Syövän syntyyn vaikuttavat geneettiset ja epigeneettiset tekijät Lynchin ja Lynch-like syndroomassa

Lynch syndrome is the most prevalent cancer predisposition syndrome that causes significantly increased lifetime risk of cancer in multiple organs such as colorectum, endometrium and ovarium. The predisposition is caused by germline mutations in DNA mismatch repair (MRR) genes MLH1, MSH2, MSH6, and PMS2. Lynch-like syndrome colorectal tumors, like Lynch syndrome tumors, are MMR-deficient. Nevertheless, Lynch-like syndrome tumors do not bear germline mutations in MMR genes nor methylation of the promoter regions of MMR genes that would explain the deficiency. Instead, MMR deficiency in majority of Lynch-like syndrome tumors is caused by two somatic mutations in the MMR genes. Dysfunctional MMR protein complex enables accumulation of mutations in the genome (mutator phenotype) and, eventually, microsatellite instability and cancer. The reasons behind organ selectivity in MMR-deficient tumors are unknown, and whether breast cancer is part of the Lynch syndrome spectrum is under debate. The germline mutations in MMR genes are well studied, but molecular characteristics of Lynch syndrome tumors remain to be studied further. Lynch-like syndrome tumors remain less well characterized: besides the double somatic MMR mutations as a cause of MMR deficiency, their molecular and clinicopathological features as well as their incidence in the population remain poorly known. Currently, the only possibility to diagnose Lynch-like syndrome is by ruling out the possibility of germline mutations in MMR genes (Lynch syndrome) or methylation of the promoter regions of MMR genes. Synchronous ovarian and endometrial carcinomas are common in Lynch syndrome; whether they are of same origin (metastatic cancer) or two independently developed primary cancers remains to be resolved. We aimed to characterize the epigenetic and somatic mutation profiles in Lynch syndrome representing colorectal, ovarian, endometrial and breast carcinomas, and to identify new unique features that could be used in evaluating cancer risk, diagnosis and targeted treatment. We used targeted high-throughput sequencing of 578 known cancer genes to investigate the somatic mutation profiles, and methylation-specific multiplex ligation-dependent probe amplification to study the epigenetic profiles of the tumors. Non-synonymous somatic mutations were detected from sequencing data of the paired tumor and normal tissues to determine mutation signatures and identify potential driver genes. The data was also compared statistically between tumors of different origin, epigenetic status, and between breast carcinomas from Lynch syndrome mutation carriers and their known non-carrier family members. We observed that Lynch- and Lynch-like syndrome tumors have unique somatic mutation and methylation profiles. We were able to link the methylator phenotype to high somatic mutation rates, and Lynch-like colorectal tumors to hypermethylated CpG island methylator phenotype (CIMP), which are novel findings. Our discovery of high mutation burden in genes associated with epigenetic regulation provides a new link between genetic and epigenetic factors in tumorigenesis. Genetic and epigenetic characterization of synchronous ovarian and endometrial carcinomas indicated shared origin, in analogy to sporadic cases. Molecular characteristics and especially mutational signatures of breast tumors of Lynch syndrome mutation carriers indicated that breast carcinoma is likely to be part of the Lynch syndrome tumor spectrum. These findings bear potential clinical relevance since the molecular tumor profiles may be used in diagnosis and may guide tailored management of the patients. Many of the mutated genes are part of signaling routes to which targeted molecules either exist or can be developed.Lynchin syndrooma on yleisin syövälle altistava perinnöllinen syndrooma, joka lisää muun muassa suolisto-, kohtu- ja munasarjasyövän riskiä. Alttiuden aiheuttavat perinnölliset mutaatiot DNA:n emäspariutumisvirheitä korjaavissa (engl. DNA mismatch repair, MMR) geeneissä MLH1, MSH2, MSH6, and PMS2. MMR-proteiineista koostuvan koneiston puutteellinen toiminta johtaa mutaatioiden kertymiseen genomiin ja lopulta mikrosatelliitti-epätasapainoon ja syövän syntyyn. Lynchin kaltaisen (engl. Lynch-like) syndrooman suolistokasvainten MMR-proteiinien puutos ei selity ituratamutaatioilla tai MMR-geenien promoottorialueiden hypermetylaatiolla. Tiedetään, että valtaosassa tapauksista Lynch-like -kasvainten MMR-geenien normaali toiminta