ALBERT

Description: Background: Graft-versus-host disease (GVHD) represents a major hurdle impeding the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT). JAK3 plays pivotal roles in the initiation of cytokine-triggered signaling transduction. Recent studies about the murine GVHD model show that targeting JAK3 in donor lymphocytes with a chemical inhibitor such as WHI-P131 may be useful in the prevention of severe GVHD. However, the level of JAK3 expression in patients with GVHD has not been reported. The current study is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using Taqman Probe for detecting JAK3 in allo-HSCT recipients. Methods: 20 patients who received myeloablative-conditioning regimen for hematological malignancies and cyclosporine for GVHD prophylaxis have been investigated in this still ongoing study clinically. One of these patients developed acute GVHD (aGVHD), 9 patients developed chronic GVHD (cGVHD), and 10 patients showed no evidence of GVHD during the first 365 days post-transplant. RNA was extracted from white blood cells and cDNA templates were synthesized using 1μg RNA. The standard curve method was used for relative quantitation of JAK3. Results: There is a clear trend for a increasing expression of JAK3 in 9 patients with GVHD. Interestingly, a significant decrease of JAK3 gene expression when the severity of GVHD was remission (P=0.04). One of the patient with cGVHD, the level of JAK3 gene expression has no changed before and after treatment. However, these patients post HSCT without GVHD have a comparable level of JAK3 expression before myeloablative conditioning regimen(P〉0.05). Conclusion: Based on our preliminary results, JAK3 mRNA level was obviously correlated with the development of GVHD. Analysis JAK3 mRNA expression may be useful in evaluating GVHD patients with allogeneic stem cell transplantation.

Description: Purpose To observe the immunological responses of recipients to infused lymphocytes from donors of different genetic backgrounds. Methods Mouse models were set up by mating female BALB/c mice with male C57BL mice. Splenic lymphocytes from donors of different genetic backgrounds were labeled with CFSE, and 1∙107 of the cells were intravenously injected into a recipient. At 6, 24, 72 and 120 h after the infusion, mononuclear cells in recipient spleen, liver, thymus, lymph node and peripheral blood were collected, and CFSE+, CFSE-, CD3+, CD8+, CD4+, CD19+, NK1.1+, CD25+, CD127+ lymphocytes in these samples were analyzed by flow cytometry. The distribution of donor T cells, B cells, NK cells, helper T cells, cytotoxic T cells, and recipient regulatory T cells in these tissues were then obtained. Results Maternal lymphocytes were more likely to survive in offspring. At 120 h after the infusion, the ratio of maternal cells was 0.52¡À0.11% in lymph node, 0.97¡À0.04% in peripheral blood, and 0.97¡À0.11% in spleen of the offspring. In aunt to child, father to child, and unrelated allogeneic groups at 120 h after the infusion, few donor cells, if any, were detected in these tissues. The subtype proportion of donor lymphocytes changed significantly in recipient tissues. Recipient Treg cells increased in mother to child group but not in aunt to child, father to child, and unrelated allogeneic groups, suggesting the decrease of cellular immune responses to allogeneic cells in mother to child group. At 120 h after the infusion, no donor cells were detected in recipient liver and thymus in all groups, implying that donor cells colonized hardly in liver and thymus. Conclusion Specific immune tolerance to maternal lymphocytes existed in offspring. Donor lymphocyte infusion of maternal origin may obtain a relatively persistent effect of adoptive immunotherapy with less side-effect. Figure 1. CFSE labeled donor splenic lymphocytes were infused to recipients of different kinships Figure 1. CFSE labeled donor splenic lymphocytes were infused to recipients of different kinships Black is male C57BL£¨H-2b£©mouse; white are female BALB/c mice (H-2d); the gray is offspring produced by mating C57Bland BALB/c. The lymphocyte infusion relationship between donor and recipient: ¢Ùmother to child group; ¢Úfather to child group; ¢Ûaunt to child group; ¢Üunrelated allogeneic group; ¢Ýsyngeneic group. Figure 2. The donor cells distribution according to different tissues in a case of mother¡¯ to child group infusion after 120 hours. Figure 2. The donor cells distribution according to different tissues in a case of mother¡¯ to child group infusion after 120 hours. M1 are CFSE-positive cells; M2 are the total cells. A. spleen£¨0.97¡À0.11%£©; B. liver£¨0.20¡À0.06%£©; C. thymus£¨0.01¡À0.01%£©; D. lymph nodes£¨0.52¡À0.11%£©; E. peripheral blood£¨0.97¡À0.04%£© Figure 3. Distribution of Treg cells at 120 h after infusion Figure 3. Distribution of Treg cells at 120 h after infusion Proportion of Treg cells in lymph node, peripheral blood and spleen. Treg cells increased significantly in lymph node, peripheral blood and spleen in mother to child group, as compared with those in aunt to child group, unrelated allogeneic group and control group. Disclosures No relevant conflicts of interest to declare.

