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BACKGROUND Currently, the prognostic stratification and therapeutic evaluation systems for multiple myeloma (MM) lack specific molecular indicators. OC-STAMP is a new gene and is also highly expressed in MM. METHODS A total of 160 MM patients have been investigated with both quantitative reverse transcription PCR (RT-qPCR), flow cytometry (FCM) and cytogenetic FISH on the same mononuclear cells isolated from bone marrow specimens. RESULTS We found that OC-STAMP mRNA levels were significantly higher in newly diagnosed cases of MM than in healthy donors (median, 0.52% vs. 0.02%, P

About 25% of patients with newly diagnosed acute myeloid leukaemia (AML) have normal cytogenetics and no nucleophosmin 1 (NPM1) mutation or Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD). The prognosis and best therapy for these patients is controversial. We evaluated 158 newly diagnosed adults with this genotype who achieved histological complete remission within two cycles of induction therapy and were assigned to two post-remission strategies with and without an allotransplant. Targeted regional sequencing at diagnosis was performed and data were used to estimate their prognosis, including relapse and survival. In multivariable analyses, having wild-type or mono-allelic mutated CCAAT/enhancer-binding protein alpha (CEBPA) [hazard ratio (HR) 2·39, 95% confidence interval (CI) 1·08-5·30; P = 0·032), mutated NRAS (HR 2·67, 95% CI 1·36-5·25; P = 0·004), mutated colony-stimulating factor 3 receptor (CSF3R) (HR 2·85, 95% CI 1·12-7·27; P = 0·028) and a positive measurable residual disease (MRD)-test after the second consolidation cycle (HR 2·88, 95% CI 1·32-6·30; P = 0·008) were independently correlated with higher cumulative incidence of relapse (CIR). These variables were also significantly associated with worse survival (HR 3·02, 95% CI 1·17-7·78, P = 0·022; HR 3·62, 95% CI 1·51-8·68, P = 0·004; HR 3·14, 95% CI 1·06-9·31, P = 0·039; HR 4·03, 95% CI 1·64-9·89, P = 0·002; respectively). Patients with ≥1 of these adverse-risk variables benefitted from a transplant, whereas the others did not. In conclusion, we identified variables associated with CIR and survival in patients with AML and normal cytogenetics without a NPM1 mutation or FLT3-ITD.

A novel hyperbranched phosphorous containing poly(amidoamine-organosilicon) (P@PAMAMOS) was successfully synthesized and the structure was characterized using Fourier transform infrared spectra (FT-IR), 1H NMR and 31P NMR. The cotton fabrics was grafted with P@PAMAMOS by sol-gel methods. The results of P@PAMAMOS modified cotton fabrics were proved by FT-IR and scanning electron microscopy (SEM). The thermal degradation of blank cotton fabrics and P@PAMAMOS modified cotton fabrics were probed by thermogravimetric (TG) analysis, the phenomenon showed that modified cotton fabrics has exquisite thermal stability, when the temperature reached 700℃，the char residues of A10 treated cotton, B10 treated cotton, C10 treated cotton were 29.5%, 34.5%, 28.8%, respectively. The combustion properties of cotton fabrics were tested by limiting oxygen index (LOI) and vertical burning test. The untreated cotton fabrics was completely burned and the LOI value reached 18.0%, however the char diameter of 20wt% P@PAMAMOS modified cotton fabrics was reduced 3.2 cm and LOI value increase to 31.2%, this means that P@PAMAMOS modification shows desirable flame retardancy.

Ya-Lan Zhou,1 Qiu-Mei Yao,1 Jiao Zhou,1 Yan Chang,1 Jin-Lan Li,1 Ya-Zhe Wang,1 Hong-Ping Wu,2 Yu-Hong Chen,1 Yan-Rong Liu,1 Xiao-Jun Huang,1 Guo-Rui Ruan1 1Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Peking University People’s Hospital and Institute of Hematology, Beijing, China; 2Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Second Military Medical University, Shanghai, China Background: Daunorubicin is a traditional chemotherapeutic agent that plays a pivotal role in leukemia therapy. However, the dose-related toxicity remains a considerable challenge. The apoptosis-regulating gene, PDCD5, is downregulated in various tumors, including leukemias, and may provide a potential target for the diagnosis and treatment of leukemia. The purpose of this study was to construct a triple-regulated oncolytic adenovirus carrying a PDCD5 gene expression cassette (SG611-PDCD5) and explore the combined antitumor efficacy of SG611-PDCD5 in combination with low dose daunorubicin on leukemic cells. Materials and methods: A variety of leukemic cell lines, including K562, MEG-01, KG-1a, HL-60, SUP-B15, and BV-173, were cultured according to the providers’ instructions. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and PCR. The tumor-selective replication of the constructed conditionally replicating SG611-PDCD5 and its antitumor efficacy in combination with daunorubicin were characterized in leukemic cell lines in vitro and in a nude mouse xenograft model. Cell viability was detected using cell-counting kit-8. Apoptosis was detected in whole living cells using flow cytometry and in paraffin-embedded tumor tissues using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The triple-regulated CRAd carrying SG611-PDCD5 and nude mouse xenograft models of K562 cells were successfully constructed. In vitro treatment with SG611-PDCD5 in combination with low-dose daunorubicin elicited more potent anti-proliferative and proapoptotic effects in leukemic cells in a dose-dependent manner. The Chou-Talalay analysis revealed synergistic anti-proliferative effects in all of the above cell lines. In the nude mice xenograft model, the tumor size in the control, daunorubicin, SG611-PDCD5, and combined treatment groups on day 10 were 170.1±47.8, 111.9±81.1, 60.7±12.3, and 33.2±17.5mm3, respectively (all P

Introduction Cysteine and glycine-rich protein 2 (CSRP2) is gaining increasing attention as a therapeutic target due to its high expression in acute leukemias and its involvement in the development of cancer. However, whether it can be used as a reliable marker for minimal residual disease (MRD) remains unknown. Methods A total of 155 adult B-cell acute lymphoblastic leukemia (ALL) patients who received at least two cycles of consolidation chemotherapy were enrolled. Their leukemia-associated aberrant immune phenotypes (LAIPs) and CSRP2 transcript levels at the second consolidation chemotherapy (CON2) were detected by flow cytometry (FCM) and real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR). According to our published work, 1.80% and 0.01% were set as the positive threshold of CSRP2 transcript level and the FCM test for diagnosis, respectively. Pearson correlation coefficient was calculated to describe the relationship between the CSRP2 transcript level and the FCM test. Competing risk model and Cox proportional hazard regression model were conducted to estimated associations between the CSRP2 transcript level at CON2 and the prognosis. Results The median CSRP2 transcript level of all 155 patients was 0.2% (0.02%-108.17%). Among them, 108 patients were negative for both FCM and CSRP2, and 8 patients were positive for both FCM and CSRP2. The coincidence rate was 74.84%. There was a significant positive correlation between FCM and CSRP2 (r=0.73, 95% CI 0.64-0.79; P Patients were divided into a high CSRP2 group (N=17) and a low CSRP2 group (N=138) based on the transcript level of 1.00% settled by the ROC curve. Nine of 17 patients with high transcript level of CSRP2 suffered from leukemia relapse during the follow-up. Moreover, among the nine relapse patients, three patients had positive CSRP2 and negative FCM at CON2. In univariate analysis, patients with high CSRP2 transcript level showed a significantly lower 5-year leukemia-free survival (LFS) (33.0% vs. 48.6%, P =0.014) and 5-year survival (OS) (28.6% vs. 72.5%, P Conclusions Our study suggested that patients with a high CSRP2 transcript level at CON2 had poor survival and was an independent risk factor for relapse. The transcript level of CSPR2 at CON2 may be a valuable marker to complement the MRD assessment system and improve the number of evaluable patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

