Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors

Alisa OlkinuoraSatu Mäki-NevalaSanjeevi UkwattageAri RistimäkiMaarit AhtiainenJukka-Pekka MecklinPäivi Peltomäki

Background: Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment.

Aim: Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients.

Methods: We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation.

Results: All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor.

Conclusion: Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

PMID: 38725616

Mitotic abnormalities precede microsatellite instability in lynch syndrome-associated colorectal tumourigenesis

Marjaana Pussila, Aleksi Laiho, Petri Törönen, Pauliina Björkbacka, Sonja Nykänen, Kirsi Pylvänäinen, Liisa Holm, Jukka-Pekka Mecklin, Laura Renkonen-Sinisalo, Taru Lehtonen, Anna Lepistö, Jere Linden, Satu Mäki-Nevala, Päivi Peltomäki, Minna Nyström

Lynch syndrome (LS) is one of the most common hereditary cancer syndromes worldwide. Dominantly inherited mutation in one of four DNA mismatch repair genes combined with somatic events leads to mismatch repair deficiency and microsatellite instability (MSI) in tumours. Due to a high lifetime risk of cancer, regular surveillance plays a key role in cancer prevention; yet the observation of frequent interval cancers points to insufficient cancer prevention by colonoscopy-based methods alone. This study aimed to identify precancerous functional changes in colonic mucosa that could facilitate the monitoring and prevention of cancer development in LS.

The study material comprised colon biopsy specimens (n = 71) collected during colonoscopy examinations from LS carriers (tumour-free, or diagnosed with adenoma, or diagnosed with carcinoma) and a control group, which included sporadic cases without LS or neoplasia. The majority (80%) of LS carriers had an inherited genetic MLH1 mutation. The remaining 20% included MSH2 mutation carriers (13%) and MSH6 mutation carriers (7%). The transcriptomes were first analysed with RNA-sequencing and followed up with Gorilla Ontology analysis and Reactome Knowledgebase and Ingenuity Pathway Analyses to detect functional changes that might be associated with the initiation of the neoplastic process in LS individuals.

With pathway and gene ontology analyses combined with measurement of mitotic perimeters from colonic mucosa and tumours, we found an increased tendency to chromosomal instability (CIN), already present in macroscopically normal LS mucosa. Our results suggest that CIN is an earlier aberration than MSI and may be the initial cancer driving aberration, whereas MSI accelerates tumour formation. Furthermore, our results suggest that MLH1 deficiency plays a significant role in the development of CIN.

The results validate our previous findings from mice and highlight early mitotic abnormalities as an important contributor and precancerous marker of colorectal tumourigenesis in LS.


Lynch Syndrome Genetics and Clinical Implications

Päivi Peltomäki, Minna Nyström, Jukka-Pekka Mecklin, Toni T Seppälä.

Lynch syndrome (LS) is one of the most prevalent hereditary cancer syndromes in humans and accounts for some 3% of unselected patients with colorectal or endometrial cancer and 10%-15% of those with DNA mismatch repair-deficient tumors. Previous studies have established the genetic basis of LS predisposition, but there have been significant advances recently in the understanding of the molecular pathogenesis of LS tumors, which has important implications in clinical management. At the same time, immunotherapy has revolutionized the treatment of advanced cancers with DNA mismatch repair defects. We aim to review the recent progress in the LS field and discuss how the accumulating epidemiologic, clinical, and molecular information has contributed to a more accurate and complete picture of LS, resulting in genotype- and immunologic subtype-specific strategies for surveillance, cancer prevention, and treatment.

PMID: 36706841


Identification of DHX40 as a candidate susceptibility gene for colorectal and hematological neoplasia

Alisa Olkinuora, Taina T Nieminen, Suvi Douglas, Anni Kauppinen, Mika Kontro, Juho Väänänen, Matti Kankainen, Ari Ristimäki, Markus Mäkinen, Päivi Lahermo, Caroline Heckman, Janna Saarela, Milla Salonen, Anna Lepistö, Heikki Järvinen, Jukka-Pekka Mecklin, Outi Kilpivaara, Ulla Wartiovaara-Kautto, Kimmo Porkka, Päivi Peltomäki.

Causative genes are mostly unknown for FCCTX, the mismatch repair-proficient category of familial colorectal cancers. Recent evidence suggests shared susceptibility factors between colorectal and hematological malignancies. We investigated 28 FCCTX families by exome sequencing, supplemented with genome sequencing, RNA-sequencing, and tumor studies to identify the predisposing genes. Guided by the findings, the exomes of ~400 patients with acute leukemia, myelodysplastic syndrome, and myeloma were examined. A family with hematological malignancies and cancers of the intestine and breast revealed a truncating variant in the DEAH-box RNA helicase gene DHX40 co-segregating with disease in seven family members. Neoplastic tissues revealed no apparent “second hit”, implying a haploinsufficiency model of tumorigenesis. DHX40 siRNA-treated cell lines exhibited a 13% increase in aberrantly spliced transcripts vs. GAPDH-siRNA or non-target siRNA-treated cells. In the hospital-based hematological series, 18% of germline and 28% of somatic exomes revealed possibly pathogenic DEAD/H box gene variants, including somatic variants of DHX40 in four. This study emphasizes aberrant RNA metabolism behind FCCTX and hematological neoplasia. Our observations on DHX40 suggest analogy to DDX41, a DEAD-box RNA helicase gene previously linked to myeloid malignancies.

