TNFAIP3 mutation is an independent poor overall survival factor for patients with T-cell acute lymphoblastic leukemia.

BACKGROUND: It is imperative to explore potential biomarkers for predicting clinical outcome and developing targeted therapies for T-cell acute lymphoblastic leukemia (T-ALL). This study aimed to investigate the mutation patterns of tumor necrosis factor-alpha-inducing protein 3 (TNFAIP3, also known as A20) and its role in the prognosis of T-ALL patients. METHODS: Polymerase chain reaction (PCR) and Sanger sequencing data from T-ALL (n = 49, JNU) and targeted sequencing data from T-ALL (n = 54, NFH) in our clinical center and a publicly available dataset (n = 121, PRJCA002270), were used to detect TNFAIP3 mutation. RESULTS: Three TNFAIP3 single nucleotide polymorphisms (SNPs; g.3033 C > T, g.3910 G > A, and g.3904 A > G) were detected in T-ALL in the JNU dataset, and g.3033 C > T accounted for the highest proportion, reaching 60% (6/10). Interestingly, TNFAIP3 mutation mainly occurred in adults but not pediatric patients in all three datasets (JNU, NFH, and PRJCA002270). T-ALL patients carrying a TNFAIP3 mutation were associated with a trend of poor overall survival (OS) (p = 0.092). Moreover, TNFAIP3 mutation was also an independent factor for OS for T-ALL patients (p = 0.008). Further subgroup analysis suggested that TNFAIP3 mutation predicted poor OS for T-ALL patients who underwent chemotherapy only (p < 0.001), and it was positively correlated with high risk and early T-cell precursor ALL (ETP-ALL) in two independent validation datasets (NFH and PRJCA002270). CONCLUSION: TNFAIP3 mutation mainly occurs in adult T-ALL patients, and it was associated with adverse clinical outcomes for T-ALL patients; thus, it might be a biomarker for prognostic stratification.

Low Expression of CD5 and CD6 Is Associated with Poor Overall Survival for Patients with T-Cell Malignancies.

Background: T-cell malignancies (TCMs), including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL), are highly aggressive and have a poor prognosis. To further understand prognostic stratifications and to design targeted therapies, this study aims to explore novel, potential biomarkers based on alterations in immune costimulatory molecules (CMs) for TCMs. Methods: Peripheral blood from 25 de novo T-ALL patients in our clinical center and transcriptome data from 131 to 162 patients with peripheral TCL (PTCL) from the GSE19069 and GSE58445 dataset, respectively, were obtained to assess the expression levels of CMs and their prognostic significance. Results: Seven CMs were associated with overall survival (OS). Among these CMs, CD5 and CD6 had the highest pairwise positive correlation (R = 0.69). CD5 and CD6 were significantly down-regulated in TCM patients compared with healthy individuals (HIs), and lower CD5 and CD6 expression was associated with poor OS for both T-ALL and TCL patients, particularly for patients greater than 60 years old. Furthermore, CD5 was positively correlated with CD6 in TCM patients. Compared with patients who were CD5highCD6high, T-ALL and TCL patients who were CD5lowCD6low had poor OS. Importantly, CD5highCD6high was an independent prognostic predictor for OS in T-ALL (HR = 0.39, 95% CI: 0.23-0.65, P < 0.001) and TCL (HR = 0.35, 95% CI: 0.19-0.62, P < 0.001) patients. Conclusions: Low expression of CD5 and CD6 was associated with poor OS for TCM patients, and this may be a potential immune biomarker panel for prognostic stratification of TCM patients.

High expression of TMEM244 is associated with poor overall survival of patients with T-cell lymphoma.

