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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.

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.

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 non-hypermutated 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. This article is protected by copyright. All rights reserved.

Screening of anaplastic lymphoma tyrosine kinase (ALK) gene fusions in non-small cell lung cancer (NSCLC) patients enables the identification of the patients likely to benefit from ALK-targeted therapy. Our aim was to assess the prevalence of ALK fusion in Finnish NSCLC patients, which has not been reported earlier, and to study the presence of ALK fusion in relation to clinicopathological characteristics and other driver gene mutations. A total of 469 formalin-fixed paraffin-embedded tumor tissue specimens from Finnish NSCLC patients were screened for ALK fusion by immunohistochemistry (IHC). For confirmation of IHC results, fluorescence in situ hybridization (FISH) was conducted for 171 specimens. Next-generation sequencing was performed for all ALK-positive specimens to characterize the association of ALK fusion with mutations in targeted regions of 22 driver genes. Of the 469 tumors screened, 11 (2.3%) harbored an ALK fusion, including nine adenocarcinomas and two large cell carcinomas. The IHC results for all 11 ALK-positive and 160 random ALK-negative specimens were confirmed by FISH. ALK fusion was significantly associated with never/ex-light smoking history (P

Non-small cell lung carcinoma (NSCLC) is the most common subtype of lung cancer. The oncogenic potential of receptor tyrosine kinases (RTKs) is widely known and they are potential targets for tailored therapy. Ephrin receptors (Ephs) form the largest group of RTKs. Nevertheless, Ephs are not widely studied in NSCLC so far. The aim of our study was to investigate novel mutations of Eph genes (EPHA1–8, EPHB1–4, EPHB6) and their association with clinically relevant mutations in BRAF, EML4-ALK, EGFR, INSR, KDR, KRAS, MET, PDGFRA, PDGFRB, PIK3, PTEN, RET, and TP53 in NSCLC patients. Targeted resequencing was conducted on 81 formalin-fixed, paraffin-embedded NSCLC tumor specimens. We analyzed missense and nonsense mutations harbored in the coding regions of the selected genes. We found 18 novel mutations of Ephs in 20% (16 of 81) of the patients. Nearly half of these mutations occurred in the protein kinase domain. The mutations were not mutually exclusive with other clinically relevant mutations. Our study shows that Ephs are frequently mutated in NSCLC patients, and occur together with other known mutations relevant to the pathogenicity of NSCLC. © 2013 Wiley Periodicals, Inc.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

Objectives: Non-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features. Materials and methods: Sequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM). Results: Data analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PlIC3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure. Conclusion: Finnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Nowaday screening of non-small cell lung cancer (NSCLC) patients for epidermal growth factor receptor (EGFR) activating mutations is carried out in routine diagnostics to select patients who could benefit from EGFR inhibitor therapies. We aimed to compare EGFR mutation testing by Ion Torrent PGM technology, using AmpliSeq Colon and Lung Cancer panel, with real-time PCR in order to evaluate the accuracy of next generation sequencing (NGS) in detecting clinically relevant EGFR mutations in NSCLC. In total, 368 NSCLC patient samples were tested for EGFR by PCR and were also sequenced by Ion Torrent PGM by using AmpliSeq Colon and Lung panel. Samples were formalin-fixed, paraffin-embedded tumor specimens of Finnish NSCLC patients. The mutations studied for comparison were G719X, S768I, T790M, L858R, L861Q, deletions in exon 19 and insertions in exon 20. Comparison of EGFR mutations detectable by both PCR kit and NGS panel, showed a high degree of concordance between the two methods. Out of 368 samples, 31 out of 32 positive by PCR were also positive by NGS, and 336 out of 336 negative by PCR for these mutations were also negative by NGS giving a concordance of 99.7%. Two negative samples by PCR showed insertions in exon 20, which were not detectable by PCR. In one sample NGS failed to detect G719X mutation that had a very weak signal in PCR. Our study shows that the Ion Torrent PGM technology gives highly comparable results with the golden standard PCR. Thus, this NGS methodology is sensitive and reliable while testing clinically and diagnostically significant EGFR mutations in FFPE samples.