About 25% of patients with newly diagnosed acute myeloid leukaemia (AML) have normal cytogenetics and no nucleophosmin 1 (NPM1) mutation or Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD). The prognosis and best therapy for these patients is controversial. We evaluated 158 newly diagnosed adults with this genotype who achieved histological complete remission within two cycles of induction therapy and were assigned to two post-remission strategies with and without an allotransplant. Targeted regional sequencing at diagnosis was performed and data were used to estimate their prognosis, including relapse and survival. In multivariable analyses, having wild-type or mono-allelic mutated CCAAT/enhancer-binding protein alpha (CEBPA) [hazard ratio (HR) 2·39, 95% confidence interval (CI) 1·08-5·30; P = 0·032), mutated NRAS (HR 2·67, 95% CI 1·36-5·25; P = 0·004), mutated colony-stimulating factor 3 receptor (CSF3R) (HR 2·85, 95% CI 1·12-7·27; P = 0·028) and a positive measurable residual disease (MRD)-test after the second consolidation cycle (HR 2·88, 95% CI 1·32-6·30; P = 0·008) were independently correlated with higher cumulative incidence of relapse (CIR). These variables were also significantly associated with worse survival (HR 3·02, 95% CI 1·17-7·78, P = 0·022; HR 3·62, 95% CI 1·51-8·68, P = 0·004; HR 3·14, 95% CI 1·06-9·31, P = 0·039; HR 4·03, 95% CI 1·64-9·89, P = 0·002; respectively). Patients with ≥1 of these adverse-risk variables benefitted from a transplant, whereas the others did not. In conclusion, we identified variables associated with CIR and survival in patients with AML and normal cytogenetics without a NPM1 mutation or FLT3-ITD.

Background: About 25% of persons with new-diagnosed acute myeloid leukemia (AML) have normal cytogenetics and no NPM1 or FLT3-ITD mutation. The prognosis and best therapy of these persons is controversial. Methods: We evaluated 809 consecutive newly diagnosed adult with normal cytogenetics and 231 of whom had no NPM1 or FLT3-ITD mutation identified by targeted regional sequencing. 158 achieved a complete remission within 2 cycles of induction therapy and were assigned to 2 different postremission strategies: (1) 6 courses of consolidation chemotherapy (N=95); or (2) 2-4 courses of consolidation chemotherapy and an allotransplant (N=63). Findings: In multi-variable analyses a WBC ≥13·6×10E+9/L, mutated IDH2, not having a bi-allelic CEBPA mutation at diagnosis, a positive measurable residual disease (MRD)-test during consolidation and not receiving an allotransplant were independently associated with a higher cumulative incidence of relapse (CIR) and worse event-free survival (EFS). Amongst subjects with IDH2 mutations, non-bi-allelic CEBPA mutations or a positive MRD-test, subjects receiving an allotransplant had a lower 5-year CIR (16% [95% confidence interval, 6, 26%]; vs. 83% [72, 95%]; hazard ratio, HR=8·77 [4·05, 13·49]; P < 0·001) and better 5- year EFS (74% [60, 88%] vs. 15% [5, 25%]; HR=0·16 [0·09, 0·29]; P < 0·001). In contrast, in subjects with none of these adverse predictive variables there was no difference in CIR and EFS between those receiving an allotransplant and those who did not. Interpretation: Our data suggest a strategy to identify which persons with AML with normal cytogenetics and no NPM1 or FLT3-ITD mutation benefit from an allotransplant. Trial Registration: Registered in the www.clinicaltrials.gov, NCT01455272 and NCT02185261. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China [Grant 81570182 and Grant 81770156], the Peking University People’s Hospital Research and Development Funds [Grant RDF2018-02], the Beijing Municipal Science and Technology Commission [Z18111000960000], the Peking University Clinical Scientist Program[BMU2019LCKXJ003] and the Fundamental Research Funds for the Central Universities Declaration of Interests: RPG is a part-time employee of Celgene Corp. Ethics Approval Statement: The study was approved by the Ethics Committee of Peking University Peoples Hospital and written informed consent obtained from all study participants according to tenets of the Declaration of Helsinki.

V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.

Background About one-half of persons with acute myeloid leukemia have normal cytogenetics but have diverse outcomes which might be explained, at least in part, by specific mutations. We focused on DDX11, the budding yeast ortholog of ChlR1, which encodes an ATP-dependent RNA- and DNA-helicase involved in diverse cell processes such as sister chromatid exchange cohesions and implicated in other cancers and which is associated with telomere shortening. Methods DNA from 359 consecutive, newly-diagnosed adults with AML and normal cytogenetics were interrogated by deep target regional sequencing (TRS). Average effective sequencing depth was 1678.5X (1000.0-1800.0X) and median coverage, 99.9% (98.0, 100.0%). Outcomes studied included cumulative incidence of relapse (CIR) and event-free survival (EFS). DDX11 mutants were transfected into a human AML cell line which was studied in vitro and in immune deficient mice. Results We identified 2909 non-synonymous somatic variants. 284 subjects (79%; 95% confidence interval [CI], 75, 83%) had ≥5 mutations with a median of 8 mutations per subject (range, 1-20). We identified mutations at 4 new loci including DDX11 in 29 subjects (8%), NPIPA5 in 15 (4%), CYP2F1 in 13 (4%), and MED12 in 9 (3%). In multi-variable analyses DDX11 mutation was independently associated with CIR (Hazard Ratio [HR]= 2.276 [1.318, 3.931]; P=0.003) and EFS (HR=2.319 [1.391, 3.867], P=0.001). Two missense DDX11-mutant alleles (DDX11P316L/WT or DDX11Q363K/WT) were generated and introduced to the kasumi-1 leukemia cells by lentivirus transfection. Both mutants showed proliferative and anti-apoptotic activities in functional analyses and increased resistance to killing by Cytarabine and Daunorubicin in vitro and in transplanted immune deficient mice. Conclusions Subjects with AML with normal cytogenetics who have a DDX11 mutation have a higher CIR and worse EFS compared with subjects with wild-type DDX11. Functional studies of a transfected DDX11 human AML cell line provide a functional explanation of our clinical findings. * Correspondence Profs. Guo-Rui Ruan and Xiao-Jun Huang Peking University Peoples Hospital and Institute of Hematology No.11 Xi-Zhi-Men South Street, Beijing 100044, China T 86-10-88324672 F 86-10-88324672 E ruanguorui@pkuph.edu.cn OR huangxiaojun@bjmu.edu.cn Disclosures No relevant conflicts of interest to declare.