T-cell lymphoma (TCL) is an aggressive and genetically heterogeneous malignancy with adverse clinical outcomes; thus, it is worth exploring biomarkers for risk stratification. Previous studies have demonstrated that transmembrane protein 244 gene (TMEM244) is ectopically expressed in Sézary syndrome (SS). In this study, the expression level of TMEM244 and its prognostic value for TCL patients was explored by analyzing RNA-seq data of two large datasets (GSE132550 and GSE113113) containing 129 TCL patients and 13 healthy individuals (HIs) from the Gene Expression Omnibus (GEO) database, the PRJCA002270 dataset containing 124 patients with T-cell acute lymphoblastic leukemia (T-ALL) from the BioProject database, and peripheral blood (PB) samples of 24 TCL and 29 T-ALL patients, as well as 11 normal CD3 + T-cells from our clinical center (JNU). The results suggested that TMEM244 was significantly up-regulated in TCL patients compared with normal CD3 + T-cells or T-ALL in the JNU, GSE132550 and GSE113113 datasets (P < 0.05). However, TMEM244 shows no expression in patients with T-ALL in the JNU-T-ALL and PRJCA002270 datasets. The receiver operating characteristic (ROC) curve analysis indicated that TMEM244 expression had a very high accuracy in diagnosing TCL compared with T-ALL (area under the curve (AUC): 99.4%; P < 0.001). Importantly, high TMEM244 expression was significantly associated with poor OS and shorter 5-year restricted mean survival time (RMST) in TCL patients, especially those treated with chemotherapy. In summary, TMEM244 is also expressed in other types of TCL besides SS, but not in T-ALL. High TMEM244 expression is associated with poor OS in TCL patients, which might be a novel biomarker for prognostic stratification in TCL patients and facilitate the design of novel therapies.

TNFAIP3 mutation may be associated with favorable overall survival for patients with T-cell lymphoma.

BACKGROUND: T-cell lymphoma (TCL) is highly aggressive and has a poor prognosis; thus, it is worth exploring biomarkers that may predict clinical outcomes and investigate their potential role in developing targeted therapies. In this study, we characterized the mutation pattern of tumor necrosis factor-alpha-inducing protein 3 (TNFAIP3) and its role in the prognosis of TCL patients. METHODS: Coding sequence (CDS) mutations in TNFAIP3 in TCL patients was explored using exome-sequencing data from 79 patients in our center (Guangdong Provincial People's Hospital, GDPH) and 544 samples from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Additionally, non-CDS mutations in TNFAIP3 in 41 TCL patients from our center (JNU) were investigated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, non-CDS mutations in TNFAIP3 in 47 TCL patients from Gene Expression Omnibus (GEO) dataset were explored. RESULTS: In the COSMIC database, TNFAIP3 mutations in TCL patients were located in the CDS, and the overall mutation frequency was 2.2%. However, TNFAIP3 mutations were not detected in the CDS of any of the samples in our center's datasets. Interestingly, non-CDS TNFAIP3 mutations were found in 14.6% and 4.3% of TCL patients in the JNU and GSE15842 dataset, respectively. Importantly, there was a clear trend showing that TCL patients with a TNFAIP3 mutation were associated with a longer 5-year restricted mean survival time (RMST) and favorable OS rate compared with those without a TNFAIP3 mutation in the JNU dataset [hazard ratio (HR) = 0.29, 95% confidence interval (CI) 0.07 to 1.31, P = 0.089]. Furthermore, TNFAIP3 mutations significantly correlated with T-cell large granular lymphocytic leukemia (T-LGLL) with a favorable prognosis in the JNU dataset (P = 0.002). Notably, the different mutation patterns of TNFAIP3 when comparing our center and the COSMIC datasets might be due to different ethnic and genetic backgrounds. CONCLUSIONS: To the best of our knowledge, we for the first time describe that TNFAIP3 mutations in non-CDS regions are associated with favorable OS for TCL patients, which might be a potential biomarker for the prognostic stratification of Chinese TCL patients.

Inhibition of BCL11B induces downregulation of PTK7 and results in growth retardation and apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of leukemia with poor prognosis, and biomarkers and novel therapeutic targets are urgently needed for this disease. Our previous studies have found that inhibition of the B-cell leukemia/lymphoma 11B (BCL11B) gene could significantly promote the apoptosis and growth retardation of T-ALL cells, but the molecular mechanism underlying this effect remains unclear. This study intends to investigate genes downstream of BCL11B and further explore its function in T-ALL cells. We found that PTK7 was a potential downstream target of BCL11B in T-ALL. Compared with the healthy individuals (HIs), PTK7 was overexpressed in T-ALL cells, and BCL11B expression was positively correlated with PTK7 expression. Importantly, BCL11B knockdown reduced PTK7 expression in T-ALL cells. Similar to the effects of BCL11B silencing, downregulation of PTK7 inhibited cell proliferation and induced apoptosis in Molt-4 cells via up-regulating the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p27. Altogether, our studies suggest that PTK7 is a potential downstream target of BCL11B, and downregulation of PTK7 expression via inhibition of the BCL11B pathway induces growth retardation and apoptosis in T-ALL cells.

