The Characteristics Of Cytogenetics And Molecular Genetics In T Cell Acute Lymphoblastic Leukemia

Objective:1 To analyze systematically the clinical and biological characteristics of 297 cases of de novo T cell acute lymphoblastic leukemia patients who received conventional cytogenetics (CC) analysis in our laboratory from 1997 to 2011.2 To evaluate the prevalence, correlation and clinical characteristics of mutated PHF6, NOTCH1, FBXW7, WT1 and the rearrangement of SET-NUP214, SIL-TAL1, CALM-AF10.3 To establish a novel human mixed phenotype acute leukemic (MPAL) cell line and analyze its biologic characteristics.Methods:1 297 cases were studied by R-band karyotypic analysis using direct method and/or short-term culture for chromosomes preparation. We investigated the cytogenetic features in T-ALL patients. 2 We analyzed the incidence and prognostic value of PHF6, NOTCH1, FBXW7, WT1 mutations in 101 Chinese T-ALL patients by polymerase chain reaction and direct sequencing. Expression of SIL-TAL1, SET-NUP214, and CALM-AF10 was assessed using the corresponding primers by reverse transcription-PCR (RT-PCR). We investigated the effection of gene mutations and rearrangement with clinical features and prognostic in T-ALL. 3 Mononuclear cells were isolated from the bone marrow of a MPAL patient with coexpression of B lineage and myeloid at her relapse, and were cultured in a 24-well cell culture cluster at a density of 1×106/ml in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% heat-inactivated fetal calf serum (FCS) at 37℃in a 5% CO2 humidified in an incubator. And the basic biologic features of JIH-5 were characterized. The growth feature of the JIH-5 cell line in flask was studied under the inverted microscope. The ultrastructure was assayed by the transmitted electronic microscope. The cell line was routinely stained with the Wright's staining method. Cytochemical studies were performed by perosidase staining. The growth curve of the JIH-5 cell line was obtained by incubating the cells in 24-well plate and the doubling time was calculated from the curve. The immunoprofile and the cell cycle distribution of JIH-5 were studied by fiow cytometry (FCM). RHG banging was employed for dynamic karyotypic analysis in the process of establishment of the cell line. The clonality was assayed by semi-solid methylcellulose clonogenic culture. Quantitative fluorescent polymerase chain reaction was used to detect Epstein-Barr virus (EBV) genomic DNA. PCR was used to detect mycoplasma. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR). The tumorigenicity in mice was evaluated in six nude mice subcutaneous injected with the JIH-5 cell line.Results:1 The clinical, laboratory and cytogenetic features of 297 newly diagnosed patients with T-ALLBetween July 1997 and January 2011, 297 newly diagnosed patients with T-ALL were registered in this study. T-ALL was more commonly seen in males: nearly three-quarters of T-ALL patients were male. According to the Franch-American- British (FAB) classification, 112, 94 and 91 cases were diagnosed as L1, L2 and not otherwise specified (NOS), respectively. All were analyzed for their clinical and biologic characteristics. The median ages at diagnosis of NOS cases were significantly older than those of L1 and L2 cases(P<0.05). There was an association between L1 patients and a high median WBC compared with L2 and NOS patients(P<0.05). No significant differences between L1, L2 and NOS in median Hb levels, platelets (PLT) counts and LDH levels.Of the 297 T-ALL patients, 9 patients (3%) had an unsuccessful cytogenetic evaluation. Of 288 successfully karyotyped patients, abnormal karyotypes were found in 122 patients (42.4%). The detection rate and recurring rate of chromosome abnormalities both are low, 18 recurring chromosome abnormalities including: 2p-, 5q-, 6q-, i(7q), 9p-, 9q-, der(11), 12p-, 12q-, 13q-, 13q+, 14q+, i(17q), t(11;14)(p13;q11), t(4;11)(q21;p15), t(9;22)(q34;q11), t(2;14)(p15;q32) and t(1;19)(q23;p13.3) were detected. The frequencies of hypodiploid, 47-50 chromosome hyperdiploid, >50 chromosome hyperdiploid and tetraploid in this series were 3.5%, 9.4%, 1.4% and 1.7%, respectively.2 The clinical and laboratory features of PHF6, NOTCH1, FBXW7, WT1 mutations and SET-NUP214, SIL-TAL1 and CALM-AF10 rearrangements in T-ALL patientsPHF6 mutations were identified in 13.9% (14/101) T-ALL patients analyzed. Among these, nonsense, frameshift, and missense mutations respectively accounted for 42.8% (6/14), 35.7% (5/14) and 21.5% (3/14) of all PHF6 mutations. PHF6 mutations identified in our series were clustered in exons 8, 9, and 10. Two nonsense mutations and two missense mutations involved the second (C-terminal) PHD-like domain of PHF6. NOTCH1 mutations were detected in 49.5% (50/101) T-ALL patients in the following domains and frequencies: HD, 30.7%; PEST, 11.8%; HD+PEST, 5.0%; TAD, 1.0%; HD+TAD, 