ABSTRACT
Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.
Subject(s)
Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung , Circulating Tumor DNA , Lung Neoplasms , Mutation , Neoplasm Metastasis , Small Cell Lung Carcinoma , Humans , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Circulating Tumor DNA/blood , Circulating Tumor DNA/genetics , Cohort Studies , Lung Neoplasms/blood , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasm Metastasis/diagnosis , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Phylogeny , Small Cell Lung Carcinoma/pathology , Liquid BiopsyABSTRACT
Human telomerase RNA (hTR) is transcribed as a precursor that is then posttranscriptionally modified and processed. A fraction of the transcripts is oligoadenylated by TRAMP and either processed into the mature hTR or degraded by the exosome. Here, we characterize the processing of 3' extended forms of varying length by PARN and RRP6. We show that tertiary RNA interactions unique to the longer transcripts favor RNA degradation, whereas H/ACA RNP assembly stimulates productive processing. Interestingly, the H/ACA complex actively promotes processing in addition to protecting the mature 3' end. Processing occurs in two steps with longer forms first being trimmed by RRP6 and shorter forms then being processed by PARN. These results reveal how RNA structure and RNP assembly affect the kinetics of processing and degradation and ultimately determine the amount of functional telomerase produced in cells.
Subject(s)
Exoribonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex/metabolism , RNA/metabolism , Ribonucleoproteins/metabolism , Telomerase/metabolism , Cell Cycle Proteins/metabolism , Humans , Nuclear Proteins/metabolismABSTRACT
Telomerase reverse transcriptase (TERT) and the non-coding telomerase RNA subunit (TR) constitute the core of telomerase. Here we now report that the putative F-box protein Pof8 is also a constitutive component of active telomerase in fission yeast. Pof8 functions in a hierarchical assembly pathway by promoting the binding of the Lsm2-8 complex to telomerase RNA, which in turn promotes binding of the catalytic subunit. Loss of Pof8 reduces TER1 stability, causes a severe assembly defect, and results in critically short telomeres. Structure profile searches identified similarities between Pof8 and telomerase subunits from ciliated protozoa, making Pof8 next to TERT the most widely conserved telomerase subunits identified to date.
Subject(s)
RNA Recognition Motif Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Telomerase/metabolism , Amino Acid Sequence , Molecular Sequence Data , Protein Binding , RNA/genetics , RNA/metabolism , RNA Recognition Motif Proteins/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , Schizosaccharomyces/chemistry , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Sequence Alignment , Telomerase/chemistry , Telomerase/genetics , Telomere/genetics , Telomere/metabolismABSTRACT
The non-coding RNA subunit of telomerase provides the template for telomerase activity. In diverse fungi, 3' end processing of telomerase RNA involves a single cleavage by the spliceosome. Here, we examine how human telomerase RNA (hTR) primary transcripts are processed into the mature form of precisely 451 nt. We find that the splicing inhibitor isoginkgetin mimics the effects of RNA exosome inhibition and causes accumulation of long hTR transcripts. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly(A)-specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and degradation and suggest treatment options for telomerase insufficiency disorders.
