ALBERT

Description: Abstract 4848 Background Myelodysplastic syndrome(MDS) belong to the most common hematological diseases however epidemiological data on MDS are sparse. Until 2008 there were no data about epidemiology of MDS in Poland. Methods From 03.2008-05.2009 we have registered 966 patients in Polish MDS Registry. We have included only alive patients of various time of diagnosis. Patients from 22 centers were diagnosed according to WHO 2001 criteria. Results There were 508(53%)males and 458(47%) females. Median age at diagnosis was 70(range 19-99). Under 50 were 83(9%) cases with preponderance of females- 51 cases( males 32cases), between 50-70 there were 353(41%) cases, half of the patients-432(50%) were above 70( 247 males and 185 females).Prior chemotherapy and/or radiotherapy had 37((3,8%) patients. Distribution of MDS subtypes was as follows: RA-170(20%) cases, RARS-58(7%), RCMD-244(28%), RCMD-RS-18(2%), RAEB-1-120(14%), RAEB-2-169(19,5%), 5q- -40(4,6%), MDS-U-44(5%).In 103(10%) subtype was not done. Karyotype was available in 276(28%) cases. Cytogenetic risk groups were: low risk-182(68%), intermediate-52(20%) and high risk-33(12%). The most frequent cytogenetic results were: normal karyotype 44%, isolated 5q deletion 19%, complex karyotype 6%, 5q deletion + another one change 3% and 5q deletion with at least 2 changes 3%. According to IPSS risk groups low risk was found in 61( 22%) of cases, intermediate-1 -130(48%), intermediate-2-47(17%) and high risk in 31(11,5%). Median values of Hb was 9,1 g/dL, plts 129 G/L, ANC 1,7 G/L. RBC transfusion dependent were 429(44%) patients and platelet transfusion dependent were 100( 11%) pts. At least 2 U/month RBC transfusion requirement was 140(14%) patients. Serum ferritin level was assessed in 530 cases-171 of them( 32%) had higher than 1000μg/L level. Conclusions We have observed predominance of females among MDS patients under 50. Half of the patients had RA or RCMD subtype. Isolated 5 q deletion was the most frequent cytogenetic abnormality. Forty four percentage of patients was RBC transfusion dependant. Serum ferritin level was significantly elevated in 32% of assessed patients at the moment of MDS diagnosis. Disclosures No relevant conflicts of interest to declare.

Description: IL-17 is involved in chronic inflammation and autoimmunity, this cytokine producing cells are well characterized in the mouse model. In man, situation is more complex due to the genetic variability, co-morbidities and environmental factors influencing the immune response. In patients post HSCT alloreactivity with the presence of aGvHD is a major factor affecting the outcome of transplantation. Understanding of the role of cells involved in regulation of the immune system is crucial for the treatment tailoring to favour tolerance but not making the recipient more prone to opportunistic infections and leukaemia relapse. In this study 27 patients post HSCT were investigated for the presence of regulatory cells and those producing IFNgamma and IL-17 in blood. Blood was collected at the time of haematological recovery or when the first clinical symptoms of aGvHD became apparent and then in one week intervals until +60 day post transplantation. PBMC were stimulated for 4 hrs with BD Leukocyte Activation Cocktail in the presence of Golgi Stop (BD, Erembodegen, Belgium) and then stained extracellularly with anti-CD4, permeabilized with Fixation/Permeabilization Concentrate and Diluent (eBioscience, San Diego, CA) and finally stained with anti-IFNgamma (BD), anti-IL-17 (eBioscience) anti FoxP3 (eBioscience). CD4+ cells subpopulations were analysed according to the expression of the stained features. It was found: FoxP3+CD4+ cells were in higher proportions in pts with aGvHD (results from all time-points taken together) (9.93%±0.61 vs 8.2%±0.50, n=98, p=0.040, U Mann-Whitney test) IFNgamma producing CD4+ lymphocytes were in higher proportions (0,34 vs 0.14, ns) in blood samples taken from patients lacking as compared to those having aGvHD. CD4+IL-17+ lymphocytes proportions increased from 1.39%±0.42 to 5.33%±2.45 (p=0.04, Wilcoxon Test for pairs) one week before aGvHD. Notably, at the time of full blown aGvHD the proportions of CD4+IL-17+ cells were lower as compared to the results of previous measurements (0.74%±0.29, p=0.008, Wilcoxon Test for pairs). Taking all results together the proportion of CD4+IL17+ lymphocytes were lower in patients having as compared to those lacking aGVHD (0.93%±0.27 vs 1,53%±0,41, p=0.05, U Mann-Whitney test). It appears that: FoxP3 positive cells expand during aGvHD likely as a response to alloreactive stimulation. INFgamma+ CD4+ cells benefit the course post HSCT making the patients less susceptible to aGvHD. CD4+IL17+ cells are likely involved at the early stage of aGvHD patho-mechanism heralding the clinical manifestation of this complication, but then they disappear from blood, possibly being marginalized in the inflamed tissues.