Introduction-About 25% of persons with new-diagnosed acute myeloid leukemia (AML) have normal cytogenetics and no NPM1 or FLT3-ITD mutation. The prognosis and best therapy of these persons is controversial. Methods-We evaluated 809 consecutive newly diagnosed adult with normal cytogenetics and 231 of whom had no NPM1 or FLT3-ITD mutation identified by targeted regional sequencing. 158 achieved a complete remission within 2 cycles of induction therapy and were assigned to 2 different post-remission strategies: (1) 6 courses of consolidation chemotherapy (N=95); or (2) 2-4 courses of consolidation chemotherapy and an allotransplant (N=63). Results-In multi-variable analyses a WBC ≥13·6×10E+9/L, mutated IDH2, not having a bi-allelic CEBPA mutation at diagnosis, a positive measurable residual disease (MRD)-test during consolidation and not receiving an allotransplant were independently associated with a higher cumulative incidence of relapse (CIR) and worse event-free survival (EFS). Amongst subjects with IDH2 mutations, non-bi-allelic CEBPA mutations or a positive MRD-test, subjects receiving an allotransplant had a lower 5-year CIR (16% [95% confidence interval, 6, 26%]; vs. 83% [72, 95%]; hazard ratio, HR=8·77 [4·05, 13·49]; P < 0·001) and better 5-year EFS (74% [60, 88%] vs. 15% [5, 25%]; HR=0·16 [0·09, 0·29]; P < 0·001). In contrast, in subjects with none of these adverse predictive variables there was no difference in CIR and EFS between those receiving an allotransplant and those who did not. Conclusions-Our data suggest a strategy to identify which persons with AML with normal cytogenetics and no NPM1 or FLT3-ITD mutation benefit from an allotransplant. Trial Registration: Registered in the www.clinicaltrials.gov, NCT01455272 and NCT02185261. Keywords: Acute myeloid leukemia, mutations, prognosis, targeted regional sequencing, measurable residual disease, risk stratification. *Correspondence Profs. Guo-Rui Ruan and Xiao-Jun Huang Peking University Peoples Hospital and Institute of Hematology No.11 Xi-Zhi-Men South Street, Beijing 100044, China T 86-10-88324672 F 86-10-88324672 Disclosures No relevant conflicts of interest to declare.

CALR mutations are detected in about 50% of persons of predominately European descent with essential thrombocythemia (ET) or primary myelofibrosis (PMF) with wild-type alleles of JAK2 and MPL. We studied 1088 Chinese with diverse myeloproliferative neoplasms including ET (N=234) and PMF (N=50) without JAK2(V617F) or MPL exon 10 mutations. CALR mutation was detected in 53% (95% CI, 46-60%) of subjects with ET and 56% (95% CI, 41-70%) of subjects with PMF. 152 CALR mutations were identified clustering into 15 types including deletions (N=8), insertions (N=3) and complex indels (N=4). We also identified 9 new mutations. Mean (±SD) mutant allele burden was 31±12% (range, 0.5-69%). Persons with PMF had higher CALR mutant allele burdens than those with ET (38±8% vs. 29±12%; P0.001). Amongst persons with CALR mutations, those with PMF had different clinical features from those with ET. These data may be useful for diagnosing ET and PMF in Chinese who are about 40% of all persons with ET and PMF and for monitoring therapy-response. They also highlight similarities and differences in CALR mutations between Chinese and persons of predominately European descent with these diseases.

Mandarin Chinese is a lexical tone language that has four tones, with a change in tone denoting a change in lexical meaning. There are few studies regarding lexical tone identification abilities in deafened children using either cochlear implants (CIs) or hearing aids (HAs). Furthermore, no study has compared the lexical tone identification abilities of deafened children with their hearing devices turned on and off. The present study aimed to investigate the lexical tone identification abilities of deafened children with CIs or HAs.Forty prelingually deafened children (20 with CIs and 20 with HAs) participated in the study. In the HA group, 20 children were binaurally aided. In the CI group, all of the children were unilaterally implanted. All of the subjects completed a computerized lexical tone pairs test with their hearing devices turned on and off. The correct answers of all items were recorded as the total score and the correct answers of the tone pairs were recorded as subtotal scores.No significant differences in the tone pair identification scores were found between the CI group and HA group either with the devices turned on or off (t=1.62, p=0.11; t=1.863, p=0.07, respectively). The scores in the aided condition were higher than in the unaided condition regardless of the device used (t=22.09, p0.001, in the HA group; t=20.20, p0.001, in the CI group). Significantly higher scores were found in the tone pairs that contained tone 4. Age at fitting of the devices was correlated with tone identification abilities in both the CI and HA groups. Other demographic factors were not correlated with tone identification ability.The hearing device, whether a hearing aid or cochlear implant, is beneficial for tone identification. The lexical tone identification abilities were similar regardless of whether the subjects wore a HA or CI. Lexical tone pairs with different durations and dissimilar tone contour patterns are more easily identified. Receiving devices at earlier age tends to produce better lexical tone identification abilities in prelingually deafened children.

