Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified ...subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.
Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.
Abstract Tumor evolution is a highly heterogeneous process where multiple oncogenic pathways can lead to host defense evasion. Advancements in genome sequencing are allowing us to better understand ...these processes and their impact. However, critical genomic variations that could be pivotal to the development of tumors may have remained undetected due to technological constraints of short-read sequencing. PacBio HiFi sequencing generates accurate (>99.9%) long reads (>15 kb) with native 5-methylcytosine information that can comprehensively delineate many variations, including ones which were previously inaccessible with short-read sequencing. Unlike standard whole-genome analysis, cancer variant calling must account for somatic variation, where the assumptions of a diploid genome are violated. We have developed and optimized a bioinformatics workflow for somatic variant detection with HiFi sequence data. To validate this pipeline, we sequenced two well-studied cancer cell lines (COLO829, HCC1395) to 60-fold depth and their matched normal lymphoblast cells (40-fold to 60-fold depth) and called all classes of variations (SNVs, INDELS, and structural variants). Next, we compared our calls against the Valle-Inclan (structural variants) and SEQC2 (small variants: SNV/INDEL) datasets. We achieved 92% F1 score for small variants across all variant allele frequencies (VAF) in the HCC1395 experiment. We observed a maintained accuracy (F1=91%) even at lower variant frequencies (10-20), highlighting the ability of HiFi sequencing to detect subclonal mutations. The workflow calls structural variants with high sensitivity, capturing 57/62 (92%) of all previously validated SVs in COLO829 at all VAF. Lastly, we applied our pipeline to five diverse tumor samples that exhibit complex oncogenic phenotypes including homologous recombination deficiency (HRD) and high genomic instability, e.g. unresolved rearrangement breakpoints in repetitive regions and unknown oncogenic drivers when prior sequencing efforts did not provide full clarity as to the molecular drivers of tumorigenesis. Notably, we were able to identify and phase 5mC methylation of promoters together with large-scale structural variants in cancer genes involved in HRD. Taken together, we demonstrated the high accuracy of HiFi reads in resolving somatic variants and its potential to provide novel insights in complex cancer samples. The ability for long reads to resolve complex genomics aberrations and epigenetic markers that underly the tumorigenesis process will become indispensable in the realm of precision oncology. Citation Format: Khi Pin Chua, Ian McLaughlin, Oliver Hofmann, Kym Pham Stewart, Primo Baybayan, Jonathan Bibliowicz, Sean Grimmond, Zev Kronenberg, Michael A. Eberle. Somatic variant workflow with HiFi sequencing provides new insights in highly challenging cancer cases abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2934.
The B-type Raf kinase (BRAF) V600E mutation is a well-established biomarker for poor prognosis in metastatic colorectal cancer (mCRC) and is a highly attractive drug target. A barrier to the ...development of new therapies targeting BRAF V600E in mCRC is the low prevalence of mutations (approximately 10 %) and the current need for access to sequencing-based technologies which are not routinely available outside of large cancer centres. Availability of a standardised immunohistochemistry (IHC) test, more suited to routine pathology practice, would provide much broader access to patient identification. We sought to evaluate the accuracy and clinical utility of a recently developed BRAF V600E IHC method as a prognostic biomarker in a large cohort of community-based CRC patients. Archival tumour samples from 505 patients with stage I–IV CRC were immunohistochemically tested with two antibodies, pBR1 for total BRAF and VE1 for BRAF V600E. Cases were assessed by two blinded pathologists, and results were compared to
BRAF
V600E mutation status determined using DNA sequencing. Discordant cases were retested with a
BRAF
V600E SNaPshot assay.
BRAF
mutation status was correlated with overall survival (OS) in stage IV CRC. By DNA sequencing and IHC, 505 and 477 patients were respectively evaluable. Out of 477 patients, 56 (11. 7 %) had
BRAF
V600E mutations detected by sequencing and 63 (13.2 %) by IHC. Using DNA sequencing results as the reference, sensitivity and specificity for IHC were 98.2 % (55/56) and 98.1 % (413/421), respectively. IHC had a positive predictive value (PPV) of 87.3 % (55/63) and a negative predictive value (NPV) of 99.8 % (413/414). Compared to DNA sequencing plus retesting of available discordant cases by SNaPshot assay, IHC using the VE1 antibody had a 100 % sensitivity (59/59), specificity (416/416), NPV (416/416) and PPV (59/59). Stage IV CRC patients with BRAF V600E protein detected by IHC exhibited a significantly shorter overall survival (hazard ratio = 2.20, 95 % CI 1.26–3.83,
p
= 0.005), consistent with other published series. Immunohistochemistry using the BRAF V600E VE1 antibody is an accurate diagnostic assay in CRC. The test provides a simple, clinically applicable method of testing for the
BRAF
V600E mutation in routine practice.
Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination ...(HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting.
A cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting.
All 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs.
Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.
Abstract Background Minimally invasive techniques, including endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), yield small specimens that are adequate for cytological ...diagnosis of lung cancer, but also need to provide material for molecular analysis to guide treatment. The number of EBUS-TBNA passes needed for mutation testing remains unclear. We sought to assess the adequacy of a single pass for genomic profiling of actionable mutations. Methods In a prospective observational study, paired samples from the same lesion were obtained from patients undergoing EBUS-TBNA for lung cancer diagnosis/staging. Following tumour cell confirmation by rapid on-site evaluation, a “reference” sample comprising ≥3 passes was obtained and formalin-fixed paraffin-embedded. A “study” sample comprising a single pass was taken and snap-frozen. Primary outcome was DNA yield and quality from a single pass. Secondary outcome was diagnostic accuracy of a single pass for detecting actionable mutations. Results In 40 patients, single pass specimens yielded a mean 3.98μg of highly intact DNA well above the minimum threshold for targeted sequencing, which was performed in adenocarcinoma cases (n=24). In 23 cases, there was 100% agreement in mutation status between reference and study samples. In 1 case, the reference sample failed to generate a molecular diagnosis due to insufficient tumour cells, however the study specimen identified a KRAS mutation. Tumour cell percentage in mutation-positive specimens was 1 to 70%, suggesting that single pass samples detect mutations even when tumour cell content is low. Conclusion Single EBUS-TBNA passes yield DNA of high quantity and quality with high accuracy for molecular profiling, irrespective of tumour cell content.
Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for ...genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (
KRAS
) mutation demonstrated the same
KRAS
mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known
KRAS
mutation, ICECOLD PCR detected the same
KRAS
variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.
Modification of cytokine production by gender hormones has been postulated to affect disease susceptibility and outcome. Here we investigate the effect of gender and the menstrual cycle on production ...of cytokines. Mononuclear cells were isolated every week for 10 consecutive weeks from healthy pre-menopausal women and men. TNF and IL-10 mRNA and protein levels were measured as well as membrane CD14 and intracellular TLR4 protein. Endotoxin stimulation of mononuclear cells from men produced more TNF and IL10 mRNA than cells from women. TLR4 expression was also significantly higher in cells from men. These gender differences in the immune response may help to elucidate the sexual dimorphism observed in infectious diseases.
Wilms’ tumors have characteristic chromosomal abnormalities, such as the 11p13 deletion, in a subset of cases. This is one of the very few reports comparing single nucleotide polymorphism (SNP) array ...analysis with conventional karyotyping of Wilms’ tumors. A total of 43 frozen tumor samples were analyzed using the Affymetrix Cytogenetics Whole-Genome 2.7 M array. The findings from the SNP array analysis were then compared with those from conventional karyotyping. A comparison between SNP array and conventional karyotype findings was possible in 38 of 43 specimens (88.4%). The SNP array and classic cytogenetic results were concordant in 33 of 38 specimens (87%). SNP array analysis was able to support the findings of classic cytogenetics. The SNP array detected regions of loss of heterozygosity (LOH) in 41 of 43 (95%) specimens. However, it did not detect balanced translocations and inversions that were observed by conventional cytogenetics. Our results show that the data generated from these platforms are complementary. The SNP array also detected additional gains and losses as well as regions of LOH with associated disomy, which are likely to represent segmental uniparental disomy. The observed discrepancies can be explained by the inherent limitations of each technique.
Are international and Asian regional institutions serving the development goals of Asian and Pacific Economies as well they should? The global economy, led by the Asia Pacific region, has undergone ...immense change and growth. Have the existing institutions and arrangements been able to keep pace with those changes in the global economy? International Institutions and Asian Development tackles these questions and is an essential book for the assessment of regional and international institutions, as well as policy prescriptions for reforming them to ensure they deliver on sustainable, peaceful growth and development in the region.
Drawing from papers presented to the 32nd Pacific Trade and Development conference in Hanoi in 2007, the contributions by distinguished authors add to the understanding of the purpose, evolution, relevance and gaps in regional and global institutions and their arrangements.
Shiro Armstrongis a Research Fellow at the Crawford School of Economics and Government at the Australian National University.
Vo Tri Thanhis Director of the Department for International Economic Integration Studies of the Central Institute of Economic Management in Vietnam
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