The c-Myc-regulated lncRNA NEAT1 and paraspeckles modulate imatinib-induced apoptosis in CML cells.

Chronic myeloid leukemia (CML) is a clonal disease characterized by the presence of the constitutively active tyrosine kinase BCR-ABL oncoprotein. Although BCR-ABL is crucially important for pathogenesis and treatment response, it is thought that some additional factors might be involved in the regulation of these processes. Aberrant expression of long noncoding RNAs (lncRNAs) has recently been identified to be involved in various diseases including cancer, suggesting that lncRNAs may play a role in BCR-ABL-mediated CML. In this study, we found that nuclear-enriched abundant transcript 1 (NEAT1), a lncRNA essential for the formation of nuclear body paraspeckles, is significantly repressed in primary CML cells. NEAT1 expression could be restored by inhibiting BCR-ABL expression or its kinase activity in K562 cells. We also demonstrated that NEAT1 is regulated by c-Myc. Knockdown of NEAT1 could promote imatinib (IM)-induced apoptosis, and we demonstrated that the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for NEAT1-mediated apoptosis in K562 cells. RNA-seq analysis revealed that SFPQ regulates cell growth and death pathway-related genes, confirming its function in IM-induced apoptosis. Collectively, these results assign a biological function to the NEAT1 lncRNA in CML apoptosis and may lead to fuller understanding of the molecular events leading to CML.

Memory T cells skew toward terminal differentiation in the CD8+ T cell population in patients with acute myeloid leukemia.

Stem cell memory T (TSCM) and central memory T (TCM) cells can rapidly differentiate into effector memory (TEM) and terminal effector (TEF) T cells, and have the most potential for immunotherapy. In this study, we found that the frequency of TSCM and TCM cells in the CD8+ population dramatically decreased together with increases in TEM and TEF cells, particularly in younger patients with acute myeloid leukemia (AML) (< 60 years). These alterations persisted in patients who achieved complete remission after chemotherapy. The decrease in TSCM and TCM together with the increase in differentiated TEM and TEF subsets in CD8+ T cells may explain the reduced T cell response and subdued anti-leukemia capacity in AML patients.

Downregulated miR-17, miR-29c, miR-92a and miR-214 may be related to BCL11B overexpression in T cell acute lymphoblastic leukemia.

AIM: BCL11B overexpression is a characteristic of most T cell acute lymphoblastic leukemia (T-ALL) cases, and downregulated BCL11B in leukemic T cells inhibits cell proliferation and induces apoptosis. The purpose of this study was to analyze the miRNA expression pattern that may be related to BCL11B regulation in T-ALL. METHODS: Quantitative real-time PCR was used to detect the miRNAs miR-17-3p, miR-17-5p, miR-29c-3p, miR-92a-3p, miR-214-3p and miR-214-5p, the BCL11B expression level in peripheral blood mononuclear cells which was obtained from 17 de novo and untreated T-ALL patients, and 15 healthy individuals (HIs) served as control. Correlations between the relative miRNA expression levels and BCL11B were analyzed. RESULTS: Based on the computational prediction that certain miRNAs bind the BCL11B 3'-UTR, miR-17-3p, miR-17-5p, miR-29c-3p, miR-92a-3p, miR-214-3p and miR-214-5p were found to be candidates for regulating BCL11B. The expression levels of the six miRNAs were decreased compared with HIs, and with the exception of miR-17-5p, statistically significant differences in expression levels were found in the T-ALL group. Moreover, while significantly higher BCL11B expression was found in the T-ALL group, a negative trend in the correlation level for all six miRNAs could be found in all groups; however, statistical significance was only found for miR-214-3p in the T-ALL group. CONCLUSION: miRNA downregulation together with BCL11B upregulation suggests that miR-17, miR-29c, miR-92a and miR-214 might be involved in BCL11B regulation. The therapeutic promise of regulating the expression of these miRNAs for T-ALL therapy may be considered in the future.

