Blood

The ecotropic viral integreation site 1 (EVI1 or MECOM) gene mapping to chromosome 3q26 exerts significant oncogene biological effects of anti-apoptosis, proliferation and differentiation on hematopoietic cells. It is considered as a poor prognostic factor in acute myeloid leukemia (AML). EVI1 positive AML accounts for about 10% of de novo AML pts. The remission rate and long time survive are poor. Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) could salvage such kind of malignancy is still unknown. We conducted a survey of 161 AML pts (median age, 35 years[y]; range 8-60y) after first complete remission undergoing myeloblastic allo-HSCT at the first affiliated hospital of Soochow University. Source of hematopoietic stem cells came from match or partial matched related, unrelated or umbilical cord blood (UCB) donors (Table 1). Pretreated specimens were obtained and real-time polymerase chain reaction was performed to detect expression of EVI1-1d (one major splice of EVI1) and common EVI1 (cEVI) (normalized to PBGD gene). Standard plasmids of EVI1 and PBGD were prepared as calibrators. 13 nonmalignant single lineage cytopenia pts were tested as baseline. Quantities of EVI1-1d and cEVI ranges from negative to 154.2%PBGD and negative to 1214.3%PBGD in each group. Above five and ten folds to baseline in EVI1-1d and cEVI respectively were considered as positive. Accordingly 27 pts were EVI1 positive while 134 pts were negative (16.8% vs 83.2% separately). Unfavorable cytogenetic abnormalities showed a high proportion in EVI1 pos (11/27) than neg group (12/134). 5 and 40 mutations were detected in each group. Three cytogenetic abnormality concerning 11p15 pts expressed high level of EVI1.

Positive expression of EVI1 indicated significant shorter progress-free survival and overall survival (P= 0.0352 and 0.0436 respectively) comparing with negative group (Figure 1).

Table 1. Sources of donors Matched (n) Partial mathed (n) siblings 82 5 Parents or sons/daughters / 30 Unrelated PBSC 26 10 Unrelated UCB 3 5

Figure 1 Figure 1.

Figure 2 Figure 2.

Progress-free Survival (upper) and Overall Survival (below) in myeloblastic allo-HSCT pts according to EVI1 expression

In conclusion, high level of evi1 expression predicts worse prognosis even after myeloblastic allo-HSCT. New treatment strategies post allo-HSCT are needed to improve long time survival in such kinds of pts.

No relevant conflicts of interest to declare.

Introduction: CALLISTO, a comprehensive programme of research on cancer-associated thrombosis (CAT) , included 3 randomized trials of rivaroxaban versus low molecular weight heparin (LMWH) for the treatment of venous thrombosis in patients with solid and haematological cancers (SELECT-D, CASTA-DIVA and CONKO-11). A meta-analysis of these studies was conducted to improve the precision of current estimates of the efficacy and safety of rivaroxaban in this patient group and investigate how patient characteristics impact the treatment effects.

Methods: The primary endpoint was the cumulative incidence of venous thromboembolism (VTE) recurrence at the end of the treatment period (≥3 months). Other endpoints included major bleeding (MB), a composite of MB or clinically relevant non-major bleeding (clinically relevant bleeding [CRB]) and deaths from any cause. All endpoints were assessed by 8 pre-defined subgroup analyses: age, gender, creatinine clearance, type of index VTE, index VTE localization, cancer localization, performance status and presence of metastases (Prospero submission 266227). Patient-level data were used in this analysis.

The cumulative incidences of VTE recurrence, CRB were estimated using the Kalbfleisch and Prentice model, while the Kaplan-Meier model was used to estimate the incidence of death. Comparisons between rivaroxaban and LMWH for VTE recurrence, MB and CRB were assessed by sub-distribution hazard ratios (SubHR) and 95% confidence intervals (CI), whereas hazard ratios and 95% CIs were used for the all-cause death endpoint. The pooled treatment effect size of each study was estimated using fixed-effect and random-effects models.