PMID: 37696923

Defective maintenance of genomic integrity in hereditary colorectal cancer

Public PhD defence on June 16th, 2023, at 13 o’clock

Haartman Institute, hall 2

Docent Taina Nieminen, Dr. Laura Valle, Prof. Päivi Peltomäki, MSc. Alisa Olkinuora

Author: MSc Alisa Olkinuora

Department of Medical and Clinical Genetics, University of Helsinki

Opponent: Dr. Laura Valle

Hereditary Cancer Program, Catalan Institute of Oncology, IDIBELL, Barcelona, Spain


Mono- and biallelic germline variants of DNA glycosylase genes in colon adenomatous polyposis families from two continents

Alisa Olkinuora, Andrea Constanza Mayordomo, Anni Katariina Kauppinen, María Belén Cerliani, Mariana Coraglio, Ávila Karina Collia, Alejandro Gutiérrez, Karin Alvarez, Alessandra Cassana, Francisco Lopéz-Köstner, Federico Jauk, Hernán García-Rivello, Ari Ristimäki, Laura Koskenvuo, Anna Lepistö, Taina Tuulikki Nieminen, Carlos Alberto Vaccaro, Walter Hernán Pavicic, Päivi Peltomäki

Recently, biallelic germline variants of the DNA glycosylase genes MUTYH and NTHL1 were linked to polyposis susceptibility. Significant fractions remain without a molecular explanation, warranting searches for underlying causes. We used exome sequencing to investigate clinically well-defined adenomatous polyposis cases and families from Finland (N=34), Chile (N=21), and Argentina (N=12), all with known susceptibility genes excluded. Nine index cases (13%) revealed germline variants with proven or possible pathogenicity in the DNA glycosylase genes, involving NEIL1 (mono- or biallelic) in 3 cases, MUTYH (monoallelic) in 3 cases, NTHL1 (biallelic) in 1 case, and OGG1 (monoallelic) in 2 cases. NTHL1 was affected with the well-established, pathogenic c.268C>T, p.(Gln90Ter) variant. A recurrent heterozygous NEIL1 c.506G>A, p.(Gly169Asp) variant was observed in two families. In a Finnish family, the variant occurred in trans with a truncating NEIL1 variant (c.821delT). In an Argentine family, the variant co-occurred with a genomic deletion of exons 2 – 11 of PMS2. Mutational signatures in tumor tissues complied with biological functions reported for NEIL1. Our results suggest that germline variants in DNA glycosylase genes may occur in a non-negligible proportion of unexplained colon polyposis cases and may predispose to tumor development.

Front Oncol. 28;12:870863.

PMID: 36387175

Immunoprofiles and DNA Methylation of Inflammatory Marker Genes in Ulcerative Colitis-Associated Colorectal Tumorigenesis

Mäki-Nevala S, Ukwattage S, Wirta E-V, Ahtiainen M, Ristimäki A, Seppälä TT , Lepistö A, Mecklin J-P, Peltomäki P.

Immunological and epigenetic changes are interconnected and contribute to tumorigenesis. We determined the immunoprofiles and promoter methylation of inflammation-related genes for colitis-associated colorectal carcinomas (CA-CRC). The results were compared with Lynch syndrome (LS)-associated colorectal tumors, which are characterized by an active immune environment through inherited mismatch repair defects. CA-CRCs (= 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274NTSR1PPARGPTGS2PYCARDSOCS1, and SOCS2), the repair gene MGMT, and eight standard marker genes for the CpG Island Methylator Phenotype (CIMP) were investigated for promoter methylation in CA-CRCs, LS tumors (n = 29), and paired normal mucosae by multiplex ligation-dependent probe amplification. All but one CA-CRCs were microsatellite-stable and all LS tumors were microsatellite-unstable. Most CA-CRCs had a high ICS (55%) and a positive CD274 expression in immune cells (52%). NTSR1 revealed frequent tumor-specific hypermethylation in CA-CRC and LS. When compared to LS mucosae, normal mucosae from patients with CA-CRC showed significantly higher methylation of NTSR1 and most CIMP markers. In conclusion, CA-CRCs share a frequent ICShigh/CD274pos expression pattern with LS tumors. Elevated methylation in normal mucosa may indicate field cancerization as a feature of CA-CRC-associated tumorigenesis.

