Colon ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2019-319438 on 19 November 2019. Downloaded from Genomic and transcriptomic profiling of carcinogenesis in patients with familial adenomatous polyposis Jingyun Li,1,2,3,4 Rui Wang,1,2,4 Xin Zhou,1 Wendong Wang ,1 Shuai Gao,1 Yunuo Mao,1 Xinglong Wu,1 Limei Guo,5 Haijing Liu,5 Lu Wen,1 Wei Fu,1 Fuchou Tang 1,2,3,4 ► Additional material is ABSTRact published online only. To view, Objective Familial adenomatous polyposis (FAP) Significance of this study please visit the journal online is characterised by the development of hundreds to (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2019- 319438). thousands of adenomas at different evolutionary stages in the colon and rectum that will inevitably progress to ► Familial adenomatous polyposis (FAP) is an For numbered affiliations see autosomal dominant syndrome primarily end of article. adenocarcinomas if left untreated. Here, we investigated the genetic alterations and transcriptomic transitions caused by inherited mutations in APC (adenomatous polyposis coli). Correspondence to from precancerous adenoma to carcinoma. Professor Fuchou Tang, Beijing Design Whole- exome sequencing, whole- genome ► Patients with FAP will develop hundreds Advanced Innovation Center sequencing and single- cell RNA sequencing were to thousands of adenomas in the colon for Genomics, Department of performed on matched adjacent normal tissues, and rectum that will inevitably progress to General Surgery, College of Life adenocarcinomas if left untreated. Sciences, Third Hospital, Peking multiregionally sampled adenomas at different Early prevention of FAP is challenging, and the University, Beijing 100871, stages and carcinomas from six patients with FAP ► China; and one patient with MUTYH- associated polyposis standard of care for classical FAP is prophylactic tangfuchou@ pku. edu. cn (n=56 exomes, n=56 genomes and n=8,757 single colectomy or proctocolectomy. and Professor Wei Fu, Beijing cells). Genomic alterations (including copy number Advanced Innovation Center What are the new findings? alterations and somatic mutations), clonal architectures for Genomics, Department of ► Spatially separated tumours can originate from General Surgery, College of Life and transcriptome dynamics during adenocarcinoma the same cancer- primed cell in patients with Sciences, Third Hospital, Peking carcinogenesis were comprehensively investigated. FAP, indicating that the pathogenic events University, Beijing 100871, Results Genomic evolutionary analysis showed that China, Beijing, China; may happen long before the appearance of http://gut.bmj.com/ fuwei@ bjmu. edu. cn adjacent lesions from the same patient with FAP can clinically identifiable adenomas, even in a originate from the same cancer- primed cell. In addition, macroscopically normal epithelium. JL, RW, XZ and WW contributed the tricarboxylic acid cycle pathway was strongly ► By comparing the transcriptomic signatures of equally. repressed in adenomas and was then slightly alleviated the epithelial cells between the normal colon in carcinomas. Cells from the ’normal’ colon epithelium Received 9 July 2019 tissues from patients with FAP and the normal Revised 22 October 2019 of patients with FAP already showed metabolic colon tissues from patients with sporadic Accepted 24 October 2019 reprogramming compared with cells from the normal colorectal cancer, we found that the normal on October 2, 2021 by guest. Protected copyright. Published Online First colon epithelium of patients with sporadic colorectal 19 November 2019 epithelium of patients with FAP already exhibits cancer. enhanced metabolic processes and proliferative Conclusions The process described in the previously activity. reported field cancerisation model also occurs in ► Reprogramming of carbohydrate metabolism patients with FAP and can contribute to the formation has already happened in precancerous of adjacent lesions in patients with FAP. Reprogramming adenomas. of carbohydrate metabolism has already occurred at the precancerous adenoma stage. Our study provides an accurate picture of the genomic and transcriptomic will inevitably progress to adenocarcinomas if left landscapes during the initiation and progression of untreated.3–5 Colectomy is the main prophylactic carcinogenesis, especially during the transition from treatment for FAP.3 4 6 APC is a tumour suppressor adenoma to carcinoma. gene regulating cell adhesion and migration, main- tenance of genome stability and apoptosis. The © Author(s) (or their employer(s)) 2020. Re-use most important role of the APC gene is negatively permitted under CC BY-­NC. No regulating the WNT signalling pathway by medi- commercial re-use . See rights INTRODUCTION ating the