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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.

Abstract Intraductal papillary mucinous neoplasms (IPMNs), often found incidentally, are potentially malignant cystic tumors of the pancreas. Due to the precancerous nature, IPMNs lacking malignant features should be kept on surveillance. The follow-up relies on magnetic resonance imaging, which has a limited accuracy to define the high-risk patients. New diagnostic methods are thus needed to recognize IPMNs with malignant potential. Here, aberrantly expressed glycans constitute a promising new area of research. We compared the N-glycan profiles of non-invasive IPMN tissues (n = 10) and invasive IPMN tissues (n = 10) to those of non-neoplastic pancreatic controls (n = 5) by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Both IPMN subgroups showed increased abundance of neutral composition H4N4 and decrease in H3N5F1, increase in sialylation, and decrease in sulfation, as compared to the controls. Furthermore, invasive IPMN showed an increase in terminal N-acetylhexosamine containing structure H4N5, and increase in acidic complex-type glycans, but decrease in their complex fucosylation and sulfation, as compared to the controls. In conclusion, the N-glycan profiles differed between healthy pancreatic tissue and non-invasive and invasive IPMNs. The unique glycans expressed in invasive IPMNs may offer interesting new options for diagnostics.

Abstract Universal testing for microsatellite instability (MSI) is recommended in colorectal cancer (CRC) to screen for Lynch syndrome and to guide optimal treatment and follow-up of the patients. Especially in neoadjuvant setting, where immuno-oncological treatments have recently shown excellent responses, identification of MSI status at biopsy is a prerequisite. Idylla MSI test offers a rapid and automated test to assess MSI-status from formalin-fixed paraffin-embedded tumor tissue sections. In this study, we compared the performance of the Idylla MSI test to mismatch repair (MMR) protein immunohistochemistry (IHC) using 117 CRC biopsies with previously known deficient MMR status. The concordance between Idylla and IHC was 99.0% (95/96) for biopsies with the recommended ≥ 20% tumor cell content. Further, 85.7% (18/21) of suboptimal CRC biopsy specimens (tumor cell content 5–15%) were diagnosed as MSI. Overall, we identified four discrepant cases of which three had tumor cell content less than 20%, explaining the discordant result. Our study shows that the Idylla MSI test offers a competent tool for MSI screening in CRC biopsy specimens.

Abstract Background Because of the premalignant nature of intraductal papillary mucinous neoplasms (IPMNs), patients should undergo surveillance as long as they remain fit for surgery. This surveillance, with imaging and laboratory tests every 6 to 12 months, is expensive and may psychologically burden patients. This study aimed to determine the effects of IPMN surveillance on patients´ health-related quality of life (HRQoL) and anxiety levels. Methods We included a random subgroup of all IPMN patients undergoing a follow-up check-up at Helsinki University Hospital (HUH) between August 2017 and November 2018. Patients were asked to complete the 15D HRQoL and state-trait anxiety inventory (STAI) questionnaires just before and three months after an IPMN control. Results Among 899 patients in IPMN follow-up, 232 participated. The 15D HRQoL results showed differences in some IPMN patients’ 15 analyzed dimensions compared to a sex- and age-standardized general population cohort, but the clinical relevance of these differences appear doubtful. We detected no significant difference in the anxiety levels determined using the STAI questionnaires before or three months after the IPMN control. Conclusion Surveillance should be less harmful than the risk of disease. Among our patients, the recommended IPMN follow-up carried minimal negative impact on patients’ HRQoL or anxiety levels. This result is important, because the number of patients under IPMN surveillance is rapidly increasing and the cancer risk among the majority of these patients remains small. Trial registration: The Surgical Ethics Committee of Helsinki University Hospital approved this study (Dnro HUS 475/2017) and it was registered at ClinicalTrials.gov (NCT03131076) before patient enrollment began.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies, is increasing in incidence. However, the stromal reaction pathophysiology and its role in PDAC development remain unknown. We, therefore, investigated the potential role of histological chronic pancreatitis findings and chronic inflammation on surgical PDAC specimens and disease-specific survival (DSS). Methods Between 2000 and 2016, we retrospectively enrolled 236 PDAC patients treated with curative-intent pancreatic surgery at Helsinki University Hospital. All pancreatic transection margin slides were re-reviewed and histological findings were evaluated applying international guidelines. Results DSS among patients with no fibrosis, acinar atrophy or chronic inflammation identified on pathology slides was significantly better than DSS among patients with fibrosis, acinar atrophy and chronic inflammation [median survival: 41.8 months, 95% confidence interval (CI) 26.0–57.6 vs. 20.6 months, 95% CI 10.3–30.9; log-rank test p = 0.001]. Multivariate analysis revealed that Ca 19–9 > 37 kU/l [hazard ratio (HR) 1.48, 95% CI 1.02–2.16], lymph node metastases N1–2 (HR 1.71, 95% CI 1.16–2.52), tumor size > 30 mm (HR 1.47, 95% CI 1.04–2.08), the combined effect of fibrosis and acinar atrophy (HR 1.91, 95% CI 1.27–2.88) and the combined effect of fibrosis, acinar atrophy and chronic inflammation (HR 1.63, 95% CI 1.03–2.58) independently served as unfavorable prognostic factors for DSS. However, we observed no significant associations between tumor size (> 30 mm) and the degree of perilobular fibrosis (p = 0.655), intralobular fibrosis (p = 0.587), acinar atrophy (p = 0.584) or chronic inflammation (p = 0.453). Conclusions Our results indicate that the pancreatic stroma is associated with PDAC patients’ DSS. Additionally, the more severe the fibrosis, acinar atrophy and chronic inflammation, the worse the impact on DSS, thereby warranting further studies investigating stroma-targeted therapies.

