Objective:Use the droplet digital PCR (ddRCR) technology to establish, optimize and evaluate the method of EGFR-T790M mutation detection.Methods:The relevant probes and primers were designed for EGFR-T790M mutations. The ddPCR reaction system was established, the optimal annealing temperature was set and the basic performance of the method was tested. On this basis, from January 2019 to October 2019, 72 cell-free DNA (cfDNA) samples from NSCLC patients were collected from Shandong Cancer Hospital Affiliated to Shandong First Medical University, and clinically verified. The consistency of the gene mutation detections with Bole ddPCR products was analyzed using Kappa test.Results:The ddPCR reaction system was established and optimized. Linear evaluation showed the R2 value was greater than 0.99. Using ddPCR, the blank detection limit was determined to be the numbers of mutant droplets≥2, with excellent specificity. For the sensitivity analysis, the lower limit of mutation detection was determined to be at least 0.05%. In the repeatability and inter-assay precision tests, the results had a coefficient of variation( CV)<20%. The relative deviation of the results was within the range of±10% for the accuracy analysis. Using the established T790M mutation detection method, 72 samples from the NSCLC patients were tested for genetic mutation in cfDNA, and the overall agreement with the Bole ddPCR products was 91.67% (66/72, Kappa=0.749; P<0.001). Conclusion:Using ddPCR, the method of EGFR-T790M mutation detection for NSCLC was successfully established.

Programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) checkpoint blockades have dramatically changed the treatment of non-small cell lung cancer (NSCLC). But we still have no definite biomarkers that may predict the efficacy of treatment by PD-1/PD-L1 inhibitors. In the 18th World Conference on Lung Cancer, the biomarkers that may predict the efficacy of treatment by PD-1/PD-L1 inhibitors in patients with lung cancer has been a popular topic, and it has huge potential in the future. In order to enable more patients to get more benefits from treatment, researchers are looking forward to finding the optimum biomarkers. By organizing and summarizing the information about the biomarkers predicting PD-1/PD-L1 in patients with lung cancer, this review mainly focused on the following six aspects to introduce: expression of PD-L1; tumor mutational burden and the ability of mutation repair, malignant tumor driver mutation, biomarker of immunological effect, blood cell account, comprehensive analysis model. We are hoping to help doctors to find the best biomarker, then much more lung cancer patients could obtain antitumor effects in PD-1/PD-L1 inhibitors treatment.
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Antineoplastic Agents ; pharmacology ; therapeutic use ; B7-H1 Antigen ; antagonists & inhibitors ; Biomarkers, Tumor ; metabolism ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Programmed Cell Death 1 Receptor ; antagonists & inhibitors

BACKGROUND: During the occurring and developing of tumor, tumor-educated platelets mRNA profiles were altered. Since platelets are anuclear, the level of mRNAs is probably post-transcriptional regulated by the splicing maturation of pre-mRNA and alternative splicing. Apoptotic chromatin condensation inducer 1 (ACIN1) has been shown to be a component of a splicing-dependent multiprotein exon junction complex (EJC) and was involved in mRNA metabolism associated with splicing. This study analyzed the expression of ACIN1 mRNA in platelets, and explored its potential as a biomarker of lung cancer. METHODS: 156 patients with lung cancer and 58 healthy controls in Shandong Cancer Hospital were collected. We isolated platelet pellets by low-speed centrifugation and extracted total RNA. The expression of ACIN1 mRNA in platelets was detected by RT-PCR, the results were analyzed statistically. And the relationship between expression of ACIN1 mRNA and clinical factors were also analyzed. RESULTS: The expression level of ACIN1 mRNA in platelets of patients with lung cancer was significantly higher than that in platelets of healthy controls (P=0.015). The ROC curve showed that the area under the curve of ACIN1 mRNA for detecting lung cancer were 0.608. The expression of ACIN1 mRNA in platelets of lung cancer has no significant relationship with age, gender, pathological type and metastasis or not (P>0.05). CONCLUSIONS ACIN1 mRNA was highly expressed in platelets of lung cancer patients, and the detection of its expression level might have potential clinical value for the diagnosis of lung cancer.
Blood Platelets ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; blood ; genetics ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism

The advantages of saliva-based biomarkers detection in cancer were accurate,simple and noninvasive.Currently,biomarkers validated for saliva detection include protein,mRNA,miRNA and DNA,using PAGE,microarray and sequencing,respectively.Analysis of literatures shows;that saliva biopsy plays an important role in cancer diagnosis,clinical treatment and prognosis.Due to the design of experiments reported werediversification,a large number of validations are necessary to standardize saliva collecting,processing,testing methods and quality assurance.

Circulating DNA is defined as a kind of extracellular DNA that exists in plasma,cerebrospinal fluid and synovial fluid.The concentration of circulating DNA of cancer patients is significantly higher than that in healthy people.The genetic and epigenetic alterations of circulating cell-free nucleic acids are relevant to cancer development and progression,for example,gene mutation,DNA methylation and microsatellite instability and so on.The quantitative and qualitative detection of circulating DNA shows promising potential value in cancer screening,diagnosis,disease monitoring treatment and prognosis.

