Molecular genetic changes in colorectal and small bowel cancer

Colorectal cancer (CRC) is the third most common cancer and the second main cause of cancer mortality worldwide. The colorectum and the small bowel are part of a continuous passageway called the gastrointestinal (GI) tract, however, the cancer incidence varies greatly between these two organs. Although the small bowel constitutes three quarters of the length of the GI tract, only 3% of GI cancers are located in the small bowel. These cancers develop due to both somatic and inherited germline mutations. Thus, characterizing the genetic events that drive tumorigenesis is crucial to provide ways to improve prevention and clinical management of the disease. The general aim of this thesis was to gain new insights into the molecular genetic backgrounds of CRC and small bowel adenocarcinoma (SBA). The first aim of the thesis was to characterize the somatic mutation patterns of the AT-Rich Interaction Domain (ARID) family genes in CRCs with microsatellite instability (MSI). Approximately 15% of CRCs display MSI which arises due to a defective DNA mismatch repair system. These tumors accumulate a high number of mutations, especially small insertions and deletions in repetitive genomic areas called microsatellites. The ARID gene family comprises 15 members, including a known tumor suppressor gene ARID1A. We utilized exome sequencing data of 25 MSI CRCs and their corresponding normal tissues and identified 12 of the ARID genes to display mutations with a frequency of 4-52%. Four genes were selected for further analysis in 21 additional MSI CRCs. We found that, in addition to ARID1A, also ARID1B, ARID2, and ARID4A were frequently mutated and might play a role in MSI CRC. However, additional studies are warranted to further scrutinize the function of these mutations in MSI CRC genesis. The second aim of the thesis was to identify novel oncogenes in MSI CRC. These tumors represent a sensitive system for studying the generation and selection of oncogenic mutations. In contrast to many reported MSI target genes, few oncogenes are known in MSI CRC and they often display specific mutation hotspots. Thus, we used the exome sequencing data of 25 MSI CRCs and their corresponding normal tissues to search for genes with recurrent somatic missense mutations. We identified 33 novel candidate oncogenes of which the following fourteen genes displayed hotspot mutations also in the validation set of 254 MSI CRCs: ANTXR1, CEP135, CRYBB1, MORC2, SLC36A1, GALNT9, PI15, KRT82, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1, and TTPAL. This work revealed a variety of novel recurrent candidate oncogene mutations that might potentially be used to develop personalized therapies. Further research is still needed to confirm their pathogenic role and detailed function in tumorigenesis. The third aim of the thesis was to study the genetic overlap within synchronous CRCs (SCRCs). Approximately 4% of CRC patients display multiple simultaneous primary cancers in the colorectum. Understanding whether SCRCs within a patient are genetically similar or distinct is essential when designing personalized treatments. Exome sequencing data of 23 SCRC pairs and their corresponding healthy tissues revealed that the paired tumors shared a maximum of only a few somatic mutations. This indicated that the tumors have independent origins. Furthermore, paired tumors favored different somatic mutations in known CRC genes and signaling pathways. Variation was observed among clinically relevant genes, such as the discordant KRAS mutation status in a quarter of patients. Tumors within pairs also displayed variation in their mutational signature content suggesting that, regardless of the shared environment, some pairs might have undergone different mutational processes. Finally, by analyzing immune cell counts, we observed that the intratumor immune response varied within most tumor pairs. This was not explained by mutation burden or clinicopathological