Description: Background: Based on the cells' antigen differentiation expression patterns, most cases of acute leukemia (AL) are classified as either myeloid or lymphoid lineage. However, there are patients with leukemic blast population that co-express both lymphoid and myeloid characteristics, known as biphenotypic acute leukemia (BAL) or mixed-phenotype acute leukemia (MPAL). BAL is a rare subgroup of acute leukemia with a poor prognosis. Currently, a standard chemotherapy treatment has yet to be established. Aims: This study aims to retrospectively investigate the incidence, pathological characteristics, and clinical outcome of BAL patients from the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China, between January, 2014 and June 2019. Methods: From January 2014 to June 2019, the medical records of newly diagnosed BAL based on the EGIL criteria, or ALAL based on the 2008/2016 WHO criteria and who were admitted at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) were retrospectively reviewed. The diagnostic workup of BAL was based on initial morphological, cytochemical, and immunophenotypic evaluation of the BM. Using the EGIL scoring system and 2008/2016 WHO criteria, treatment methods and outcome data, including induction chemotherapy, complete remission (CR), relapse, and death, were collected and reviewed. This study was an observational, retrospective, and descriptive study of the clinical aspects of BAL, which was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Results: Among a total of 6100 newly diagnosed patients with AL, 10 (0.16%) patients satisfied the definition of BAL based on the EGIL criteria, or MPAL based on the WHO criteria, including 7 males and 3 females. The median age of these patients at diagnosis was 19 years (range 3-67 years). One patient (Pt #1) had extramedullary invasion, including neck, mediastinum (area 8), posterior septal group, left axillary, peritoneal and retroperitoneal lymph nodes. Another patient (Pt #7) had extramedullary invasion with central nervous system leukemia. Immunophenotypic characteristics showed that among 10 BAL patients, 4 cases carried B/Myeloid phenotype, 4 cases carried T/Myeloid phenotype and 2 case carried T/B phenotype. For 8 patients with myeloid lineage differentiation, MPO was positive in 6 (75%), CD13 in 4 (50%), CD33 in 4 (50%), CD38 in 6 (75%), CD58 in 3 (37.5%), CD117 in 4 (50%) patients. In 5 patients with B-lymphoid lineage differentiation, CD19 was positive in 4 (80%), CD79a in 5 (100%) patients. The most frequently T-lymphoid lineage positive markers were CD7 and cCD3, which were positive in 4 of 5 (80%) patients. The stem cell markers HLA-DR and CD34 were both positive in 7 (7/10, 70%) patients, while CD117 was positive in 4 (4/10, 40%) patients. Cytogenetic analysis results showed that 7 of 10 patients had normal karyotypes (Pt#1, 2, 6, 7, 8, 9,10) while the other 3 patients had clonal abnormalities. Pt#4 had 46, xx, t(9;22)(q34;q11) aberration; Pt#6 had 45, XY, -7/46, idem,+8 aberration; and Pt#7 had 45, X,-Y, del(7)q32, t(8,21) (q22;q22) aberration. Two patients (Pt# 3, 5) had RUNX1 gene mutation, one patient (Pt#4) had BCR/ABL fusion gene mutation, and one patient (Pt#9) had JAK1, JAK3, FBXW7 mutation. Six patients received ALL-directed induction therapy (VDLP), whereas two patients received AML-directed induction therapy (MEA and IA regimens). Overall, 4 of 8 (50%) patients with chemotherapy achieved complete remission (CR) after initial induction therapy. In the AML-directed therapy group, 1 of 2 (50%) patients achieved CR. Meanwhile, 3 of 6 (50%) patients achieved CR after ALL-directed induction chemotherapy. Two patients received HSCT after initial CRs, one patient (Pt#7) died two months after transplantation due to the infection, and another patient (Pt#5) is still alive. With an average of 14.3 (4.0-42.0) months follow-up, the median survival time was 7 months. Conclusion: We reported 10 cases of BAL, including 4 cases of B/Myeloid phenotype, 4 cases of T/Myeloid phenotype and 2 case of T/B phenotype. 4 of 8 patients achieved CR (50%) after initial chemotherapy. Although many patients achieved CR after initial chemotherapy, but the relapse rate was very high and the CR rate after relapse was very low. Our results confirmed that BAL is a rare malignancy with a very poor prognosis. Disclosures No relevant conflicts of interest to declare.