Background Bone disease is an important determinant of quality-of-life and survival of persons with plasma cell myeloma (PCM). Biomarkers of bone disease could be useful to predict risk and monitor therapy. In our prior analyses OCSTAMP (osteoclast stimulatory trans-membrane protein) mRNA levels were identified as increased above normals in persons with PCM. OCSTAMP encodes a membrane-anchored cell surface receptor promoting nucleation of osteoclasts involved in bone resorption and osteoclast differentiation. Aims Measure levels of OCSTAMP mRNA in subjects with PCM and interrogate clinical associations. Methods OCSTAMP mRNA levels were quantified by quantitative real-time polymerase chain reaction (RT-qPCR) in 224 bone marrow samples from 160 subjects with PCM including 160 newly-diagnosed; 55 in remission and 9 with recurrent PCM. Results were compared with 42 normals and data expressed as ratio of OSSTAMP mRNA/ABL mRNA. Associations with clinical variables were interrogated and comparisons analyzed using the Chi-square test. Results OCSTAMP mRNA levels were significantly greater than normals in 111 subjects (69%, [95% confidence interval l[CI], 62-77%], P Conclusions OCSTAMP is highly transcribed in persons with newly-diagnosed and recurrent PCM compared with normals whereas persons in remission have similar levels to normals. OCSTAMP mRNA levels correlate with several clinical variables including Durie/Salmon stage, serum albumin C-reactive protein levels and likelihood of pathological bone fractures. OCSTAMP mRNA levels are potentially useful as a biomarker if our data are validated. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Biodegradable polymeric scaffolds have been widely used in tissue engineering as a platform for cell proliferation and subsequent tissue regeneration. Conventional microextrusion methods for three-dimensional (3D) scaffold fabrication were limited by their low resolution. Electrospinning, a form of electrohydrodynamic (EHD) printing, is an attractive method due to its capability of fabricating high-resolution scaffolds at the nanometer/micrometer scale level. However, the scaffold was composed of randomly orientated filaments which could not guide the cells in a specific direction. Furthermore, the pores of the electrospun scaffold were small, thus preventing cell infiltration. In this study, an alternative EHD jet printing (E-jetting) technique has been developed and employed to fabricate 3D polycaprolactone (PCL) scaffolds with desired filament orientation and pore size. The effect of PCL solution concentration was evaluated. Results showed that solidified filaments were achieved at concentration >70% (w/v). Uniform filaments of diameter 20 μm were produced via the E-jetting technique, and X-ray diffraction and attenuated total reflectance Fourier transform infrared spectroscopic analyses revealed that there was no physicochemical changes toward PCL. Scaffold with a pore size of 450 μm and porosity level of 92%, was achieved. A preliminary in vitro study illustrated that live chondrocytes were attaching on the outer and inner surfaces of collagen-coated E-jetted PCL scaffolds. E-jetted scaffolds increased chondrocytes extracellular matrix secretion, and newly formed matrices from chondrocytes contributed significantly to the mechanical strength of the scaffolds. All these results suggested that E-jetting is an alternative scaffold fabrication technique, which has the capability to construct 3D scaffolds with aligned filaments and large pore sizes for tissue engineering applications.

The molecular response of chronic myeloid leukemia (CML) patients to tyrosine kinase inhibitor treatment can be evaluated either by BCR-ABL mRNA levels on international scale (IS) or by log reduction from the baseline level of the laboratory. Both methods were compared in 248 newly diagnosed chronic phase CML patients treated with imatinib. The major molecular responses (MMR) obtained by both methods predict progression-free survival (PFS, all P0.0001). Thirty-six patients, who were identified as MMR patients by the IS method but as non-MMR patients by the log reduction method, had the same PFS as MMR patients identified by both methods. The molecular responses of patients at 3 and 6 months, as evaluated by the two methods, have similar predictive values on their cytogenetic responses at 12 months and on their molecular responses at 18 months. Both ≤ 10%(IS) and ≥ 1 log reduction at 3 months and ≤ 1%(IS) at 6 months were significantly associated with PFS (P=0.0011, 0.0090, and 0.0064). The percentages of patients with BCR-ABL(IS) of ≤ 1%,1-10%, and of10% at 3 months and 6 months in the German CML Study IV were similar with those with corresponding BCR-ABL(IS) in our center, but was significantly different with those evaluated by the log reduction method. Therefore, the molecular response evaluated by BCR-ABL(IS) has similar trends in PFS and in response prediction, but can better differentiate patients than that by the log reduction method. Furthermore, the IS method allows comparison among molecular response results from different laboratories.

nanohydroxyapatite (nHA) and collagen were utilized to fabricate the bio-inspired organic-inorganic composite coating (OICC) via the Drop-on-Demand (DoD) micro-dispensing technique, which could flexibly construct multi-layer structures with varied materials composition within a layer and /or among layers reliably. This technique has been further investigated on its capability of OICC fabrication with regards to various materials (hydroxyapatite and collagen) as well as its dispensing parameters. A four-layered structure was formed, with the sequence of nHA-collagen-nHA-collagen from bottom to top. The dispensing parameters were also investigated with regards to the characteristics of the OICC fabrication. The coating was then subjected to various characterizations including scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS) and adhesion test. SEM and XRD results revealed that the DoD micro-dispensing technique did not change the morphology and phase of these two coating materials. And the results of EDS further demonstrated the corresponding elemental distributions within the four-layered coating structure which demonstrated the feasibility of the DoD micro-dispensing technique for the fabrication of thin-layered OICC.

The FEM positive analysis is made using H-K non-stationary creep constitutive model in this paper, the finite element program for non-stationary viscoelastic-plastic displacement back analysis is compiled combining the non-stationary viscoelastic-plastic program with the complex shape optimization method, and the displacement back analysis of soft tunnel engineering is conducted. The result indicates that the viscous aging characteristics of rock mass can be reflected objectively if rock mass is regarded as non-stationary viscoelastic-plastic model, and the plastic zone development of surrounding rock can be predicted considering the plastic flow of rock mass and regarding the back analysis results as the calculation parameters.

Objectives To investigate the appearance of the pediatric larynx on ultrasound images and the diagnostic potential of ultrasound in cases of pediatric vocal fold paralysis. Methods First, we confirmed the ultrasonographic features of the laryngeal landmarks in 2 pediatric cadaveric larynxes. Secondly, 45 children were enrolled in a clinical study (13 patients with vocal fold paralysis and 32 normal children). Quantitative analysis of vocal fold mobility was carried out by measuring the maximum glottic angle (MGA) and vocal fold-arytenoid angle (VAA). Results All the paralyzed vocal folds showed abnormal mobility, and were flaccid during breathing and phonation. The rima glottis appeared as a hyperechoic air-column band in ultrasound images during phonation. The mean value of the MGA was 61.47 ± 9.00 in the normal larynx and 42.25 ± 10.41 in the paralyzed larynx. In the affected side of the paralyzed larynx, the VAA in maximum abduction was smaller than that in the normal larynx or in the unaffected side. The median difference of the VAA between maximum abduction and maximum adduction was less than that in the normal larynx. The kappa value was 0.96. Conclusion MGA and VAA are quantitative indicators of vocal fold immobility. Ultrasound is a reliable method of diagnosis of pediatric VFP. To diagnose VFP from an ultrasound image, the criteria are: (1) abnormal mobility (this was the most important and direct evidence), (2) hyperechoic air-column band of the glottic rima during phonation, (3) flaccid vocal fold and (4) asymmetry of the glottal structures.

CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias.

Recently, mutations in the nucleophosmin (NPM1) gene were detected in 50-60% of adult acute myelogenous leukemia (AML) patients, mainly with a normal karyotype. In this study, we detected typical NPM1 mutations (types A, B, D) in untreated Chinese AML patients using real-time quantitative polymerase chain reaction (RQ-PCR) followed by sequence analysis. The detection rate of NPM1 mutations in 220 AML patients was 16.4%, including 107 (14.2%) with the French-American-British (FAB) subtype M2, 43 (2.3%) with M3, and 52 (30.8%) with M4/M5. Only one case each with an NPM1 mutation was detected in four M0, seven M1, five M6, and two M7 cases. Eight patients were followed up after treatment, and five patients in hematologic remission continued to test negative for NPM1 mutations within 2-14 months of follow-up. Sequence analysis revealed that all the 36 positive cases were heterozygous for the mutation with 4-bp insertions at nt 959; the 36 cases included 29 (80.6%) cases with type A, four (11.1%) cases with type B, and one rare DD-3 mutation. We also detected two new mutations, namely, CTCG and CAAG insertions, named BJ-01 and BJ-02, respectively. Further, 38.9% (14/36) patients with NPM1 mutations simultaneously exhibited internal tandem duplications in the FLT3 gene, and 66.7% (22/33) patients did not express CD34. The results demonstrated that RQ-PCR was a reliable and sensitive method for detecting NPM1 mutations, for screening AML, and for the quantitative analysis of minimal residual diseases.

