Genomic and transcriptomic profiling of carcinogenesis in patients with familial adenomatous polyposis

Abstract

Objective: Familial adenomatous polyposis (FAP) is characterised by the development of hundreds to thousands of adenomas at different evolutionary stages in the colon and rectum that will inevitably progress to adenocarcinomas if left untreated. Here, we investigated the genetic alterations and transcriptomic transitions from precancerous adenoma to carcinoma.

Design: Whole-exome sequencing, whole-genome sequencing and single-cell RNA sequencing were performed on matched adjacent normal tissues, multiregionally sampled adenomas at different stages and carcinomas from six patients with FAP and one patient with MUTYH-associated polyposis (n=56 exomes, n=56 genomes and n=8,757 single cells). Genomic alterations (including copy number alterations and somatic mutations), clonal architectures and transcriptome dynamics during adenocarcinoma carcinogenesis were comprehensively investigated.

Results: Genomic evolutionary analysis showed that adjacent lesions from the same patient with FAP can originate from the same cancer-primed cell. In addition, the tricarboxylic acid cycle pathway was strongly repressed in adenomas and was then slightly alleviated in carcinomas. Cells from the 'normal' colon epithelium of patients with FAP already showed metabolic reprogramming compared with cells from the normal colon epithelium of patients with sporadic colorectal cancer.

Conclusions: The process described in the previously reported field cancerisation model also occurs in patients with FAP and can contribute to the formation of adjacent lesions in patients with FAP. Reprogramming of carbohydrate metabolism has already occurred at the precancerous adenoma stage. Our study provides an accurate picture of the genomic and transcriptomic landscapes during the initiation and progression of carcinogenesis, especially during the transition from adenoma to carcinoma.

Keywords: MUTYH-associated polyposis (MAP); Familial adenomatous polyposis (FAP); Field cancerization; Single-cell transcriptome profiling; Tumor heterogeneity; colon carcinogenesis; colorectal adenomas.

Figure 1

Landscapes of genomic alterations in FAP. (A) Workflow. Adjacent normal tissue, adenomas and carcinomas from colon and rectum of FAPs were obtained for dissociation into single cells. Multiple-region sampling was used for adenocarcinomas larger than 10 mm. Parts of the dissociated cells were used for single-cell RNA-seq, and the remaining parts and matched peripheral blood were used for whole-exome sequencing and whole-genome sequencing. (B) Potential driver events in the five patients with FAP and one patient with MAP(including 8 adjacent normal tissues, 31 regions from 20 low-grade adenomas, 9 regions from 4 high-grade adenomas and 19 regions from 7 carcinomas). Top, the patient origin, somatic mutation frequency per mega-base, inherited or postzygotic mutations on APC or MUTYH, and tumour grade for each sample are indicated. Middle, graph shows detailed information about the mutations of potential driver genes. The samples from the same patient were arranged together. The genes were sorted by the frequency of genomic alterations in the cohort. Different types of genomic alterations, including stop gain, frameshift, splice variant, missense, inframe, APC cnLOH and copy number alterations, are shown by different colours. Lesions using multiregion sampling strategy are indicated. The black dot line separates different multiregion sampled lesions. APC, adenomatous polyposis coli; cnLOH, copy neutral loss of heterogeneity; CNV, copy number variation; FAP, familial adenomatous polyposis; MAP, MUTYH-associated polyposis.

Figure 2

Clonal architectures of lesions at different evolutionary stages from FAP1. (A) Heatmap showing the regional distribution of somatic mutations in all samples from FAP1. All mutations were classified into three types: lesion-shared mutations (dark blue), branched/truncal mutations (deep orange) and private mutations (light green). The number of private mutations for each sample is shown. Samples from different lesions are separated by a black line. (B) Phylogenetic tree of lesions at different evolutionary stages from FAP1 by using maximum parsimony algorithm. The colours of the lines in the phylogenetic tree correspond to the mutation types as mentioned previously. Potential driver mutations and cnLOH of APC genes are shown. (C) Schematic diagram indicating that all the lesions of FAP1 originated from the same cell. (D) Clonal or individual CNAs and cnLOHs were presented on the phylogenetic tree of lesions from FAP1 constructed from somatic mutations. Ade, adenoma; APC, adenomatous polyposis coli; Car, carcinoma; cnLOH, copy neutral loss of heterogeneity; FAP, familial adenomatous polyposis; HGIN, high-grade intra-epithelial neoplasia; Nor, adjacent normal tissue; R, region.

Figure 3

Transcriptome differences between the ‘normal’ epithelium of patients with FAP and the normal epithelium of sporadic patients with CRC. (A) Heatmap showing the top DEGs with the highest p-value between epithelial cells from adjacent normal tissue of patients with FAP (30 genes) and epithelial cells from adjacent normal tissue of patients with sporadic CRC (15 genes). The full list of DEGs is summarised in online supplementary table 7. (B) Gene ontology analysis of genes that show higher expression levels in FAP colon epithelium compared to CRC normal colon epithelium. (C) Immumohistochemical staining of MKI67 in adjacent normal tissue of FAP2 and adjacent normal tissue from patient with CRC (non-FAP). Scale bar, 100 µm. (D) Five views (×400) were randomly chosen from immumohistochemical staining to calculate the percentage of MKI67-positive epithelial cells (MKI67-positive epithelial cells/all epithelial cells) for each normal mucosa from three patients with sporadic CRC (patients without FAP) and three patients with FAP. Significance analysis was also done between the normal mucosa from patients with FAP and from the normal mucosa of patients without FAP. CRC, colorectal cancer; DEG, differentially expressed gene; FAP, familial adenomatous polyposis.

Figure 4

Transcriptome heterogeneity of lesions at different evolutionary stages from FAP1. (A) Clustering analysis of all epithelial cells from FAP1 by using tSNE. Four clusterswere identified (top) and the tissue origin for each single cell was indicated (bottom). (B) Heatmap showing the specifically highly expressed genes in each cluster. (C) GO analysis of genes highly expressed in cluster 3. (D) Immumohistochemical staining of CD8 in four regions of carcinomas from FAP1. Scale bar, 100 µm. (E) GO analysis of genes highly expressed in cluster 4. Car, carcinoma; Ade, adenoms; FAP, familial adenomatous polyposis; GO, gene ontology; MAPK, mitogen-activated protein kinase.

Figure 5

Transcriptome dynamics during carcinogenesis. (A) Heatmap showing scaled expression of dynamic genes along the pseudotime. The definition of dynamic genes was described in online supplementary Materials and methods.The full list of the dynamic genes is summarised in online supplementary table 8. Rows of the heatmap represent genes that show dynamic changes along the pseudotime, and these genes were clustered into four groups according to their expression pattern along the pseudotime. We sorted the cells along the pseudotime into 100 windows. The colour scheme represents the z-score distribution from −3 (blue) to 3 (red). The pie plot shows the percentages of cells from multigrade lesions at early, mid and late stage of the pseudotime. (B) Gene ontology analysis of genes from group 1 in (A). (C) Heatmap showing scaled expression of TCA-related genes along the pseudotime. The colour scheme represents the z-score distribution from −3 (blue) to 3 (red). TCA, tricarboxylic acid cycle.