The clonal relationships between pre‐cancer and cancer revealed by ultra‐deep sequencing

Abstract

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognised, especially now the extent of clonal heterogeneity in fully invasive disease is being realised. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they too are heterogeneous is unknown.We performed ultra-deep sequencing of thousands of selected mutations together with copy number analysis from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient.We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas, and ultimately, the diversity of processes by which tumours are initiated, spread and metastasise.Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly-related genomes. We show that oral pre-cancer exhibits considerable sub-clonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable.

The genomic road to invasion—examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples

“…As mutation calls with a VAF <0.12 were more likely to be caused by sequencing errors or FFPE artefacts [6], these analyses were performed both with and without those calls with a VAF <0.12. The validity of this cutoff was confirmed by analysis of PCR validation of exome data from a similar cohort of previously published HNSCC FFPE samples [15] (Additional file 1: Figure S2).…”

“…This could be due to the fact that only one dysplasia sample was taken for each patient. More extensive sampling might have detected these mutations in a region of the dysplasia more directly ancestral to the SCC, as we sometimes found when we sampled multiple regions of a smaller group of patients [15]. An alternative explanation is that these driver mutations only affected the invasive potential of the lesions and had less effect on their growth rate.…”

“…Seminal work in colorectal cancer has suggested a simple step-wise genomic development from normal tissue, through pre-cancerous stages of increasing grade to fully invasive disease [2]. In previous work we have examined clonal relationships between dysplasia and carcinoma in five patients, which suggested that the relationships are sometimes complex and are not always a simple progression [15]. By working with larger numbers of patients, we were able in this study to examine trends between the different grades of disease.…”

“…The genomic pathways from normal tissue through pre-cancerous stages to fully invasive disease has not been characterised in HNSCC. Work on small numbers of patients shows considerable variety in both the genomic evolutionary relationships and the differentially expressed pathways between HNSCC and their related dysplasias [15, 16]. …”

Genomic approaches to accelerate cancer interception

“…Cancer is thought to proceed through a multi-step process where accumulating alterations in driver genes allow cells to clonally expand and invade surrounding tissues 34 . A limited number of RNA and DNA sequencing studies interrogating a range of precancerous tissues from the breast 35–39 , colon 40–44 , lung 45,46 , head and neck 47 , skin 48,49 , and esophagus 50–55 have indicated that the path to cancer development is context-specific and involves diverse processes. Thesey studies suggest that early lesions are polyclonal and driver mutations present in late-stage disease, and that tumor development occurs via large-scale genomic rearrangements and copy number changes.…”