TCR spectratyping revealed T lymphocytes associated with graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Clonal expansion of T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been observed, but their characteristics remain to be fully elucidated. We report here that CD8(+) T cells were the dominant T lymphocytes seen and T-cell repertoire diversity decreased dramatically during the first 3 months after allo-HSCT. Patients with GVHD grade II - IV had significantly lower T-cell repertoire diversity compared with non-GVHD patients. TCR beta variable gene (TCRBV) subfamily 8, 5.1, 5.2, 4, and 13 were the five most frequently expanded subfamilies among these patients. Among the 49 over-expanded clones identified, clonotype "TCR3-5" and "TCR18-5" were isolated from four patients with HLA-A2 allele and skin GVHD. Their frequencies correlated well with skin symptoms (i.e. rash). Moreover, they were detected in donors but not detected in recipients before transplantation. Lastly, three common TCRBV CDR3 motifs shared by T cells related with GVHD were discovered: TGDS, GLAG, and GGG. These findings suggest that TCR spectratyping is helpful for revealing GVHD-related T cells and may have utility in early diagnosis.

[Real-time PCR array for simultaneous detection of 37 kinds of fusion genes in leukemia].

OBJECTIVE: To develop a real-time PCR array for simultaneous quantitative detection of translocations/chromosomal aberrations in patients with leukemia, and to investigate the feasibility and utility thereof. METHODS: By construction and optimization a set of specific primes (totally 82 primers), an array containing 66 parallel PCR reactions was developed. That array was used on the specimens of bone marrow or peripheral blood from 31 patients with leukemia to detect simultaneously 37 fusion genes and 4 proto-oncogene activations often occurring in patients with leukemia. Eva Green fluorescent dye method was chosen in the protocol. Relative quantification was performed by Ct analysis and the result was expressed as the ratio of the target gene versus the internal control gene (ABL). Six patients with chronic myelocytic leukemia (CML) among the 31 cases underwent prior to and after treatment so as to study the expression changes of fusion genes and/or proto-oncogene. RESULTS: The established PCR array showed high efficiency of amplification and good sensibility (232 copies/microl) in the fusion gene detected. The standard curve had a satisfying linear range (10(2) approximately 10(8) copies/microl), showing a good reproducibility. Fourteen fusion genes, including PML/RARalpha, PLZF/RARalpha, BCR/ABL, MLL/AF1, MLL/AF6, MLL/AF10, AML/Eto, CBFbeta/MYH11, TLS/ERG, TEL/AML1, MOZ/CBP, MLL/hCDCrel, LAF4/MLLT2, and FIP1L1/PDGFRalpha, and activation of all 4 proto-oncogenes were found in the 31 samples. In one patient, 5 fusion genes and activation of 2 proto-oncogenes were observed. Such results were compared with those of RT-nested PCR in 28 samples. The comparison showed that this array was a bit less sensitive than RT-nested PCR, however, without significant difference between them (P = 0.009). The expression of BCR/ABL fusion gene, WT1 gene, and EVI1 gene decreased after treatment in the 6 CML patients, which was in accordance with the clinical features. CONCLUSION: The PCR array newly-established successfully detects various leukemia related fusion genes and proto-oncogene activation. It is useful in molecule diagnosis and monitoring minimal residual disease in leukemia, and therapeutic effect monitoring.

[Role of molecular screening for common fusion genes in the diagnosis and classification of leukemia].

