Molekulaariset markkerit suomalaisilla keukosyöpäpotilailla

Lung cancer is a common cancer with a poor prognosis. Prognostic and predictive molecular markers for lung cancer have become available and along those treatment options have multiplied. Treatments can be targeted to aberrant molecules driving the tumorigenesis. Thus, clarification of molecular characteristics of a tumor is important for the optimized therapy. Further development in this field would be aided by the identification of (novel) significant markers and understanding how their incidence is associated with clinical characteristics. The aim of this thesis work was to examine known and novel molecular markers, more specifically mutations in selected genes which could be potential molecular markers of cancer, and to link findings with clinical data in Finnish lung cancer patients. Mutations were studied in genes encoding ephrin receptors (Ephs), EGFR, and in 22 other lung cancer related genes. In addition, coding regions of the genome, i.e. exons, were studied for mutations potentially associated with asbestos-exposure. The study material consisted of more than 600 patients, their tumor specimens and clinical data. The majority of the specimens were formalin-fixed, paraffin-embedded non-small cell lung cancer (NSCLC) and malignant mesothelioma (MM) samples. Most of the specimens were subjected to next generation sequencing (NGS); the suitability of this technology in cancer diagnostics was also assessed. Mutations in Ephs were common; 18 % of the patients carried one or more novel mutation. In MM, in particular EPHB1 was found to be mutated. The mutations did not associate with any particular clinical characteristic and they were found often concurrently with known pathogenic driver mutations, which points to a probable passenger mutation nature for these mutations. However, when considering their diverse role in cellular function, as well as their oncogenic and tumor suppressive properties, therapeutically they represent a very intriguing group of molecules. Clinically significant EGFR mutations were found in 11 % of tumors from NSCLC patients. The mutations were associated with adenocarcinoma histology, female gender and never-smoking status, as has been reported in previous studies. The incidence of EGFR mutations resembled that described in previous studies conducted on other Western patients. Similarly, screening of mutational hot spot regions in 22 genes revealed the mutation profile to be rather similar to that described in Western NSCLC patients with some exceptions, such as the higher BRAF mutation and lower STK11 mutation frequency. TP53 and KRAS as the most frequently mutated genes, being mutated in 46 % and 26 % of the NSCLC patients, respectively. In particular, TP53 mutations were found to co-occur recurrently with other mutations, also with pathogenic EGFR and KRAS mutations. Of the 425 patients, 77 % carried one or more mutations. Statistically significant associations were found between the following mutated genes and clinical characteristics: TP53 and PIK3CA and squamous cell carcinoma, KRAS and adenocarcinoma, and CTNNB1 and light ex-smoking status. In the study comparing asbestos-exposed and non-exposed lung cancer, eight candidate genes (BAP1, COPG1, INPP4A, MBD1, SDK1, SEMA5B, TTLL6 and XAB2) were found to be recurrently mutated exclusively in the asbestos-exposed patients. Mutations in BAP1 and COPG1 were found exclusively in MM. However, the well-established lung cancer-related, pathogenic clinically relevant mutations, such as EGFR and KRAS, do not associate with asbestos-exposure. Finally, the application of NGS technology proved to be very suitable for cancer diagnostics. One major advantage of this technology is the possibility to test for different alterations in multiple genes simultaneously as well as the ability to detect and characterize both known and novel alterations.Keuhkosyöpä on yleinen ja huonoennusteinen syöpä, jonka taudinkulun ennusteeseen (prognostinen) ja hoitovasteen ennusteeseen (prediktiivinen) vaikuttavista molekulaarisista markkereista tiedetään yhä enemmän, ja sen myötä syövän hoito on muuttunut. Käytössä on hoitomuotoja, jotka kohdentuvat syövän taustalla olevaan muuttuneeseen molekyyliin. Kasvaimen molekulaaristen ominaisuuksien tunteminen mahdollistaa parhaan nykyaikaisen hoidon. Kohdennettujen hoitojen edelleen kehittäminen edellyttää tutkimusta merkittävien poikkeamien tunnistamiseksi, esiintyvyyden selvittämiseksi ja niiden yhteydestä taudin kliinispatologisiin omaisuuksiin. Tämän väitöskirjatyön tavoitteena oli tutkia valikoitujen geenien uusia ja jo tunnettuja molekulaarisia markkereita, mutaatioita DNA:ssa eli perimän emässekvenssissä, suomalaisilla keuhkosyöpäpotilailla, ja niiden yhteyttä potilaiden kliinisiin ominaisuuksiin. Etnisen taustan on huomattu vaikuttavan keuhkosyöpään liittyvien mutaatioiden esiintymiseen ja siten suomalainen potilasaineisto tarjoaa kiinnostavan tutkimuskohteen suomalaisen perimähistorian eli geneettisen eristäytymisen ja/tai pienen perustajaväestön takia. Mutaatioita tutkittiin seuraavissa geeneissä: efriinireseptorit, EGFR, ja 22 keuhkosyöpään liittyvää geeniä. Lisäksi tutkittiin koko genomin proteiinia koodaavilta alueilta, eli eksoneista, mahdollisia asbestialtistukseen liittyviä mutaatioita. Materiaalina tutkimuksessa oli yhteensä yli 600 potilaan aineisto, sisältäen tuumorinäytteet ja kliiniset tiedot. Näytteet olivat ei-pienisolukeuhkosyöpä (NSCLC)- ja mesotelioomanäytteitä (MM). Tutkimuksessa käytettiin pääsääntöisesti uuden polven sekvensointimenetelmiä (NGS), jotka mahdollistavat perimän emässekvenssin tehokkaan tutkimisen. Menetelmien luotettavuutta ja sopivuutta syöpädiagnostiikassa arvioitiin. Mutaatiot efriinireseptori-geeneissä olivat yleisiä; 18 % potilaista todettiin vähintään yksi uusi mutaatio. Mutaatiot eivät olleet kytköksissä mihinkään tiettyyn kliiniseen ominaisuuteen, ja ne esiintyivät usein yhdessä tunnettujen, syövän kehityksen kannalta merkittävien, ns. aloitusmutaatioiden kanssa, mikä viittaa efriinireseptorimutaatioiden matkustajamutaatio-luonteeseen l. ne ovat osa kasvaimen genomista monimuotoisuutta ilman syövän synnyn kannalta merkittävää ominaisuutta. Efriinireseptorit ovat kuitenkin hoidollisesti hyvin kiinnostava proteiiniryhmä, koska niillä on tärkeä ja monimuotoinen rooli solun toiminnassa, ja koska ne voivat toimia joko syöpää estävästi tai sitä edistäen. Siten niiden poikkeamien laajempi tunteminen olisi tärkeää. Kliinisesti merkittäviä EGFR-geenin mutaatioita, jotka ennustavat hoitovastetta kohdennetulle EGFR-estäjälle, löydettiin 11 % NSCLC-potilaista. Mutaatiot olivat kytköksissä adenokarsinooma-histologiaan, naissukupuoleen ja tupakoimattomuuteen, kuten aiemmissakin tutkimuksissa on kuvattu. Sekä EGFR-mutaatioiden että 22 keuhkosyöpään liittyvän geenin mutaatioprofiili oli suuresti samankaltainen kuin länsimaalaisilla potilailla aiemmin kuvattu. Mutaatiokartoituksessa kuitenkin joitakin eroavaisuuksia löydettiin. Yleisimmin mutatoituneet geenit olivat TP53 (46 % potilaista) ja KRAS (26 %). Noin kolmella neljästä NSCLC-potilaista ilmeni vähintään yksi mutaatio. Tilastollisesti merkittäviä yhteyksiä löydettiin seuraavien geenimutaatioiden ja kliinispatologisten ominaisuuksien välillä: TP53 ja PIK3CA ja levyepiteelikarsinooma-histologia, KRAS ja adenokarsinooma-histologia, sekä CTNNB1 ja kevyt tupakointihistoria. Asbestialtistuneen ja altistumattoman keuhkosyövän vertailututkimuksessa löysimme kahdeksan kandidaattigeeniä (BAP1, COPG1, INPP4A, MBD1, SDK1, SEMA5B, TTLL6 ja XAB2), jotka olivat toistuvasti mutatoituneet vain asbestialtistuneilla potilailla. Hyvin tunnetut syövän kannalta merkittävät mutaatiot, kuten EGFR and KRAS, eivät kuitenkaan näytä liittyvän asbestialtistukseen. Lisäksi, NGS-menetelmät sopivat hyvin syöpädiagnostiikkaan. Niiden yksi ehdoton etu on mahdollisuus testata monenlaisia poikkeamia samanaikaisesti, ja tunnistaa sekä jo tunnettuja että täysin uusia poikkeamia yksityiskohtaisesti

