[Clinical Value of Detecting ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Based on High-Throughput Sequencing Technology].

OBJECTIVE: To compare the efficacy and clinical value of high-throughput sequencing (HTS) and Sanger sequencing in detecting ABL kinase domain mutations in patients with chronic myeloid leukemia (CML). METHODS: A total of 198 samples of 147 CML patients from July 2017 to March 2021 in Henan Cancer Hospital were collected and underwent high-throughput sequencing and Sanger sequencing to detect the mutations in ABL kinase domain, and the relevant clinical data were collected for comparative analysis. RESULTS: The proportion of total mutations and ≥2 mutations detected by high-throughput sequencing were significantly higher than those detected by Sanger sequencing (P =0.01; P =0.046). ≥2 mutations were detected in 22 cases, of which 5 cases (22.7%) had compound mutations. High-throughput sequencing can detect low level mutations that cannot be detected by Sanger sequencing. In 198 samples, 25 (12.6%) were low level mutations, 33 (16.7%) were high level mutations and 10 (5.1%) were mixed high and low level mutations. In the analysis of related clinical factors, the total mutation rate and the low level mutation rate in the optimal period, failure period and warning period were gradually increased (total mutation rate, P =0.016; low level mutation rate, P =0.005). The mutation rate of the samples with additional chromosomal abnormalities was also significantly increased (P =0.009). The mutation rate of patients who received first- and second-line treatment was significantly lower than that of patients who received third- or higher-line treatment (P =0.006). Analysis based on variant allele frequency (VAF) of the mutation site was helpful to visually evaluate the clonal evolution status of TKI-resistance CML cells. CONCLUSION: High-throughput sequencing is more sensitive and accurate than Sanger sequencing in mutation detection, which is helpful to accurately and visually evaluate TKI treatment response and optimize treatment strategy for CML.

[Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation].

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing. METHODS: The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method. RESULTS: The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance. CONCLUSION: MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Effect of the Recombinant Adenovirus-Mediated HIF-1 Alpha on the Expression of VEGF in the Hypoxic Brain Microvascular Endothelial Cells of Rats.

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated HIF-1 alpha (HIF-1α) on the expression of vascular endothelial growth factor (VEGFA) and HIF-1α in hypoxic brain microvascular endothelial cells (BMEC) in rats. METHODS: Primary cultured rat BMEC in vitro were treated without or with either recombinant adenovirus-mediated hypoxia-inducible factor-1 alpha (AdHIF-1α) or recombinant adenovirus empty vector (Ad) in the presence of CoCl2 (simulating hypoxia conditions), or were grown under normoxia conditions. The expression of VEGFA and HIF-1α was analyzed at 12h, 24h, 48h and 72h incubation time, respectively. We also accessed a GEO dataset of stroke to analyze in vivo the alteration of HIF-1α and VEGFA expression, and the correlations between HIF-1α, VEGFA and CD31 mRNA levels in vascular vessels after stroke. RESULTS: VEGFA and HIF-1α expression were significantly higher in at each time point in the AdHIF-1α than other groups (p<0.05), whereas the Ad group and hypoxia group, showed no statistically significant difference (p>0.05). Moreover, VEGFA and HIF-1α levels were significantly higher in BMEC under hypoxia conditions than normoxia conditions (p <0.05). Both HIF-1α and VEGFA expression significantly increased after stroke in vivo with 1.30 and 1.57 fold-change in log2, respectively. There were significantly positive associations between HIF-1α, VEGFA and CD31 mRNA levels in vivo after stroke. CONCLUSION: Hypoxia-induced HIF-1α and VEGFA expression in vascular vessels, and recombinant AdHIF-1α could up-regulate VEGFA, and enhance HIF-1ααlevels in BMEC in vitro, which may play an important role in the recovery of stroke.

Pathological Pattern of Intrahepatic HBV in HCC is Phenocopied by PDX-Derived Mice: a Novel Model for Antiviral Treatment.

