Does breast carcinoma belong to the Lynch syndrome tumor spectrum? – Somatic mutational profiles vs. ovarian and colorectal carcinomas.

Porkka NK, Olkinuora A, Kuopio T, Ahtiainen M, Eldfors S, Almusa H, Mecklin JP, Peltomäki P.


Inherited DNA mismatch repair (MMR) defects cause predisposition to colorectal, endometrial, ovarian, and other cancers occurring in Lynch syndrome (LS). It is unsettled whether breast carcinoma belongs to the LS tumor spectrum. We approached this question through somatic mutational analysis of breast carcinomas from LS families, using established LS-spectrum tumors for comparison. Somatic mutational profiles of 578 cancer-relevant genes were determined for LS-breast cancer (LS-BC, n = 20), non-carrier breast cancer (NC-BC, n = 10), LS-ovarian cancer (LS-OC, n = 16), and LS-colorectal cancer (LS-CRC, n = 18) from the National LS Registry of Finland. Microsatellite and MMR protein analysis stratified LS-BCs into MMR-deficient (dMMR, n = 11) and MMR-proficient (pMMR, n = 9) subgroups. All NC-BCs were pMMR and all LS-OCs and LS-CRCs dMMR. All but one dMMR LS-BCs were hypermutated (> 10 non-synonymous mutations/Mb; average 174/Mb per tumor) and the frequency of MMR-deficiency-associated signatures 6, 20, and 26 was comparable to that in LS-OC and LS-CRC. LS-BCs that were pMMR resembled NC-BCs with respect to somatic mutational loads (4/9, 44%, hypermutated with average mutation count 33/Mb vs. 3/10, 30%, hypermutated with average 88 mutations/Mb), whereas mutational signatures shared features of dMMR LS-BC, LS-OC, and LS-CRC. Epigenetic regulatory genes were significantly enriched as mutational targets in LS-BC, LS-OC, and LS-CRC. Many top mutant genes of our LS-BCs have previously been identified as drivers of unselected breast carcinomas. In conclusion, somatic mutational signatures suggest that conventional MMR status of tumor tissues is likely to underestimate the significance of the predisposing MMR defects as contributors to breast tumorigenesis in LS.


DNA mismatch repair; Lynch syndrome; MSI; breast carcinoma; somatic mutation

Oncotarget. 2020 Apr 7;11(14):1244-1256.

PubMed ID: 32292574

Biallelic germline nonsense variant of MLH3 underlies polyposis predisposition.

Olkinuora A, Nieminen TT, Mårtensson E, Rohlin A, Ristimäki A, Koskenvuo L, Lepistö A, Swedish Extended Genetic Analysis of Colorectal Neoplasia (SWEN) Study Group, Gebre-Medhin S, Nordling M, Peltomäki P.

PURPOSE: Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes.

METHODS: Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of ~1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility.

RESULTS: Three homozygous carriers of a truncating variant in MLH3, c.3563C>G, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability.

CONCLUSION: Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

Genet Med. 2018 Dec 21. [Epub ahead of print]

PubMed ID: 30573798

Epidemiological, clinical and molecular characterization of Lynch‐like syndrome – A population‐based study

Noora Porkka, Laura Lahtinen, Maarit Ahtiainen, Jan Böhm, Teijo Kuopio, Samuli Eldfors, Jukka-Pekka Mecklin, Toni Seppälä, Päivi Peltomäki

Colorectal carcinomas that are mismatch repair (MMR)‐deficient in the absence of MLH1 promoter methylation or germline mutations represent Lynch‐like syndrome (LLS). Double somatic events inactivating MMR genes are involved in the etiology of LLS tumors. Our purpose was to define the clinical and broader molecular hallmarks of LLS tumors and the population incidence of LLS, which remain poorly characterized. We investigated 762 consecutive colorectal carcinomas operated in Central Finland in 2000 – 2010. LLS cases were identified by a stepwise protocol based on MMR protein expression, MLH1 methylation, and MMR gene mutation status. LLS tumors were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations in 578 cancer‐relevant genes. Among 107 MMR‐deficient tumors, 81 (76%) were attributable to MLH1 promoter methylation and 9 (8%) to germline mutations (Lynch syndrome, LS), leaving 14 LLS cases (13%) (3 remained unclassified). LLS carcinomas were diagnosed at a mean age of 65 years (vs. 44 years in LS, p < 0.001), had a proximal to distal ratio of 1:1, and all were BRAF V600E‐negative. Two somatic events in MMR genes were identifiable in 11 tumors (79%). As novel findings, the tumors contained an average of 31 nonsynonymous somatic mutations/Mb and 13/14 were CIMP‐positive. In conclusion, we establish the epidemiological, clinical, and molecular characteristics of LLS in a population‐based study design. Significantly more frequent CIMP‐positivity and lower rates of somatic mutations make a distinction to LS. The absence of BRAF V600E mutation separates LLS colorectal carcinomas from MLH1‐methylated colorectal carcinomas with CIMP‐positive phenotype.