variables. Overall, this work revealed major diversity within SCRCs and highlights the need to evaluate all synchronous lesions within an individual for optimized therapeutic approach. Additional studies are still required to further elucidate the reasons underlying tumor multiplicity. The fourth aim of the thesis was to characterize the somatic mutation content in SBA. Due to its rarity, knowledge on the genetic background of SBA has remained somewhat elusive. We conducted the first large exome sequencing effort of 106 population-based SBAs representing all three small bowel segments. This work revealed significantly mutated genes previously associated with SBA (TP53, KRAS, APC, SMAD4, and BRAF), as well as novel candidate drivers, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We identified clear mutation hotspot patterns in ERBB2 and BRAF. Interestingly, we observed no V600E mutations, the most common BRAF mutation hotspot in CRC. Other clinically relevant aspects included mutations in ERBB family genes in over a quarter of SBAs as well as mutations in multiple genes that could predict anti-EGFR treatment resistance. We performed the first comprehensive mutation signature analysis on SBA that highlighted four signatures: 1A, 6, 17, and U2. Comparison of the three small bowel segments unveiled some variation in tumor characteristics. Further studies are needed to robustly clarify these differences and their clinical relevance. This comprehensive characterization provided further evidence that SBA is a distinct tumor type and singled out many potential therapeutic targets that could be utilized in SBA treatment development.Paksusuolisyöpä on maailman kolmanneksi yleisin syöpä. Ohut- ja paksusuoli ovat molemmat osa ruuansulatuskanavaa, mutta syövän esiintyvyys on paljon matalampi ohutsuolessa. Syövän syntyyn vaikuttavat niin perinnölliset kuin ympäristötekijät. Suolistosyövän taustalla olevien geneettisten muutosten selvittäminen on tärkeää ymmärtääksemme paremmin kasvainten kehitystä. Tätä tietoa voidaan myös hyödyntää syövän ennaltaehkäisyn ja tehokkaampien hoitojen kehittämisessä. Tämän väitöskirjantyön tavoite oli löytää geenejä, joiden muutokset edistävät suolistosyövän kasvua sekä saada lisää tietoa potilaan yhtäaikaisten kasvainten kehityksestä molekyylitasolla. Keskimäärin 15 % paksusuolisyövistä on mikrosatelliitti-instabiileja (MSI). Nämä kasvaimet syntyvät yhteensopimattomien nukleotidien korjausmekanismin viasta, jonka seurauksena DNA:han kertyy paljon mutaatioita erityisesti lyhyisiin toistojaksoihin, mikrosatelliitteihin. Kartoitimme kaikkien ARID-perheeseen kuuluvien geenien roolia MSI-paksusuolikasvaimissa. Osoitimme, että tunnetun kasvunrajoitegeenin ARID1A:n lisäksi myös ARID1B, ARID2 ja ARID4A ovat melko usein mutatoituneita ja osallistuvat mahdollisesti kasvainten kehitykseen. Etsimme aineistosta myös uusia syöpägeenejä, joissa sama nukleotidi oli mutatoitunut vähintään kahdessa kasvaimessa. Tällainen pistemutaatiokeskittymä antaa viitettä geenin merkittävyydestä syövän kehityksessä. Löysimme 33 ehdokasgeeniä, joista 14 oli mutatoitunut myös jatkotutkimuksissa laajemmassa paksusuolisyöpäryhmässä. Osalla potilaista esiintyy vähintään kaksi yhtäaikaista paksusuolikasvainta. Vertailimme kasvainparien mutaatiosisältöjä ja selvitimme onko kasvaimilla yhteinen alkuperä. Potilaan kasvainten välillä oli huomattavasti eroja, mikä viittasi syöpien syntyneen eri soluista. Löydökset korostivat myös yksilön kaikkien kasvainten arvioimisen tarvetta yksilöidyn hoidon optimoimiseksi. Ohutsuolisyöpää ei ole harvinaisuutensa vuoksi tutkittu laajasti. Selvitimme tämän syövän mutaatiokirjoa kattavasti laajassa populaatiotason tutkimuksessa. Määritimme ohutsyövälle keskeisimmät geenit, jotka sisälsivät sekä aiemmin tunnettuja että uusia ehdokkaita. Löydökset osoittivat ohutsuolisyövän olevan oma syöpätyyppinsä ja vahvistivat aiempaa tietoa. Havaitsimme myös useita muutoksia, joita voisi mahdollisesti hyödyntää tulevaisuudessa ohutsuolisyövän hoidon kehityksen kohteina