OBJECTIVE: To assess the value of common fusion genes analysis in the diagnosis and classification of leukemia by multiplex RT-PCR. METHODS: The multiplex RT-PCR, including 8 parallel PCR reactions, could screen 86 mRNA breakpoints or splice variants at the same time, which was important for the diagnosis and prognosis of leukemia. Bone marrow samples from 161 cases of leukemia and 8 cases of myelodysplastic syndrome (MDS) were involved in the study. The distribution of common fusion genes in leukemia was analyzed by the method mentioned above in combination with clinical and morphological features. RESULTS: Ten fusion genes were detected in 115 cases of leukemia, including AML1/ETO, PML/RAR alpha, PLZF/RAR alpha, dupMLL, MLL/AF6, MLL/AF10, CBFbeta/MYH11, BCR/ABL, Hox11, and EVI1 BCR/ABL was positive in all the 52 cases of chronic myeloid leukemia; PML/RAR alpha was found in 21 of 25 acute promyelocytic leukemia (APL), and PLZF/RAR alpha was detected in one case of APL. Sixteen cases of 17 AML1/ETO-positive acute leukemia (AL) belonged to FAB-M2 subtype, and one case was mixed leukemia. Three of 4 AL cases carrying CBFbeta/MYH11 were M4 subtype, and one was M5 subtype. MLL aberrations were found in 16 AL, in which all MLL/AF6 translocation existed in M5 subtype with classic monoblastic characters. Furthermore, BCR/ABL was detected in 5 acute lymphoblastic leukemia (ALL) cases. Fusion genes were also found in 2 MDS cases, of which AML1/ETO positive-MDS-RAEB progressed to AML rapidly. CONCLUSION: Screening of common fusion genes by multiplex RT-PCR is an important tool which could provide useful and reliable molecular genetic information for the diagnosis and treatment of leukemia.

[The characteristics of anti-B-cell malignance cytotoxic T lymphocytic clones induced by lymphoma relative peptides].

OBJECTIVE: To analyze if the cytotoxic T-cell (CTLs) are peptide-special and HLA restrict, and what is the sequence characteristic of TCRbeta genes. METHODS: Using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMCs) from healthy HLA-A0201 positive donor were stimulated by PBMCs and T2 cells loading the IgHV1-QLVQSGAEV nonapeptide or IgHV3-QLVQSGAEV, B-lymphoma-related nonapeptides, as antigen presenting cells (APCs) once a week for four weeks so as to obtain peptide-specific CTLs. PBMCs from non-HLA-A * 0201 positive donors were used as controls. The immunophenotypes of the CTLs (CD3, CD4, or CD8) were identified by flow cytometry. The proportions of CD8 and peptide/tetramer double positive cells were assayed by using peptide/ HLA tetramer method, The IFN-gamma-releasing capacity of the CTLs incubated together with different target cells was assayed by ELISA. The changes of lymphocyte clones were analyzed TCR beta genes were identified by RT-PCR and spectral type method and then sequenced. RESULTS: After four times stimulation, the CD4/CD8 ratio of the cultured cell decreased obviously from 1.43 to 0.10 (P < 0.05), showing a proliferation of CD8(+) CTLs. The frequency of CD8 and IgHV1-QLVQSGAEV/ HLA-A * 0201 tetramer double positive CTLs was 49.83%, significantly higher than that before stimulation (0.04%). The IFN-gamma secretion detected by ELISA indicated that these CTLs were capable of recognizing the target cells in a peptide-specific and MHC-restricted way. Spectral type method showed that the TCRbeta repertoires were skewed in only a few TCR families. CONCLUSION: The peptides derived from IgHV succeeds in generation of peptide-specific CTLs in vitro in a clonality manner. These CTLs are capable of recognizing the target cells in a peptide-specific and MHC-restricted way, Understanding the function of these CTLs and the molecular structures of these TCR identification-related antigen peptides help discover the interaction between the B-cell and T-cell clones.

[Comparison of therapeutic effects of low-dose versus high-dose interferon alpha-2b treatment on chronic myelocytic leukemia: a prospective randomized study].