Results: When considering the prevention of VTE recurrence in the 3 randomized trials (N=804), an overall reduction of 48% was observed with rivaroxaban compared with LMWH (SubHR = 0.52, 95% CI 0.28─0.98). The estimation appeared to be homogeneous across subgroups of patients. In comparison with LMWH, rivaroxaban was associated with an increased risk of CRB (SubHR = 2.03, 95% CI 1.34─3.09), without significant difference in MB (SubHR = 1.24, 95% CI 0.60─2.57), and no difference was observed for death.

Conclusions: This pooled analysis suggests that rivaroxaban may be an alternative treatment option for the prevention of VTE recurrence in cancer patients with VTE. The gain in statistical power has shown significant benefit, as well as some risk associated with rivaroxaban treatment in this complex patient population. The impact of patient characteristics on these treatment effects will be presented at the meeting.

Laporte: Bayer Healthcare: Other: personal fees and non-financial support; Pfizer: Other: non-financial support; LEO Pharma: Other: non-financial support. Marshall: Bayer: Research Funding. Young: BMS/Pfizer Alliance: Honoraria; Leo Pharma: Honoraria; Chugai: Honoraria; Bayer: Honoraria, Research Funding. Riess: Bristel Myers Squibb: Honoraria; Bayer: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; ASPEN: Honoraria; Leo Pharma: Honoraria; Pfizer: Honoraria. Sinn: BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Pfizer: Honoraria; Servier: Consultancy, Honoraria, Research Funding; Astra Zenica: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; MSD: Consultancy, Research Funding; Sanofi: Consultancy; Bayer: Research Funding. Girard: Bayer Healthcare: Other: Personal fees, Research Funding; LEO Pharma: Other: Perconal fees, Research Funding. Sanchez: BAYER: Other: reports grants, personal fees and non-financial suppor; BMS: Other: grants, personal fees and non-financial support; PFIZER: Other: personal fees and non-financial support; BOEHRINGER INGELHEIM: Other: personal fees and non-financial support; CHIESI: Other: personal fees; BOSTON SCIENTIFICS: Other: grants and personal fees.

Abstract 1906

Reactivation of latent viruses such as cytomegalovirus (CMV) and adenovirus (AdV) is responsible for infections which may be life-threatening in HSCT recipients. In the post-transplantation period, severity and frequency of these infections depend on (a) the degree of donor-recipient HLA incompatibility and (b) the intensity of immunosuppressive therapy used to prevent immunological complications. Antiviral drugs may be partially effective, often toxic and cannot always control those viral infections.T cell immunity plays a major role in the control of viral infections. It has been demonstrated that the transfer of donor T lymphocytes specifically directed against viral antigens is capable of preventing, controlling and clearing viral infection (Feuchtinger T et al., 2004 and 2010). The present project aimed the evaluation of specific, cell-based immunity against CMV and AdV by injection of IFN-g-positive CD4+and CD8+ donor T lymphocytes isolated ex vivo after stimulation with viral peptides.

Our protocol was designed for pediatric or adult patients treated by allogeneic HSCT and matching the following inclusion criteria: (1) biological and/or clinical symptoms of CMV and/or AdV infection 2) no response or contraindication to conventional antiviral treatment and (3) no or low grade pre-existing aGvHD at inclusion (≤ grade II) controlled by corticoids (<1 mg/kg). Antiviral treatments are allowed during the inclusion period.

Donor IFN-g-positive T lymphocytes are isolated with the CliniMACS Cytokine Capture System (Miltenyi Biotech) after incubation with viral peptide pools.

Primary evaluation criterion is the efficacy of the treatment on CMV viral load 21 days after the first injection. In the event of a negative or partial response and the absence of aGvHD, a second injection may be scheduled.