1.0%. FBXW7 mutations were identified in 9.9% (10/101) T-ALL cases; 9 cases haboring missense mutations and frameshift mutations located in exon 9, 1 patient harboring missence mutation located in exon 10; alone in 3, and associated with NOTCH1 HD mutations in 7 cases. WT1 mutations were found in 4.0% (4/101) cases; one patient harboring a missense mutation at codon 462 (R462W) located in exon 9, while others all harboring frameshift mutations located in exon 7.SET-NUP214 fusion transcript was detected in 5/64 (7.8%) patients, SIL-TAL1 was positive in 12/64 (18.8%) patients. The 12 cases with SIL-TAL1 fusion were younger than the 52 cases without SIL-TAL1 fusion (P=0.002). In contrast, no CALM-AF10 fusion was detected in 64 T-ALL patients tested.Peripheral WBC counts and LDH level were lower for PHF6-mutated (PHF6-mut) patients compared with PHF6-wild type (PHF6-wt) patients(P<0.05). PLT counts and percentage of blast cell in bone marrow were higher for PHF6-mut compared with PHF6-wt(P<0.05). The hemoglobin level, platelet counts and sex ratio of patients with PHF6-mut and PHF6-wt were similar. PLT counts were lower for FBXW7-mut patients compared with FBXW7-wt patients(P<0.05). WBC counts were lower for SET-NUP214-positive compared with SET-NUP214-negative. No significant differences between the NOTCH1-mut and NOTCH1-wt, WT1-mut and WT1-wt, SIL-TAL1-positive and SIL-TAL1-negative were found for median WBC counts, Hb level, PLT counts, LDH level and percentage of blast cell in bone marrow. PHF6-mut were identified in 12/62 (18.6%) adult and 2/39 (5.4%) pediatric T-ALL patients analyzed(P<0.05). SIL-TAL1 rearrangement is more common in children compared with adult(P<0.05). PHF6-mut was more likely than PHF6-wt patients to harbor the following: NOTCH1-mut (P=0.004), JAK1-mut (P=0.005) and SET-NUP214 fusion (P=0.006).The complete remission rate was higher for children T-ALL compared with adult T-ALL (85.2% vs 60.5%,P<0.05). The Relapse-free survival time was lower for patients with PHF6-mut compared with PHF6-wt (11 vs 24.8 months, P<0.05). No significant differences between the PHF6-mut and PHF6-wt, NOTCH1-mut and NOTCH1-wt, WT1-mut and WT1-wt, SET-NUP214-positive and SET-NUP214- negative, SIL-TAL1-positive and SIL-TAL1-negative were found for complete remission rate and overall survival(P>0.05).3 Establishment and characterization of a human mixed phenotype acute leukemic cell line, JIH-5The JIH-5 cell line could be cultured in IMDM medium with 20% fetal Calf Serum (FCS) and without the supernatant of the 5637 cell line or any cytokines. It grows as single cell in suspension with a few cells adhering to flask. The cell line has proliferated continuously in vitro for 10 months. After suffering frozen and thawed repeatedly, it still successfully expands in culture. The cultured cells showed a slow but stable proliferation. The doubling time of JIH-5 was 97 hours. The morphology of JIH-5 cells showed a regular cell shape, folded nucleus with prominent nucleoli, finely reticular chromatin. The JIH-5 cell line was heterogeneous in ultrastructual morphology. Most cells have numerous mitochondria, endoplastic reticulum, lysosome, vacuole and folded nucleus with prominent nucleoli. All of the JIH-5 cells were negative for peroxidase staining. The results of immunoprofile showed that the JIH-5 cell line expressed antigens of myeloid and B lymphocytic lineage. A few markers of megakaryocytic lineage were also presented. The JIH-5 cell line showed negative result of EBV and mycoplasma detection, and the complete correspondence with primary leukemic cells by STR-PCR. The karyotypic results of the JIH-5 cell line showed 46,X,?t(X;20)(q25;p13),?t(2;2)(q33;q37),?t(6;10)(p22;q24),t(12;22)(p13;q11)[8]. And, the JIH-5 can form the colony in semi-solid methylcellulose clonogenic assay, had tumorigenicity in nude mice.Conclusions:1 Of 297 de novo T-ALL patients, the detection rate of abnormal karyotype was 42.4%, and the most frequent structural abnormality was rearrangement in the 14q11 region (6.3%), followed by del(6q). The detection rate of t(9;22)(q34;q11) in T-ALL was 2.4%. The frequency of karyotype aberration was improved by detection of concealable chromosome rearrangement.2 These results identify the presence of PHF6 mutations as a recurrent genetic alteration in T-ALL. The Relapse-free survival time was lower for patients with PHF6-mut compared with PHF6-wt. NOTCH1 mutations are the most common in T-ALL patients. The incidence of FBXW7 and WT1 mutations significantly lower than that reported abroad.3 Our results point to a strong genetic interaction among PHF6, NOTCH1, JAK1 mutations and SET-NUP214 rearrangement in the pathogenesis of T-ALL.4 We established a new human mixed phenotype acute leukemic cell line, JIH-5, with coexpression of B lymphoid and myeloid markers. It has clear biological characteristic and provide a powerful tool.