ALBERT

Description: Abstract 4487 HLA mismatch constitute the risk of aGvHD. However, environmental factors play a significant role what is exemplified by the protective effect of germ free environment. This association can be discussed on the ground of a significant role of cytokine milieu which determines lymphocytes subsets differentiation. Our previous work showed a decrease of proportions of Th17 cells in blood at the onset of aGvHD (Dlubek et al., Transplantation Proceedings 2010; 42(8):3277-9). These cells, however, were seen at the tissue site likely being marginalized during aGvHD process. In this study we looked at Th17 lymphocytes proportions in blood of patients post alloHSCT in the context of an increase of C-reactive protein in serum and NOD2/CARD15 gene mutations. Eighty four patients (median age: 41 yrs, range: 1–64 yrs) entered the study. All of them were clinically followed for the presence of infections complications and aGvHD. Blood work was done at the very first day of the later complications and in addition routinely in one week intervals beginning from the day of transplantation. The cells were labeled for CD4 (Becton Dickinson, San Jose, CA, USA), intracellular IL-17A (e-biosciences, San Diego, CA, U04)SA) and FoxP3 (Becton Dickinson, San Jose, CA, USA) detection. CRP was measured in serum using a Behring BN Prospect nephelometer (Dade Behring Inc., Marburg, Germany). All studied cases were genotyped for NOD2/CARD15 mutations: 104C-T (SNP8, Arg702W; rs.2066844), 2722G-C (SNP12, Gly908Arg; rs.2066845), and 3020insC (SNP13, Leu1007fsinsC; rs.2066847) with the use of RFLP-PCR technique. It was found: Acute GvHD manifestation was seen in 34 (40%) patients among them 22 (26%) had only skin symptoms and 12 (14%) presented gut manifestation. Gut aGvHD was seen at later time post HSCT than skin aGvHD (median 33 vs. 21 days, M-W U test p=0.035).Twelve patients had NOD2/CARD15 mutations. They had lower proportions of Th17 lymphocytes in blood as compared to those lacking these mutations (median 0.03 vs. 0.07%, M-W U test p=0.052) and 6 (50%) of them presented aGvHD.CRP levels measured from 3 to 5 days before clinically apparent aGvHD were significantly higher in patients with gut symptoms as compared to those presented only skin lesions (median 66.8 vs. 7.61 mg/L, M-W U test p=0.020). Notably, patients having only skin symptoms had rather lower CRP levels in sera than those without aGvHD (median 7.61 vs. 27.6 mg/L, M-W U test p=0.028).In patients with gut manifestation FoxP3+CD4+ cells proportions were negatively correlated with serum CRP levels (Spearman r −0.664, p=0.018) what was not seen in patients presented only skin symptoms aGvHD (0.068, p=0.769). In conclusion: CRP elevation constituted the risk of gut aGvHD and was associated with rather exhausted T regulatory cell function. NOD2/CARD15 mutations associated factors contributed to the lowering of Th17 cells in blood what was previously found to be associated with the overt manifestation of aGvHD (Dlubek et al., Transplantation Proceedings 2010; 42(8):3277-9). These both findings are in line with the hypothetical role of microbial pathogens which via inflammation favor differentiation of lymphocytes to Th17 positive cell subset. Disclosures: No relevant conflicts of interest to declare.

