Outcome after allogeneic hematopoietic stem cell transplantation following Venetoclax-based therapy among AML and MDS patients.

The use of Bcl-2 inhibitor Venetoclax (VEN) combined with hypomethylating agents or chemotherapy has shown efficacy in treating acute myeloid leukemia (AML) as frontline treatment and for relapse, allowing more patients to bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the influence of VEN-based therapy on the prognosis of subsequent allogeneic HSCT remains unknown. We retrospectively collected data from patients who proceeded to allo-HSCT between November 2018 and November 2020 after VEN-based therapy at five transplant centers in Zhejiang Province, China. A total of 39 patients were analyzed. Thirty-one patients were diagnosed with AML (28 de novo, 3 secondary to MDS), 6 with MDS, and 2 with CMML. The majority (74.4%) of patients received VEN-based therapy for the treatment of relapse (38.5%) or refractory disease (35.9%); 5 (12.8%) received it as an initial treatment, and 5 (12.8%) patients who were already in complete remission (CR) received VEN for further consolidation or deep remission before HSCT. Twenty-seven (69.2%) patients were in CR at the time of HSCT. Day + 100 cumulative incidences of grade I-IV acute graft-versus-host disease (aGVHD) and grade II-IV aGVHD were 43.6% and 15.4%, respectively. Of 34 evaluable patients, 6.4% and 25.6% developed chronic GVHD at 1 year and 2 years. The 100-day cytomegalovirus (CMV) reactivation occurred in 76.3% of patients and Epstein-Barr virus (EBV) reactivation occurred in 29.7% of patients. With a median follow-up of 14.7 months, overall survival, progression-free survival, relapse, and non-relapse mortality incidence at 1 year were 75.5%, 61.6%, 16.7%, and 21.7%, respectively. Both univariate and multivariate analysis revealed that relapsed/refractory (R/R) disease was associated with inferior PFS (HR 4.849, 95% CI 1.009-23.30; p = 0.049). Prior poor response to VEN was found to be a significant factor predicting higher risk of relapse (HR 4.37, 95% CI 1.130-16.9; p = 0.033). Our results showed that VEN-based regimen therapy followed by allo-HSCT in AML patients is feasible and does not increase the risk of transplant-related mortality and toxicity.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway.

We investigated the oncogenic role of SETDB1, focusing on non-small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT-ß-catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1.

Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers.

Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) down-regulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.

The learning curve of lateral access lumbar interbody fusion in an Asian population: a prospective study.

PURPOSE: Lateral access lumbar interbody fusion (LLIF) is a minimally invasive technique that has an increasing popularity. It offers unique advantages and circumvents risk of certain serious complications encountered in other conventional spinal approaches. This study provides a statistical analysis defining the lateral access learning curve in the Asian population. METHODS: This prospective study included 32 consecutive patients who underwent LLIF from April 2012 to August 2014. The surgeries were performed by two senior spine surgeons and follow-up was conducted at 6 weeks, 3, 6, 9 months and 1 year post-operation. RESULTS: The breakpoint in operating time occurred at the 22nd level operated, from a mean of 71 min in the early phase group to a mean of 42 min in the steady state group. LLIF at L4/5 level is technically more demanding but technically feasible as competency is achieved. CONCLUSIONS: During the learning process, there was no compromise of perioperative or clinical outcomes. It should be feasibly incorporated into a spine surgeon's repertoire of procedures for the lumbar spine.

Viral contamination on the surfaces of the personal protective equipment among health care professionals working in COVID-19 wards: A single-center prospective, observational study.