BACKGROUND: Hepatitis B virus (HBV) is one of the most prominent risk factors for hepatocellular carcinoma (HCC) development and virus-mediated cases represents more than 80% of HCC in East Asia, where it is endemic. Currently, the HBV status of pathological HCC is not fully clarified, especially by comparison to nontumorous tissues. Lack of clinicopathological animal models of HCC impedes clinical application of antiviral treatment in the field. MATERIALS AND METHODS: A cohort sample of 14 HCC and corresponding stroma tissues were analyzed for pathological patterns of HBV antigens using immunohistochemistry; 10 fresh primary tumor tissues were inoculated into NOD/SCID mice and risk factors for patient-derived xenograft (PDX) model were identified by the univariate F test. Consistency of HBV features and cellular biomarkers between patient tissues and tumor grafts were examined. RESULTS: In HCC, HBV surface antigen (HBsAg) was mainly absent. Only 9.9% of samples showed HBsAg positivity in the tumor tissue that was limited to benign hepatocytes. In contrast, HBV core antigen (HBcAg) exhibited positive staining in all HCC tissues, located mainly in the cytoplasm of tumor cells. Of 14 HCC cases, three were diagnosed as occult infection of HBV based on HBcAg expression. The successful rate for the PDX model was 20% (2/10). Tumor lesions on hepatic lobes of V and VI, severe liver dysfunction and higher CA125 showed p-values of 0.01, 0.035, and 0.01, respectively. HBsAg absence in original tumors of #6 and 8 patients were faithfully reproduced by engraftments. Mixed distribution of HBcAg in cellular compartments of original tumor cells was also observed in mice. ki67 was dramatically increased in tumor grafts. CONCLUSION: We delineated pathological HBV profiles of HCC specimens and perilesional areas, which provided evidence for virus-based therapy in the future. PDX mice may phenocopy virological and cellular features of patient tissues, which is novel in the virus-related hepatocarcinogenesis field.

NDRG1 facilitates the replication and persistence of Kaposi's sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects host cells and establishes lifelong persistence as an extra-chromosomal episome in the nucleus. To persist in proliferating cells, the viral genome typically replicates once per cell cycle and is distributed into daughter cells. This process involves host machinery utilized by KSHV, however the underlying mechanisms are not fully elucidated. In present study, we found that N-Myc downstream regulated gene 1 (NDRG1), a cellular gene known to be non-detectable in primary B cells and endothelial cells which are the major cell types for KSHV infection in vivo, was highly upregulated by KSHV in these cells. We further demonstrated that the high expression of NDRG1 was regulated by latency-associated nuclear antigen (LANA), the major viral latent protein which tethers the viral genome to host chromosome and plays an essential role in viral genome maintenance. Surprisingly, knockdown of NDRG1 in KSHV latently infected cells resulted in a significant decrease of viral genome copy number in these cells. Interestingly, NDRG1 can directly interact with proliferating cell nuclear antigen (PCNA), a cellular protein which functions as a DNA polymerase clamp during DNA replication. Intriguingly, we found that NDRG1 forms a complex with LANA and PCNA and serves as a scaffold protein bridging these two proteins. We further demonstrated that NDRG1 is critical for mediating LANA to recruit PCNA onto terminal repeat (TR) of KSHV genome, and facilitates viral DNA replication and episome persistence. Taken together, our findings suggest that NDRG1 plays an important role in KSHV viral genome replication, and provide new clues for understanding of KSHV persistence.

Male hormones activate EphA2 to facilitate Kaposi's sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi's sarcoma.

There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi's sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells.

Guanylate-Binding Protein 1 Inhibits Nuclear Delivery of Kaposi's Sarcoma-Associated Herpesvirus Virions by Disrupting Formation of Actin Filament.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV.IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.

Prognostic significance of TET2 mutations in myelodysplastic syndromes: A meta-analysis.

Tet methylcytosine dioxygenase 2 (TET2) mutations occur frequently in myelodysplastic syndromes (MDS), but its prognostic impact has not been fully assessed and was controversial. Therefore, we performed a meta-analysis to evaluate the prognostic significance of TET2 mutations in MDS. PubMed, EMBASE databases and Cochrane Library were searched for studies reporting TET2 mutations and overall survival in MDS. Hazard ratios (HR) with 95% confidence interval (CI) were determined using random-effect modeling. A total of 1494 patients from nine studies were subjected to meta-analysis. The frequency of TET2 mutations was 18.34% (274/1494) in the study. MDS with TET2 mutations had similar overall survival compared to patients without the mutations (hazard ratio 1.13, 95% CI: 0.81-1.5).Our findings suggest that TET2 mutations have no prognosis impact on OS of patients with MDS. Therefore, the status of TET2 mutations cannot be served as a prognostic marker in MDS.