Different genetic alteration of A20 in a Sézary syndrome case with Vα2-Jα22 T cell clone.

BACKGROUND: The comprehensive genetic alterations underlying the pathogenesis of Sézary syndrome (SS) remains largely unknown. Previous studies showed that alterations of tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) are frequent in SS. In this study, we characterized the mutation and polymorphisms of A20 in a case with SS and compared with the genetic feature of A20 in T-cell acute lymphoblastic leukemia (T-ALL). METHODS: Using a novel approach based on the combination of fine-tiling array comparative genomic hybridization ( and ligation-mediated polymerase chain reaction (LM-PCR) to identify SS clone, the polymorphisms in the A20 gene (promoter, exons 2-9 [coding region] and 3'UTR) were detected by PCR and sequencing. RESULTS: The malignant SS clone was identified as TCR Vα2-Jα22 rearrangement without deletion at the A20 loci (6q23-27 region) in the SS case. Six polymorphisms were identified, all of them are belonging to single nucleotide polymorphisms (SNPs) that are recorded in genebank: rs5029924, rs5029937, rs2230926, rs582757 and rs77191406, while rs2307859 was not identified in the SS sample, which is found in all T-ALL. The alteration pattern of A20 in this case seemed different from the T-ALL samples, in contrast, it is similar to the alteration of A20 in samples from rheumatoid arthritis or systemic lupus erythematosus with poor clinical outcome and cancer developing. CONCLUSIONS: The genetic alteration of A20 in the SS case was different from the T-ALL samples and similar to the cases with refractory autoimmune disease and related to tumorigenesis. The findings lead to discuss whether such SNPs of A20 may link the refractory autoimmune inflammation and the tumorigenesis.

The roles of stem cell memory T cells in hematological malignancies.

Adoptive cell therapy (ACT) is rapidly migrating from bench to clinical therapy for hematological malignancies. Recently, a new subtype of memory T cells, stem cell memory T (TSCM) cells, was shown to be one of the most favorable subsets for ACT. TSCM has high self-renewal capacity and is associated with superior T cell engraftment, persistence, and antitumor immunity. In this review, we focused on the characteristics of antigen-specific TSCM cells and discussed their potential for immunotherapy targeting hematological malignancies.

Overexpression of MALT1-A20-NF-κB in adult B-cell acute lymphoblastic leukemia.

BACKGROUND: A20 is a dual inhibitor of NF-κB activation and apoptosis in the tumor necrosis factor receptor 1 signaling pathway, and both are related to tumorigenesis. A20 is frequently inactivated by deletions and/or mutations in several B and T cell lymphoma subtypes; however, knowledge of the role of A20 in B-cell acute lymphoblastic leukemia (B-ALL) remains limited. In this study, we characterized the A20 gene expression pattern, the expression level of its upstream regulating factor MALT1, and its downstream target NF-κB in adult B-ALL. METHODS: The expression level of MALT1, A20 and NF-κB1 was detected in peripheral blood mononuclear cells (PBMCs) from 20 patients with adult B-ALL (including 12 de novo B-ALL and 8 refractory/relapse B-ALL cases), and nine patients with B-ALL in complete remission (CR) using real-time PCR. Sixteen healthy individuals served as controls. RESULTS: Significant A20 overexpression was found in the B-ALL (median: 13.489) compared with B-ALL CR (median: 3.755) (P = 0.003) patients and healthy individuals (median: 8.748) (P = 0.002), while there was no significant difference in A20 expression between B-ALL CR patients and healthy individuals (P = 0.107). Interestingly, the A20 expression level in the B-ALL samples was relatively different with approximately 50% of the B-ALL cases showing a relatively high A20 expression level, while the remaining 50% cases demonstrated slight upregulation or a similar expression level as the healthy controls. However, there was no significant difference in the A20 expression level between de novo B-ALL (median 12.252) and refractory/relapse B-ALL patients (median 21.342) (P = 0.616). Similarly, a significantly higher expression level of NF-κB1 was found in the B-ALL (median 1.062) patients compared with healthy individuals (median 0.335) (P < 0.0001), while the NF-κB1 expression level was downregulated in the B-ALL CR group (median 0.339), which was significantly lower than that in those with B-ALL (P = 0.001). Moreover, the MALT1 expression level in B-ALL was upregulated (median 1.938) and significantly higher than that in healthy individuals (median 0.677) (P = 0.002) and B-ALL CR patients (median 0.153) (P = 0.008). The correlation of the expression levels of all three genes was lost in B-ALL. CONCLUSIONS: We found that MALT1-A20-NF-κB is overexpressed in adult B-ALL, which may be related to the pathogenesis of B-ALL, and this pathway may be considered a potentially attractive target for the development of B-ALL therapeutics.