on hiljentynyt kahden somaattisen eli hankitun mutaation seurauksena. Kudosspesifisyyden syitä syövissä, joissa MMR-koneiston toiminta on puutteellista, ei vielä tunneta. Rintasyövän kuulumisesta Lynchin syndrooman syöpäspektriin on ristiriitaisia tutkimustuloksia, ja synkronisten munasarja- ja kohdunrungonsyövän alkuperästä (metastasoitunut kasvain vai itsenäisesti kehittyneet primaarikasvaimet) ei löydy julkaistua tutkimustietoa. MMR-geenien perinnölliset mutaatiot tunnetaan jo melko hyvin, mutta Lynchin syndrooman kasvainten muista molekulaarisista ominaisuuksista tarvitaan vielä lisää tutkimustietoa. Lynch-like -kasvaimista on niukemmin tutkimustietoa: kahden somaattisen MMR-mutaation lisäksi niiden molekulaarisista ja kliinispatologisista ominaisuuksista sekä kyseisen tautimuodon esiintyvyydestä populaatiotasolla tiedetään hyvin vähän. Toistaiseksi Lynch-like -syndrooma on mahdollista diagnosoida vain poissulkemalla Lynchin syndrooma ja MMR-geenien hypermetylaatio. Tavoitteenamme oli kartoittaa Lynchin ja Lynch-like -syndroomaan kuuluvien suolisto-, munasarja-, kohdunrungon ja rintasyöpien somaattisia ja epigeneettisiä muutoksia. Näiden piirteiden tunnistaminen eri syöpätyypeissä mahdollistaisi syövän riskin arvioinnin, tarkemman diagnosoinnin sekä kohdennetun hoidon kehittämisen. Tutkimme somaattisia mutaatioita sekvensoimalla kasvainten DNA:ta paneelilla, joka kattaa 578 tunnettua syöpägeeniä, sekä niiden metylaatioprofiilia ns. MS-MLPA (eng. methylation-specific multiplex ligation-dependent probe amplification) -tekniikan avulla. Tunnistimme ei-synonyymiset mutaatiot parittaisesta kasvain- ja normaalikudosten sekvenssidatasta määrittääksemme mutaatioprofiilit sekä syövänkehityksen ajurigeenit (engl. driver genes). Tilastolliset vertailut tehtiin eri kudosten ja metylaatiostatusten kesken. Lisäksi Lynchin syndrooman mutaationkantajilta peräisin olevia rintasyöpiä verrattiin rintasyöpiin sukulaisilta, jotka eivät olleet perineet sukunsa alttiusmutaatiota. Havaitsimme, että Lynchin ja Lynch-like syndroomien syövillä on niille luonteenomaiset epigeneettiset ja somaattiset mutaatioprofiilit. Uusina löydöksinä osoitimme yhteyden yleistyneen hypermetylaatio- ja mutaatiotaipumuksen välillä, sekä yhteyden Lynch-like -kasvainten ja hypermetyloituneen fenotyypin välillä. Epigeneettiseen säätelyyn osallistuvien geenien sekä DNA:n korjausgeenien lisääntynyt mutaatioherkkyys liittää geneettiset ja epigeneettiset tekijät uudella tavalla toisiinsa syövänkehityksessä. Se, että synkroniset munasarjan ja kohdunrungon kasvaimet olivat geneettisesti ja epigeneettisesti yhteneväisiä, osoittaa, että kasvaimet ovat samaa alkuperää, eli edustavat metastasoitunutta kasvainta, kuten vastaavissa sporadisissa syövissä on aiemmin osoitettu. Lynchin syndrooman kantajien ja ei-kantajien rintasyöpien molekulaariset tutkimukset ja erityisesti mutaatioprofiilit toivat viitteitä siitä, että rintasyöpä kuuluu Lynchin syndrooman kasvainspektriin. Tutkimus tuo tärkeää uutta tietoa Lynchin ja Lynch-like syndrooman syöpien syntymekanismeista sekä ominaispiirteistä, joita on mahdollista hyödyntää syövän diagnosoinnissa sekä kohdennettua hoitoa suunniteltaessa. Monet mutatoituneista geeneistä kuuluvat säätelyreitteihin, joihin on joko olemassa tai kehitettävissä kohdennettuja lääkemolekyylejä

Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

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.Peer reviewe

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

: ; ; ; ;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.Peer reviewe

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Instituto Multidisciplinario de Biología Celula

Epidemiological, clinical and molecular characterization of Lynch-like syndrome : A population-based study

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.Peer reviewe

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

AbstractObjective 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.Peer reviewe

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Instituto Multidisciplinario de Biología Celula

The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Fil: Nieminen, Taina T.. University of Helsinski; FinlandiaFil: Pavicic, Walter Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. University of Helsinski; FinlandiaFil: Porkka, Noora. University of Helsinski; FinlandiaFil: Kankainen, Matti. University of Helsinski; FinlandiaFil: Järvinen, Heikki J.. University of Helsinski; FinlandiaFil: Lepistö, Anna. University of Helsinski; FinlandiaFil: Peltomäki, Päivi. University of Helsinski; Finlandi