A Novel Recombinant Multi-Epitope Vaccine Could Induce Specific Cytotoxic T Lymphocyte Response In Vitro and In Vivo.

BACKGROUND: Malignant tumor is still one of the important diseases worldwide, cytotoxic CD8+ T lymphocytes (CTLs) play an important role in killing tumor cells. OBJECTIVE: To enhance the immune response of our previously identified HLA-A2-restricted CTL epitopes, we designed a multiepitope YL66. METHOD: The fusion protein GST-YL66 and DNA vaccine pcDNA3.1(+)-YL66 were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*02+ healthy donors and and in HLA-A2.1/Kb transgenic(Tg) mice. and the activity of induced CTLs were tested by IFN-γ relesde ELISPOT assay and LDH cytotoxicity assay. RESULTS: GST-YL66 induced CTL could lysis tumor cells and release IFN-γ both in vitro and in vivo, and pcDNA3.1(+)-YL66 could also induce significant CTL response in vivo. CONCLUSION: The designed fusion multiepitope YL66 could be used as a vaccine against patients with tumors expressing COX-2 and/or MAGE-4.

The immunogenicity of a novel cytotoxic T lymphocyte epitope from tumor antigen PL2L60 could be enhanced by 4-chlorophenylalanine substitution at position 1.

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer­testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe1]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe1]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.

A novel baseline-correction method for standard addition based derivative spectra and its application to quantitative analysis of benzo(a)pyrene in vegetable oil samples.

In the present work, a baseline-correction method based on peak-to-derivative baseline measurement was proposed for the elimination of complex matrix interference that was mainly caused by unknown components and/or background in the analysis of derivative spectra. This novel method was applicable particularly when the matrix interfering components showed a broad spectral band, which was common in practical analysis. The derivative baseline was established by connecting two crossing points of the spectral curves obtained with a standard addition method (SAM). The applicability and reliability of the proposed method was demonstrated through both theoretical simulation and practical application. Firstly, Gaussian bands were used to simulate 'interfering' and 'analyte' bands to investigate the effect of different parameters of interfering band on the derivative baseline. This simulation analysis verified that the accuracy of the proposed method was remarkably better than other conventional methods such as peak-to-zero, tangent, and peak-to-peak measurements. Then the above proposed baseline-correction method was applied to the determination of benzo(a)pyrene (BaP) in vegetable oil samples by second-derivative synchronous fluorescence spectroscopy. The satisfactory results were obtained by using this new method to analyze a certified reference material (coconut oil, BCR(®)-458) with a relative error of -3.2% from the certified BaP concentration. Potentially, the proposed method can be applied to various types of derivative spectra in different fields such as UV-visible absorption spectroscopy, fluorescence spectroscopy and infrared spectroscopy.

A novel synchronous fluorescence spectroscopic approach for the rapid determination of three polycyclic aromatic hydrocarbons in tea with simple microwave-assisted pretreatment of sample.

Many polycyclic aromatic hydrocarbons (PAHs) are carcinogenic, and some have been reported to be present in tea. People can be exposed to PAHs through tea consumption. Therefore, there is real importance for the determination of PAHs in tea. Because of the complex matrix of tea, it is hard to detect PAHs in tea without cleanup and chromatographic separation procedures. In this research, for the first time, a novel synchronous fluorescence spectroscopic approach coupling nonlinear variable-angle synchronous and matrix-isopotential synchronous scanning modes has been developed for the rapid determination of benzo(a)pyrene (BaP), benzo(k)fluoranthene (BkF), and anthracene (AN) in tea with simple microwave-assisted pretreatment of samples. This novel technique is able to resolve the spectra of the three PAHs well, even with interference from other EPA PAHs. The detection limits for BaP, BkF, and AN in tea were 0.18-0.28, 0.55-0.89, and 0.64-3.58 µg/kg, respectively, depending on various teas, with satisfactory recoveries ranging from 77.1 to 116%. The relative standard deviations achieved for BaP, BkF, and AN were 1.5, 6.6, and 8.5% for green tea; 2.9, 7.4, and 2.1% for oolong tea; and 5.6, 5.4, and 5.8% for black tea, respectively. Our results showed good correlation with those of gas chromatography-mass spectrometry. The approach developed is simple, reliable, and cost-efficient, providing an attractive alternative for the rapid selective screening of PAHs in tea.