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair (MMR) genes resulting in an increased risk of colorectal cancer (CRC) and other malignancies. Those germline defects with other genetic and epigenetic changes accelerate multistep tumorigenesis. However, early events leading to polyp formation and the timing and order of the molecular “hits” remain unknown. Studies on sporadic CRC have indicated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations at early stages of tumorigenesis, but its importance in hereditary CRC is obscure. In this study we aimed to define 1) methylation changes that occur at different stages of LS-associated colorectal tumor progression, and 2) somatic mutations that occur in adenomas comparing the status of low- and high-grade dysplasia and MMR deficiency (MMR-D) and proficiency (MMR-P). Colorectal biopsies including normal mucosae, adenomas and carcinomas were prospectively collected from 104 LS patients during colonoscopy surveillance, supplemented with retrospective tumor specimens from 56 patients. Promoter methylation was analyzed using the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) including selected tumor suppressor genes (TSGs) associated with early colon oncogenesis. Hypermethylation tendency was evaluated based on the frequency of CpG island methylator phenotype (CIMP) according to the methylation status of eight established CIMP marker genes. LINE-1 methylation was studied as a surrogate marker for global hypomethylation. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. As an ongoing study mutation status of adenomas are studied using the Ion AmpliSeqTM Colon and Lung Cancer Panel targeting mutational hot spots in 22 genes. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression, suggesting the presence of other somatic events that drive neoplastic transformation. Taken together, methylation of TSGs and frequency of CIMP increased and LINE-1 methylation decreased in adenomas and carcinomas along with dysplasia. However, a significant increase of methylation in some genes was detected already in adenomas with low-grade dysplasia compared to normal mucosae. In addition, a proportion of low-grade adenomas could already be classified CIMP positive. When comparing MMR-P and MMR-D adenomas, LINE-1 methylation decreased along the loss of MMR protein expression, and interestingly, higher methylation of SFRP1 was observed in MMR-P adenomas compared to MMR-D cases and normal mucosae. These findings emphasize the importance and early appearance of epigenetic changes in LS-associated tumorigenesis. Citation Format: Satu Mäki-Nevala, Satu Valo, Ari Ristimäki, Laura Renkonen-Sinisalo, Anna Lepistö, Jukka-Pekka Mecklin, Päivi Peltomäki. Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3081.

Lynch syndrome (LS) is the most prevalent cancer predisposition syndrome in which germline mutation in one of four DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes significantly increased lifetime risks of colorectal, endometrial, ovarian, and other cancers. Whether or not breast cancer is part of LS tumor spectrum is currently debated. Moreover, the reasons for organ selectivity in germline mutation carriers are unknown. We have reported that breast carcinoma from LS individuals resembles common breast carcinoma in many respects, but differs with respect to genomic instability (Lotsari et al. 2012, Cancer Res.). To extend these preliminary observations, we are now carrying out an in-depth investigation into the spectra of somatic mutations in LS and sporadic breast carcinomas. All available breast carcinomas among 300 LS families from the nation-wide Hereditary Colon Cancer Registry of Finland have been identified and the tumors investigated for MMR status, DNA methylator phenotype, mechanisms of two-allele inactivation for MMR genes, and somatic mutation status. For the latter, we use massive parallel sequencing as described in our recent publication (Porkka et al. 2017, Oncotarget). To define the unique vs. shared molecular features of LS breast tumorigenesis, profiles of somatic alterations in LS breast carcinomas are compared to those of established LS spectrum carcinomas (colorectal and ovarian) as well as sporadic breast carcinomas with sequence information available from large electronic data-sets. Of 14 LS breast carcinoma samples, 9 (64 %) were MMR-deficient and 5 (36 %) MMR-proficient. This contrasts with LS colorectal and ovarian carcinomas, all of which were MMR-deficient (p=0.0012). MMR-deficient LS breast carcinomas harbored an average of 783 non-synonymous mutations and MMR-proficient tumors 555 mutations (statistically non-significant). In contrast to colorectal and ovarian carcinomas, in which loss of heterozygosity (LOH) or somatic MMR gene mutation provided a second hit consistent with two-hit inactivation in 89 % (25/28) of the tumors, a detectable second hit (mainly LOH) was present in only 43 % (6/14) of breast carcinomas (p=0.0025). Genes harboring high-frequency mutations in at least one-third of tumors were regarded candidates for driver genes. There were 133 such genes in LS colorectal carcinomas, 10 in ovarian carcinomas, and 18 in breast carcinomas. Genes involved in epigenetic regulation were significantly enriched in all three tumor types. In addition, a significant enrichment of NOTCH signaling associated genes characterized LS breast carcinomas. This research is likely to shed light to the mechanisms of organ-specific cancer susceptibility in germline carriers of MMR gene mutations. Our results are expected to guide the surveillance and other clinical management of LS individuals with breast carcinoma. Citation Format: Noora Porkka, Alisa Olkinuora, Satu Mäki-Nevala, Samuli Eldfors, Päivi Peltomäki. Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5369.

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.