Biomolecules 2021, 11(10):1440.

PMID: 34680073

From APC to the genetics of hereditary and familial colon cancer syndromes

Olkinuora A, Peltomäki P, Aaltonen L, Rajamäki K.

Hereditary colorectal cancer syndromes attributable to high penetrance mutations represent 9-26% of young-onset colorectal cancer cases. The clinical significance of many of these mutations is understood well enough to be used in diagnostics and as an aid in patient care. However, despite the advances made in the field, a significant proportion of familial and early-onset cases remains molecularly uncharacterized and extensive work is still needed to fully understand the genetic nature of colorectal cancer susceptibility. With the emergence of next generation sequencing and associated methods, several predisposition loci have been unravelled but validation is incomplete. Individuals with cancer predisposing mutations are currently enrolled in life-long surveillance, but with the development of new treatments, such as cancer vaccinations, this might change in the not so distant future for at least some individuals. For individuals without a known cause for their disease susceptibility, prevention and therapy options are less precise. Herein, we review the progress achieved in the last three decades with a focus on how colorectal cancer predisposition genes were discovered. Furthermore, we discuss the clinical implications of these discoveries and anticipate what to expect in the next decade.

Hum Mol Genet. 2021 Jul 30; ddab208.

PMID: 34329396

Somatic mutation profiles as molecular classifiers of ulcerative colitis-associated colorectal cancer

Mäki-Nevala S, Ukwattage S, Olkinuora A, Almusa H, Ahtiainen M, Ristimäki A, Seppälä T, Lepistö A, Mecklin JP, Peltomäki P

Ulcerative colitis increases colorectal cancer risk by mechanisms that remain incompletely understood. We approached this question by determining the genetic and epigenetic profiles of colitis-associated colorectal carcinomas (CA-CRC). The findings were compared to Lynch syndrome (LS), a different form of cancer predisposition that shares the importance of immunological factors in tumorigenesis. CA-CRCs (n = 27) were investigated for microsatellite instability, CpG island methylator phenotype and somatic mutations of 999 cancer-relevant genes (“Pan-cancer” panel). A subpanel of “Pan-cancer” design (578 genes) was used for LS colorectal tumors (n = 28). Mutational loads and signatures stratified CA-CRCs into three subgroups: hypermutated microsatellite-unstable (Group 1, n = 1), hypermutated microsatellite-stable (Group 2, n = 9) and nonhypermutated microsatellite-stable (Group 3, n = 17). The Group 1 tumor was the only one with MLH1 promoter hypermethylation and exhibited the mismatch repair deficiency-associated Signatures 21 and 15. Signatures 30 and 32 characterized Group 2, whereas no prominent single signature existed in Group 3. TP53, the most common mutational target in CA-CRC (16/27, 59%), was similarly affected in Groups 2 and 3, but DNA repair genes and Wnt signaling genes were mutated significantly more often in Group 2. In LS tumors, the degree of hypermutability exceeded that of the hypermutated CA-CRC Groups 1 and 2, and somatic mutational profiles and signatures were different. In conclusion, Groups 1 (4%) and 3 (63%) comply with published studies, whereas Group 2 (33%) is novel. The existence of molecularly distinct subgroups within CA-CRC may guide clinical management, such as therapy options.

Keywords: Lynch syndrome; Ulcerative colitis; colorectal cancer; microsatellite instability; somatic mutation.

Int J Cancer. 2021, Jun 15;148(12):2997-3007.

PMID: 33521965

Updates in the field of hereditary nonpolyposis colorectal cancer

Peltomäki P, Olkinuora A, Nieminen TT

Introduction: Up to one third of colorectal cancers show familial clustering and 5% are hereditary single-gene disorders. Hereditary non-polyposis colorectal cancer comprises DNA mismatch repair-deficient and -proficient subsets, represented by Lynch syndrome (LS) and familial colorectal cancer type X (FCCTX), respectively. Accurate knowledge of molecular etiology and genotype-phenotype correlations are critical for tailored cancer prevention and treatment.

Areas covered: The authors highlight advances in the molecular dissection of hereditary non-polyposis colorectal cancer, based on recent literature retrieved from PubMed. Future possibilities for novel gene discoveries are discussed.

Expert commentary: LS is molecularly well established, but new information is accumulating of the associated clinical and tumor phenotypes. FCCTX remains poorly defined, but several promising candidate genes have been discovered and share some preferential biological pathways. Multi-level characterization of specimens from large patient cohorts representing multiple populations, combined with proper bioinformatic and functional analyses, will be necessary to resolve the outstanding questions.

Keywords: DNA mismatch repair; Hereditary non-polyposis colorectal cancer; constitutional epimutation; familial colorectal cancer type X; genomic instability; germline mutation; lynch syndrome.

Expert Rev Gastroenterol Hepatol. 2020 Aug 5;1-14.

PMID: 32755332