Secondary evaluation criteria are (1) the occurrence of de novo aGvHD or aggravation of existing aGvHD, (2) the evolution of clinical symptoms potentially related to the infection, (3) the demonstration of biological in vivo expansion of injected T lymphocytes (as evidenced by the IFN-g secretion capacity and specific tetramer assays) and (4) for AdV infection, evaluation of efficacy (viral load, in vivo expansion of transfused lymphocytes, clinical symptoms) and the safety (occurrence of aGvHD) of this immunotherapy.

From September 2010 to July 2012, 9 patients were included: 3 male adults (46–54 years, 1 CLL, 1 CML and 1 AA, 2 geno- and 1 pheno-identical transplantation) and 6 children (age: 7–25 months, sex ratio F/M: 4/2, 4 FLH, 1 SCID and 1AA, 4 haplo, 1 geno- and 1 pheno-id transplantation).

4/9 patients were treated for CMV, 3/9 for AdV and 2/9 for CMV and AdV reactivation. 5/9 patients received 2 cytotoxic T lymphocytes (CTL) injections. Mean number of CD3 IFN-g positive cells injected was 4206/kg (1167–6000/kg) with 55% and 69% of CD4 and CD8 anti CMV-T cells and 56% and 61% of CD4 and CD8 anti AdV T cells respectively.

Mean delay of first immunotherapy was 109 days (28–270) after transplantation.

2/9 patients were not evaluable due to early death (<21 days post injection) and 1/9 patient died of graft failure 43 days after CTL injection without efficacy on infectious evolution. 6 patients are still alive: 4 with complete, 1 with partial remission of virus replication and 1 recently included, is still under evaluation. An in vivo expansion of transfused CTL was observed (mean expansion was 33 and 35 fold for CD8-IFN-g and CD4-IFN-g positive cells respectively 42 days after injection) in parallel with the decrease of viral load in all alive patients. No aGvHD was detected in the 5/6 evaluated patients. One of 6 presenting cGvH at inclusion need increase of corticotherapy 3 months after second injection of CTL One patient presenting with CMV retinitis received 2 CTL injections without worsening of retina lesions which healed.

The CliniMACS Cytokine Capture System allows the isolation of virus-specific T cells in a brief delay (24 hours) with a satisfactory enrichment of both CD4 and CD8 T cells.

First results show efficacy of virus-specific T cells injection on viral load without signs of aGvHD in 5/6 evaluable patients. More patients need to be included in this trial in order to confirm these encouraging results.

Cambouris: Miltenyi Biotec: Employment.

Background: Human leukocyte antigen (HLA) matching between donor and recipient is a key part of successful allogeneic hematopoietic cell transplantation (allo-HCT). The HCT from the unrelated donor (UD) with one allele/antigen mismatch (MM) can be as beneficial as HCT from perfectly matched donor. For the remaining patients, the donors with permissive mismatches may be the option. In HLA-mismatched transplantation, the patient and donor can also be mismatched for their killer cell immunoglobulin-like receptor (KIR) ligands that recognize allotypic determinants shared by certain HLA class I allele groups. Recent research has accumulated evidence of the role of each HLA locus and KIR ligand MM on clinical outcomes for UD-HCT. However, HCT outcomes of the patients with permissive MM depending on KIR ligand MM (KIR-L-MM) status remain obscure in UD-HCT.

In the current study, we identified permissive and nonpermissive MM allele combinations and analyzed the effects of these mismatches in combination of KIR ligand mismatches in patients with acute myeloid leukemia (AML).

Methods: A total of 438 patients with AML who underwent allo-HCT from UD from 2007 to 2014 were analyzed. Alleles of patients and donors at the HLA-A, -B, -C, and -DRB1 loci were identified by the high resolution DNA typing. Nonpermissive HLA allele combinations were defined as a significant HLA risk factor for severe acute graft-versus-host disease (aGVHD). KIR-L-MM among patient-donor pairs were searched in the Immuno Polymorphism Database available at www.ebi.ac.uk/ipd/kir.