Description: Macrophage activation syndrome (MAS) /Hemophagocytic syndrome (HPS) is characterized by proliferation of activated macrophages under conditions such as infection(C Clin Infect Dis 2004)lymphoma(Aouba A Am J Hematol 2004), autoimmune disease(Kaneko K Clin Rheumatol 2005), solid organ transplantation(Akamatsu N,Transplant Proc 2006;). There have been several reports of MAS /HPS after hematopoietic stem cell transplantation, involving not only allogeneic,but also autologous transplantation(Sreedharan A Bone Marrow Transplantation,2006). Generally, MAS /HPS is a cytokine-related disorder.But at present, its clinical characteristics remain unknown. We firstly study here the T-cell receptor repertoire diversity and flow cytometric analysis in MAS /HPS after unrelated peripheral blood stem cell transplantation. The CDR3 of TCR Vα and Vβ subfamily genes were amplified in peripheral blood mononuclear cells from the patient with MAS/HPS after unrelated peripheral blood stem cell using RT-PCR for detection of the distribution of TCR Vα and Vβ repertoire, the PCR products were further analyzed by genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR Vα and VβT cells. Lymphocyte subsets in the peripheral blood were detected by monoclone antibody and flow cytometry including T lymphocyte subsets and NK cells. Flow-cytometric analysis showed CD56+ CD16+ cell 68.65% and CD3+ cell 11.79% in the lymphocyte population;CD16+CD69+ cell 68.51% and CD25+CD16+ cell 31.59% in NK cell. In the T lymphocytic subsets, CD25 + CD3+ cell 62%; CD69+CD3+ cell 75.81%; CD25CD4+ cell 0.81%,CD25CD8+ cell 3.48%; CD69CD4+ cell 0.31%, CD69+CD8+ cell 16.86%.The results show that the main activated lymphocytes is NK cell in patient at diagnosed with MAS/HPS. Of interest, it was only after the addition of high-dose IVIG 1g/kg/d for two days (Ostronoff et al BMT2006) to the treatment that MAS remitted. There are 23 Vα and 15Vβ subfamily T cells could be identified in this time, and the clonal expansion T cells could be found in TCR Vα5, 13, 20; TCR Vβ4, 11, 15 and 21subfamilies. Billiau et al (Blood 2005)describes the immunohistochemical findings on liver tissues from 5 children with MAS in the context of a different type of hemophagocytic syndrome (HPS) in liver transplantation. This study is the first directly to substantiates the presumed immunopathogenesis of MAS by documenting in situ expression of IFN-γ+ by activated CD8+ lymphocytes, and of IL-6 and TNF-α+ by hemophagocytosing macrophages, on liver tissues of patients with MAS. We found no evidence of potential infectious, autoimmune or malignant triggers of R-HPS in our patient, despite extensive investigations. We conclued that the skew distribution and clonal expansion of TCR Vα and Vβ subfamily T cells underscore the primary role of T cells in the pathogenesis of MAS/HPS.