To explore the long-term efficacy of imatinib to chronic myeloid leukemia, a total of 46 late chronic phase (CP) patients were assessed after achieving complete cytogenetic response (CCyR). The median duration of imatinib treatment was 68 (61-74) months. Two hundred fifty-three bone marrow samples were detected BCR-ABL messenger RNA (mRNA) levels by TaqMan-based real-time quantitative reverse transcription-polymerase chain reaction. The median time when CCyR was first achieved was eight (3-72) months. Thirty-four patients achieved major molecular response (MMoR), and their median time when MMoR was first achieved was 35 (3-65) months. More patients achieving CCyR within 18 months obtained MMoR than those after 18 months (85% vs 42%, p = 0.006). One patient progressed into blastic crisis, and four patients suffered cytogenetic relapse later. The estimated 6-year event-free survival (EFS) rate of all patients was 81%. The BCR-ABL mRNA levels at the time of first CCyR of relapsed patients were significantly higher than those in continuous CCyR (p = 0.011). The 6-year estimated EFS rate of MMoR patients was significantly higher than that of non-MMoR patients (100% vs 44%, p = 0.0001). Achieving CCyR within 18 months had a higher probability of achieving MMoR within 6 years. The 6-year estimated EFS rate was significantly higher for patients achieving CCyR within 12 months than those after 12 months (97% vs 55%, p = 0.05). The time when MMoR was first achieved did not affect 6-year estimated EFS. Therefore, imatinib could induce most late CP patients to achieve long-term durable responses after achieving CCyR. Both the time when CCyR was first achieved and the depth of BCR-ABL reduction after CCyR are relevant to long-term EFS.

Background: The vast majority of chronic myeloid leukemia (CML) patients express the BCR-ABL transcript with the b2a2 (e13a2) and/or b3a2 (e14a2) junctions. However, some rare cases have atypical breakpoints. Methods and Results: We identified a CML patient with a unique e8a2 BCR-ABL transcript variant. It contained the first 114 nucleotides of BCR exon 8, with an insertion of 16 nucleotides from the 3′ end of ABL intron 1a, followed by ABL exon 2. Due to her uncontrolled thrombocytosis after 3 years of interferon-α treatment, the patient received imatinib at a dosage of 400 mg/day. Though achieving a sustained and complete hematological response after 3 months, she was resistant to imatinib during the entire 65-month imatinib treatment. That is, she failed to achieve major cytogenetic response and there was no significant decrease in her BCR-ABL transcript levels. Meanwhile, an M351T mutant was detected at 18 months after the start of imatinib treatment. Conclusion: ABL point mutation is also a mechanism of imatinib resistance for CML patients with the BCR-ABL transcript variant.

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed.We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden.We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background.PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

To further elucidate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF(121)cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The growth of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow endothelial cells (BMECs), and examined the synergic effects of antisense VEGF(121)cDNA and IFNalpha or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF(121)cDNA showed growth retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF(121)cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNalpha. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.

The purpose of the present study was to compare the reliability of microchip electrophoresis and capillary electrophoresis for screening FLT3-ITD gene mutation in acute myeloid leukemia. The FLT3-ITD mutation in the genomic DNA samples from 214 untreated AML patients were separately detected by PCR-microchip electrophoresis and PCR-capillary electrophoresis, then the DNA direct sequencing analysis was carried out. The results from PCR-microchip electrophoresis showed that there were 151 FLT3-ITD mutation negative, 58 FLT3-ITD mutation positive (58/214, 27.1%) and 5 FLT3-ITD mutation doubtful positive (5/214, 2.3%), while the outcomes from PCR-capillary electrophoresis displayed that there were 147 FLT3-ITD mutation negative and 67 FLT3-ITD mutation positive (67/214, 31.3%) without doubtful positive. In the 67 FLT3-ITD mutation positive samples detected by using PCR-capillary electrophoresis, 4 samples were detected as the negative while 5 samples were measured as the doubtful positive by using PCR-microchip electrophoresis. The followed sequencing analysis demonstrated that the above 9 samples were all FLT3-ITD mutation positive, indicating that PCR-capillary electrophoresis was more accurate and sensitive in screening the FLT3-ITD mutation, although statistic analysis showed that there were no significant differences in the detected results between PCR-microchip electrophoresis and PCR-capillary electrophoresis groups (Pearson Chi-squared Test, P0.05). It is concluded that both PCR-microchip electrophoresis and PCR-capillary electrophoresis were convenient and fast for screening FLT3-ITD mutation, but the accuracy of PCR-microchip electrophoresis awaits further improvement.

Biodegradable polymeric scaffolds have been widely used in tissue engineering as a platform for cell proliferation and subsequent tissue regeneration. Conventional microextrusion methods for three-dimensional (3D) scaffold fabrication were limited by their low resolution. Electrospinning, a form of electrohydrodynamic (EHD) printing, is an attractive method due to its capability of fabricating high-resolution scaffolds at the nanometer/micrometer scale level. However, the scaffold was composed of randomly orientated filaments which could not guide the cells in a specific direction. Furthermore, the pores of the electrospun scaffold were small, thus preventing cell infiltration. In this study, an alternative EHD jet printing (E-jetting) technique has been developed and employed to fabricate 3D polycaprolactone (PCL) scaffolds with desired filament orientation and pore size. The effect of PCL solution concentration was evaluated. Results showed that solidified filaments were achieved at concentration70% (w/v). Uniform filaments of diameter 20 μm were produced via the E-jetting technique, and X-ray diffraction and attenuated total reflectance Fourier transform infrared spectroscopic analyses revealed that there was no physicochemical changes toward PCL. Scaffold with a pore size of 450 μm and porosity level of 92%, was achieved. A preliminary in vitro study illustrated that live chondrocytes were attaching on the outer and inner surfaces of collagen-coated E-jetted PCL scaffolds. E-jetted scaffolds increased chondrocytes extracellular matrix secretion, and newly formed matrices from chondrocytes contributed significantly to the mechanical strength of the scaffolds. All these results suggested that E-jetting is an alternative scaffold fabrication technique, which has the capability to construct 3D scaffolds with aligned filaments and large pore sizes for tissue engineering applications.

