Endobronchial Ultrasound Doppler Image Features Correlate with mRNA Expression of hif1-α and vegf-c in Patients with Non–Small-Cell Lung Cancer

IntroductionWe attempted to assess the correlation between the Doppler mode image patterns during endobronchial ultrasound-guided (EBUS) transbronchial needle aspiration and the expression of angiogenesis-related molecules within lymph nodes in patients with non–small-cell lung cancer.MethodsThirty-eight archived EBUS- transbronchial needle aspiration samples of lymph nodes (27 metastatic and 11 nonmetastatic) in patients with non–small-cell lung cancer with Doppler mode ultrasound image were analyzed. The Doppler mode image of the vasculature of the targeted lymph node was categorized into the following groups: normal blood flow, low blood flow (LBF), and high blood flow (HBF). Vascular index ratio (vascular area/lymph node area) of each metastatic lymph node was calculated. Total RNA and protein was extracted and analyzed for expression of HIF-1α, VEGF-A, and VEGF-C by quantitative RT-PCR and enzyme-linked immunosorbent assay.ResultsWithin the 27 metastatic lymph nodes, eight were categorized into the LBF group and 19 into the HBF group. Vascular index ratio was significantly higher in HBF than LBF (p = 0.0003). mRNA expression of HIF-1α and VEGF-A was significantly higher in metastatic lymph nodes than in benign lymph nodes (p < 0.0001). Compared with LBF and HBF, HIF-1α mRNA expression was significantly higher in LBF (p = 0.01) and VEGF-C mRNA expression was significantly higher in HBF (p = 0.0315). There was no significant difference in protein expression by enzyme-linked immunosorbent assay analysis.ConclusionsThe vascularity of metastatic lymph nodes observed by EBUS correlates with the mRNA expression of HIF-1α and VEGF-C (not VEGF-A). This correlation is a clinical utility that needs to be evaluated further

Targeting interstrand crosslink repair to improve chemotherapeutic strategies

Many clinically effective chemotherapeutic agents exert their toxicity through formation of DNA interstrand cross-links (ICL). These prevent the replication and transcription of DNA and if unrepaired lead to cell death. Despite the success of ICL inducing agents in treating cancers, issues remain with a lack of sensitivity in some tumours, therapy-limiting toxicity and acquired resistance and tumour recurrence. The modulation of ICL repair could improve chemotherapy outcomes and the specific targeting of ICL repair in quiescent cells may selectively target cancer stem cells and ameliorate issues with recurrence. This thesis seeks to identify potential druggable targets that may sensitise cancer cells to ICL inducing agents and elucidate the role of ICL repair in quiescence. Previous studies have suggested ICL repair occurs chiefly in S-phase when DNA replication forks collide with ICLs. However, recent research in budding yeast, using Xenopus cell-free extracts and using genetic reporter assays in mammalian cells suggests that ICL repair can occur in the G1 phase of the cell cycle via the nucleotide excision repair (NER) and translesional synthesis (TLS) pathways. Previously this G1 pathway had not been well explored in mammalian cells. In this thesis a model for G1 ICL repair was created using a mammalian cell line. Flow cytometry analysis (FACS) of cell cycle dynamics following ICL induction in phase G1 demonstrated that repair of ICL can occur in G1 and this appears time dependent. Further analysis by FACS, comet assays and cell survival analysis confirmed repair of ICL in G1 and suggested that the NER factors XPF, XPB and XPD and the exonucleases SNM1A and EXO1 may be required for ICL repair in G1 but not the XPG, FAN1 or CSB repair proteins. Along-side these studies, a large-scale siRNA screen of 640 DNA damage repair genes was performed in a lung cancer cell line utilising colony formation assays to identify potential cisplatin and radiotherapy sensitising genes. Following a secondary screen, eight potential radiation and cisplatin sensitising genes were identified and examined in further detail, with the gene PIF1 finally confirmed in CRISPR/Cas9 knockout cells as a potential cisplatin sensitising gene. The findings in this thesis confirm ICL repair can occur in G1 in mammalian cells and identify PIF1 as a potential cisplatin sensitiser in human cells. In the future, targeting of the specific endonucleases responsible for G1 ICL repair or PIF1 may offer a potential clinical cancer treatment option.</p