[Effects of Down-regulating PPP2R5C Expression on Expression Profile of TAL1-related Regulating Genes in Jurkat Cells].

OBJECTIVE: Based on our previous study showing the inhibition of lenkemia T cell proliferation by down-regulating PPP2R5C expression, this study was aimed to analyze the influence of down-regulating PPP2R5 expression via RNA interference on genes relatied with TAL1 signaling pathway by using gene chip technique. METHODS: The PPP2R5C-siRNA799 was transduced into Jurkat cells by nucleofection, the total RNA was isolated from treated Jurkat cells after culture for 48 hours; the target sequences were prepared by revevse transcription after mRNA purification, and were hybridized with affymetrix gene expression profile chip 3' IVT. The original image data were collected using affymetrix gene chip scanner 3 000, and the gene expression profile was analyzed using gene spring GX 11.0 soflware. RESULTS: The expression of all 26 genes related with TAL1 signaling pathway was changed, out of which the expression of 15 genes were up-regulated and the expression of 11 genes was down-regulated in PPP2R5C-siRNA 799-transfected Jurkat cells. The genes with significantly up-regulated expression were GATA1, TCF4, XRCC6 and TCF3, while the genes with significantly down-regulated expression were SIN3A and RUNX1. CONCLUSION: The down-regulation of PPP2R5C gene expression in Jurkat cells via RNA interference to a certain degree can inhibit TAL1 signaling pathway genes, thereby suppresses the proliferation of Jurkat cells.

Characteristics of CARMA1-BCL10-MALT1-A20-NF-κB expression in T cell-acute lymphocytic leukemia.

BACKGROUND: Knowledge of the oncogenic signaling pathways of T-cell acute lymphoblastic leukemia (T-ALL) remains limited. Constitutive aberrant activation of the nuclear factor kappa B (NF-κB) signaling pathway has been detected in various lymphoid malignancies and plays a key role in the development of these carcinomas. The zinc finger-containing protein, A20, is a central regulator of multiple NF-κB-activating signaling cascades. A20 is frequently inactivated by deletions and/or mutations in several B-and T-cell lymphoma subtypes. However, few A20 mutations and polymorphisms have been reported in T-ALL. Thus, it is of interest to analyze the expression characteristics of A20 and its regulating factors, including upstream regulators and the CBM complex, which includes CARMA1, BCL10, and MALT1. METHODS: The expression levels of CARMA1, BCL10, MALT1, A20, and NF-κB were detected in peripheral blood mononuclear cells (PBMCs) from 21 patients with newly diagnosed T-ALL using real-time PCR, and correlations between the aberrant expression of these genes in T-ALL was analyzed. Sixteen healthy individuals, including 10 males and 6 females, served as controls. RESULTS: Significantly lower A20 expression was found in T-ALL patients (median: 4.853) compared with healthy individuals (median: 8.748; P = 0.017), and significantly increased expression levels of CARMA1 (median: 2.916; P = 0.034), BCL10 (median: 0.285; P = 0.033), and MALT1 (median: 1.201; P = 0.010) were found in T-ALL compared with the healthy individuals (median: 1.379, 0.169, and 0.677, respectively). In contrast, overexpression of NF-κB (median: 0.714) was found in T-ALL compared with healthy individuals (median: 0.335; P = 0.001). A negative correlation between the MALT1 and A20 expression levels and a positive correlation between CARMA1 and BCL10 were found in T-ALL and healthy individuals. However, no negative correlation was found between A20 and NF-κB and the MALT1 and NF-κB expression level in the T-ALL group. CONCLUSIONS: We characterized the expression of the CARMA-BCL10-MALT1-A20-NF-κB pathway genes in T-ALL. Overexpression of CARMA-BCL10-MALT in T-ALL may contribute to the constitutive cleavage and inactivation of A20, which enhances NF-κB signaling and may be related to T-ALL pathogenesis.