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.

Abstract Podocalyxin overexpression associates with poor survival in pancreatic cancer (PDAC). We investigated whether podocalyxin expression correlates with treatment response or survival in neoadjuvant-treated PDAC. Through immunohistochemistry, we evaluated podocalyxin expression in 88 neoadjuvant and 143 upfront surgery patients using two antibodies. We developed a six-tier grading scheme for neoadjuvant responses evaluating the remaining tumor cells in surgical specimens. Strong podocalyxin immunopositivity associated with poor survival in the patients responding poorly to the neoadjuvant treatment (HR 4.16, 95% CI 1.56–11.01, p = 0.004), although neoadjuvant patients exhibited generally low podocalyxin expression (p = 0.017). Strong podocalyxin expression associated with perineural invasion (p = 0.003) and lack of radiation (p = 0.036). Two patients exhibited a complete neoadjuvant response, while a strong neoadjuvant response (≤ 5% of residual tumor cells) significantly associated with lower stage, pT-class and grade, less spread to the regional lymph nodes, less perineural invasion, and podocalyxin negativity (p

BackgroundKRAS mutations, present in over 40% of metastatic colorectal cancer (mCRC), are negative predictive factors for anti-EGFR therapy. Mutations in KRAS-G12C have a cysteine residue for which drugs have been developed. Published data on this specific mutation are conflicting; thus, we studied the frequency and clinical characteristics in a real-world and population-based setting.MethodsPatients from three Nordic population-based cohorts and the real-life RAXO-study were combined. RAS and BRAF tests were performed in routine healthcare, except for one cohort. The dataset consisted of 2,559 patients, of which 1,871 could be accurately classified as KRAS, NRAS, and BRAF-V600E. Demographics, treatments, and outcomes were compared using logistic regression. Overall survival (OS) was estimated with Kaplan–Meier, and differences were compared using Cox regression, adjusted for baseline factors.ResultsThe KRAS-G12C frequency was 2%–4% of all tested in the seven cohorts (mean 3%) and 4%–8% of KRAS mutated tumors in the cohorts (mean 7%). Metastasectomies and ablations were performed more often (38% vs. 28%, p = 0.040), and bevacizumab was added more often (any line 74% vs. 59%, p = 0.007) for patients with KRAS-G12C- vs. other KRAS-mutated tumors, whereas chemotherapy was given to similar proportions. OS did not differ according to KRAS mutation, neither overall (adjusted hazard ratio (HR) 1.03; 95% CI 0.74–1.42, reference KRAS-G12C) nor within treatment groups defined as “systemic chemotherapy, alone or with biologics”, “metastasectomy and/or ablations”, or “best supportive care”, RAS and BRAF wild-type tumors (n = 548) differed similarly to KRAS-G12C, as to other KRAS- or NRAS-mutated (n = 66) tumors.ConclusionsIn these real-life and population-based cohorts, there were no significant differences in patient characteristics and outcomes between patients with KRAS-G12C tumors and those with other KRAS mutations. This contrasts with the results of most previous studies claiming differences in many aspects, often with worse outcomes for those with a KRAS-G12C mutation, although not consistent. When specific drugs are developed, as for this mutation, differences in outcome will hopefully emerge.