Int J Cancer. 2019 Jul 1;145(1):87-98.

PubMed ID 30575961

DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression

Satu Mäki-Nevala, Satu Valo, Ari Ristimäki, Virinder Sarhadi, Sakari Knuutila, Minna Nyström, Laura Renkonen-Sinisalo, Anna Lepistö, Jukka-Pekka Mecklin, Päivi Peltomäki.

Background: DNA mismatch repair (MMR) defects are a major factor in colorectal tumorigenesis in Lynch syndrome (LS) and 15% of sporadic cases. Some adenomas from carriers of inherited MMR gene mutations have intact MMR protein expression implying other mechanisms accelerating tumorigenesis. We determined roles of DNA methylation changes and somatic mutations in cancer-associated genes as tumorigenic events in LS-associated colorectal adenomas with intact MMR.Methods: We investigated 122 archival colorectal specimens of normal mucosae, adenomas and carcinomas from 57 LS patients. MMR-deficient (MMR-D, n = 49) and MMR-proficient (MMR-P, n = 18) adenomas were of particular interest and were interrogated by methylation-specific multiplex ligation-dependent probe amplification and Ion Torrent sequencing.Findings: Promoter methylation of CpG island methylator phenotype (CIMP)-associated marker genes and selected colorectal cancer (CRC)-associated tumor suppressor genes (TSGs) increased and LINE-1 methylation decreased from normal mucosa to MMR-P adenomas to MMR-D adenomas. Methylation differences were statistically significant when either adenoma group was compared with normal mucosa, but not between MMR-P and MMR-D adenomas. Significantly increased methylation was found in multiple CIMP marker genes (IGF2, NEUROG1, CRABP1, and CDKN2A) and TSGs (SFRP1 and SFRP2) in MMR-P adenomas already. Furthermore, certain CRC-associated somatic mutations, such as KRAS, were prevalent in MMR-P adenomas.

Interpretation: We conclude that DNA methylation changes and somatic mutations of cancer-associated genes might serve as an alternative pathway accelerating LS-associated tumorigenesis in the presence of proficient MMR.

EBioMedicine 2019 Jan;39:280-291.
PubMed ID: 30578081

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer.

Pussila M, Törönen P, Einarsdottir E, Katayama S, Krjutškov K, Holm L, Kere J, Peltomäki P, Mäkinen MJ, Linden J, Nyström M.

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/-) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC.

Carcinogenesis. 2018 May 28;39(6):788-797.

Pubmed ID:29701748

Converging endometrial and ovarian tumorigenesis in Lynch syndrome: Shared origin of synchronous carcinomas

Niskakoski A, Pasanen A, Porkka N, Eldfors S, Lassus H, Renkonen-Sinisalo L, Kaur S, Mecklin JP, Bützow R, Peltomäki P.

OBJECTIVE: The diagnosis of carcinoma in both the uterus and the ovary simultaneously is not uncommon and raises the question of synchronous primaries vs. metastatic disease. Targeted sequencing of sporadic synchronous endometrial and ovarian carcinomas has shown that such tumors are clonally related and thus represent metastatic disease from one site to the other. Our purpose was to investigate whether or not the same applies to Lynch syndrome (LS), in which synchronous cancers of the gynecological tract are twice as frequent as in sporadic cases, reflecting inherited defects in DNA mismatch repair (MMR).

METHODS: MMR gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nationwide registry. Endometrial (n = 35) and ovarian carcinomas (n = 23), including 13 synchronous carcinoma pairs, were collected as well as endometrial hyperplasias (n = 56) and normal endometria (n = 99) from a surveillance program over two decades. All samples were studied for MMR status, ARID1A and L1CAM protein expression and tumor suppressor gene promoter methylation, and synchronous carcinomas additionally for somatic mutation profiles of 578 cancer-relevant genes.

RESULTS: Synchronous carcinomas were molecularly concordant in all cases. Prior or concurrent complex (but not simple) endometrial hyperplasias showed a high degree of concordance with endometrial or ovarian carcinoma as the endpoint lesion.

CONCLUSIONS: Our investigation suggests shared origins for synchronous endometrial and ovarian carcinomas in LS, in analogy to sporadic cases. The similar degrees of concordance between complex hyperplasias and endometrial vs. ovarian carcinoma highlight converging pathways for endometrial and ovarian tumorigenesis overall.