To investigate the comparability of bcr-abl (P210) transcript levels detected in different hospitals.Ten hospitals in China took part in the four times of sample exchange and comparisons from April, 2010 to August, 2011. The exchange samples were prepared by Peking University People's Hospital. Firstly, the BCR-ABL (P210)(+) cells from a newly diagnosed chronic myeloid leukemia patient were 10-fold serially diluted by BCR-ABL (P210)(-) cells and they covered 4 magnitudes. Then, TRIzol reagents were thoroughly mixed with cells in each tube. Every 12 samples (three samples per magnitude) were sent to the other 9 hospitals. The cell number of each sample was 8×10(6). The detection of bcr-abl transcript levels by real-time quantitative PCR were performed in every hospital according to their own protocols. Conversion factors (CF) were calculated using regression equation.Differences in bcr-abl transcript levels did exist among results of 10 hospitals in each comparison. In general, the results of the most of hospitals were in line with the dilutions of cells. CF of every hospital fluctuated. Three hospitals had relatively stable CF, and their ranges were 2.8 - 5.2, 1.2 - 2.8 and 2.2 - 6.8, respectively; two hospitals had unstable CF with ranges 0.76 - 7.0 and 2.1 - 18.7; three hospitals couldn't be calculated CF one or two times because of the significant deviation of the results from the actually bcr-abl transcript levels, and their ranges of CF which could be calculated were 1.9 - 19.2, 3.6 - 7.6 and 0.18 - 14.7; One hospital only had two CF (3.3 and 5.0) because of the replacement of an important reagent during the period of comparisons.Comparability of bcr-abl (P210) transcript levels between different hospitals could be achieved through CF which acquired by sample exchange and comparison. The stable and reliable detection system is the premise to acquire correct CF.

To compare the immunophenotypic and clinical characteristics between NPM1 mutated acute myeloid leukemia (AML) (NPM1m(+)AML) and unmutated AML(NPM1m(-)AML) not otherwise characterized (NOS) under similar FAB subtypes constituent ratio.Immunophenotyping and NPM1 gene mutation type-A, B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 104 AML patients with NPM1m(+)AML and performed immunophenotyping assay were included, 97 with NPM1m(-)AML.There were significant difference between the two groups at presentation in terms of sex, white blood count(WBC), platelet counts (PLT), blast ratio, normal karyotype ratio, WT1 expression level, FLT3-ITD mutation positive rate and remission rate of first course of induction therapy (P0.05). On the immunophenotype, the expression of early differentiation antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2), myeloid and monocytic differentiation-associated antigens (CD13, CD14, CD15) were lower, and that of CD33 as well as CD123 were higher in NPM1m(+)AML patients. Among them, only CD34, HLA-DR, CD7, and CD4 positive cases were significantly lower in NPM1m(+)AML group than in NPM1m(-)AML group (P0.05), the rest of them had significant difference in the number of positive cells (P0.05). Above features were further analyzed between the M1/M2 and M4/M5 subgroups. M1/M2 cases retained the women prominent and had a higher WT1 expression level (P0.05). The expression of monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens were higher and that of CD117 were lower in M4/M5 subtype (P0.05). Among them, the positive rates of HLA-DR, CD64, CD11b, CD10, CD15, and CD4 were significantly higher in M4/M5 than in M1/M2 in NPM1m(+)AML group (P0.05).The most clinical characteristics in NPM1m(+)AML patients are consistent with reports, but some immunophenotype are different to the previous reports under similar FAB subtypes constituent ratio. The major immunophenotypic features of NPM1m(+)AML patients are lower expression of progenitor, myeloid and lymphoid lineage antigens. Monocytic differentiation-associated antigens are only higher expression in M4/M5 cases when comparison with M1/M2 cases within NPM1m(+)AML group.

The aim of this study was to compare the basic characters of the speech-evoked auditory brainstem response (speech-ABR) between young adults and school-age children.Speech-ABR of thirty eight normal hearing subjects including eighteen school-age children and twenty young adults were recorded. The speech syllable/da/as stimulus sound was transmitted to right ear by insertion the earphones in speech-ABR test.Response waves of speech-ABR in school-age children were similar to those in young adults, which contained the onset response (peak V and A), the transition (peak C), the frequency following responses (peak D, E and F) and the offset response (peak O). Both the latency and amplitude showed no significant difference in all waves between young adults and school-age children, except the latency of wave O and amplitude of wave F. The latency of O wave in school-age children (47.80 ± 0.38) ms were significantly shorter than that in adults (48.10 ± 0.40) ms (t = 2.330,P = 0.026). The amplitude of F wave in school-age children (-0.21 ± 0.15) µV were significantly larger than that in adults (-0.12 ± 0.08) µV (t = 2.146,P = 0.043).Both the latency and amplitude of the speech ABR in school age children at 6 - 11 years old show the great similarity with the young adults, which indicate that the ability of speech processing of brainstem in children has completely reached maturity.

This study was purpose to investigate the biological characteristics of B lymphoblastic leukemia (B-ALL) between CD34 positive CD38 positive (CD34(+)CD38(+)) and CD34(+)CD38(low/-) subgroups and their clinical significance. Immunophenotyping of B cells in bone marrow of 54 patients with newly diagnosed CD34(+)B-ALL were analyzed by 4 color multiparametric flow cytometry (FCM). According to the different expression of CD38, the newly diagnosed patients with B-ALL were divided into two groups: CD34(+)CD38(+) subgroup and CD34(+)CD38(low/-) subgroup. BCR-ABL, TEL-AML1 fusion genes and WT1 gene were detected by real time RT-PCR simultaneously. After chemotherapy, minimal residual disease (MRD) was monitored by one tube of 7 color FCM. The average follow-up time was 12 months (range 1 - 28), the average follow-up interval was 2 months (range 1 - 5). The results showed that there was no significant differences such as WBC, Plt count and Hb level between the two groups at diagnosis, the positive rate of BCR-ABL, TEL-AML1 and WT1 gene was also no significantly different. After clinical complete remission (CR), MRD positive (MDR(+)) case rates were 28.57% (10/35) in CD34(+)CD38(+) subgroup and 68.42% (13/19) in CD34(+)CD38(low/-) subgroup (P0.01). The relapse rate between the two groups was 5.71% (2/35) in CD34(+)CD38(+) subgroup (relapse time at 94 and 245 d respectively) and 36.84% (7/19) in CD34(+)CD38(low/-) group [median relapse time was 263 d (range 46 - 468), P0.01]. The age distribution was analyzed in these two subgroups (16 or ≤ 16 years old), there was 8 (8/35) adult patients (16 years old) in CD34(+)CD38(+)group and 10 (10/19) adult patients in CD34(+)CD38(low/-) group (P0.05). It is concluded that CD34(+)CD38(low/-) phenotype is more often presented in adult patients and the CD34(+)CD38(low/-) patients with B-ALL are more likely to have MRD(+)and relapse after treatment.