Abstract Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. Few previous mass spectrometry‐based studies have analyzed differences in protein expression depending on the tumor location. In this study, we have used mass spectrometry‐based proteomics to analyze plasma samples from 83 CRC patients to study if differences in plasma protein expression can be seen depending on primary tumor location (right colon, left colon, or rectum). Differences were studied between the groups both regardless of and according to tumor stage (II or III). Large differences in plasma protein expression were seen, and we found that plasma samples from patients with rectal cancer separated from samples from patients with colon cancer when analyzed by principal component analysis and hierarchical clustering. Samples from patients with cancer in the right and left colon also tended to separate from each other. Pathway analysis discovered canonical pathways involved in lipid metabolism and inflammation to be enriched. This study will help to further define CRC as distinct entities depending on tumor location, as shown by the widespread differences in plasma protein profile and dysregulated pathways.

Abstract Colorectal cancer (CRC) stands for 10% of the worldwide cancer burden and has recently become the second most common cause of cancer death. The 5‐year survival rate depends mainly on stage at diagnosis. Mass spectrometric proteomic analysis is widely used to study the plasma proteome, which is complex and contains multitudes of proteins. In this study, we have used Ultra Performance Liquid Chromatography‐Ultra Definition Mass Spectrometry (UPLC‐UDMSE)‐based proteomics to analyze plasma samples from 76 CRC patients. We identified several plasma proteins, such as CP, TVP23C, FETUB, and IGFBP3, of which altered levels led to significant differences in survival, as seen by Cox regression and Kaplan‐Meier analysis. Additionally, during Cox regression analysis, samples were adjusted for age and/or tumor stage, enabling stringent analysis. These proteins, although in need of further validation, could be of use during patient follow‐up, as their levels can non‐invasively be measured from blood samples, and could be of use in predicting patient outcome. Several of these proteins additionally have roles in metabolism and inflammation, two processes central to the development and progression of cancer, further indicating their importance in cancer.

Retrotransposons are usually dormant in healthy tissue, but become activated during malignancy. Here, in colorectal cancer, Cajuso et al. show that retrotransposon activity associates with clinical features of the disease.

Abstract Background Colorectal cancer (CRC) is the third most common cancer worldwide, and its incidence is expected to increase to over 2.2 million new cases in 2030. Stage II CRC is classified as localized disease, while stage III CRC has spread to regional lymph nodes. The 5-year survival rate is over 80% for patients with stage II CRC, but less than 60% for patients with stage III CRC. Proteins, especially plasma proteins that are detectable in easily obtained blood samples, that differ between stage II and III CRC could be useful for predicting and monitoring disease progression. CRC displays differences depending on primary tumor location (right colon, left colon, or rectum), and how plasma protein expression changes during CRC progression from stage II to III depending on primary tumor location is not well-characterized. Methods In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 83 patients with stage II or III CRC, followed by statistical and pathway analysis (data are available via ProteomeXchange). The patients were divided into groups according to tumor stage (II or III) and changes in plasma protein expression between stage II and III (localized and regional disease) samples were studied both regardless of primary tumor location and also within each primary tumor location (right colon, left colon, rectum). Results We discovered differences in plasma protein expression within all groups analyzed and identified proteins whose levels changed in one, two, or all three primary tumor locations between stage II and III CRC. Proteins were identified that could separate the groups compared and pathway analysis by IPA discovered altered pathways involved in lipid metabolism and inflammation, among others. Conclusions Plasma protein expression changes significantly as CRC progresses from stage II to III. While the levels of certain plasma proteins changed during cancer progression in only one or two primary tumor locations, the levels of 13 proteins changed in all primary tumor locations and are therefore common to CRC progression.

Abstract Background Prognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities. Methods Gene expression data was analyzed from three independent colon cancer microarray gene expression data sets (N = 1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 (L1TD1). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients. Results We found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer. Conclusions Our results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients.