OBJECTIVE: To evaluate potential viral contamination on the surfaces of personal protective equipment (PPE) in COVID-19 wards. METHODS: Face shields, gloves, the chest area of PPE and shoe soles were sampled at different time points. The samples were tested for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by PCR, and the cycle threshold (CT) values were recorded. RESULTS: The positive rate was 74.7% (239/320) for all PPE specimens. The CT values of the samples were ranked in the following order: face shields > chests > gloves > shoe soles (37.08±1.38, 35.48±2.02, 34.17±1.91 and 33.52±3.16, respectively; P for trend < .001). After disinfection, the CT values of shoe soles decreased compared with before disinfection (32.78±3.47 vs. 34.3±2.61, P = .037), whereas no significant effect of disinfection on the CT values of face shields, chests and gloves was observed. After disinfection, the CT values of specimens collected from shoe soles gradually increased; before disinfection, the CT values of shoe sole specimens were all less than 35. CONCLUSIONS: SARS-CoV-2 can attach to the surfaces of the PPE of healthcare professionals in COVID-19 wards, especially the shoe soles and undisinfected gloves. Shoe soles had the highest SARS-CoV-2 loads among all tested PPE items.

A novel risk model for predicting early relapse in acute myeloid leukemia patients undergoing allogeneic hematopoietic stem-cell transplantation.

Relapse remains the leading cause of death in acute myeloid leukemia (AML) patients following allogeneic hematopoietic stem-cell transplantation (allo-HSCT), limiting the efficacy of allo-HSCT. Thus, the ability to identify high-risk patients in a manner that permits early intervention has the potential to improve survival outcomes. We retrospectively enrolled 414 younger patients (aged 14-60 years) with AML who received allo-HSCT between January 2014 and May 2020. From June 2020 to June 2021, 110 consecutive patients were included prospectively in the validation cohort. The primary outcome was early relapse (relapse within 1 year). The cumulative incidence of early relapse after allo-HSCT was 11.8%. The overall survival rate for patients who relapsed within 1-year was 4.1% at 3 years after relapse. After multivariable adjustment, statistically significant associations between primary resistance, pre-transplantation measurable residual disease, DNMT3A mutation, or white blood cell count at diagnosis and early relapse were observed. An early relapse prediction model was developed based on these factors and the model performed well. Patients deemed to have a high risk or a low risk of early relapse had early relapse rates of 26.2% and 6.8%, respectively (P < 0.001). The prediction model could be used to help identify patients at risk for early relapse and to guide personalized relapse prevention.

Quinoline-cored Poly(Aryl Ether) Dendritic Organogels with Multiple Stimuli-Responsive and Adsorptive Properties.

Functional supramolecular gel materials have potential applications in sensors, optical switches, artificial antennae, drug delivery and so on. In this paper, quinoline-cored poly(aryl ether) dendritic organogelators were designed, synthesized and fully characterized. The gelation behaviour of the dendritic organogelator was tested in organic solvents, mixed solvents and ionic liquids. The dendron Q-G1 was found to be an efficient and versatile organogelator toward various apolar and polar organic solvents with the critical gelation concentrations (CGCs) approaching 1.2×10-2  mol/L, indicating one dendritic organogelator could immobilize 1.2×103 solvent molecules in the organogel network. Interestingly, these dendrons exhibited excellent gel formation in ionic liquids. Notably, these dendritic organogels were found to display multiple stimuli-responsive properties toward external stimuli including heat, ultrasound and shear stress, with a reversible sol-gel phase transition. In addition, the dendritic organogel could effectively adsorb heavy metals and organic dyes. The removal rate of Pb2+ was up to 20% and the adsorption rate for Rhodamine B was as high as 89%.

[2,9-Bis(3,5-dimethyl-1H-pyrazol-1-yl-κN)-1,10-phenanthroline-κN,N'](methanol-κO)(nitrito-κO,O')cadmium(II) perchlorate.