Results: Median age of the patients was 45 (range 15-60) years and 117 patients (40.4%) were female. Eighty-five (19.4%) patients were high risk at the time of HCT. Reduced intensity conditioning was performed in 131 patients (29.9%) and anti-thymocyte globulin was used in 324 patients (74.0%). Primary graft source was peripheral blood stem cells (n=369, 84.2%) and median 6.0 x 106/kg cells were infused. Severe aGVDH occurred in 43 patients (9.8%) and chronic GVHD (cGVHD) in 193 (44.1%). With median follow-up duration of 19 (range, 2-96) months, treatment-related mortality (TRM) occurred in 111 patients (25.3%), relapse in 119 (27.2%) and death in 214 (48.9%).

Two-hundred sixty-four patients (60.3%) were HLA full matched in the 4 loci. Mismatches in HLA-A loci observed in 64 patients, HLA-B in 35, HLA-C in 98, and HLA-DRB1 in 60. Five nonpermissive MM pairs in 33 patients were identified as donor/patient pair: A*02:06/A*02:01, C*03:03/C*08:01, C*08:01/C03:04, C*08:01/C*15:02, and DRB1*04:03/DRB1*04:05. Among 98 patients with HLA-C loci MM, 16 patients showed KIR ligand MM (KIR-L-MM) as GvH direction, which was observed in the permissive MM group.

Severe aGVHD occurred in 30.4%, 22.4%, 13.4%, and 10.8% in nonpermissive, permissive MM and KIR-L-MM, permissive MM and KIR-L-M, and full match group, respectively (p=0.003). The 3-year overall survival (OS) rate was inferior in permissive MM and KIR-L-MM group (30.0%) compared to full match (53.5%), permissive MM and KIR-L-M (51.8%), and nonpermissive (42.4%) group (p=0.067). The 3-year TRM was higher in permissive MM and KIR-L-MM group (57.5%) than full match (21.0%), permissive MM and KIR-L-M (27.7%), and nonpermissive (33.3%) group (p=0.006). In the multivariate analysis, high risk at HCT (HR 2.087, p<0.001), severe aGVHD (HR 3.851, p<0.001), and cGVHD (HR 0.321, p<0.001) were identified as variables affecting the OS. The following variables adversely affected on TRM: permissive MM and KIR-L-MM group (HR 2.699, p=0.007), severe aGVDH (HR 2.204, p=0.001), and cGVHD (HR 2.052, p<0.001). Non-permissive MM (HR 7.487, p=0.001) and CD34+ cells >6x106/kg (HR 4.113, p=0.017) were high risk factors on severe aGVHD.

Conclusion: Permissive MM for HLA could be further classified into high risk groups with regard to TRM by KIR-L matching in UD-HCT. The evaluation of KIR-L matching is warranted to reduce unfavorable outcomes among the patients with permissive MM in UD-HCT.

Figure 1 Figure 1. Figure 2 Figure 2.

No relevant conflicts of interest to declare.

Nền tảng: Chế độ ăn dinh dưỡng nguyên tố (ED) là dạng dinh dưỡng lỏng dễ hấp thu, bao gồm các axit amin, đường, vitamin và khoáng chất. Nó giúp giảm tải tiêu hóa từ ruột và chủ yếu được sử dụng để hỗ trợ dinh dưỡng qua đường ruột trong điều trị bệnh nhân mắc bệnh Crohn. Các tác nhân kiềm hóa liều cao và chiếu xạ toàn thân (TBI) thường được sử dụng trong các chế độ điều trị chuẩn bị ghép tế bào gốc huyết học (HSCT) được biết đến là gây ra độc tính tiêu hóa nghiêm trọng, bao gồm viêm niêm mạc miệng, làm cản trở việc tiếp nhận thức ăn của bệnh nhân. Dinh dưỡng không đủ, yêu cầu dinh dưỡng qua đường tĩnh mạch hoàn toàn (TPN) làm kéo dài thời gian nằm viện và có thể dẫn đến nhiễm trùng đe dọa tính mạng do sự di chuyển vi khuẩn qua các màng niêm mạc hỏng.