Description: Phosphoinositide-3 kinase (PI3K) plays a vital role in T-cell signaling pathway including cell proliferation, differentiation, migration and so on. Scholars have found that PI3K expression is limited to cells related with transplant rejection activation of the immune cells. PI3K plays a crucial role in GVHD occurrence: 1 crucial, PI3K are located in main areas in T-cell activation signal, resulting in affecting GVHD significantly; 2 selective, PI3K expression is limited to the participation of activated cells with immune rejection. But the expression of PI3K in GVHD patients has been not clear. In order to give the more experimental evidence for prophylaxis and treatment of GVHD, PI3K gene expression in patients with different state of GVHD was detected. This research collected the peripheral blood lymphocytes in 32 patients after allogeneic hematopoietic stem cell transplantation from January 2007 to January 2008. The cases include 15 male and 17 female cases, the median age was 27.9 (14–43) years. In accord with the classification of GVHD, all cases were divided into ALL (12), AML (7), CML (10), CMML (2), MDS (1). After one year after transplantation, the number of patients with no-GVHD is 9, simply aGVHD is 8, single-cGVHD is 8, aGVHD and cGVHD is 7. The total sample number is 122(state of blood reconstruction 32, no GVHD 37, aGVHD 27, cGVHD 26). By real-time PCR, PI3K expression were determined of patients at normal blood reconstruction, and at the 30th day, the 60th day, the 90th day, the 180th day, the 270th day, the 360th day after allogeneic hematopoietic stem cell transplantation. Matching with own blood reconstruction, PI3K expression in acute or chronic GVHD patients is statistical difference significantly. At the state of No-GVHD, aGVHD and cGVHD, it was 2.51 ± 1.16, 7.15 ± 5.92, 4.35 ± 3.14 respectively. Results suggest that, comparing with no-GVHD, PI3K expression with aGVHD increase significantly. And PI3K expression increase significantly when occuring cGVHD, but it is less than with aGVHD. In the experiment we also found, PI3K expressed in lymphocytes correlated with the increasing degree of GVHD. Particularly in the cGVHD, PI3K expression increased significantly when GVHD becoming more serious; with access to stable cGVHD, PI3K expression gradually become flat. We can draw the conclusions from this study: PI3K gene expression increased significantly in patients with GVHD than that of patients with no-GVHD, and related to different stages of GVHD. PI3K expression increased in cGVHD patients than that of patients with normal hematopoietic reconstruction, but the amplitude is not significant than that of aGVHD.

Description: Abstract 2533 CD71 (transferring receptor 1) is an integral membrane glycoprotein that plays an important role in cellular uptake of iron. It is well known as a marker for cell proliferation and activation. Although all proliferating cells in hematopoietic system express CD71, however, CD71 has been considered as a useful erythroid-associated antigen. The expression proportion on nucleated red blood cells was significantly higher than other cells, approximately 80% of all CD71 positive cells were of CD71 positive erythroid cells in normal bone marrow. CD71 was usually considered as the representative marker for differentiating erythroblasts and diagnosing acute erythroid leukemia (AEL) by flow cytometry. At the ISAC 2000 Congress, most experts agreed that at least one or more B, T, myeloid, erythroid and megakaryocytic reagents should be included in the essential panel. The reagents recommended for erythroid cells included CD36, CD71 and glycophorin A (GlyA). However, there was no agreement on how to choose and group these antibodies. In the practical analysis of immune phenotypes of leukemic cells we noted that no CD71 expression was detected on blasts of some cases of AEL with typical morphological and cytochemical findings, but other types of acute myeloblastic leukemia (AML) cells may express CD71. Thus, we speculated that CD71 expression may associate with the abnormal antigen expression resulting from hematopoietic disorders. In this study, we evaluated CD71 expression on different acute leukemia cells in association with a variety of other antibodies. In this study we aimed