OBJECTIVE: To compare the therapeutic effects of low-dose and high-dose interferon alpha-2b (IFN) treatment on chronic myelocytic leukemia (CML). METHODS: A real-time quantitative reverse transcriptase PCR (RQ-PCR) method was established to detect the fusion gene bcr-abl expression, thereby studying the reduction of leukemic cells. Thirty newly diagnosed CML patients, 21 males and 9 females, aged 14 - 69, were treated with hydroxyurea to keep the white blood cell count less than 20 x 10(9)/L, and then randomized into 2 groups: high-dose IFN group receiving IFN alpha-2b 5MIU 6 times per week for 3 - 6 months and low-dose IFN group receiving IFN alpha-2b 3MIU every other day for 3 - 6 months. Bone marrow was collected every month to Real-time PCR was used to detect the expression of bcr-abl mRNA. Mononuclear cells were isolated and RNA was extracted to detect the expression of fusion gene bcr-abl and a control gene GAPDH. The results were reported as the number of bcr-abl copies/GAPDH copy. RESULTS: The established real-time quantitative PCR method could detect the bcr-abl molecules as low as 50 copies. The intra-assay coefficient of variation (CV) was less than 5% and the inter-assay CV was 5.13%. The median bcr-abl fusion gene expression level of 30 CML patients before IFN therapy was 0.098 (range: 0.010 - 5.799). The bcr-abl expression level decreased by 19.37% and 24.86% in the low-dose and high-dose IFN groups respectively after 3 months' therapy. No significant difference was observed between the two groups (P = 0.398). Relatively more side effects were observed in the high-dose IFN group than in low-dose group. CONCLUSION: RQ-PCR is a reliable method to monitor CML therapy by analyzing fusion gene bcr-abl expression. There is a difference in bcr-abl fusion gene expression levels among the newly diagnosed patients, and low-dose IFN is as effective as high-dose IFN in reducing bcr-abl expression but with less side effects.

[Expression of PRAME gene in acute leukemia and its clinical significance].

This study was aimed to detect the expression levels of preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia (AL) and to evaluate the clinical significance of PRAME gene. The quantitative detection method was established by SYBR Green I real-time quantitative RT-PCR, then PRAME mRNA was measured by this method in 55 cases of acute leukemia, out of which 43 cases were acute myeloid leukemia (AML), 9 cases were acute lymphocytic leukemia (ALL) and other types leukemia were 3 cases. In addition, expression of PRAME gene was also analyzed in 7 cases of non-malignant hematological diseases and 8 healthy volunteers. K562 cell line was used as a positive control. The results showed that the expression of PRAME gene was found in 35 cases of acute leukemia, the positive percentage was 64%. No expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. In 35 PRAME positive cases, 28 cases were AML, which mainly belonged to M3, M4 and M2 subtypes, and 5 cases was ALL. In 31 fusion gene positive cases, 23 cases were PRAME positive, and in 24 fusion gene negative cases 12 cases were PRAME positive. No significant relationship was found between PRAME expression level and clinical characteristics (age, sex, WBC count, blast cells in BM). The expression of PRAME gene decreased or disappeared in 6 patients achieving complete remission (CR). It is concluded that the PRAME gene expresses in 64% AML patients, which mainly belonged to M3, M4 and M2 subtypes, no expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. There is remarkable difference in the level of PRAME transcript of the 35 cases and the expression of PRAME gene decreases or disappears when the patients achieved complete remission. These results suggest that PRAME expression in acute leukemia may be a useful marker to detect the minimal resi-dual disease (MRD) and to determine the response to therapy in AL patients.

[Characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigens].

To understand the characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigen, the characteristics of T cells were analyzed using T cell receptor beta variable region (TCRbetaV) gene spectrotyping in a case of antiphospholipid syndrome (APS). The results indicated that in the case of APS there were 2 dominant T cell clones. The TCRbetaVs sequences of the 2 T cell clones showed the TCRbetaVs belonged to 8 and 23 gene families respectively. The peptides of third complementarity-determining regions (CDR3) in the TCRbetaVs were CASSLLVAGGPRAYNEQFFGPG and CASSLAGFGQPQHFGDG. Comparing the motifs in CDR3 with another autoimmune disease, the motif YNEQFFGPG in TCRbetaV8 and motif QHFGDG in TCRbetaV23 were identical with that of idiopathic thrombocytopenic purpura and systemic lupus erythematosus reported before. In conclusion, some T cell clones proliferating in these autoimmune diseases may recognize the same antigens.