Mục tiêu: Theo thông tin chúng tôi biết, chưa có báo cáo nào về hiệu quả của ED ở bệnh nhân ghép tế bào gốc huyết học. Chúng tôi đã tiến hành một nghiên cứu đoàn hệ tiến cứu để xác định liệu ED có cải thiện kết quả điều trị của bệnh nhân không.

Bệnh nhân: Chúng tôi đã tuyển chọn 73 bệnh nhân liên tiếp thực hiện HSCT từ tháng 3 năm 2011 đến tháng 3 năm 2013 tại Bệnh viện Anjo Kosei. Năm mươi bệnh nhân đã thực hiện HSCT đồng loại, và 23 bệnh nhân đã thực hiện HSCT tự thân. Nguồn gốc của HSCT đồng loại bao gồm tủy xương không liên quan (n=16), tế bào gốc máu ngoại vi liên quan (n=16), và máu cuống rốn không liên quan (n=18). Độ tuổi trung vị là 47 tuổi (khoảng, 17-72 tuổi). Có 41 bệnh nhân nam và 32 bệnh nhân nữ. Bệnh nhân mắc bệnh bạch cầu (n = 38), u lympho ác tính (n = 24), đa u tủy (n = 7), và các loại khác (n = 3). Tại thời điểm ghép, 52 bệnh nhân đạt tình trạng thuyên giảm hoàn toàn (CR), 21 bệnh nhân không đạt CR. Hai mươi mốt bệnh nhân được ghép trước tháng 11 năm 2011 không nhận ED (nhóm không dùng ED; NEG), và 52 bệnh nhân ghép sau tháng 11 năm 2011 được nhận ED như là liệu pháp tiêu chuẩn (nhóm ED; EG).

Phương pháp: Việc sử dụng ED được bắt đầu từ ngày -8 đến ngày -4 cho tất cả bệnh nhân khi các chế độ chuẩn bị cho HSCT bắt đầu, và kết thúc vào ngày +28 sau HSCT. Liều khuyến nghị là 80g (1255 kJ) hàng ngày cho tổng lượng ED qua đường miệng. Chúng tôi không loại trừ việc tiêu thụ sau khi việc sử dụng ED không thành công, việc tiêu thụ thực phẩm bổ sung và dinh dưỡng qua đường tĩnh mạch hoàn toàn (TPN). Thực hành tiêu chuẩn của chúng tôi cho HSCT đồng loại là bắt đầu TPN vào ngày -1 và tiếp tục cho đến khi bệnh nhân đạt lượng calo ghi nhận là 6279 kJ mỗi ngày sau khi được cấy ghép. Đối với HSCT tự thân, quy trình của chúng tôi là bắt đầu TPN khi bệnh nhân không đạt được lượng calo ghi nhận là 6279 kJ mỗi ngày qua việc tiêu thụ thực phẩm và tiếp tục cho đến khi đạt mức này. Nghiên cứu này đã được phê duyệt bởi ủy ban xét duyệt của chúng tôi.

Abstract 3192

Iron-induced cardiotoxicity remains the leading cause of morbidity and mortality in patients with transfusion-dependent thalassemia major (TM). Heart failure in these patients, which may be reversible but has a poor prognosis, is characterized by myocardial iron deposition-related early diastolic dysfunction. N-terminal-proBNP (amino-terminal) (NT-proBNP) is a sensitive biomarker for the detection of asymptomatic left ventricular (LV) dysfunction.

Patients and Methods: In this study, we prospectively evaluated plasma NT-proBNP levels determined on the Roche cobas e411immunoassay analyzer using electrochemiluminescence technique in 187 adult patients aged 19–54 years with TM. Possible correlations with the proposed recently cardiac iron concentration [Fe] based on an equation derived from heart T2* assessment by MRI: [Fe] = 45.0 × [T2*]−1.22 with [Fe] in milligrams per gram dry weight and T2* in milliseconds (Carpenter et al Circulation 2011;123;1519–1528) were explored.