to define CD71 as a flow cytometric marker for the diagnosis of acute leukemia. Bone marrow samples were collected from 82 newly diagnosed acute leukemia patients as well as 13 normal controls. The diagnosis were made according to the WHO 2008 diagnostic criteria. All 6 cases of AEL were erythroid/myeloid subtype (acute erythroid/myeloid leukemia, M6a). The samples were then analyzed using a four-color flow cytometer with antibody panels against a variety of lymphoid, myelomonocytic, erythroid and megakaryocytic antigens. The antibodies included anti-CD3, CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD45, CD56, CD61, CD64, CD71, CD117, GlyA, HLA-DR, IgG, IgM, MPO. Subpopulations of bone marrow cells were gated based on CD45 intensities and side scatter (SSC) value to further analyze the expression of antigens in different cell populations. Positive CD71 expression were identified on bone marrow blast cells of 41 (50%) acute leukemia patients and 9 (69.23%) normal controls. The mean expression level on normal controls was 35.99±19.06%. The mean CD71 expression level on blasts of AML with blasts differentiation at early stage of myelopoiesis (including FAB-M0/M1/M2/M4) was significantly higher than AML with partial differentiation of leukemic cells (FAB-M3/M5) and acuteB lymphoblastic leukemia (B-ALL) (p

Description: Chronic graft-versus-host disease (cGVHD) continue to cause significant morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). It remains the most frequent complication following allogeneic hematopoietic stem cell transplantation, occurring in 30% to 70% of long-term survivors.[Lee SJ, Blood 2002 ]. An experienced clinician may be able to make a diagnosis of chronic GVHD based on classic manifestations such as lichenoid or sclerodermatous skin changes. If the findings are not typical, a tissue biopsy is warranted to aid the diagnosis and to determine if infection is present (A.L. Gilman Gilman and J. Serody seminhematol.2005). Recently, there are many research in lymphocyte subsets on cGVHD by using flowcytometry.It helps us to understand the mechsim about it, and could be help for diagnosis, staging, and response criteria in this disorder. However, the change of lymphocytes subsets is still unknown in the different stage of cGVHD. We are here to study the lymphocyte subsets in the peripheral blood with different stage of cGVHD after allo-HSCT. Peripheral blood mononuclear cells were isolated from anticoagulated venous blood using lymphocyte separation medium according to manufacturer’s nstructions.Freshly isolated peripheral blood mononuclear cells were analyzed. Fluorescein sothiocyanate conjugated monoclonal antibodies (Becton Dickinson, mAb) By using FACScalibur, (Becton Dickinson).The data of the two-group comparison was done on the analysis of variance and Students t test.According to the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in chronic Graft-versus-Host Disease in 2005, we analysis the diffierent groups with non-cGVHD, mild, moderate and severe cGVHD. The proportions of the lymphocyte subsets are different with the different stage of cGVHD. The more serious of cGVHD, the more proportions of the total lymphoctyes. B lymphocyte has significant difference with different stage of cGVHD, although the percentage of the total lymphocytes is 2.14%∼16.14% (P=0.0023). There is significant difference on the percent of CD8+ CD45RO+ cell among the non-cGVHD, mild cGVHD, moderate cGVHD or severe cGVHD(44.56%,46.59%,56.77% and 61.68%, respectively. P=0.0475). But the percent of CD4+CD25+ cell is higher than that of CD8+CD25+ cell (31.86%∼43.75%/4.50%∼7.08%)The percent of CD69+CD4+,CD69+CD8+ (CD95+CD4+, CD95+CD8+, CD28+ CD4+, CD28+CD8+ cell are not significant different within the different statue cGVHD. We can draw a conclusion as below: It may help for clinician to early diagnosis,stage using routine monoclone antibodies by flowcytometry. the percents of the lymphocyte subsets are different with the different status of cGVHD. With the more serious of cGVHD, the more proportions of the total lymphocytes; CD4+CD25+ cell ; CD3+CD8+ cell and B cell increase significantly, the percent of CD45RO+CD8+ cell and CD3+CD4+ cell drease at the same time. It may help for early diagnoses and treatment for the cGVHD.