Description: Immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post alloHSCT. The rate of success depends on the characteristics of leukemia which is more beneficial if the cells are recognized in the way of alloreactivity. Intra venous DLI is associated with a high risk of aGvHD and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. The previous reports documented a lower risk of aGvHD if the transplant material was given to the bone marrow cavity which at relapse is usually infiltrated by the blasts. To address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (IB-DLI) in patients post alloHSCT at relapse. This technique was employed in 4 patients, 3 with AML and one with CLL, all relapsed post alloHSCT: 3 alloSIB: 50-year-old female AML patient with normal karyotype (relapsed 2 years post HSCT) and 22-year-old AML male 7q31 del (relapsed at 3 years post HSCT) and 64-year-old CLL male, TP53 del, recurrent EBV reactivation with vasculitis (progressed 7 years), and 25-year-old male AML FLT3 ITD+ received MUD HSCT (relapsed 9 months). Two patients with a proportion of 26% and 12% blasts in the marrow respectively received IB-DLI up-front and two others due to the excess (52.50%, 57.70% (CD5+CD19+)) of leukemic cells received either FLAG (AML case) or anti-CD20 MoAB (CLL case) followed by IB-DLI. The patients received from 3 to 5 IB-DLIs according to the escalating dose regimen starting from 10E6 and ending with a dose of 10E8 of CD3+ cells/kg b. w. (blood MNC were harvested with use of Spectra Optia (Terumo BTC) for SIB transplantation, in MUD HSCT they were taken from the PBPC material (J Hasskarl et al. 2012). The intervals between IB DLI varied from 1 to 2 months. One patient received azacitidine between the DL infusions (Schroeder T et al. 2013) which was associated with longer intervals. The cells were injected directly to the bone marrow cavity under local anaesthesia with a low molecular Heparin prophylaxis. The blood and marrow specimens were taken prior each IB-DLI for: cytology, cytometry (including CD8, CD279, CD26, CD28 MoAb in addition to the routine staining used for blast cells), genetic work (chimerism, mutations associated with the disease). Clinical outcome:No side effects were noticed (including GvHD).The patients are alive.Anti-leukemic effect: 3.1 Responding AML ITD+ case was free of blasts 6 months after the initiation of the treatment. However, after the third IB-DLI blasts appeared in the marrow but the patient responded favorably to the Sorafenib treatment and the following course of IB-DLI. 3.2 Partial remission (stable proportion of blasts and sustained hematopoiesis); the patient (22-year-old AML male) was transplanted a second time but blasts at the range of 38% were still observed in the marrow without progression after 9 months. 3.3 One case which failed to respond to IB-DLI (female AML patient) was transplanted a second time but this approach also failed and this patient now has full blown leukemia. The laboratory work up:Proportions of lymphocytes in the marrows tended to increase after completion of each IB-DLI (32.0 ± 4.6% vs 37.1 ± 3.7%, Wilcoxon test for pairs, p=0.078).Collectively CD279+ cells contributed to the lymphocyte pool in the marrow to a greater extent than it was seen in the blood at the same time (16.6 ± 2.9% vs 33.1 ± 4.0%, p

Description: Abstract 4552 It is known that CMV infection/reactivation influences the survival of patients post HSCT. Risk factors are associated with both (i) the CMV serostatus of donors and recipients and (ii) the immunological potential of the recipients post transplantation. In this study 142 patients - 69 males and 73 females suffering from hematological malignancies, SAA and inborn errors at the age from 0.6 to 64 (median 40) - were followed for the presence of CMV and EBV DNA copies and the presence of CMV and EBV IgM antibodies in blood post HSCT (61 sibling and 81 matched unrelated donors) with 625 days of median observation time. In addition the number of lymphocytes in blood and the proportions and numbers of CD4, CD8, CD20 positive cells in blood were analyzed in association with the CMV and EBV findings. CMV and EBV DNA copies were determined with the use of qPCR at 1-week intervals until 30 day post HSCT, then monthly until one year post HSCT and then at routine follow-up examinations (usually in 3 months intervals), and always when clinically suggested. Values exceeding 50 copies/10^5 cells were recognized as positive and clinically significant. At the same time the presence of IgM and IgG antibodies specific for CMV and EBV were examined with the use of ELISA. The first positive results for CMV DNA copies were seen at median time of 48 days post-transplant and IgM antibodies if present were detected in a majority of cases within 30 days after DNA copies finding. EBV copies were detected at the similar time post transplantation (median day of positive results: 48 days) and IgM antibodies if present were usually seen within 2 months after DNA copies detection. Notably, in 9 patients EBV reactivations were associated by the appearance of IgM CMV but not EBV antibodies. Therefore the presence of IgM antibodies against either CMV or EBV was recognized as a sign of the ability of a given patient to respond immunologically to CMV or EBV viruses. For the sake of this study the patients group was divided into subgroups as follow: (1) CMV and/or EBV copies present but IgM antibodies absent, (2) CMV and/or EBV DNA copies as well as CMV and/or EBV antibodies present, (3) neither CMV or EBV copies nor IgM antibodies present, (4) CMV and EBV DNA copies absent but IgM CMV and/or EBV antibodies present. Patients lacking DNA copies or having both copies and IgM antibodies against CMV and/or EBV had significantly better survival than those having DNA copies but lacking IgM antibodies any time during observation period (2 yrs survival 70% vs 41%, p