(Tn5-)fish-based imaging in the era of 3D/spatial genomics

3D genomics mainly focuses on the 3D position of single genes at the cell level, while spatial genomics focuses more on the tissue level. In this exciting new era of 3D/spatial genomics, half-century old FISH and its derivative methods, including Tn5-FISH, play important roles. In this review, we introduce the Tn5-FISH we developed recently, and present six different applications published by our collaborators and us, based on (Tn5-)FISH, which can be either general BAC clone-based FISH or Tn5-FISH. In these interesting cases, (Tn5-)FISH demonstrated its vigorous ability of targeting sub-chromosomal structures across different diseases and cell lines (leukemia, mESCs (mouse embryonic stem cells), and differentiation cell lines). Serving as an effective tool to image genomic structures at the kilobase level, Tn5-FISH holds great potential to detect chromosomal structures in a high-throughput manner, thus bringing the dawn for new discoveries in the great era of 3D/spatial genomics

Mutation status analysis of 58 patients with advanced ALK fusion gene positive non small cell lung cancer

Abstract Purpose To analyze the characteristics and prognostic values of Anaplastic Lymphoma Kinase (ALK) fusion gene partner, gene subtype and abundance in tumor tissues of advanced Non Small Cell Lung Cancer (NSCLC) patients with positive ALK fusion gene and to explore the best treatment mode of ALK-Tyrosine Kinase Inhibitors(TKIs). Methods Cases of advanced NSCLC patients with ALK positive confirmed by both Next Generation Sequencing (NGS) and immunohistochemistry were retrospectively collected. The relationships of Overall Survival (OS)/Progression Free Survival (PFS) between different mutation subtypes, mutation abundance, clinicopathological features were analyzed. OS/PFS between different treatment mode of ALK inhibitors were compared. Results Fifty-eight patients were enrolled. There were diverse fusion partners. Five subtypes of Echinoderm Microtubule-associated protein-Like 4 gene (EML4)-ALK fusion mutation were detected: V1,V2,V3,V5 and V7. The mutation abundance ranged from 0.13 to 27.77%, with a median of 5.34%. The abundance of V2 and V5 was higher than V1 and V3 respectively. There was no difference in OS between the low abundance group(≤ 5.34%) and the high abundance group(>5.34%) (P = 0.434). PFS of second-generation ALK inhibitors as first-line treatment was longer than that of Crizotinib as first-line (P<0.001). Never smokers had longer OS than current smokers(P = 0.001). Conclusions There are differences in abundance between different fusion partners and subtypes in advanced NSCLC with positive ALK. OS is not associated with subtypes, mutation abundance and first line treatment option of either generation of ALK inhibitors. Smoking is a poor prognostic factor

Clinical characteristics from co-infection with avian influenza A H7N9 and Mycoplasma pneumoniae: a case report

Abstract Background More and more cases of human infections with avian influenza A H7N9 have been reported since it was first mentioned in 2013 in China, but concurrence of influenza A H7N9 with Mycoplasma pneumoniae, however, has never been described. Here, we reported the case of a woman co-infected by influenza A H7N9 and Mycoplasma pneumoniae, whose treatment process was a little bit longer and a little bit complicated as well. Case presentation Our patient was an 80-year-old Chinese woman who presented with fever, cough, chest tightness, and shortness of breath. A computed tomography scan showed obvious infiltrations at lower parts of both lungs. Arterial blood gas analysis confirmed a severe respiratory failure (type I). Her sputum and throat swabs were checked for nucleic acid of influenza A and the result was positive for influenza A H7N9. She was diagnosed as having severe influenza A H7N9 and acute respiratory distress syndrome, and was admitted to an intensive care unit. She was given comprehensive treatment, including oseltamivir, methylprednisolone, immunoglobulin, gastric protection, and noninvasive mechanical ventilation. Her condition improved 4 days later. However, some symptoms exacerbated again 2 days later with ground-glass changes appearing in upper area of right lung and the titer of antibody to Mycoplasma pneumoniae rising from 1:80 to 1:640. She was reasonably considered to be infected with Mycoplasma pneumoniae as well, and azithromycin and moxifloxacin were added to her treatment. Oseltamivir was discontinued because of three consecutive negative results of nucleic acid for influenza A H7N9, but anti-Mycoplasma treatment was continued. Although her symptoms and abnormal changes on computed tomography scan slowly went away, she finally recovered from the mixed infection after a total of 33 days of management. Conclusion In patients with confirmed influenza A H7N9 infection whose condition worsens again, especially with new infiltration or lung ground-glass infiltration, one should suspect infection by other pathogens such as Mycoplasma pneumoniae

Post-therapeutic circulating tumor cell-associated white blood cell clusters predict poor survival in patients with advanced driver gene-negative non-small cell lung cancer