Description: Abstract 5058 The role of T cells residing in the marrow is poorly understood. However, some literature data suggest that the presence of CD8+ lymphocytes in the marrow plays a role in keeping the tumor cells in a dormant state. In chronic myeloid leukemia a significant role of lymphocytes in controlling the malignant clones is well documented. The aim of this study was to define whether there is a specific enrichment of some CD4+ lymphocytes subpopulations in the marrows of newly diagnosed patents with essential thrombocythemia (ET). Seven patients (F/M: 6/1, age 28–72 - median 6 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets 425–809 ×10∧3/ul (median 676 × 10∧3), NAP 74–138 score (median 112 score), 6 of them were positive for JAK 2 V617F mutation (heterozygotes). Marrow cellularity was from 18 – 64 ×10∧3/ul (median 44 × 10∧3/ul) Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters was seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Lymphocytes in the marrows were in proportions from 4.5 – 15,7% (median 10,6%) what resulted with lymphocytosis from 2640/ul to 19405/ul (median 5645/ul). The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, USA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. We found:Proportions of lymphocytes in the marrows were rather in a normal value range but in numbers they prevailed over blood lymphocytes (2640-19405/ul, median 5645/ul vs 1300–3100/ul, median 1800/ul, p= 0,003 Wilcoxon Test for pairs),Among T cells subpopulations effector/memory CD4+ lymphocytes CD45RO+, CCR7-) were significantly higher in numbers in the marrows of ET patients as compared to blood (744-3241/ul, median 1126/ul vs 406–2437/ul, median 592/ul, p=0,04 Wilcoxon Test for pairs). There was no difference when numbers of naïve (CD45Ro-, CCR7+) and central memory (CD45RO+, CCR7+) CD4+ lymphocytes were analyzed.The marrows of ET patients had higher numbers of CD4+CD25++ lymphocytes as compared to blood (8,1-40,7/ul, median 11,4/ul vs 1,2-14,8/ul, median 7,3/ul; p=0,02 Wilcoxon Test for pairs). It appeared that in ET patients there was a selective enrichment of marrow lymphocyte populations in CD4+EM cells and CD4+CD25++ lymphocytes what strongly suggests the presence of active immune response in the marrows affected by this myeloproliferative disorder. Disclosures: No relevant conflicts of interest to declare.

