One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell.

Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.

A CpG Methylation Classifier to Predict Relapse in Adults with T-Cell Lymphoblastic Lymphoma.

PURPOSE: Adults with T-cell lymphoblastic lymphoma (T-LBL) generally benefit from treatment with acute lymphoblastic leukemia (ALL)-like regimens, but approximately 40% will relapse after such treatment. We evaluated the value of CpG methylation in predicting relapse for adults with T-LBL treated with ALL-like regimens. EXPERIMENTAL DESIGN: A total of 549 adults with T-LBL from 27 medical centers were included in the analysis. Using the Illumina Methylation 850K Beadchip, 44 relapse-related CpGs were identified from 49 T-LBL samples by two algorithms: least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE). We built a four-CpG classifier using LASSO Cox regression based on association between the methylation level of CpGs and relapse-free survival in the training cohort (n = 160). The four-CpG classifier was validated in the internal testing cohort (n = 68) and independent validation cohort (n = 321). RESULTS: The four-CpG-based classifier discriminated patients with T-LBL at high risk of relapse in the training cohort from those at low risk (P < 0.001). This classifier also showed good predictive value in the internal testing cohort (P < 0.001) and the independent validation cohort (P < 0.001). A nomogram incorporating five independent prognostic factors including the CpG-based classifier, lactate dehydrogenase levels, Eastern Cooperative Oncology Group performance status, central nervous system involvement, and NOTCH1/FBXW7 status showed a significantly higher predictive accuracy than each single variable. Stratification into different subgroups by the nomogram helped identify the subset of patients who most benefited from more intensive chemotherapy and/or sequential hematopoietic stem cell transplantation. CONCLUSIONS: Our four-CpG-based classifier could predict disease relapse in patients with T-LBL, and could be used to guide treatment decision.

A gene-expression-based signature predicts survival in adults with T-cell lymphoblastic lymphoma: a multicenter study.

We aimed to establish a discriminative gene-expression-based classifier to predict survival outcomes of T-cell lymphoblastic lymphoma (T-LBL) patients. After exploring global gene-expression profiles of progressive (n = 22) vs. progression-free (n = 28) T-LBL patients, 43 differentially expressed mRNAs were identified. Then an eleven-gene-based classifier was established using LASSO Cox regression based on NanoString quantification. In the training cohort (n = 169), high-risk patients stratified using the classifier had significantly lower progression-free survival (PFS: hazards ratio 4.123, 95% CI 2.565-6.628; p < 0.001), disease-free survival (DFS: HR 3.148, 95% CI 1.857-5.339; p < 0.001), and overall survival (OS: HR 3.790, 95% CI 2.237-6.423; p < 0.001) compared with low-risk patients. The prognostic accuracy of the classifier was validated in the internal testing (n = 84) and independent validation cohorts (n = 360). A prognostic nomogram consisting of five independent variables including the classifier, lactate dehydrogenase levels, ECOG-PS, central nervous system involvement, and NOTCH1/FBXW7 status showed significantly greater prognostic accuracy than each single variable alone. The addition of a five-miRNA-based signature further enhanced the accuracy of this nomogram. Furthermore, patients with a nomogram score ≥154.2 significantly benefited from the BFM protocol. In conclusion, our nomogram comprising the 11-gene-based classifier may make contributions to individual prognosis prediction and treatment decision-making.

Prognostic and predictive value of a microRNA signature in adults with T-cell lymphoblastic lymphoma.

New prognostic factors are needed to establish indications for haematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) for T-cell lymphoblastic lymphoma (T-LBL) patients. We used microarray to compare T-LBL tissue samples (n = 75) and fetal thymus tissues (n = 20), and identified 35 differentially expressed miRNAs. Using 107 subjects as the training group, we developed a five-miRNA-based classifier to predict patient survival with LASSO Cox regression: lower risk was associated with better prognosis (disease-free survival (DFS): hazard ratio (HR) 4.548, 95% CI 2.433-8.499, p < 0.001; overall survival (OS): HR 5.030, 95% CI 2.407-10.513, p < 0.001). This classifier displayed good performance in the internal testing set (n = 106) and the independent external set (n = 304). High risk was associated with more favorable response to HSCT (DFS: HR 1.675, 95% CI 1.127-2.488, p = 0.011; OS: HR 1.602, 95% CI 1.055-2.433, p = 0.027). When combined with ECOG-PS and/or NOTCH1/FBXW7 status, this classifier had even better prognostic performance in patients receiving HSCT (DFS: HR 2.088, 95% CI 1.290-3.379, p = 0.003; OS: HR 1.996, 95% CI 1.203-3.311, p = 0.007). The five-miRNA classifier may be a useful prognostic biomarker for T-LBL adults, and could identify subjects who could benefit from HSCT.