Altered expression pattern of miR-29a, miR-29b and the target genes in myeloid leukemia.

OBJECTIVES: The miR-29 family have been demonstrated acting as vital tumor suppressor in multiple cancers as well as regulators in the adaptive immune system. Little is known about their role in leukemogenesis. The purpose of this study is to analyze the expression pattern of miR-29a/29b and its target genes Mcl-1 (myeloid cell leukemia sequence 1) and B-cell lymphoma 2 (Bcl-2) in myeloid leukemia. METHODS: Quantitative real-time PCR was used for detecting genes expression level in peripheral blood mononuclear cells (PBMCs) from 10 cases with newly diagnosed, untreated acute myeloid leukemia (AML) and 14 cases with newly diagnosed, untreated chronic myeloid leukemia (CML) in chronic phase, and 14 healthy individual (HI) served as controls. Correlation between the relative expression levels of different genes have been analyzed. RESULTS: Significant lower expression of miR-29a/29b and higher expression level of two potential target genes Bcl-2 and Mcl-1 were found in PBMCs from AML and CML patients compared with HI group. In addtion, miR-29a expression in AML was significantly lower than that in CML. Moreover, negative correlation between miR-29a/29b and its target genes have been found. While, positive correlation between relative expression level of miR-29a and miR-29b or Bcl-2 and Mcl-1 were presented in the total 38 research objects. CONCLUSION: Down-regulated miR-29a and miR-29b, and accompanying up-regulated Bcl-2 and Mcl-1 are the common feature in myeloid leukemias. These data further support the role for miR-29a/29b dysregulation in myeloid leukemogenesis and the therapeutic promise of regulating miR-29a/29b expression for myeloid leukemia in the future.

Abnormal expression of A20 and its regulated genes in peripheral blood from patients with lymphomas.

BACKGROUND: Cell-mediated immunity is often suppressed in patients with hematological malignancies. Recently, we found that low T cell receptor (TCR)-CD3 signaling was related to abnormal expression of the negative regulator of nuclear factor kappa B (NF-κB) A20 in acute myeloid leukemia. To investigate the characteristics of T cell immunodeficiency in lymphomas, we analyzed the expression features of A20 and its upstream regulating factor mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) and genes downstream of NF-κB in patients with different lymphoma subtypes, including T cell non-Hodgkin lymphoma (T-NHL), B cell non-Hodgkin lymphoma (B-NHL) and NK/T cell lymphoma (NK/T-CL). METHODS: Real-time PCR was used to determine the expression level of the MALT1, MALT-V1 (variant 1), A20 and NF-κB genes in peripheral blood mononuclear cells (PBMCs) from 24 cases with T-NHL, 19 cases with B-NHL and 16 cases with NK/T-CL, and 31 healthy individuals (HI) served as control. RESULTS: Significantly lower A20 and NF-κB expression was found in patients with all three lymphoma subtypes compared with the healthy controls. Moreover, the MALT1 expression level was downregulated in all three lymphoma subtypes. A significant positive correlation between the expression level of MALT1 and A20, MALT1-V1 and A20, MALT1-V1 and NF-κB, and A20 and NF-κB was found. CONCLUSIONS: An abnormal MALT1-A20-NF-κB expression pattern was found in patients with lymphoma, which may result a lack of A20 and dysfunctional MALT1 and may be related to lower T cell activation, which is a common feature in Chinese patients with lymphoma. This finding may at least partially explain the molecular mechanism of T cell immunodeficiency in lymphomas.

[Overexpression of eIF4E gene in acute myeloid leukemia and its relation with disease progression].