Description: Cytokines belonging to interleukin -17 family (IL-17A, B, C, D, E and F) are produced mainly by activated and memory CD4+ cells which are described as Th17. These cells (IL-17+CD4+) promote autoimmune reaction (mouse model), were found in tissues involved in autoimmune process in man and produce IFNgamma. Therefore, Th17 cells constitute a population which may contribute to aGvHD together with Treg cells (FoxP3+). In this study we examined the cytoplasmic expression of IL-17A and FoxP3 in CD4+ lymphocytes in 6 pts post HSCT (3 MUD and 3 SIB, median (mean) age: 35 (33,9) years, 3 AML, 2 ALL-T, 1 SCID). Three of them developed aGvHD. All consecutive pts were included to the study. Peripheral blood lymphocytes were examined or at the very first day of aGvHD manifestation either at the beginning of hematological reconstitution and thereafter in one week intervals. Nine healthy individuals served as controls. PBMC were stimulated with BD Leukocyte Activation Cocktail containing mixture of PMA, Ionomycin and brefeldin A in the presence of BD GolgiStop then stained with CD4, IL-17A and FoxP3 monoclonal antibodies and analyzed in CD4+ and CD4- lymphocytes gates by three colour flow cytometry. Therefore, all results reflect the situation in stimulated cells. FoxP3 mRNA was measured in unstimulated PBMC with the use of qPCR. We found: Patients post HSCT (during 6 weeks period post transplant) had higher percentages of IL-17A+ cells in CD4+ lymphocytes as compared to healthy controls (2.0%±1.08 vs 0.2%±0.06, p=0.006) independently whether they developed or not aGvHD. In contrast to the lower proportion of total CD4+ cells in pts post HSCT then in controls (18.5%±2.1 vs 34.9%±0.7, p=0.0006) proportions of a subset of CD4+ lymphocytes which produced IL-17A+ were rather higher in pts post HSCT as compared to controls (0.2%±0.06 vs 0.07%±0.02, ns). IL-17A+ cells contributed rather to CD4+ then CD4- lymphocytes in controls (0.2%±0.06 vs 0.05%±0.01, p=0.007) as well as in pts post HSCT (2.0%±1.08 vs 0.05%±0.01, p=0.00004) FoxP3 positive cells in stimulated CD4+ lymphocytes were in lower proportions in pts post HSCT then in healthy individuals (51.6%±3.9 vs 62.2%±2.9, ns). Percentages of FoxP3+CD4+ in stimulated lymphocytes (cytometry) correlated with quantities of FoxP3 mRNA in unstimulated PBMC (qPCR) (R=0,42, p=0,065). Notably, there was a negative correlation between proportions of FoxP3 and IL-17A positive cells in CD4+ lymphocytes in patients post HSCT (R= − 0,55; p=0,012). In conclusion, HSCT was associated with: an increase in proportion of Th17 cells, a higher contribution of IL-17A+ cells to the pool of CD4+ cells as compared to healthy individuals and a network in the immune system in which CD4+ FoxP3 + cells and Th17 cells play an opposite role.

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: Thirty-one patients with essential thrombocytosis (ET, F/M: 20/11 age 19-86, median 60 years, platelets: 499-1724x10^3/μl (median: 652x10^3), JAK2 V617F mutation: 26 positive, marrow cellularity: 6.1–184x10^3/μl (median: 31x10^3/μl)) were studied at the time of the diagnostic procedure for the presence in the marrow (i) cells and (ii) some transcripts associated with the immune response. Five techniques were employed: (i) marrow smears analysis, (ii) four color cytometric (BD, San Jose; e-biosciences, San Diego, CA, USA) analysis, (iii) RT-PCR relative quantification of IL-17, ROR gamma t, IFN gamma and CXCL10 mRNA against four housekeeping genes (ABL, HPRT, b-actin, beta 2 microglobulin), (iv) trephine biopsies specimens were in addition to the routine analysis (PAS, HE and reticulin silver stain) stained for CD34, CD15, CD68, CD3, CD4, CD8, IL-17 and (v)immunofluorescence double staining (IL-17/CD15). For transcripts study the ET patients results were compared with those of CML patients (6 patients at diagnosis). Nine healthy volunteers served as a control group for the peripheral blood mononuclear cells IL-17 study. Statistical analysis employed the paired sample Wilcoxon Signed Rank Test and Mann-Whitney U Test, as appropriate. The following observations were made: (i) The marrow lymphocyte population (gated according to the physical parameters and CD45 positivity) differed as compared to blood with respect to: lower CD4+/CD8+ ratio (1.30±0.115 vs 2.125±0.202, p 40 CD8+ cells/HPF), • high proportions of cells positive for IL-17 in 25 patients out of 30 (30 to 180 IL-17+ cells/HPF). Double staining for IL-17 and CD15 documented that IL-17+ cells were in the majority also CD15 positive cells. We conclude: • in the lymphocyte marrow population of ET patients there is an increase of cytotoxic lymphocytes and those of effector/memory cells phenotype what may suggest the presence of an active immune response at the marrow site in at early stage of the disease prior to the treatment, • IL-17 engagement in ET associated processes is suggested by an increase of IL-17+ lymphocytes in blood and high proportions of IL-17 producing cells in the marrow the majority of which are CD15 positive. The latter observation suggests that the innate immunity is involved in initiation of the immune system recognition of ET associated factors. Supported by N N402 43 0039 grant from the Polish Ministry of Science & Higher Education. Disclosures: No relevant conflicts of interest to declare.

