Inger Marie Bowitz Lothe

Polymorphisms of the XRCC1, XRCC3 and XPD genes and risk of colorectal adenoma and carcinoma, in a Norwegian cohort: a case control study

BackgroundGenetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with risk of developing cancer. For colorectal cancer the importance of mutations in mismatch repair genes has been extensively documented. Less is known about other DNA repair pathways in colorectal carcinogenesis. In this study we have focused on the XRCC1, XRCC3 and XPD genes, involved in base excision repair, homologous recombinational repair and nucleotide excision repair, respectively.MethodsWe used a case-control study design (157 carcinomas, 983 adenomas and 399 controls) to test the association between five polymorphisms in these DNA repair genes (XRCC1 Arg194Trp, Arg280His, Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln), and risk of colorectal adenomas and carcinomas in a Norwegian cohort. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by binary logistic regression model adjusting for age, gender, cigarette smoking and alcohol consumption.ResultsThe XRCC1 280His allele was associated with an increased risk of adenomas (OR 2.30, 95% CI 1.19–4.46). The XRCC1 399Gln allele was associated with a reduction of risk of high-risk adenomas (OR 0.62, 95% CI 0.41–0.96). Carriers of the variant XPD 751Gln allele had an increased risk of low-risk adenomas (OR 1.40, 95% CI 1.03–1.89), while no association was found with risk of carcinomas.ConclusionOur results suggest an increased risk for advanced colorectal neoplasia in individuals with the XRCC1 Arg280His polymorphism and a reduced risk associated with the XRCC1 Arg399Gln polymorphism. Interestingly, individuals with the XPD Lys751Gln polymorphism had an increased risk of low-risk adenomas. This may suggest a role in regression of adenomas.

Expression of prostasin and its inhibitors during colorectal cancer carcinogenesis

BackgroundClinical trials where cancer patients were treated with protease inhibitors have suggested that the serine protease, prostasin, may act as a tumour suppressor. Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of hepatocyte growth factor activator inhibitor-1 (HAI-1), HAI-1A, and HAI-1B.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels for prostasin and PN-1 in colorectal cancer tissue (n = 116), severe dysplasia (n = 13), mild/moderate dysplasia (n = 93), and in normal tissue from the same individuals. In addition, corresponding tissues were examined from healthy volunteers (n = 23). A part of the cohort was further analysed for the mRNA levels of the two variants of HAI-1, here denoted HAI-1A and HAI-1B. mRNA levels were normalised to β-actin. Immunohistochemical analysis of prostasin and HAI-1 was performed on normal and cancer tissue.ResultsThe mRNA level of prostasin was slightly but significantly decreased in both mild/moderate dysplasia (p < 0.001) and severe dysplasia (p < 0.01) and in carcinomas (p < 0.05) compared to normal tissue from the same individual. The mRNA level of PN-1 was more that two-fold elevated in colorectal cancer tissue as compared to healthy individuals (p < 0.001) and elevated in both mild/moderate dysplasia (p < 0.01), severe dysplasia (p < 0.05) and in colorectal cancer tissue (p < 0.001) as compared to normal tissue from the same individual. The mRNA levels of HAI-1A and HAI-1B mRNAs showed the same patterns of expression. Immunohistochemistry showed that prostasin is located mainly on the apical plasma membrane in normal colorectal tissue. A large variation was found in the degree of polarization of prostasin in colorectal cancer tissue.ConclusionThese results show that the mRNA level of PN-1 is significantly elevated in colorectal cancer tissue. Future studies are required to clarify whether down-regulation of prostasin activity via up regulation of PN-1 is causing the malignant progression or if it is a consequence of it.

The level of claudin-7 is reduced as an early event in colorectal carcinogenesis

BackgroundCompromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both in vivo and in vitro. We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression. We have previously shown that the matriptase expression level decreases during colorectal carcinogenesis. In the present study we investigated whether claudin-7 expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue.MethodsThe mRNA level of claudin-7 (CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings.ResultsA 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the claudin-7 mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.ConclusionsOur results show that the claudin-7 mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.

Collagen mRNA levels changes during colorectal cancer carcinogenesis

BackgroundInvasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different α(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (α1/α4/α6) and type VII collagen (α1) during colorectal cancer carcinogenesis.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels for α1(IV), α4(IV), α6(IV), and α1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals. In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to β-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients.ResultsThe α1(IV) and α1(VII) mRNA levels were statistically significantly higher in colorectal cancer tissue (p < 0.001) as compared to corresponding tissue from healthy controls. This is an early event as tissue from adenomas also displayed a higher level. There were small changes in the levels of α4(IV). The level of α6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p < 0.01). The localisation of type IV and type VII collagen was visualized by immunohistochemical staining.ConclusionOur results suggest that the down-regulation of α6(IV) mRNA coincides with the acquisition of invasive growth properties, whereas α1(IV) and α1(VII) mRNAs were up-regulated already in dysplastic tissue. There are no differences in collagen expression between tissues from healthy individuals and normal tissues from affected individuals.