Abstract Background Intraductal Papillary Mucinous Neoplasia (IPMN) are potentially malignant cystic tumors of the pancreas. IPMN can progress from low to moderate to high grade dysplasia and further to IPMN associated carcinoma. Often the difference between benign and malignant nature of the IPMN is not clear preoperatively. We aim to elucidate molecular expression patterns of various grades of IPMN and pancreatic carcinoma. Additionally we suggest potential novel biomarkers to differentiate IPMN from healthy individuals and pancreatic carcinoma to enable early detection as well as help in differential diagnosis in future. Methods We have performed retrospective label-free proteomic analysis of the serum samples from 44 patients with various grades of benign IPMN or IPMN associated carcinoma and 11 healthy controls. Proteomic data was further analyzed by various multivariate statistical methods. Four groups of samples (low-grade, high-grade IPMN, pancreatic carcinoma and age- and sex-matched healthy controls) were compared with ANOVA. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) modeling gave S-plot for feature selection. Stringently selected potential markers were further evaluated with ROC curve analysis and area under the curve was calculated. Differentially expressed proteins were used for pathway analysis. Linear trend analysis (Mann Kendall test) was used for identifying significant increasing or decreasing trends from healthy-low grade-high grade IPMN-pancreatic carcinoma. Results Based on protein expression (436 proteins quantified), PCA separated most sample groups from each other. S-Plot selected biomarker panels with moderate to very high AUC values for differentiating controls from Low-, High-Grade IPMN and carcinoma. Linear trend analysis identified 12 proteins which were consistently increasing or decreasing trend among the groups. We found potential biomarkers to differentiate healthy controls from different degrees of dysplasia and pancreatic carcinoma. These biomarkers can classify IPMN, carcinoma and healthy controls from each other which is an unmet clinical need. Data are available via ProteomeXchange with identifier PXD009139. Conclusion Kininogen-1 was able to differentiate healthy persons from low and high-grade IPMN. Retinol binding protein-4 could classify the low-grade IPMN from pancreatic carcinoma. Twelve proteins including apolipoproteins and complement proteins had significantly increasing or decreasing trends from healthy to low to high-grade IPMN to pancreatic carcinoma.

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. Fund: Jane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid Juselius Foundation, and HiLIFE. Keywords: Lynch syndrome, Colorectal adenoma, DNA methylation, DNA mismatch repair, LINE-1 methylation, Mutation, Tumorigenesis, Tumor suppressor

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.

Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45-63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

In this study the authors examine the allelic imbalance (AI) landscape of colorectal cancer, reporting loss of TP53 as a driver of AI. They use CRISPR-Cas9 screens to identify 79 genes (within AI regions) regulating cell growth and identify a network of transcription factors that may contribute to 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.

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.

Over 1.4 million people are diagnosed with colorectal cancer (CRC) each year, making it the third most common cancer in the world. Increased screening and therapeutic modalities including improved combination treatments have reduced CRC mortality, although incidence and mortality rates are still increasing in some areas. Serum-based biomarkers are mainly used for follow-up of cancer, and are ideal due to the ease and minimally invasive nature of sample collection. Unfortunately, CEA and other serum markers have too low sensitivity for screening and preoperative diagnostic purposes. Increasing interest is focused on the possible use of biomarkers for predicting treatment response and prognosis in cancer. In this study, we have performed mass spectrometry analysis (UPLC-UDMSE) of serum samples from 19 CRC patients. Increased levels of C-reactive protein (CRP), which occur during local inflammation and the presence of a systemic inflammatory response, have been linked to poor prognosis in CRC patients. We chose to analyze samples according to CRP values by dividing them into the categories CRP 30, and, separately, according to short and long 5-year survival. The aim was to discover differentially expressed proteins associated with poor prognosis and shorter survival. We quantified 256 proteins and performed detailed statistical analyses and pathway analysis. We discovered multiple proteins that are up- or downregulated in patients with CRP >30 as compared to CRP

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma mainly restricted to the peritoneal cavity and most commonly originating from the appendix. The genetic background of PMP is poorly understood and no targeted treatments are currently available for this fatal disease. While RAS signaling pathway is affected in most if not all PMP cases and over half of them also have a mutation in the GNAS gene, other genetic alterations and affected pathways are, to a large degree, poorly known. In this study, we sequenced whole coding genome of nine PMP tumors and paired normal tissues in order to identify additional, commonly mutated genes and signaling pathways affected in PMP. These exome sequencing results were validated with an ultra-deep amplicon sequencing method, leading to 14 validated variants. The validated results contain seven genes that contribute to the protein kinase A (PKA) pathway. PKA pathway, which also contains GNAS, is a major player of overproduction of mucin, which is the characteristic feature of PMP. In addition to PKA pathway, we identified mutations in six genes that belong to the transforming growth factor beta (TGF-β) pathway, which is a key regulator of cell proliferation. Since either GNAS mutation or an alternative mutation in the PKA pathway was identified in 8/9 patients, inhibition of the PKA pathway might reduce mucin production in most of the PMP patients and potentially suppress disease progression.

The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.