Description: Normal karyotype AML patients with FLT3 ITD have a higher risk of relapse than those lacking this mutation (Estey EH, Am J Hematol. 2013). Our comparative genomic hybridization study (77 patients) showed that AML cases with as compared to those without FLT3 ITD had significantly lower number of amplifications or deletions (number of total CNV aberrations: 18±4 vs 51±8, p=0.003). The latter observation in concert with that of others (Thiede, Blood 2002) suggest that FLT3 ITD mutation if present in normal karyotype patients is a key player in the pathogenesis of leukaemia and targeting this mutation may be used successfully in FLT3 ITD positive patients relapsing post HSCT. This was also the case in the two patients presented in this study. Patient UPN 952, male, 53 years old, AML M4 (FAB), normal karyotype, received alloHSCT in 2 CR after completion of two lines of remission induction therapies (first: DA3+7 (Daunorubicin, Ara-C), HAM, high dose Ara-C; second: ICE (IDA, Ara-C, VP-16)) and then promptly transplanted. He relapsed 56 days after transplantation. Patient UPN 938, female, 50 years old, AML, normal karyotype, received as an induction DA 3+7, and for consolidation HAM and high dose Ara-C and completed only one course of the maintenance therapy and transplanted in CR. Both cases were transplanted from unrelated donors (10/10 HLA A, B, C, DR and DQ alleles matched), they received myeloablative conditioning (i.v. Busulfan and Cyclophosphamide) and Cyclosporin A as GvHD prophylaxis. At relapse they received salvage chemotherapy tailored to their biological performance (UPN 938 having Age Adjusted Charlson Comorbidity Index 3 received FLAG and UPN 952 with the index 9, ECOG 3, DA2+5). Both cases received in addition Sorafenib (two times 400 mg per day). The response was prompt and the marrow was free from blasts beginning from 11 day post chemotherapy. UPN 952 received as a maintenance only one course of 6-TG with low dose of Ara-C. Due to the substantial comorbidity and liver toxicity WHO3 further chemotherapy treatment was terminated. The patient was left only on Sorafenib (2 times 200 mg per day). UPN 938 was receiving the maintenance therapy (AML protocol) in 6 – 8 weeks intervals based on low dose Ara-C with 6TG or DNR and also Sorafenib (2 times 200 mg per day). In both cases FLT3 signalling pathway (FLT3 Pathway Mutation PCR Array, SABiosciences, Qiagen) revealed a lack of any additional mutation at the check points in FLT3, KRAS, HRAS, NRAS, MEK1, PIK3CA, BRAF and PTEN genes. UPN 938 had in addition to FLT3 ITD mutation c.1807_1808insATGAATATGATCTCAAAT (p.K602_W603insYEYDLK) insertion which relevance was not so far describe. Sorafenib resistance mutations were not found. The patients were on Sorafenib for 8 (UPN 938) and 9 months (UPN 952). The course of UPN938 was uncomplicated. The second case showed mild aGvHD symptoms evolving into cGvHD (skin lesions and dry eye) slowed down with rapamicine. Up to now the patients are in CR and free from FLT3 ITD. The main observation points: - Sorafenib with chemotherapy tailored to the biologic performance of patients contributes to the efficient salvage in patients with relapsing FLT3 ITD positive AML post HSCT and is also effective given alone as a maintenance. - Side effects were seen in one out of two patients likely associated with the blocking of VEGFR signalling transmission (hand foot skin rash, von Willebrandt factor activity – 388%). Conclusion: The use of multikinase inhibitor (Sorafenib) contributes effectively to salvage therapy in FLT3+ AML patients relapsing post alloHSCTSorafenib given alone is able to maintain long-lasting remissionSide effects are individually dependent Supported by NBiR CellsTherpy grant and Byer Health Care Disclosures No relevant conflicts of interest to declare.