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

Concordant Results of Epidermal Growth Factor Receptor Mutation Detection by Real-Time Polymerase Chain Reaction and Ion Torrent Technology in Non-Small Cell Lung Cancer

Peer reviewe

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

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 (1612, NEUROGI,CRABP1, and CDKN2A) and TSGs (SERPI 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. Fund: Jane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid juselius Foundation, and HiL1FE. (C) 2019 Published by Elsevier B.V.Peer reviewe

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

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 (n = 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274, NTSR1, PPARG, PTGS2, PYCARD, SOCS1, 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

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

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 (n = 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274, NTSR1, PPARG, PTGS2, PYCARD, SOCS1, 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

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

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.Peer reviewe

Epidermal Growth Factor Receptor Mutations in 510 Finnish Non–Small-Cell Lung Cancer Patients

Introduction:Among the driver gene mutations in non–small-cell lung cancer, mutations in epidermal growth factor receptor (EGFR) are the most important because of their predictive role in selecting patients eligible for targeted therapy. Our aim was to study EGFR mutations in a Finnish non–small-cell lung cancer cohort of 528 patients.Methods:Mutation testing was conducted on DNA extracted from paraffin-embedded, formalin-fixed tumor material using the following real-time polymerase chain reaction-based kits: Therascreen EGFR PCR Kit and cobas EGFR Mutation Test.Results:EGFR mutation frequency was 11.4% and all positive cases were adenocarcinomas, of which a majority had an acinar predominant pattern. Mutations were seen significantly more often in females and never-smokers than in males and smokers. The most frequent mutations were L858R in exon 21 and deletions in exon 19. Overall survival of the patients, not treated with EGFR inhibitor, did not differ between EGFR mutation-positive and EGFR mutation-negative patients.Conclusion:EGFR mutation profile in this Finnish non–small-cell lung cancer cohort resembles in many respect with that of other Western European cohorts, even though the overall frequency of mutations is slightly higher. We show the occurrence of EGFR mutations in patients with occupational asbestos exposure and also in those diagnosed with chronic obstructive pulmonary disease who have not been often investigated before

Table_4_Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.xlsx

BackgroundLynch 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.AimCurrently, 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.MethodsWe 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.ResultsAll 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.ConclusionFrequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.</p

Table_3_Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.xlsx

BackgroundLynch 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.AimCurrently, 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.MethodsWe 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.ResultsAll 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.ConclusionFrequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.</p