In the title complex, [Cd(NO(2))(C(22)H(20)N(6))(CH(3)OH)]ClO(4), the Cd(II) ion is in a distorted penta-gonal-bipyramidal CdN(4)O(3) coordination geometry. The dihedral angles formed between the mean planes of the pyrazole rings and the phenanthroline ring system are 4.37 (19) and 5.84 (21)°. In the crystal, the anions and cations are connected by inter-molecular O-H⋯O hydrogen bonding, while pairs of weak inter-molecular C-H⋯O hydrogen bonds connect the cations into centrosymmetric dimers. In addition, there is a π-π stacking inter-action involving two symmetry-related benzene rings, with a centroid-centroid distance of 3.437 (3) Å.

[Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the effects of mycophenolic acid (MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). METHODS: MSCs were treated with MPA at the concentration of 1 µ mol/L, 10 µ mol/L, 50 µ mol/L, and 100 µ mol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5'-monophosphate dehydrogenase (IMPDH) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining, Ca(2+) quantification and real-time PCR. RESULTS: In the range of 1 µ mol/L to 100 µ mol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH I and IMPDH II. von Kossa staining and Ca(2+) quantification indicated that MPA inhibited osteogenic differentiation of MSCs, and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix. CONCLUSION: MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration-and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.

[Changes of the concentrations and stoichiometry of carbon, nitrogen and phosphorus in soil aggregates along different altitudes of Helan Mountains, Northwest China.] / è´ºå °å±±ä¸åŒæµ·æ‹”åœŸå£¤å›¢èšä½“ç¢³æ°®ç£·å«é‡åŠå ¶åŒ–å­¦è®¡é‡ç‰¹å¾å˜åŒ–.

Exploring the distribution patterns of soil nutrients in aggregates of forests along different altitudes in arid and semi-arid areas can provide a theoretical basis for understanding nutrient cycling in vulnerable mountain ecosystems. In this study, we analyzed the distribution and stability of aggregates in the 0-20 cm soil layer along different altitudes (1380-2438 m) of Helan Mountains and measured the storage and stoichiometric characteristics of organic carbon, total nitrogen, and total phosphorus in soil aggregates. Results showed that the main soil aggregates of Helan Mountains changed from micro-aggregates (0.25-0.053 mm) to macro-aggregates (>0.25 mm) with increa-sing elevation. The mean weight diameter (MWD) and geometric mean diameter (GMD) of soil aggregates in high altitude (2139-2248 m) were significantly higher than those in low altitude (1380-1650 m). The content and storage of organic carbon and total nitrogen in soil aggregates of different size fractions were positively correlated with altitude, while the content of total phosphorus fluctuated with the increase in elevation and distributed uniformly in aggregates. Macro-aggregates and micro-aggregates had more contribution to soil nutrient storage than the silt and clay fractions, indicating that the proportion of aggregates with different size fractions was the key factor affecting soil nutrient storage and that macro-aggregates and micro-aggregates were the main carriers of soil nutrients. Moreover, the C:N ratio in aggregates of different size fractions did not change across different altitudes, whereas the C:P and N:P ratio were higher at mid and high elevations than those at low elevations. Our results indicated that the mid and high elevations of Helan Mountains had higher nutrient storage in the surface soil layer, and that higher content of macro-aggregates and micro-aggregates would help to retain organic carbon and nutrients in the soil. Soil nitrogen limitation was strong at low altitude in our study, suggesting that the appropriate amount of nitrogen addition in low altitudes could improve total nitrogen status during forest cultivation.

Lymphocyte-to-C-reactive protein ratio is a potential new prognostic biomarker for patients with lung cancer.

Aim: To compare and evaluate the prognostic value of various pretreatment combinations of inflammatory factors in patients with lung cancer (LC). Materials & methods: This study enrolled 1005 patients with LC and categorized into a discovery cohort and a validation cohort. Results: A combination of Lymphocyte-to-C-reactive protein levels (LCR) demonstrated the highest correlation with poor first-line progression-free survival (PFS) and overall survival (OS) (p < 0.05), but not disease-free survival (p > 0.05) compared with other parameters in LC patients. Decreased preoperative LCR was an independent prognostic factor for first-line PFS and OS (p < 0.05), but not disease-free survival (p > 0.05) in patients. Conclusion: Pretreatment LCR is a promising biomarker for first-line PFS and OS in patients with LC.

XPD inhibits cell growth and invasion and enhances chemosensitivity in esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a lethal disease due to its high aggressiveness. The aim of the present study was to investigate the role of xeroderma pigmentosum complementation group D (XPD) in the growth and invasion of ESCC and to elucidate the potential underlying molecular mechanisms. Western blot analysis and RT­qPCR were used to detect the expression level of XPD in ESCC tissue samples and adjacent normal esophageal tissue samples. The pEGFP­N2/XPD plasmid was transfected into human ESCC cell lines (EC9706 and EC109). The proliferation, apoptosis, migration and invasion of EC9706 or EC109 cells were assessed following transfection with the XPD overexpression plasmid. The chemosensitivity of EC9706 or EC109 cells to cisplatin or fluorouracil was evaluated by CCK­8 assay. The expression levels of phosphoinositide 3­kinase (PI3K)/AKT, nuclear factor (NF)­κB, Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and mitogen­activated protein kinase (MAPK) signaling pathway­related genes were detected by RT­qPCR and western blot analysis. The results demonstrated that the expression level of XPD was markedly lower in ESCC tissue samples than in adjacent normal esophageal tissue samples. The pEGFP­N2/XPD plasmid was successfully transfected into EC9706 or EC109 cells, inducing XPD overexpression. A High XPD expression markedly suppressed cell proliferation, migration and invasion, and increased the apoptotic rate of EC9706 and EC109 cells. Furthermore, the overexpression of XPD significantly increased the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil. Following XPD overexpression, the expression levels of PI3K, p­AKT, c­Myc, Cyclin D1, Bcl­2, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)­9 were markedly downregulated, while the expression level of p21 was markedly upregulated. On the whole, the findings of the present study demonstrate that XPD inhibits the growth and invasion of EC9706 and EC109 cells, whilst also enhancing the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil by regulating the PI3K/AKT signaling pathway. XPD may thus be an underlying target for ESCC treatment and drug resistance.

2-(3-Oxocyclo-hex-1-enylamino)acetic acid.

The six-membered ring of the title compound, C(8)H(11)NO(3), adopts an envelope shape with the C atom in the meta position of the carbonyl representing the flap. This atom is disordered over two positions in an 0.865 (6): 0.135 (6) ratio. In the crystal, a two-dimensional supra-molecular network parallel to the ac plane is built up from O-H⋯O and N-H⋯O hydrogen bonds.

[Clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 infection].

OBJECTIVE: To study the clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 (EV71) infection. METHODS: The clinical data of 32 hospitalized children with serious brainstem encephalitis caused by EV71 infection between May and December 2008 were retrospectively reviewed. RESULTS: The children whose age was younger than 3 years old accounted for 88% (22 cases). Fever(>38.5 degrees centigrade)lasting at least 3 days, frequent vomiting and limb twitch were presented as the main manifestations in the 32 children. Cyanosis, tachypnea, tachycardia and cold extremities were observed, and pulmonary edema or even pulmonary hemorrhage occurred in 8 children 3 to 4 days after the onset. The 32 children received a medical treatment: reduction of intracranial pressure with mannitol or frusemide, inhibition of inflammation reactivity with gamma globulin and methylprednisolone, and improvement of cardiac function and pulmonary edema with innotropic agents, fluid restriction and positive mechanical ventilation. CONCLUSIONS: Vegetative nerve functional disturbance is the main clinical feature of brainstem encephalitis caused by EV71 infection in children. An early identification and treatment of pulmonary edema or hemorrhage is of great importance.

TPX2 silencing exerts anti­tumor effects on hepatocellular carcinoma by regulating the PI3K/AKT signaling pathway.