Description: In our previous study (Jaskula et al., Blood 2017 130:1420) we reported that the number of CNVs within the KANSL1 gene was associated with the phenotype of AML. In the present study we looked at the presence of CNV across the whole genome. One hundred and twenty seven AML patients (diagnosed according to the FAB/WHO criteria, F/M=62/65, age median: 57, range: 21 - 84 years) were included to the present study. The patients were genetically analysed including GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). In all patients the microarray analysis of the bone marrow cells having blasts cells (median value 56%) at diagnosis Agilent - Catalog Agilent Cancer CGH+SNP 180K (74 patients) or Roche - WG Catalog NimbleGene 12x270K (53 patients) microarrays were employed and the results were analysed with the use the Partek software employing the Partek Hidden Markov Model (HMM) and segmentation algorithms. Results: The number of CNV in M0 AML marrow cells was significantly lower (median: 0 aberration), as compared to secondary to MDS AML (median: 4.5 aberrations, p=0.006). Patients with AML subtypes from M1 to M6 had higher number of CNV amplifications (median: 2) as compared to the patients with minimally differentiated blasts (M0, median: 0 amplifications, p=0.030). Knowing that (i) changes in the chromatin structure may be associated with the CNV prevalence within the genome (Gheldof et al., PLoS One. 2013 Nov 12;8(11):e79973) and (ii) the aberrant expression of CD19 in AML blasts results from the chromatin structure variations (Walter et al., Oncogene. 2010 May 20;29(20):2927-37) we looked at the presence of association between the numbers of CNV and the presence of the aberrant CD19 expression in the leukemic blasts. It appeared that 11 AML patients having aberrant expression of CD19 (within whom 4 had t(8:21)) had more frequently CNV deletions than those lacking this aberrant expression (median: 2 vs 1 deletions, p=0.018, HMM algorithm). Having the survival curves of the patients plotted accordingly to the high and low numbers of CNV, the situation is more complex and shows that: the patients having higher numbers of CNV aberrations (exceeding the mean of the whole group +SD) enjoyed better survival (20% vs 11%, p=0.090) when segmentation algorithm was employed. HMM analysis also suggested that the higher values of CNV (amplifications, exceeding the mean of the whole group +SD) was associated with better 5-year survival as compared to those with low numbers (42% vs 20%, ns). The aberrant expression of CD19 analysis was associated with higher numbers of deletions (see above) and with better 5-year survival than those lacking this aberrant expression (45% vs 20%, p=0.064). In conclusion, the prevalence of CNV within the genome shapes the phenotype of the leukemia and facilitates the survival. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5155 Patients with newly diagnosed essential thrombocythemia (ET) were investigated for selective accumulation/expansion of CD4+ lymphocytes subpopulations in the marrow, to verifie the hypothesis that the neoplastic process associated factor(s) are recognized by the immune system. Sixteen patients (F/M: 12/4, age 28–72 - median 60 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets from 496–809 x 10̂3/ul (median 644 × 10̂3 ), (12 heterozygotes, 2 homozygotes). Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters were seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Marrow cellularity was from 9 – 126 x10̂3/ul (median 42 × 10̂3/ul). The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, UmarSA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. Immunostaining of trephine biopsies was used for detection of FOXP3 cells. It was found: (i) Lymphocytes in the marrow were in proportions from 4,5%-15,7% (median 7,25%), in numbers from1377/ul to 8245/ul (median 3870/ul) what was significantly higher as compared to blood from 1300/ul to 3900/ul (median 2100/ul) (Wilcoxon Matched Pairs Test, p=0.010). (ii) CD45RO+CCR7- lymphocytes were higher in numbers in the marrow aspirates as compared to blood (median 1281/ul vs 631,2/ul, p=0,04 Wilcoxon Test for pairs). (iii) In contrast numbers in the marrow aspirates of CD45 RO-CCR7+ (median 297,4/ul vs median 238,2/ul), as well as CD45 RO+CCR7+ (median 149,6/ul vs 146,4/ul) did not differ from those in blood. (iv) It was a significant increase of CD4+CD25high+ cells in the marrow aspirates as compared to blood (median 36,2/ul vs 9,1/ul; p=0,02 Wilcoxon Test for pairs). Indeed in 11 out of 14 trephine biopsies investigated FoxP3+ cells were identified. In conclusion an increase, in the marrow of ET patients, of effector/memory CD4 lymphocytes (CD45RO+CCR7-) but not those of naïve and central memory lymphocytes phenotypes points out on the selective accumulation of these cells in the marrows. Concomitant increase of T regulatory cells (CD4+CD25+high) suggests that an active immunological is taking place at the site. It is possible that effector memory cells are attracted to come to the marrows by ET process associated factors and if so the present observation constitute the primary observation building the rationale behind immunotherapy in ET patients Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic stem cell transplantation offers complete remission in CML patients. However, transplant related mortality and long term consequences especially associated with cGvHD make patients reluctant to accept this treatment modality especially in the era of Imatinib. Therefore, in 1999 we started a transplant program for CML patients characterized with a reduced dose of Busulfan to 8 mg/kg of. b.w., Fludarabine (120 mg/m2 in sibiling and 150 mg/m2 in MUD setting) and ATG to facilitate engraftement. At the beginning ATG cumulative dose was 40 mg/kg of b.w. (5 patients), or 20 mg/kg of b.w (13 patients). In this high dose ATG group 6 patients had CMV or EBV reactivation what prompted us to decrease ATG dose to 2.5 mg/kg of b.w. given in three consecutive days and then on day 4 the dose was increased to 5 mg/kg of b.w. if patients lymphocyte count did not reach the target values (≤300/ul of WBC and 〈 0.1% of CD3+ cells in nucleated blood cells population). With the use of the above program 52 adults and two children (F/M: 22/32, age from 11 to 55 yrs (median: 34) were transplanted from sibling (20 patients) and alternative (33 MUD and 1 related haploidentical) donors. Thirty nine and 15 patients received PBPC and marrow, respectively. Seven patients died by + 100 days post transplant (6 infectious complications including 1 case with viral encephalitis and 1 blast crisis). One patient had marrow failure, 15 patients developed ≥ IIo aGvHD, 23 patients had cGvHD and 14 of them extensive form. Thirty four patients (63%) are alive with an observation time from 1 to 81 months post transplant (median 22 months). Survival was better in sibling than in MUD transplanted patients (77% vs 50%, p=0.03, Cox analysis). In alive patients group STR technique proved complete chimerism in 26 out of 31 examined patients. Five cases lacking full chimerism include: 1 patient with hematological relapse, and 4 with molecular relapse. All other cases were bcr/abl negative. In conclusion: low dose ATG was sufficient to secure the take and the employed reduced intensity conditioning was associated with: low rate of transplant related mortality, aGvHD was infrequent and mild, cGvHD was the main cause of late complications, full chimerism and bcr/abl free status were observed in a vast majority (84%) of long term survivors.