143 patients had low cardiac hemosiderosis, defined as [Fe] < 1.1 mg/g dry weight, corresponding to T2*>20 milliseconds and 44 patients with high cardiac hemosiderosis, defined as [Fe] >1.2 mg/g dry weight. The main results of the study showed that: a) NT-proBNP levels were markedly increased in thalassemic patients (152.2 26.4 pg/mL, ranged from 6.0 – 1336.0 pg/mL compared to normal control levels 40.1 19.7 pg/mL, p<0.001, b) NT-proBNP levels were significantly higher in high cardiac hemosiderosis patients compared to low cardiac hemosiderosis patients (185.1 78.2 vs 128.9 20.2 pg/mL, p<0.05), c) NT-proBNP levels correlated with [Fe] values (r= 0.387, p<0.001). This correlation was significant in patients with high cardiac hemosiderosis (r= 0.520, p<0.001), but not in patients with low cardiac hemosiderosis (p>0.1), and d) no significant correlation was found between NT-proBNP levels and left ventricular ejection fraction (LVEF) values, (p>0.3).

Our study demonstrate for first time the significant association of NT-proBNP levels and cardiac iron concentration in patients with TM linking blood chemistry and imaging techniques. Multicenter studies of these parameters during iron chelation therapies are needed to validate their association and further exploit its clinical use.

Kattamis: Novartis: Honoraria, Research Funding, Speakers Bureau. Off Label Use: Wording to add.

Paraproteins are found in 5–10% of CLL patients using conventional techniques and in a higher number using more sensitive techniques. The significance of this finding is uncertain although it has been suggested to be associated with a worse prognosis. When a paraprotein occurs with CLL it is usually considered to be a product of the leukemic clone. However there is an increased incidence of both B cell clonal expansions and monoclonal immunoglobulins (Igs) in the elderly suggesting an alternative source may exist.

We examined the clinicopathological features of 34 cases of paraproteins who had an immunophenotype consistent with CLL (CD5+ B cells and CD23+ if tested). These were untreated patients who had an elevation of one or more immunoglobulins (Igs) on routine screening and subsequently had immunofixation (IF) to determine the presence of a paraprotein.

In a database of chronic lymphoproliferative disorders 1380 patients had Ig quantitation and 168 were found to have an elevation in one or more Igs. Cases were excluded from this group if the disease was found to be T cell, CD5- or CD23-. This left 116 CLL patients with elevated Igs, of which 53 had IF. A polyclonal increase was detected in 19 and paraproteins in 34 (14 IgG, 16 IgM, 1 IgA and 3 oligoclonal). The level of paraprotein ranged from 0.2–4.4 g/dl for IgG, 0.2–2.4 g/dl for IgM and was 0.4 g/dl for IgA. Bence Jones Protein was associated with both IgG and IgM paraproteins when tested (2 patients in each group). Suppression of other Igs was observed in 12 patients (35%) with paraproteins and only one patient (5%) with a polyclonal increase.

When compared to patients with a polyclonal increase in Igs, the patients with paraproteins had more advanced disease and higher bone marrow lymphocytosis, β2-microglobulin and LDH (p<0.05). The immunophenotype in approximately half of the cases in both groups was atypical for CLL with features including CD22+, CD79b+, FMC7+ and moderate to strong expression of surface Ig. Cytogenetic abnormalities were present in 8 of 34 cases with paraproteins but were not detected in the polyclonal group. The most frequent abnormality was trisomy 12 found in 4 cases. The survival of the 2 groups was not statistically different with a median follow up of 104 months.

