Evolution of neoplastic cell lineages in Barrett oesophagus

Abstract

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

Fig. 1

Clonal evolution in patients with BE in vivo. For each cell population, ploidy, LOH (5q^-, 9p^-, 13q^-, 17p^-, 18q^-) and mutations are given below. LOH of alternate alleles is denoted (^-1, ^-2). Cancer clones are red. Epithelial populations were purified by flow sorting. Ploidies were scored as different if DNA contents differed by 0.2N or more, as described. The distribution of clones with somatic genetic abnormalities in premalignant epithelium at the baseline endoscopy and the evolution of neoplastic cell lineages are shown. a, In patient 391 (21 samples, 6 years), TP53 (CGA→TGA; R306X) and CDKN2A (CGA→TGA; R58X) mutations were found at all levels of an 8-cm Barrett segment. Endoscopic and surgical biopsies are from 1990-1996. b, Summary of cell lineages. c, In patient 772 (13 samples, three years), TP53 (CGG→CAG; R248E) mutation was found at all levels of an 11-cm Barrett segment. d, In patient 779 (17 samples, three years), TP53 (CGA→TGA; R306X) and CDKN2A (GAG→TAG; E88X) mutations were found in endoscopic and surgical biopsies from 1989 and 1992. Other somatic events may precede TP53 and CDKN2A abnormalities or lead linearly to cancer, but we found no evidence for them among the non-random LOHs detected in our allelotype of EA (ref. 8).

Fig. 2

Multiple genetic routes to cancer in BE. Each clone represents a cell population detected in this study, but the figure is not intended to be comprehensive because the lack of obligate orders creates multiple genetic pathways to cancer, only some of which are represented. Although there are multiple pathways to cancer, some general patterns appear to be consistent among many patients. LOH events arise in diploid Ki67-positive populations (2N) in Barrett metaplasia (M). We detected 2N populations that developed LOH without lesions involving either TP53 or CDKN2A in 4 patients, but these clones failed to progress. LOH at either 17p (17p^-) or 9p (9p^-) can occur in diploid populations and can precede mutations in TP53 and CDKN2A. Although both 17p and 9p LOH develop as early events, they occur in no obligate order. 2N clones with somatic genetic abnormalities involving TP53 (17p^-/TP53^-) and CDKN2A (9p^-/CDKN2A^-; 9p^-) frequently give rise to populations with elevated 4N fractions (4N) that can evolve aneuploid cell populations (An). Methylation of the CDKN2A CpG island was also detected in diploid cells before the development of aneuploidy. The 4N abnormality was not always detected, possibly from sampling limitations or failure of a genetically unstable intermediate to persist. In some cases, such as patient 391 (Fig. 1), changes in ploidy may result in a clone with a 2N or near-2N DNA content, yet with multiple genetic abnormalities. 5q, 13q and 18q LOH have no obligate order relative to aneuploidy, cancer or each other. During evolution, neoplastic cell lineages may bifurcate, giving rise to mosaics with the same TP53 and CDKN2A abnormalities but different ploidies and additional LOHs. Some clones in these mosaics develop into cancer (Ca), whereas others either are delayed in their progression or represent dead ends. Subsequently, multiple malignant clones (Ca) with different ploidies and additional LOHs evolve as the cancer progresses.