Targeting interstrand crosslink repair to improve chemotherapeutic strategies

Many clinically effective chemotherapeutic agents exert their toxicity through formation of DNA interstrand cross-links (ICL). These prevent the replication and transcription of DNA and if unrepaired lead to cell death. Despite the success of ICL inducing agents in treating cancers, issues remain with a lack of sensitivity in some tumours, therapy-limiting toxicity and acquired resistance and tumour recurrence. The modulation of ICL repair could improve chemotherapy outcomes and the specific targeting of ICL repair in quiescent cells may selectively target cancer stem cells and ameliorate issues with recurrence. This thesis seeks to identify potential druggable targets that may sensitise cancer cells to ICL inducing agents and elucidate the role of ICL repair in quiescence. Previous studies have suggested ICL repair occurs chiefly in S-phase when DNA replication forks collide with ICLs. However, recent research in budding yeast, using Xenopus cell-free extracts and using genetic reporter assays in mammalian cells suggests that ICL repair can occur in the G1 phase of the cell cycle via the nucleotide excision repair (NER) and translesional synthesis (TLS) pathways. Previously this G1 pathway had not been well explored in mammalian cells. In this thesis a model for G1 ICL repair was created using a mammalian cell line. Flow cytometry analysis (FACS) of cell cycle dynamics following ICL induction in phase G1 demonstrated that repair of ICL can occur in G1 and this appears time dependent. Further analysis by FACS, comet assays and cell survival analysis confirmed repair of ICL in G1 and suggested that the NER factors XPF, XPB and XPD and the exonucleases SNM1A and EXO1 may be required for ICL repair in G1 but not the XPG, FAN1 or CSB repair proteins. Along-side these studies, a large-scale siRNA screen of 640 DNA damage repair genes was performed in a lung cancer cell line utilising colony formation assays to identify potential cisplatin and radiotherapy sensitising genes. Following a secondary screen, eight potential radiation and cisplatin sensitising genes were identified and examined in further detail, with the gene PIF1 finally confirmed in CRISPR/Cas9 knockout cells as a potential cisplatin sensitising gene. The findings in this thesis confirm ICL repair can occur in G1 in mammalian cells and identify PIF1 as a potential cisplatin sensitiser in human cells. In the future, targeting of the specific endonucleases responsible for G1 ICL repair or PIF1 may offer a potential clinical cancer treatment option.</p

Pregnancy and neonatal outcomes of COVID -19: coreporting of common outcomes from PAN-COVID and AAP-SONPM registries

Objective Few large cohort studies have reported data on maternal, fetal, perinatal and neonatal outcomes associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection in pregnancy. We report the outcome of infected pregnancies from a collaboration formed early during the pandemic between the investigators of two registries, the UK and Global Pregnancy and Neonatal outcomes in COVID‐19 (PAN‐COVID) study and the American Academy of Pediatrics (AAP) Section on Neonatal–Perinatal Medicine (SONPM) National Perinatal COVID‐19 Registry. Methods This was an analysis of data from the PAN‐COVID registry (1 January to 25 July 2020), which includes pregnancies with suspected or confirmed maternal SARS‐CoV‐2 infection at any stage in pregnancy, and the AAP‐SONPM National Perinatal COVID‐19 registry (4 April to 8 August 2020), which includes pregnancies with positive maternal testing for SARS‐CoV‐2 from 14 days before delivery to 3 days after delivery. The registries collected data on maternal, fetal, perinatal and neonatal outcomes. The PAN‐COVID results are presented overall for pregnancies with suspected or confirmed SARS‐CoV‐2 infection and separately in those with confirmed infection. Results We report on 4005 pregnant women with suspected or confirmed SARS‐CoV‐2 infection (1606 from PAN‐COVID and 2399 from AAP‐SONPM). For obstetric outcomes, in PAN‐COVID overall and in those with confirmed infection in PAN‐COVID and AAP‐SONPM, respectively, maternal death occurred in 0.5%, 0.5% and 0.2% of cases, early neonatal death in 0.2%, 0.3% and 0.3% of cases and stillbirth in 0.5%, 0.6% and 0.4% of cases. Delivery was preterm (< 37 weeks' gestation) in 12.0% of all women in PAN‐COVID, in 16.1% of those women with confirmed infection in PAN‐COVID and in 15.7% of women in AAP‐SONPM. Extreme preterm delivery (< 27 weeks' gestation) occurred in 0.5% of cases in PAN‐COVID and 0.3% in AAP‐SONPM. Neonatal SARS‐CoV‐2 infection was reported in 0.9% of all deliveries in PAN‐COVID overall, in 2.0% in those with confirmed infection in PAN‐COVID and in 1.8% in AAP‐SONPM; the proportions of neonates tested were 9.5%, 20.7% and 87.2%, respectively. The rates of a small‐for‐gestational‐age (SGA) neonate were 8.2% in PAN‐COVID overall, 9.7% in those with confirmed infection and 9.6% in AAP‐SONPM. Mean gestational‐age‐adjusted birth‐weight Z‐scores were −0.03 in PAN‐COVID and −0.18 in AAP‐SONPM. Conclusions The findings from the UK and USA registries of pregnancies with SARS‐CoV‐2 infection were remarkably concordant. Preterm delivery affected a higher proportion of women than expected based on historical and contemporaneous national data. The proportions of pregnancies affected by stillbirth, a SGA infant or early neonatal death were comparable to those in historical and contemporaneous UK and USA data. Although maternal death was uncommon, the rate was higher than expected based on UK and USA population data, which is likely explained by underascertainment of women affected by milder or asymptomatic infection in pregnancy in the PAN‐COVID study, although not in the AAP‐SONPM study. The data presented support strong guidance for enhanced precautions to prevent SARS‐CoV‐2 infection in pregnancy, particularly in the context of increased risks of preterm delivery and maternal mortality, and for priority vaccination of pregnant women and women planning pregnancy. Copyright © 2021 ISUOG. Published by John Wiley & Sons Ltd