Prognostic Value of Immune Environment Analysis in Small Bowel Adenocarcinomas with Verified Mutational Landscape and Predisposing Conditions

Background: Small bowel adenocarcinoma (SBA) is a rare yet insidious cancer with poor survival. The abundance of tumour-infiltrating lymphocytes is associated with improved survival, but the role of the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway in tumour escape is controversial. We evaluated immune cell infiltration, PD1/PD-L1 expression and their prognostic value in a series of SBAs with previously verified predisposing conditions and exome-wide somatic mutation characterization. Methods: Formalin-fixed paraffin-embedded tissue sections stained for CD3, CD8, PD-L1 and PD-1 were analysed from 94 SBAs. An immune cell score (ICS) was formed from the amount of the CD3 and CD8 positive lymphocytes from the tumour centre and invasive margin. The PD-L1 and PD-1 positive immune cells (ICs) and ICS were combined into a variable called Immunoprofile. Results: High ICS, PD-L1IC and PD-1, individually and combined as Immunoprofile, were prognostic for better patient outcome. Sixty-five (69%) SBAs expressed ≥1% positive PD-L1IC. A high tumour mutation burden was common (19%) and associated with immune markers. Immunoprofile, adjusted for TNM stage, mismatch repair status, tumour location, sex and age were independent prognostic markers for disease-specific and overall survival. Conclusions: Analysing tumoral immune contexture provides prognostic information in SBA. Combining ICS, PD-1 and PD-L1IC as Immunoprofile enhanced the prognostic performance

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.Peer reviewe

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

Peer reviewe

Exome-wide somatic mutation characterization of small bowel adenocarcinoma

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (Al) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.Peer reviewe

Deciphering colorectal cancer genetics through multi-omic analysis of 100,204 cases and 154,587 controls of European and east Asian ancestries

In the version of this article initially published, the author affiliations incorrectly listed “Candiolo Cancer Institute FPO-IRCCS, Candiolo (TO), Italy” as “Candiolo Cancer Institute, Candiolo, Italy.” The change has been made to the HTML and PDF versions of the article

Mendelian randomisation analysis strongly implicates adiposity with risk of developing colorectal cancer

Background: Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC. Methods: We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls. Results: In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02-1.49, P = 0.033), 1.59 (95% CI: 1.08-2.34, P = 0.019) and 1.07 (95% CI: 1.03-1.13, P = 0.018), respectively. There was no evidence for association between birth weight and CRC (OR = 1.22, 95% CI: 0.89-1.67, P = 0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10-1.44, P = 7.7 x 10(-4)) and 1.40 (95% CI: 1.14-1.72, P = 1.2 x 10(-3)), respectively. Conclusions: These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.Peer reviewe

Fine-mapping analysis including over 254,000 East Asian and European descendants identifies 136 putative colorectal cancer susceptibility genes

Genome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development

Prognostic Value of Immune Environment Analysis in Small Bowel Adenocarcinomas with Verified Mutational Landscape and Predisposing Conditions

Background: Small bowel adenocarcinoma (SBA) is a rare yet insidious cancer with poor survival. The abundance of tumour-infiltrating lymphocytes is associated with improved survival, but the role of the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway in tumour escape is controversial. We evaluated immune cell infiltration, PD1/PD-L1 expression and their prognostic value in a series of SBAs with previously verified predisposing conditions and exome-wide somatic mutation characterization. Methods: Formalin-fixed paraffin-embedded tissue sections stained for CD3, CD8, PD-L1 and PD-1 were analysed from 94 SBAs. An immune cell score (ICS) was formed from the amount of the CD3 and CD8 positive lymphocytes from the tumour centre and invasive margin. The PD-L1 and PD-1 positive immune cells (ICs) and ICS were combined into a variable called Immunoprofile. Results: High ICS, PD-L1IC and PD-1, individually and combined as Immunoprofile, were prognostic for better patient outcome. Sixty-five (69%) SBAs expressed ≥1% positive PD-L1IC. A high tumour mutation burden was common (19%) and associated with immune markers. Immunoprofile, adjusted for TNM stage, mismatch repair status, tumour location, sex and age were independent prognostic markers for disease-specific and overall survival. Conclusions: Analysing tumoral immune contexture provides prognostic information in SBA. Combining ICS, PD-1 and PD-L1IC as Immunoprofile enhanced the prognostic performance.peerReviewe

Contribution of allelic imbalance to colorectal cancer

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (Al) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible Al targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within Al peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of Al. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between Al target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of Al in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.Peer reviewe