The origin of paraproteins is usually considered to be the CLL clone with CLL cells capable of secreting IgM as well as producing IgG and IgA paraproteins by isotype switching. In this cohort 5/14 patients with an IgG paraprotein had a different light chain expressed on the CLL clone. In addition 3/16 patients with IgM paraproteins had biclonal IgMs and in one case the 2 Igs had different light chains suggesting that at least one was not a product of the CLL clone. In conclusion, paraproteins in CLL are frequently not the product of the CLL clone but may reflect an associated age-related restriction in B cell repertoire and existence of other clonal expansions. Further studies are needed to determine if CLL emerges from one of these clones or develops independently.

We developed a mouse monoclonal antibody (MoAb 115–21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115–21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115–21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115–21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115– 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115–21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115–21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.

Allogeneic hematopoietic stem cell transplantation is an effective method for treatment of hematological malignancies, while GVHD and graft rejection are main complications, which seriously affect patients' survival rates and quality of life.

Establishing allo-transplantation mice model with mRFP and GFP transgenic mice, to simulate clinical hematopoietic stem cell transplantation and explore the mechanism of stem cell homing and GVHD.

1) Thirteen C57BL/6 GFP transgenic mice, used as recipients, were irradiated with 7 Gy. Each mouse was injected through caudal vein with 2*106 bone marrow cells isolated from FVB mRFP transgenic mice. 2) Symptoms like weight loss, depilation, diarrhea were observed as GVHD manifestation while survival rates were evaluated. Routine blood test and FACS were performed at different time points to confirm hematopoiesis reconstitution. 3) Mice were perfused with paraformaldehyde under anesthesia to fix the tissue, while pathological examination and real-time PCR were performed for studying donor and recipient cells interactions in different organs. 4) Semi-solid decalcification was used to treat the femora before observing under confocal microscope directly or after making frozen section, three-dimensional reconstruction were made to observe the cellular interaction, especially for cells within the bone marrow.

1) Depilation, wrinkled skin, hunchback and sharp decline of weight were observed in 8/13 mice. Routine blood test implicated hematopoietic reconstitution. FACS showed 86.1%±7.8% mRFP+ cells in peripheral blood of recipients. 2) mRFP+ cells were found distributing throughout the body's organs. mRFP+ Lymphocyte infiltration and inflammatory exudate were seen especially in the small intestine, lung, liver and skin (Fig.1). GFP+ cells were found surrounding mRFP+ cells in the bone marrow of the femora decalcified with semi-solid decalcification. Their interactions can be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction by confocal microscope (Fig.2).

Owing to RFP on donors' cells and GFP on recipients' cells, together with our novel protocol named semi-solid decalcification, we can visually observe the donor and recipient cells' location, ratio and cellular interaction, as well as morphological changes. Within various tissues especially for such tissues as bone marrow and lung, the details between cells can be studied lively by fluorescence microscope and confocal microscope. In recipient mice with GVHD, donor cells can be found in various target tissues such as intestine, lung, liver and skin. Gene marked cells with fluorescence protein can benefit morphological, immunological, cytogenetic and molecular studies in recipients after HSCT.

The allo-transplantation model with mRFP and GFP transgenic mice is powerful in study of Stem Cell Homing and Donor-Recipient Cellular Interaction. The cellular interaction can be easily observed by three-dimensional reconstruction after semi-solid decalcification, especially for bone marrow and lung.

No relevant conflicts of interest to declare.

In a 10-year study of T-cell acute lymphoblastic leukemias (T-ALL) in children, we have identified five cases expressing the T-cell receptor tau delta (TCR tau delta). The incidence (26%) of TCR tau delta+T-cell leukemias in our material was high. Clinically, the TCR tau delta+ leukemias represented a distinct subgroup of T-cell leukemias. Mean age at onset of disease, 1.8 years, was remarkably low for mature T-cell leukemias. White blood cell counts were high, lymph node enlargements were discrete, and no mediastinal tumors were seen. Four of five TCR tau delta+ leukemias carried rearrangements of the C tau 2 gene, and transcribed the T-early alpha genetic element.