Gynecol Oncol. 2018 Apr 28. doi: 10.1016/j.ygyno.2018.04.566. [Epub ahead of print]


Molecular changes preceding endometrial and ovarian cancer: a study of consecutive endometrial specimens from Lynch syndrome surveillance

Niskakoski A, Pasanen A, Lassus H, Renkonen-Sinisalo L, Kaur S, Mecklin J-P, Bützow R and Peltomäki P

Molecular alterations preceding endometrial and ovarian cancer and the sequence of events are unknown. Consecutive specimens from lifelong surveillance for Lynch syndrome provides a natural setting to address such questions. To molecularly define the multistep gynecological tumorigenesis, DNA mismatch repair gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nation-wide registry and endometrial biopsy specimens taken from these individuals during 20 years of screening were collected. A total of 213 endometrial and ovarian specimens from Lynch syndrome individuals and 197 histology-matched (non-serous) samples from sporadic cases were available for this investigation. The specimens were profiled for markers linked to endometrial and ovarian tumorigenesis, including ARID1A protein expression, mismatch repair status, and tumor suppressor gene promoter methylation. In Lynch syndrome-associated endometrial and ovarian carcinomas, ARID1A protein was lost in 61–100% and mismatch repair was deficient in 97–100%, compared to 0–17% and 14–44% in sporadic cases (P = 0.000). ARID1A loss appeared in complex hyperplasia and deficient mismatch repair and tumor suppressor gene promoter methylation in histologically normal endometrium. Despite quantitative differences between Lynch syndrome and sporadic cases, ARID1A expression, mismatch repair, and tumor suppressor gene promoter methylation divided endometrial samples from both patient groups into three categories of increasing abnormality, comprising normal endometrium and simple hyperplasia (I), complex hyperplasia with or without atypia (II), and endometrial cancer (III). Complex hyperplasias without vs. with atypia were molecularly indistinguishable. In conclusion, surveillance specimens from Lynch syndrome identify mismatch repair deficiency, tumor suppressor gene promoter methylation, and ARID1A loss as early changes in tumor development. Our findings are clinically relevant for the classification of endometrial hyperplasias and have potential implications in cancer prevention in Lynch syndrome and beyond.

Mod Pathol. 2018 Mar 27. doi: 10.1038/s41379-018-0044-4. [Epub ahead of print]

PMID: 29588532


Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

Porkka N, Valo S, Nieminen TT, Olkinuora A, Mäki-Nevala S, Eldfors S and Peltomäki P

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.

Oncotarget, 2017[]=22445&path%5B%5D=70979

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon

Dermadi D, Valo S, Ollila S, Soliymani R, Sipari N, Pussila M, Sarantaus L, Linden J, Baumann M and Nyström M

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risk of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTβ, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation.

Cancer Res; 77(12); 3352–63. ©2017 AACR.

PMID: 28416481

Review in Duodecim: Disorders of DNA repair mechanisms and their clinical significance

Kansikas M, Nyström M & Peltomäki P.

DNA repair mechanisms maintain genome stability by preventing the multiplication of genetic errors, caused by environmental factors and intracellular processes during cell division. Unrepaired damage may permanently alter the genome and cell functions, and even minor changes in DNA strand may initiate malignant transformation of the cell. Up to 25 000 changes in DNA occur daily in a single actively dividing cell, and these changes are continuously repaired. If DNA repair mechanisms are impaired, errors will accumulate into the genome. As numerous factors of different nature can cause genetic errors, several different DNA repair mechanisms are necessary to ensure genomic stability.

DNA:n korjausmekanismit pitävät yllä perimän vakautta estämällä ympäristön ja solujen sisäisten prosessien aiheuttamien vaurioiden monistumisen solunjakautumisten yhteydessä. Korjaamatta jääneet vauriot voivat muuttaa perimän ja solun toimintaa pysyvästi. Jo pienikin DNA:n emäsjuosteessa tapahtuva muutos voi aloittaa solun muuttumisen syöpäsoluksi. Yhdessä aktiivisesti jakautuvassa solussa on arvioitu päivittäin tapahtuvan jopa 25 000 DNA:n muutosta, joita koko ajan korjataan. Mikäli DNA:n korjausmekanismien toiminta häiriintyy, alkaa perimään nopeasti kasautua virheitä. Koska virheitä aiheuttavat monet eri tekijät, jotka ovat luonteeltaan erilaisia, tarvitaan myös useita DNA:n korjausmekanismeja. Näiden mekanismien selvittäminen on ollut tärkeää, ja siinä uraauurtavaa työtä tehneet tutkijat palkittiin vuoden 2015 Nobelin kemianpalkinnolla.

Duodecim, 2017. (in Finnish)