Background and purpose:Sentinel lymph node biopsy is regarded as the standard of care in pa-tients without clinical axillary lymph node metastases in early-stage breast cancer. Accurate detection of sentinel lymph node is an important step for staging, prognosis, and treatment. In this study, a new sentinel lymph node tracer was produced by the rituximab to combine with the lfuorescence tracer (indocyanine green, ICG), and to identify the most appropriate combination ratio of the two agents. Its biological property and safety limitation were evaluated.Methods:Rituximab was combined directly with ICG. The new tracer was analyzed for labeled rate by instant thin-layer chroma-tography-silica gel, molecular integrity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular immune activity by ELLAS. The safety limitation was tested according to the Chinese Pharmacopeia. The localization ability of sentinel lymph node was tested in mice.Results:The new tracer was intact and kept the immune activity of rituximab. The ICG labeled rate of rituximab was 100%. The new tracer was bacteria and pyogen free, and was safe to body with location injection. The most appropriate combination ratio of rituximab and ICG was 4∶1 and 6∶1 with the best sentinel lymph node imaging. The location of sentinel lymph node identiifed by the new tracer was accorded with the radiotracer.Conclusion:The preparation method of the new sentinel lymph node tracer is simple and no radioactive injury. The new tracer has no bacteria, no pyogen and no acute toxicity, and can be used in sentinel lymph node visual-ization.

ObjectiveTo investigate the role of OPN in human breast cancer cell line ( MDA-MB-231) by using small interfering RNA to specifically knockdown OPN expression. MethodsOPN ShRNA expression vector was stably transfected to MDA-MB-231 cell line.The expression of OPN mRNA and protein were analyzed using reverse transcription polymerase chain reaction (RT-PCR)and Western blot,respectively.The growth of MDA-MB-231 cells were observed by MTT.The effect of OPN siRNA on the transplanted tumor growth and tumor hypoxia were assessed in nude mice. ResultsThe expression level of OPN in MDA-MB-231 cells were significantly lower under hypoxia or normoxia(P ＜ 0.05 ).OPN silence with RNAi significantly inhibited the invasion ability and proliferation of MDA-MB-231 cell lines (P ＜ 0.01 ).Inhibition of OPN with RNAi significantly inhibited the growth ability of MDA-MB-231 cells in vivo(P ＜0.05).The tumor hypoxia significantly decreased(P ＜ 0.05). ConclusionsOPN silence with RNAi can effectively inhibit cell proliferation and tumor growth of MDA-MB-231 cells,and decrease the bypoxia level of MDA-MB-231 transplanted tumor in nude mice.

Objective To observe the influence of lysyl oxidase(LOX)downregulation via RNAi on hypoxic metastasis of human lung cancer cell 95D and stduy its molecular mechanism.Methods LOX siRNA was used to transfect 95D cell line in normoxia (19％ O2 ).After 24-hour incubation,the cells were cultured in hypoxic incubator (0.5％ O2 ) for 24h.Real-time PCR assay was applied to detect LOX mRNA and Snail mRNA expression.Levels of Src,phosphorylation of Src (P-Src Y418 ) and Snail protein were determined by Western blot assay.Transwell chamber was used to evaluate the cellular invasion potential.Results Compared with 95D cells under normoxic conditions,hypoixa treatment increased LOX mRNA expression by 14 times and invasion ability by 2.12 times respectively.Compared with siRNA control group,LOX siRNA transfection decreased LOX mRNA expression,the invasion ability of hypoxic cells,and the protein expression of P-Src Y418 and Snail by 70％ - 75％,about 30％,and about 40％ respectively (P ＜ 0.05).However,it didn't affect the expression level of Src protein or Snail mRNA ( P ＞ 0.05).Conclusions Impaired metastatic potential of hypoxic human lung cancer cell induced by LOX downregulation is associated with reduced expression level of Src activation and Snail protein.The present data provids experimental evidence for LOX as a potential target for prevention and treatment of lung cancer metastasis under hypoxia.

Objective To observe the radiosensitivity by targeting HIF-lα in human lung cancer and the effects on tumor growth in nude mice.Methods Radiosensitivity of A549 and A549/HIF-1α ( － ) cells were tested by clonogenic forming assay.A549/HIF-1 α( － ) cells and A549 cells were injected into the male BALB/C nude mice.Tumor growth was observed.The expression of HIF-1α and microvessel density were detected by immunohistochemistry method.Results SERs of HIF-1α gene silencing were 1.03 in normoxia and 1.65 in hypoxia.The sizes of tumor xenografts derived from A549/HIF-1α( － ) cells were significantly reduced compared to those of the xenografts derived from A549 cells.HIF-1 α protein staining result showed a dramatic decrease in tumors from A549/HIF-1α ( － ) mice.The microvessel densities (MVD) were 19.83 ± 4.09 in A549 group and 11.61 ±3.04 in A549/HIF-lα (-)group(F=15.57,P ＜0.05 ).Conclusions Hypoxia-induced radio-resistance in lung cancer A549 cells could be reversed by silencing the HIF-1α.It also retards the growth of tumor xenografts,decreases HIF-1α expression and reduces the vascularity.

Astrocyte elevated gene-1 (AEG-1) was cloned as an human immunodeficiency virus -1-inducible and tumor necrosis factor-α-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 down-regulates the expression of the glutamate transporter EAAT2, thus, it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Meanwhile, AEG-1 expression is elevated in subsets of breast cancer, prostatic cancer, glioblastoma multiforme and melanoma cells, having a dual specificity phosphatase activity. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. Recent observations indicate that AEG-1 exerts its effects by activating the nuclear factor kappa B (NF-κB) pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration.