies on eculizumab treatment and in 5/13 (38,5%) cases without the drug. Caesarean sections were performed in all of births, early surgical delivery (30-34 weeks)-in 4/7 cases (preeclampsia-2, placenta previa-1, breakthrough hemolysis-1). Adverse pregnancy outcomes occurred only in patients not receiving eculizumab and amounted to 8/13 (61,5%). Only three patients had planned the pregnancy, other 12 cases were unplanned. Consequently, in 4 (30,8%) cases of pregnancy in the midst of illness was performed the abortion for medical reasons. Spontaneous miscarriage was registered in 2 (15,4%) patients. Two pregnancies (15,4%) ended in fetal death on 27th and 20th gestation weeks. Transfusion requirements increased in two pregnancies with symptomatic therapy, but did not increase on eculizumab. One of patients had first pregnancy without eculizumab and developed complications such as preeclampsia, postpartum severe epistaxis and high transfusion requirement (an average of 1,2 units per month). During second pregnancy on eculizumab she had no obstetric complications and transfusion requirements were less (0,5 units monthly). Second patient continued to have evidence of intravascular hemolysis despite the treatment, and so received eculizumab more frequent (one time in 10 days) in the third trimester. PNH granulocyte clone size decreased in both cases of eculizumab treatment during pregnancy. The risk of complications in PNH patients during pregnancy may be minimized by applying the management algorithm with eculizumab treatment. Despite the small number of observations, we can safely conclude that pregnancy outcomes in PNH patients with eculizumab are better than with only symptomatic therapy. Our experience confirms that eculizumab can be safely used in PNH throughout pregnancy to reduce the risk of complications and adverse outcomes. There is no difference in health between infants born by mothers with PNH and the newborns from general population. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1879 Splenectomy in patients with MDS is a treatment option that is beeing applied very rare [Steensma D., et al, Leuk Res.,2003; Bourgeosis E., et al, Leukemia, 2001]. There are anecdotal reports with very few patients demonstrating its efficacy. In most cases splenectomy was indicated for MDS patients with immune related thrombocytopenia. Here we would like to report the results of 33 splenectomies in patients with MDS who have been treated in our Center during 1994–2010. Within this period of follow-up totally 155 patients were diagnosed with different forms of MDS, 35% of them presenting with hypoplasia. The MDS treatment algorithm in our Center incorporates splenectomy as one of the options for pts with hypoplastic forms of MDS with bone marrow blast count less than 10%, refractory to initial cyclosporin A treatment or refractory to transfusions. Among patients who were splenectomised there were 20 females, 13 males with a median age of 40 years (range 18–74). Median time from diagnosis to splenectomy was 12 months (range 4–107). By WHO-classification there were 2 patients with RA, 22 – with RCMD, 2 – with MDS and del (5q), 6 - with RAEB, 1 - with AML after MDS. Cytogenetic analysis was available in 32 cases, and karyotype was normal in 15 patients (47%).The most common abnormalities were: del (5q) - 3, del (20q) - 2, trisomy 8 - 2, tetrasomy 8 - 1, monosomy 7 - 2, complex karyotype - 4. Bone marrow biopsy revealed hypoplasia in 25 patients (75%), myelofibrosis – in 7 (21%). The median WBC count was 2,6*109/L (range 0,6-8,7), hemoglobin 6,9 g/dL (47-119) and platelets 26*109/L (6-170). 27 pts (82%) were RBC transfusion dependent, 22 (67%) - platelets transfusion dependent. 13 pts had received immunosuppression therapy (ATG, cyclosporine A) before splenectomy, 2 - cytotoxic chemotherapy, 3 - decitabine. The majority of splenectomies were done by laparoscopic method - 26 (79%), in one case the convertion was done. In all cases we performed liver biopsy. Postoperative complications (hemorrhage) occurred in 1 patient but there were no deaths due to operation. One death occurred in 7 days after splenectomy due to fulminant progression to AML. Median spleen weight was 180 gms (range 70–930). Median intraoperative blood loss was 250 ml (range 50–9350). Histology was available in 30 patients. Extramedullary hematopoesis was revealed in 3 cases (10%), blast infiltration - in 2 (7%), massive lymphoid infiltration was detected in 5 cases and in one patient in was proved to be clonal (marginal zone lymphoma, MZL). Hemosiderin depositions in the macrophages were seen in half of the cases -16 (53%). One case was characterized by granulomatosis in spleen and liver with negative immunohistohemical staining to Mycobacteria tuberculosis. Splenectomy lead to sustained improvement of cytopenias in 16 cases (48%): decreased transfusion dependence in 14 (42%) and transfusion independence in 2 (6%). After splenectomy 5 patients were followed by “wait and see” approach, 17 continued with immunosuppressive therapy (ATG,CyA), 3 patients were treated with cytotoxic chemotherapy, 1 – with decytabine, 2 received EPO, 1- danazol, 2 - iron chelation therapy, 2 – only transfusions therapy. We did not noticed the infections rate augmentation after splenectomy. Transformation to AML was registered 6 (18%) at median 6 months (0,3 -9). 13 splenectomized patients (39%) died at a median 12 months (range 0,3-84) and the main death reasons were: AML progression, aplasia deterioration followed by infections and hemorrhage. 20 patients are alive with a median follow-up after splenectomy 33 months (2-108). Analysis of our 15-years study data give us a confidence to conclude that splenectomy still may be an adequate option for distinct forms of MDS (hypoplastic forms with bone marrow blast count less than 10%, refractory to initial immunosupressive treatment or refractory to transfusions), producing cytopenia improvement in half of the patients with decreasing transfusion dependance also in half of the patients, sometimes bringing a clear diagnosis (MZL). The mechanism of action is not very clear but we can speculate that splenectomy removes the “cell-destroying” organ, deminishes immune pathways of cytopenias due to large lymphoid compartment deletion, provides the resustainment of sensitivity to immunosupressive agents. Disclosures: No relevant conflicts of interest to declare.

Description: Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.