[Efficacy of Decitabine Combined with Pre-Excitation Chemotherapy in the Treatment of Middle-Aged and Elderly MDS Transformed Acute Myeloid Leukemia].

OBJECTIVE: To investigate the efficacy of domestic decitabine (D) combined with pre-excitation chemotherapy consisted of Ara-c, THP and G-CSF(CTG) in treatment of middle-aged and elderly patients with MDS-transformed AML and prognosis-related factors. METHODS: Seventy-six patients with MDS-transformed AML treated in our hospital from June 2013 to June 2015 were selected according to treatment regimens, 76 patients were divided into 2 groups: CTG group(36 cases) and D+CTG group(40 cases). The patients in CTG group received treatment with Ara-C, THP and G-CSF; the patients received the treatment with decitabine plus CTG. The patients in 2 groups all received 4 course treatment, then received maintaining treatment. The therapeutic efficacy and incidence of adverse reactions in 2 group were compared, at the same time, the risk factors affecting the prognos of patients treated with D+CTG were analyzed. RESULTS: There were no siginificant differences in age, sex, initial blood cell count, bone marrow blast ratio, disease types, chromosome karyotypes and FLT3-ITD gene mutation between 2 groups. The efficacy analysis showed that the efficacy of D+CTG was superior to CTG, ORR in D+CTG group was significantly higher than that in CTG group (72、52 vs 50%) (P<0.05), moreover, no significant differences in bone marrow inhibition digree infeetion, gastroinfestinal response and liver damage were found between 2 groups (P>0.05). The follow-np for 2 years showed that the median survival time in D+CTG group was significantly longer than that in CTG group (19.9 vs 11.0 months) (P<0.05). The multivariate analysis showed that the 1 course efficacy (RR=3.926, P=0.015) and FLT3-ITD gene mutation (RR=4.347, P=0.004) were independent risk factors affecting the efficacy of D+CTG treatment. CONCLUSION: The short-and long-term efficacy of domestic decitasine combined with preexcitation chenotherapy in treatment of middec-aged and eldery patients with MDS transformed AML is superior to single pre-excitation chenothrapy, moreover the incidence of adverse reactions did not increase. The 1 course efficacy and FLT-3 ITD gene mutation are the independent risk factors affecting the prognosis of patients. .

[Killing effect of double suicide genes mediated by retroviral vector on k562 cells].

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.

[Application of pseudotyped lentivirus-mediated double suicide genes to allogeneic bone marrow transplantation in mice].

OBJECTIVE: To explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment. METHODS: The pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC). RESULTS: The pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone. CONCLUSION: Administration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.

[Killing effect of double suicide genes mediated by lentivirus on lymphoma cells].

BACKGROUND & OBJECTIVE: The lentiviral vectors can integrate interest genes into genome of the target cells that allow for stable transgenic expression even in non-dividing cells without evoking an immune response of the host. All the features have promised them to be used in vivo gene therapy. This study was designed to explore the killing effect of double suicide genes mediated by lentivirus on lymphoma cells (Raji). METHODS: The three plasmids expressed lentivirus, packaging plasmid pCMV 8.2, envelope plasmid pCMV.VSVG and target plasmid (pHR'CS. GFP as control group, pHR'CS.CDglytk as experiment group) were packaged into 293T cells using lipofectine method. Supernatant was harvested and concentrated. The Raji cells were infected with the concentrated virus. The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR. After prodrug GCV or/and 5-FC administration, MTT method was used to detect the growth inhibition rate (GIR) of Raji cells for evaluating the killing effect of CD and HSV-tk double suicide genes on Raji cells. RESULTS: The three plasmids were effectively transferred into 293T cells. Green fluorescence on the cell was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy. Double suicide genes mediated by lentivirus were effectively and stably expressed in Raji cells. The GIR of Raji cells using GCV or 5-FC was 51% or 50%, respectively, and it was apparently higher than that of untransfected cells(P< 0.01). When using GCV and 5-FC together, the GIR was 73%, which was apparently higher than that of group using GCV or 5-FC alone (P< 0.01). CONCLUSION: Double suicide genes mediated by lentiviral vector could transfect lymphoma cells effectively and stably. The double suicide gene system enhanced killing effect remarkably on lymphoma cells than CD/5FC or HSV-tk/GCV system alone.