The aim of this study was to detect the expression level of eIF4E gene in patients with non-treated, remission and non-remission/relapse acute myeloid leukemia (AML), and other non-malignant haematologic diseases so as to analyze and reveal the relationship of eIF4E gene expression with AML progression. SYBR Green I RT-PCR was used to assay the expression level of eIF4E mRNA extracted from bone marrow mononuclear cells in 30 patients with AML (6 in M2, 5 in M3, 8 in M4, 10 in M5, 1 in M6) and 20 patients with non-malignant hematologic diseases. The ß2-microglubin(ß2M) was used as internal reference and the formula 2(-ΔCt)×100% was applied to calculate the expression level of eIF4E gene. The results showed that the eIF4E expression level (7.098 ± 5.544)% in patients with non-treated and non-remitted/relapsed AML was significantly higher than that in patients with remission (0.964 ± 0.312)% (P < 0.01) and non-malignant hematologic diseases (0.248 ± 0.163)% (P < 0.01). There was no difference between latter two group patients, even though the expression level of eIF4E gene in patients with M4 and M5 was higher. As compared with non-malignant hematologic diseases, the expression level of eIF4E gene of patients with remission patients showed no significant difference. It is concluded that the over-expression of eIF4E gene has been found in patients with AML, and its level obviously decreases along with remission of disease, thus the eIF4E gene may be a surveillance parameter for disease progression.

[Analysis of CDR3 sequence of TCR Valpha6, Valpha10 and Vbeta23 oligoclonal T cells in one APL case].

AIM: To investigate the clonality and the pattern of CDR3 sequence of TCR Valpha and Vbeta repertoire in patients with acute promyelocytic leukemia (APL). METHODS: The CDR3 of TCR Valpha 29 and Vbeta 24 subfamily genes were amplified in mononuclear cells from peripheral blood of one case with APL using RT-PCR. The positive PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Valpha and Vbeta T cells. The products of the oligoclonal T cells were analyzed by sequencing to define the sequence of CDR3. RESULTS: Oligoclonal T cells expressed TCR Valpha6, Valpha10 or Vbeta23 genes were identified from the APL patient. The molecular characteristics of the CDR3 of Valpha and Vbeta genes rearrangement were Valpha6NJalpha17, Valpha10NJalpha35 and Vbeta23NDbeta1NJbeta2.7. The amino acid sequence of CDR3 region in TCR Valpha 6, Valpha10 and Vbeta23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). CONCLUSION: Three idiotype Valpha and Vbeta sequences are identified in a case with APL, which is thought that may mediate the host specific immunity response for leukemia cells in APL. It might provide data for farther research on the specific immunotherapy of APL.

T cell receptor Vbeta repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia.

BACKGROUND: In general, it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know, few studies have dealt with the distribution of oligoclonal T cells in leukemia, so we investigated the distribution and clonality of TCR Vbeta repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. METHODS: The complementarity determining region 3 (CDR3) of TCR Vbeta24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vbeta repertoire, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vbeta T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3. RESULTS: The expression pattern of TCR Vbeta repertoire in different individuals are different. Vbeta2-21 subfamilies could be detected in CML cases. The frequent usage Vbeta repertoire in CML was Vbeta1, Vbeta2 or Vbeta13. Most of the PCR products from 27 patients displayed polyclonality, while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vbeta16 subfamilies. The frequent usage of Vbeta genes in clonal expansion was Vbeta3, Vbeta13 or Vbeta21. Multiple Vbeta clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vbeta21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments. CONCLUSIONS: These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vbeta3, Vbeta13 and Vbeta21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.

[Clonal expansion of TCR Vbeta subfamily T cells induced by bcr3-abl2 peptide].

OBJECTIVE: To investigate the clonal expansion of T cell receptor (TCR) Vbeta subfamily T cells from cord blood induced by bcr3-abl2 peptide in vitro. METHODS: T cells from 3 units of cord blood were amplified by anti-CD(3) monoclonal antibody (McAb) and IL-2 with or without synthetic b3a2 peptide. T cell specific cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay, TCR Vbeta subfamilies by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan technique. RESULTS: bcr3-abl2 peptide specific cytotoxicity T cells were successfully induced from the 3 units of cord blood by synthetic b3a2 peptide. Compared with that in CD(3) McAb induced cells, distribution pattern of TCR Vbeta repertoire was different in T cells induced with b3a2 peptide. Oligoclonal and oligoclonal tendency TCR Vbeta subfamily T cells could be identified in cord blood T cells induced by b3a2 peptide in 1 or 2 weeks, whereas those induced by anti-CD(3) McAb and IL-2 were mostly polyclonal. CONCLUSION: The cytotoxicity T cells with anti-CML specificity could be induced by b3a2 peptide. The specific anti-CML cytotoxicity may be derived from the clonal expansion TCR Vbeta subfamily T cells.