degradation of CTNNB1, a key compo- and permissions. Published Familial adenomatous polyposis (FAP) is an auto- nent of the WNT signalling pathway. Enhanced by BMJ. somal dominant syndrome primarily caused by WNT signalling pathway activity in colon cells can 7–9 To cite: Li J, Wang R, Zhou X, inherited mutations in adenomatous polyposis coli lead to epithelial hyperplasia. et al. Gut (APC).1 2 Patients with FAP develop hundreds to Approximately 85% of non- FAP sporadic 2020;69:1283–1293. thousands of adenomas in the colon and rectum that colorectal cancers (CRCs) have somatic APC Li J, et al. Gut 2020;69:1283–1293. doi:10.1136/gutjnl-2019-319438 1283 Colon adaptors were pooled for whole-ex ome sequence capture using Significance of this study SureSelectXT Human All Exon v5 and SureSelectXT Human All Gut: first published as 10.1136/gutjnl-2019-319438 on 19 November 2019. Downloaded from Exon v6 kits (Agilent Technologies, cat. G7530-9000). How might it impact on clinical practice in the foreseeable future? Modified STRT-Seq ► Although the adjacent normal epithelium of patients We modified the STRT-Seq 13–15 protocol for application to with FAP patents has not accumulated potential driver a large number of single cells, as we mentioned in previously mutations and copy number alterations (CNAs), at the published papers.16–18 See online supplementary materials and transcriptomic level, these tissues have already been primed methods for more details. for carcinogenesis, suggesting the necessity of targeting the ‘“normal’” mucosa for the prevention, diagnosis, and therapy Sanger sequencing of patients with FAP patients. All APC germline mutation sites and somatic mutation sites, as ► Reprogramming of carbohydrate metabolism has already well as the germline mutation sites in MUTYH, were verified by occurred at early stages during the carcinogenesis of lesions Sanger sequencing. The primers used for Sanger sequencing are in patients with FAP patients, suggesting that carbohydrate summarised in online supplementary table 3. metabolism may be a target for early prevention of adenomas. Data analysis For further details, please see the online supplementary materials and methods. mutations, and APC was shown to be the gatekeeper gene during 7 10 the adenoma–carcinoma sequence of CRC. Unlike sporadic RESULTS CRCs, from which only one lesion can be collected, the lesions Overview of the cohort at multiple evolutionary stages in FAP make this condition an The cohort in this study comprised six patients with FAP, one ideal natural model for tracing colorectal carcinogenesis. patient with MUTYH- associated polyposis (MAP) and two Previous studies have applied bulk whole- exome sequencing patients with sporadic CRC. A total of 56 samples were collected: (WES) and whole- genome sequencing (WGS) approaches to 6 peripheral blood samples, 12 adjacent normal tissues, 23 low- investigate the differences in genomic alterations between grade adenomas (LGIN, including grade I and grade II), 5 high- adenomas and carcinomas, aiming to uncover the crucial events 11 12 grade adenomas (HGIN, grade III) and 10 carcinomas (online during the progression of CRC. But the differences among supplementary table 1 and online supplementary figure 1A). lesions obtained from a large cohort may be confounded by the The smallest adenomas were 2 mm in diameter, and the largest interindividual variations, such as genetic background, eating carcinomas were 50 mm in diameter. For most lesions larger habits and intestinal flora. In this study, we simultaneously than 10 mm in diameter, two to five regions were sampled sepa- collected adjacent normal tissue, adenomas at different stages rately (figure 1A). In total, 86 regions from the 56 samples were and carcinomas from the same patient. A multiregional sampling collected. The sequencing data for each region were summarised strategy was employed to investigate intratumour heterogeneity. (online supplementary table 2). For each lesion, three sets of data were simultaneously obtained, Four of the patients with FAP had signatures of family inher- http://gut.bmj.com/ including WES, WGS and single- cell RNA-seq data. The genomic itance (online supplementary figure 1B–G). Inherited germ- landscapes and clonal architecture of lesions at different evolu- line mutation sites in the APC gene were identified in FAP1 tionary stages from the same patient with FAP were compre- (p.E1309fs), FAP2 (p.E1397fs), FAP3 (p.W699*) and FAP4 hensively investigated. In addition, single- cell RNA- seq data (c.834+1G>C, a splice donor variant) (online supplementary were used to investigate the transcriptomic heterogeneity among figure 2A and online supplementary table 3). The clinical pheno- multiregionally sampled lesions