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Background:Determining the somatic mutations of epidermal growth factor receptor (EGFR)-pathway networks is the key to effective treatment for non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs).The somatic mutation frequencies and their association with gender, smoking history and histology was analysed and reported in this study.Methods:Five thousand one hundred and twenty-five NSCLC patients' pathology samples were collected, and EGFR, KRAS, BRAF and PIK3CA mutations were detected by multiplex testing. The mutation status of EGFR, KRAS, BRAF and PIK3CA and their association with gender, age, smoking history and histological type were evaluated by appropriate statistical analysis.Results:EGFR, KRAS, BRAF and PIK3CA mutation rates revealed 36.2%, 8.4%, 0.5% and 3.3%, respectively, across the 5125 pathology samples. For the first time, evidence of KRAS mutations were detected in two female, non-smoking patients, age 5 and 14, with NSCLC. Furthermore, we identified 153 double and coexisting mutations and 7 triple mutations. Interestingly, the second drug-resistant mutations, T790M or E545K, were found in 44 samples from patients who had never received TKI treatments.Conclusions:EGFR exons 19, 20 and 21, and BRAF mutations tend to happen in females and non-smokers, whereas KRAS mutations were more inclined to males and smokers. Activating and resistant mutations to EGFR-TKI drugs can coexist and ‘second drug-resistant mutations', T790M or E545K, may be primary mutations in some patients. These results will help oncologists to decide candidates for mutation testing and EGFR-TKI treatment.

Background: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CART cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CART cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. Methods: Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5 + malignant cell lines, primary CD5 + malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5 + T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. Results: Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5 + malignant cells in vitro and prolonged the survival of TALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5 + malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. Conclusions: Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cellassociated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

BackgroundChimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. However, treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation.MethodsThe FLT3L CAR-T using FLT3 ligand as recognizing domain was constructed. The specific cytotoxicity against FLT3+ leukemia cell lines, primary AML cells, and normal hematopoietic progenitor stem cells (HPSCs) in vitro were evaluated. In addition, FLT3+ AML mouse model was used to assess the effect of FLT3L CAR-T therapy in vivo.ResultsFLT3L CAR-T cells could specifically kill FLT3+ leukemia cell lines and AML patients’ bone marrow mononuclear cells in vitro (with or without FLT3 mutation) and have more potent cytotoxicity to FLT3-ITD cells. In a human FLT3+ AML xenograft mouse model, FLT3L CAR-T cells could significantly prolong the survival of mice. Furthermore, it was found that FLT3L CAR-T cells could activate the FLT3/ERK signaling pathway of FLT3+ leukemia cells with wild-type FLT3; meanwhile, it had no inhibitory effects on the colony formation of CD34+ stem cells derived from normal human umbilical cord blood.ConclusionsThe ligand-based FLT3L CAR-T cells could be a promising strategy for FLT3+ AML treatment, especially those carried FLT3 mutation.Electronic supplementary materialThe online version of this article (10.1186/s13045-018-0603-7) contains supplementary material, which is available to authorized users.

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Key Points• A novel RARa fusion gene, TBLR1-RARa, was found in rare cases of APL with t(3;17) chromosomal translocation.• TBLR1-RARa exhibited diminished transcriptional activity by recruiting more corepressors compared with RARa.The majority of acute promyelocytic leukemia (APL) cases are characterized by the PMLRARa fusion gene. Although the PML-RARa fusion gene can be detected in >98% of APL cases, RARa is also found to be fused with other partner genes, which are also related to all-trans retinoic acid (ATRA)-dependent transcriptional activity and cell differentiation.In this study, we identified a novel RARa fusion gene, TBLR1-RARa (GenBank KF589333), in a rare case of APL with a t(3;17)(q26;q21),t(7;17)(q11.2;q21) complex chromosomal rearrangement. To our knowledge, TBLR1-RARa is the 10th RARa chimeric gene that has been reported up to now. TBLR1-RARa contained the B-F domains of RARa and exhibited a distinct subcellular localization. It could form homodimers and also heterodimers with retinoid X receptor a. As a result, TBLR1-RARa exhibited diminished transcriptional activity by recruitment of more transcriptional corepressors compared with RARa. In the presence of pharmacologic doses of ATRA, TBLR1-RARa could be degraded, and its homodimerization was abrogated. Moreover, when treated with ATRA, TBLR1-RARa could mediate the dissociation and degradation of transcriptional corepressors, consequent transactivation of RARa target genes, and cell differentiation induction in a dose-and time-dependent manner. (Blood. 2014;124(6):936-945) IntroductionAcute promyelocytic leukemia (APL) is a special disease entity of acute myeloid leukemia. With the clinical use of all-trans retinoic acid (ATRA), APL turns into the most curable form of acute myeloid leukemia. The majority of APL cases are characterized by the fusion between the promyelocytic leukemia (PML) gene and the retinoic acid receptor a (RARa) gene, which is the consequence of t(15;17)(q22;q21) chromosomal translocation.1 Although the PML-RARa fusion gene can be detected in .98% of APL patients, 2 RARa is also found to be fused with other partner genes, 3-10 such as PML zinc finger (PLZF), nucleophosmin (NPM), and so forth. The RARa portion within fusion proteins is conserved, containing B-F domains of RARa, which cover the DNA-binding and ligand-binding motifs, so that RARa can be combined with retinoid X receptor a (RXRa) to bind the retinoic acid responsive element (RARE). 1,11 As a result, the fusion proteins usually form heterodimers with RXRa.9,12 A critical property that all RARa fusion proteins have in common is the ability to self-associate, forming homodimers. 13 The chimeric proteins, such as PML-RARa, PLZF-RARa, and NPMRARa, can robustly recruit transcriptional corepressors, including nuclear receptor corepressor/silencing mediator of retinoid and thyroid receptors (N-CoR/SMRT) and histone deacetylases (HDACs), to RARE and consequently result in ectopic repression of RARa target genes. 1,13,14 Transducin b-like 1 X-linked receptor 1 (TBLR1) ...

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