Abstract Purpose This study aimed to investigate the clinical utility of diverse aneuploid circulating tumor cell (CTC) subtypes and particularly CTC-associated white blood cell (CTC-WBC) clusters in predicting treatment response, prognosis and real-time monitoring disease progression in advanced driver gene-negative non-small lung cancer (NSCLC) patients. Materials and methods A total of 74 eligible patients were prospectively enrolled and serial blood samples were collected at pre-treatment(t0), after two cycles of therapy (t1) and at post-four-to-six treatment cycles (t2). Co-detection of diverse subtypes of aneuploid CTCs and CTC-WBC clusters was conducted in advanced NSCLC patients receiving first-line treatment. Results At baseline, CTCs were detected in 69 (93.24%) patients and CTC-WBC clusters were detected in 23 (31.08%) patients. Patients with CTCs < 5/6ml or with CTC-WBC clusters undetectable exhibited a better treatment response than patients with pre-therapeutic aneuploid CTCs ≥ 5/6ml or harboring CTC-WBC clusters (p = 0.034 and p = 0.012, respectively). Before treatment, patients bearing tetraploid CTCs ≥ 1/6ml showed significantly inferior progression-free survival (PFS) [hazard ratio (HR):2.420, 95% confidence interval (CI): 1.426–4.106; p = 0.001] and overall survival (OS) compared to patients with tetraploid CTCs < 1/6ml (HR:1.907, 95%CI: 1.119–3.251; p = 0.018). A longitudinal study demonstrated that post-therapeutic patients harboring CTC-WBC clusters displayed the reduced PFS and OS compared with those without CTC-WBC clusters, and subgroup analysis showed that the presence of CTC-WBC clusters indicated a worse prognosis in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. After adjusting for multiple significant factors, post-therapeutic CTC-WBC clusters were the only independent predictor of both PFS (HR:2.872, 95% CI: 1.539–5.368; p = 0.001) and OS (HR:2.162, 95% CI: 1.168–4.003; p = 0.014). Conclusions In addition to CTCs, longitudinal detection of CTC-WBC clusters provided a feasible tool to indicate initial treatment response, dynamically monitor disease progression and predict survival in driver gene-negative advanced NSCLC patients

Investigating the mechanism by which SMAD3 induces PAX6 transcription to promote the development of non-small cell lung cancer

Abstract Background This study investigated the function of SMAD3 (SMAD family member 3) in regulating PAX6 (paired box 6) in non-small cell lung cancer. Methods First, qRT-PCR was employed to detect SMAD3 expression in cancer tissues along with normal tissues and four cell lines, including BEAS-2B, H125, HCC827 and A549 cells. SMAD3 was knocked down by small interference RNA (siRNA), and then its expression was determined via qRT-PCR and Western blot analysis. The correlation between SMAD3 and PAX6 was determined by double luciferase reporter experiments and chromatin immunoprecipitation (ChIP) assay. Cell viability was evaluated by CCK-8 and colony forming assays, while cell migration and invasion were detected by Transwell analysis. Results SMAD3 and PAX6 were upregulated in lung cancer tissues and cancer cells. Knocking down SMAD3 and PAX6 by transfection with siRNAs specifically suppressed the expression of SMAD3 and PAX6 mRNA and protein levels. SMAD3 could promote PAX6 transcriptional activity by binding to its promoter. Reduced expression of SMAD3 led to the downregulation of PAX6 mRNA and protein levels along with decreased cell migration, invasion, proliferation and viability in A549 and HCC827 cells. PAX6 overexpression altered the si-SMAD3-induced inhibition of cell migration, invasion, proliferation and viability in A549 and HCC827 cells. Additionally, PAX6 knockdown alone also repressed the cell migration, invasion, proliferation and viability of the cell lines. Conclusions SMAD3 promotes the progression of non-small cell lung cancer by upregulating PAX6 expression

Role of aneuploid circulating tumor cells and CD31+ circulating tumor endothelial cells in predicting and monitoring anti‐angiogenic therapy efficacy in advanced NSCLC

Prognosticating the efficacy of anti‐angiogenic therapy through longitudinal monitoring and early detection of treatment resistance in cancer patients remain highly challenging. In this study, co‐detection and comprehensive phenotypic and karyotypic molecular characterization of aneuploid circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) were conducted on non‐small cell lung cancer (NSCLC) patients receiving bevacizumab plus chemotherapy. Prognostic values of the cell‐based significant univariate risk factors identified by Cox regression analyses were progressively investigated. Subjects showing an increase in total post‐therapeutic platelet endothelial cell adhesion molecule‐1 (CD31)– CTCs and CD31+ CTECs exhibited a significantly reduced median progression‐free survival (mPFS) and overall survival. Further stratification analyses indicated that pretherapeutic patients bearing vimentin (Vim)+ CTECs (mesenchymal M‐type) at baseline revealed a significantly shortened mPFS compared with patients with Vim– CTECs. Post‐therapeutic patients harboring epithelial cell adhesion molecule (EpCAM)+ CTCs and CTECs (epithelial E‐type), regardless of Vim expression or not, showed a significantly reduced mPFS. Post‐therapeutic patients possessing de novo EpCAM+/Vim+ (hybrid E/M‐type) CTECs displayed the shortest mPFS. Patients harboring either pre‐ or post‐therapeutic EpCAM–/Vim– null CTECs (N‐type) exhibited a better response to therapy compared to patients harboring EpCAM+ and/or Vim+ CTECs. The presented results support the notion that baseline Vim+ CTECs and post‐therapeutic EpCAM+ CTCs and CTECs are predictive biomarkers for longitudinal monitoring of response to anti‐angiogenesis combination regimens in NSCLC patients