Hepatocellular carcinoma (HCC) is one of the primary causes of cancer­associated deaths worldwide. Current treatment methods include surgical resection, chemotherapy and radiotherapy; however the curative rate remains low, thus novel treatments are required. The aim of the present study was to investigate the role of targeting protein for Xenopus kinesin­like protein 2 (TPX2) in the growth of HCC and its underlying molecular mechanism. Immunohistochemistry staining, reverse transcription­quantitative (RT­q)PCR and western blotting were used to detect the expression of TPX2 mRNA and protein in liver cancer tissue samples, adjacent normal liver tissue samples, and the HCC cell lines Huh7, Hep3B, PLC/PRF/5 and MHCC97­H. The recombinant plasmid pMagic4.1­shRNA­TPX2 was constructed and transfected into Huh7 and Hep3B HCC cells to silence TPX2 expression. The proliferation, apoptosis, migration and invasion of Huh7 cells and Hep3B cells were evaluated before and after TPX2 silencing. The mRNA and protein expression levels of multiple signaling pathway­associated genes were detected by RT­qPCR and western blotting. The expression levels of TPX2 mRNA and protein were significantly higher in HCC tissue samples compared with adjacent normal liver tissue sample. TPX2 mRNA and protein expression levels were detected in the different HCC cell lines. The recombinant plasmid pMagic4.1­shRNA­TPX2 was successfully transfected into Huh7 and Hep3B cells, resulting in TPX2 silencing. TPX2 knockdown significantly reduced cell proliferation, cell migration and cell invasion of Huh7 and Hep3B cells, whilst also increasing the rate of apoptosis in these cells. Following TPX2 silencing, the expression levels of PI3K, phospho­AKT, Bcl­2, c­Myc and Cyclin D1 were significantly decreased, whereas the expression levels of P21 and P27 were significantly increased. In conclusion, TPX2 may suppress the growth of HCC by regulating the PI3K/AKT signaling pathway and thus, TPX2 may be a potential target for the treatment of liver cancer.

[Response to therapy of 13 children with rhabdomyosarcoma].

OBJECTIVE: To study the clinical response to comprehensive therapy in children with rhabdomyosarcoma. METHODS: Clinical data of 13 children (8 males and 5 females) with rhabdomyosarcoma from January 1998 through October 2005 were retrospectively studied. Their ages ranged from 7 months to 12 years. The 13 cases of rhabdomyosarcoma consisted of 2 cases in stage I, 2 cases in stage II, 3 cases in stage III, and 6 cases in stage IV. Rhabdomyosarcoma was confirmed by biopsy, 12 cases (92.3%) presenting as embryonal type and 1 as alveolar type in histology. One patient underwent surgery treatment alone, one patient received surgery plus local radiation treatment, one patient received surgery plus chemotherapy and 10 patients were administered with a combination of surgery, local radiation treatment and chemotherapy. The chemotherapy protocol before 2002 was VDCA, VAC or VadrC. After 2002, the COG protocol was employed, with CDV+IE for stage III, and CT+VAC or CT+VAC+VCT for stage IV patients. RESULTS: The 2-year overall survival was 60% in the 10 patients who received a combination of surgery, local radiation treatment and chemotherapy, but the three patients died without receiving combination therapy. The 2-year overall survival in the 13 patients was 46.2%. The 2-year overall survival of the patients after 2002 (60%, 3/5) was higher than that before 2002 (37.5%, 3/8). CONCLUSIONS: Embryonal rhabdomyosarcoma dominates the histology type in children, which is highly malignant. A combination therapy of surgery, local radiation and chemotherapy can result in a satisfactory therapeutic effect in children with rhabdomyosarcoma.

[Detection of MYCN mRNA in neuroblastoma cell lines by quantitative RT-PCR].

OBJECTIVE: To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5. METHODS: MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies. RESULTS: Standard curves were linear and showed high correlations (R2>0.99). The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained (17.4 +/- 1.2). CONCLUSIONS: Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia.

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390-400. ©2016 AACR.