Chimerism analysis is an important tool for the surveillance of post-transplant engraftment. It offers the possibility of identifying impending graft rejection and recurrence of underlying malignant or non-malignant disease. Here we investigated the quantitative chimerism kinetics of 21 relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation (HSCT).A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time polymerase chain reaction (RT-PCR) to obtain the informative marker for every leukemia patient. Quantitative chimerism analysis of bone marrow (BM) samples of 21 relapsed patients and 20 patients in stable remission was performed longitudinally. The chimerisms of BM and peripheral blood (PB) samples of 14 patients at relapse were compared.Twenty-one patients experienced leukemia relapse at a median of 135 days (range, 30 - 720 days) after transplantation. High recipient chimerism in BM was found in all patients at relapse, and increased recipient chimerism in BM samples was observed in 90% (19/21) of patients before relapse. With 0.5% recipient DNA as the cut-off, median time between the detection of increased recipient chimerism and relapse was 45 days (range, 0 - 120 days), with 76% of patients showing increased recipient chimerism at least 1 month prior to relapse. Median percentage of recipient DNA in 20 stable remission patients was 0.28%, 0.04%, 0.05%, 0.05%, 0.08%, and 0.05% at 1, 2, 3, 6, 9, and 12 months, respectively, after transplantation. This was concordant with other specific fusion transcripts and fluorescent in situ hybridization examination. The recipient chimerisms in BM were significantly higher than those in PB at relapse (P = 0.001).This SP-based RT-PCR assay is a reliable method for chimerism analysis. Chimerism kinetics in BM can be used as a marker of impending leukemia relapse, especially when no other specific marker is available. Based on our findings, we recommend examining not only PB samples but also BM samples in HSCT patients.

Acute myeloid leukemia with mutated nucleophosmin (NPM1m+AML) is a heterogeneous entity. We investigated whether NPM1m+AML with monocytic or myeloid differentiation have distinct immunophenotype. The study included 160 NPM1m+AMLpatients and 178 AML patients without NPM1 mutation and recurrent cytogenetic abnormality (NPM1wt-AML). We analyzed the immunophenotype by flow cytometry. NPM1 mutation was detected by PCR. Compared with NPM1wt-AML patients, NPM1m+AML patients showed higher positive rates of CD33 and CD9 and lower positive rates of CD34, HLA-DR, CD7, CD15 and CD117 (all P

To investigate the application of auditory brainstem response (ABR) and 40 Hz auditory event related potential (40 Hz AERP) to the diagnosis of occupational noise-induced hearing impairment and to provide the evidence for diagnosis of occupational deafness.Pure tone audiometry, ABR and 40 Hz AERP were performed in 54 workers occupationally exposed to noise. The thresholds of higher frequency band, 3 kHz and 4 kHz were compared with the threshold of ABR. The thresholds of auditory frequency ban and 0.5 kHz were compared with the threshold of 40 Hz AERP.A better correlation was found between thresholds of ABR and higher frequency pure tone audiometry. There was a significant difference of thresholds between 40 kHz AERP and pure tone audiometry. The correction values of thresholds between 40 kHz AERP and pure tone audiometry in the light noise-induced hearing impairment group and the moderate noise-induced hearing impairment group were (16.43 ± 1.08) and (11.80 ± 1.12) dBn HL, respectively.In diagnosis of occupational noise-induced hearing impairment, the threshold of ABR can be used to estimate the hearing threshold of pure noise higher frequency. Because there is the significant difference of the thresholds between pure tone audiometry and 40 Hz AERP, the response threshold can not be served as the audiometry threshold, and the behavioral hearing thresholds can only be obtained by adjusting the response threshold with respective correction value.

Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed. Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Disclosures No relevant conflicts of interest to declare.

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT.A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally.Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method.This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

To investigate the effects of Shenfu (SF) injection on hemodynamics and plasma E-selectin concentrations in elderly patients undergoing total hip replacement (THR).A total of 24 ASA II/III patients aged 65 to 85 years old were divided equally into two groups (n = 12). In Group S, SF injection was administered by peripheral intravenous infusion at initially 0.2 ml/kg and then 0.8 ml×kg(-1)×h(-1) until the end of operations. In Group N, normal saline was administered similarly. All patients received a post-operative regimen of patient-controlled epidural analgesia (PCEA). Blood samples were taken pre-operation (T(0)), immediately post-operation (T(3)) and 24 hours post-operation (T(4)) so as to detect the levels of E-selectin at these time points. mean arterial pressure (MAP), heart rate (HR) and central venous pressure (CVP) were continuously monitored and recorded at T(0), 15 min post-anesthesia (T(1)), 30 min post-anesthesia (T(2)) and T(3).The concentrations of E-selectin in Group N increased at T(3) and T(4) while those decreased in Group S [(119 ± 23) mg/L and (109 ± 23) mg/L vs (86 ± 15) mg/L, (83 ± 15) mg/L and (83 ± 12) mg/L vs (92 ± 37) mg/ L, all P0.01]. As compared with Group S, they were significantly higher in Group N (P0.05 or 0.01). There was no significant difference in CVP, HR and MAP between two groups.The activation of vascular endothelial cells is manifested by an elevated plasma concentration of E-selectin in the elderly patients undergoing THR. SF injection can inhibit the activation and exert no significant effects on the hemodynamics.

This study was aimed to investigate whether difference exists between real time RT-PCR results of nucleated cells isolated by lysis method and mononuclear cells isolated by gradient concentration method. 14 bone marrow samples from leukemia patients (7 samples of AML-M(2), 1 of AML-M(4), 1 of AML-M(4)EO, 1 of AML-M(6), 1 of APL and 3 of CML) were collected. Each sample was divided into 2 parts, and was used to isolate mononuclear cells by Ficoll-Hypaque gradient centrifugation, and other was used to isolate nuclear cells by lysis method. The RNA extraction and detection of internal reference gene ABL for all of samples were performed by RT-PCR, and mRNA expression levels in 3 samples of BCR/ABL, 6 of AML/ETO, 1 of CBFβ-MHY11, 1 of WT1 and 6 PRAME were detected by RT-PCR. The results showed that ABL copies of all samples were over 3 × 10(4), and there was significant difference in ABL copies between each pair of samples by lysis and gradient centrifugation method (p0.05). There was also no significant difference in every detected mRNA levels between 14 pair samples (p0.05). It is concluded that the lysis method may be useful one for extracting RNA from nuclear cells, and can adopt as a routing detection method for simultaneously extracting RNA from many samples to detect leukemia specific mRNA by using RT-PCR.

The expression levels of programmed cell death 5 (PDCD5) are down-regulated in many malignancies. SG611-pdcd5, a recombinant conditionally replicative adenovirus carrying pdcd5 gene expression cassette, can evidently kill the leukemic cells and protect selectively the normal cells. The purpose of this study was to investigate the synergistic killing effect of SG611-pdcd5 and low-dose etoposide (VP-16) on K562 cells. K562 cells were treated with different concentrations of VP-16 or different multiplicities of infection (MOI) of SG611-pdcd5. After 48 hours of incubation the cell viability was determined by using MTT assay. The results showed that the cell viability of SG611-pdcd5 (MOI = 40) plus VP-16 (0.5 µg/ml) group significantly decreased as compared with single SG611-pdcd5 (MOI = 40) treatment group or single VP-16(0.5 µg/ml) treatment group (42.00 ± 5.75% vs 59.45 ± 4.12%; 42.00 ± 5.75% vs 82.91 ± 3.41%, respectively, both p0.05). The synergistic killing effect of SG611-pdcd5 plus VP-16 was higher than that of PDCD5 protein plus VP-16 or that of non-replicating adenovirus carrying pdcd5 (Ad-pdcd5) plus VP-16 (both p0.05). The cell viability of VP-16 (4.0 µg/ml) plus SG611-pdcd5 (MOI = 40) group, VP-16 (4.0 µg/ml) plus proPDCD5 (40 µg/ml) group and VP-16 (4.0 µg/ml) plus Ad-pdcd5 (MOI = 80) group was 37.09 ± 1.89%, 52.36 ± 1.64% and 73.64 ± 4.33%, respectively. It is concluded that SG611-pdcd5 can promote K562 cell death induced by low-dose VP-16. The combination of SG611-pdcd5 and VP-16 can enhance the killing effect on leukemic cells.

To investigate the effects of SF injection on coagulation function, p-selectin and D-dimer concentrations in plasma of elderly patients undergoing total hip replacement (THR).Twenty-four ASA II/III patients aged 65 to 85 were divided equally into two groups (n = 12). In Group S, SF injection was administered by peripheral intravenous infusion at initially 0.2 ml/kg and then 0.8 ml (kg/h) until the end of operations. In Group N, normal saline was administered similarly. All the patients were given patient-controlled epidural analgesia (PCEA) after operations. Blood samples were taken before operations (T(0)), immediately post-operation (T(1)) and 24 h post-operation to detect the levels of p-selectin, D-dimer, blood platelet count and clotting time at all time points.The concentrations of p-selectin and D-dimer in Group N increased at T(1) and T(2) while those in Group S decreased at T(1) (P0.05). Compared with Group N, they were significantly lower in Group S (P0.05). There was no significant difference at T(2) between two groups.The plasma concentrations of p-selectin and D-dimer increase obviously in elderly THR patients. SF injection can decrease the plasma concentrations of p-selectin and D-dimer in patients during surgery. Thus it may protect the vascular endothelial cells and attenuate the activation of platelet and fibrinolysis.

The aim of this study was to investigate the gene expression of programmed cell death 5 (pdcd5) in plasma and bone marrow cells from patients with multiple myeloma (MM). Enzyme liked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) were used to examine pdcd5 gene expression in plasma and marrow cells in 45 MM patients and 20 normal controls. The results showed that serum levels of PDCD5 protein in 45 MM patients were lower significantly compared with the normal controls and 20 responsive patients after chemotherapy, their plasma levels were (16.91 +/- 0.28) ng/ml, (19.11 +/- 0.29) ng/ml and (17.94 +/- 0.154) ng/ml respectively (p0.05). The pdcd5 gene expression levels detected by RQ-RT-PCR in 45 MM patients were lower significantly compared with the normal controls, their pdcd5 gene expression levels were 0.64 +/- 0.47 and 1.28 +/- 1.21 respectively (p0.05). It is concluded that the PDCD5 protein expression levels are low in patients with MM. These findings suggest that abnormal expression of pdcd5 may be involved in the pathogenesis of MM.

This study was aimed to detect the expression level of cmtm 5 (CKLF-like MARVEL transmembrane domain containing member 5) gene in the bone marrow cells from patients with multiple myeloma (MM), and to investigate the correlation between the expression level of cmtm5 and various clinical characteristics. Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) was used to measure the expression levels of cmtm5 gene in the bone marrow cells collected from MM patients, and the MM cell lines, namely, RPMI8226 and CZ1 cells. The normal donor marrow specimens were used as the reference. The ratio of cmtm5 copy number to abl (Abelson murine leukemia viral oncogene homolog) gene copy number was used for indicating the expression level. The results showed that the expression level of cmtm5 gene was significantly down-regulated in bone marrow cells of 51 untreated or relapsed/refractory MM patient as compared to those of normal donor marrow cells (0.047+/-0.062 for the untreated or relapsed/refractory MM patients versus 0.255+/-0.333 for the normal, p0.01). According to the International Staging System (ISS), the cmtm5 expression level in marrow cells of patients in ISS III stage was significantly lower than that in patients in ISS I stage (0.034+/-0.034 for the ISS III stage versus 0.103+/-0.109 for ISSI stage, p0.01). Similarly, lower expression levels of cmtm5 gene were also found in two human MM cell lines (0.014+/-0.009 for RPMI8226 cells and 0.004+/-0.006 for CZ1 cells). After the MM patients were effectively treated, their expression levels of cmtm5 gene significantly increased (0.020+/-0.005 for the untreated patients versus 0.227+/-0.038 for the effectively treated patients, p0.01). A significant negative correlation was observed between the expression level of cmtm5 gene and the number of bone marrow plasma cells (r=-0.307, p0.05). However, the correlation was not found between the expression level of cmtm5 gene and the clinical characteristics, such as gender, age, hemoglobin level, or M-protein level, etc. It is concluded that the expression level of cmtm5 gene is abnormally lower in the bone marrow cells from the MM patients, and are associated with ISS stages. Furthermore, the expression level of cmtm5 gene is negatively correlated with the number of bone marrow abnormal plasma cells in MM patients, which suggests that the abnormally lower expression of cmtm5 may be involved in the pathogenesis of the MM patients.

To explore the characteristics of BCR–ABL kinase domain point mutations in Chinese chronic myeloid leukemia (CML) patients, a cohort of 127 patients with hematologic or cytogenetic resistance to imatinib are screened by direct sequencing. Mutations are found in 74 patients (58%). More patients with hematologic resistance show mutations compared to patients with cytogenetic resistance (70% vs 44%, p = 0.002), and more patients with acquired resistance present mutations compared to patients with primary resistance (68% vs 48%, p = 0.031). Frequencies of mutations were similar in early chronic phase (ECP), late chronic phase (LCP), accelerated phase, and blast phase (BP) patients (56%, 58%, 50%, and 69%, respectively; p > 0.05). Overall, 25 mutants are found in 21 amino acid sites, and four of them (I418V, E450A, E453L, and E455K) are first reported here. All patients with these four mutants either progress to or reenter the BP. The most frequent mutant is M244V, followed by Y253H, F359C/V/I, G250E, E255K, and T315I. Only seven patients (9%) have T315I mutants, and all showed hematologic resistance. Three of them were in the ECP and three in the LCP. Look-back studies show that mutants were detected 0–20 (median 7) months ahead of the appearance of clinical resistance in 15 tested patients with acquired resistance. ABL mutations are common in Chinese imatinib-resistant CML patients and are associated with clinical resistance. Chinese patients also seem to have unique profiles in the types and frequencies of some mutants, the disease phases of patients with T315I mutation, and the frequency of mutations in CP patients.