Alterations in regulators of the extracellular matrix in non-Hodgkin lymphomas

Bone marrow angiogenesis is increased in non-Hodgkin lymphomas (NHL). Compounds affecting extracellular matrix (ECM) may modify angiogenesis. Here we investigated ECM regulators in 48 unselected NHL patients compared with 35 controls. Untreated patients had elevated (P < 0.05) serum matrix metalloproteinase (MMP) 9 and tissue inhibitor of metalloproteinase (TIMP) 1, while MMP-2, TIMP-2 and syndecan-1 were not significantly different from controls. MMP-9 mRNA was significantly up-regulated in blood mononuclear cells, while mRNA expressions of the other ECM regulators were unaltered. We found strong correlations between mRNA expressions of both vascular endothelial growth factor and fibroblast growth factor 2, and MMP-9, TIMP-1 and TIMP-2. After therapy, serum MMP-2 increased while MMP-9 decreased (P < 0.05), the others being unchanged. Several compounds affecting ECM may be involved in angiogenic activity in NHL.

Cetuximab in treatment of metastatic colorectal cancer: final survival analyses and extended RAS data from the NORDIC-VII study

Background:The NORDIC-VII study is a randomised phase III trial of cetuximab plus continuous or intermittent fluorouracil, folinic acid, and oxaliplatin (Nordic FLOX) vs FLOX alone in first-line treatment of metastatic colorectal cancer. The present report presents an updated and final survival analysis with BRAF and extended RAS mutational status, 5 years after the primary analysis.Methods:A total of 566 patients were included in the intention-to-treat (ITT) population of the NORDIC-VII study. Updated survival status was obtained from 176 patients who were alive in the primary survival analyses. Samples from 223 tumours previously found to be KRAS (exon 2) and BRAF (V600E) wild-type, were re-analysed for KRAS (exons 3 and 4) and NRAS (exons 2–4) mutations.Results:Including the extended RAS analyses, RAS and BRAF mutational status was available from 457 patients (81% of the ITT population). RAS was mutated in 46% and BRAF in 12% of the tumours. RAS and BRAF, if mutated, were negative prognostic factors. The updated analyses confirmed the finding of the primary report that cetuximab did not provide any additional benefit when added to FLOX in patients with RAS/BRAF wild-type tumours, neither on progression-free nor overall survival. However, the outcomes in a subset of patients, which, after the first eight treatment cycles, received cetuximab alone, suggested a beneficial effect of cetuximab monotherapy.Conclusions:Adding cetuximab to Nordic FLOX did not provide any clinical benefit, but the data suggested an effect of cetuximab monotherapy in patients with RAS/BRAF wild-type tumours in the NORDIC-VII cohort. The data were compatible with a negative interaction between cetuximab and the Nordic FLOX chemotherapy backbone.

The Genomic landscape of pancreatic and periampullary Adenocarcinoma

Despite advances in diagnostics, less than 5% of patients with periampullary tumors experience an overall survival of five years or more. Periampullary tumors are neoplasms that arise in the vicinity of the ampulla of Vater, an enlargement of liver and pancreas ducts where they join and enter the small intestine. In this study, we analyzed copy number aberrations using Affymetrix SNP 6.0 arrays in 60 periampullary adenocarcinomas from Oslo University Hospital to identify genome-wide copy number aberrations, putative driver genes, deregulated pathways, and potential prognostic markers. Results were validated in a separate cohort derived from The Cancer Genome Atlas Consortium (n = 127). In contrast to many other solid tumors, periampullary adenocarcinomas exhibited more frequent genomic deletions than gains. Genes in the frequently codeleted region 17p13 and 18q21/22 were associated with cell cycle, apoptosis, and p53 and Wnt signaling. By integrating genomics and transcriptomics data from the same patients, we identified CCNE1 and ERBB2 as candidate driver genes. Morphologic subtypes of periampullary adenocarcinomas (i.e., pancreatobiliary or intestinal) harbor many common genomic aberrations. However, gain of 13q and 3q, and deletions of 5q were found specific to the intestinal subtype. Our study also implicated the use of the PAM50 classifier in identifying a subgroup of patients with a high proliferation rate, which had impaired survival. Furthermore, gain of 18p11 (18p11.21-23, 18p11.31-32) and 19q13 (19q13.2, 19q13.31-32) and subsequent overexpression of the genes in these loci were associated with impaired survival. Our work identifies potential prognostic markers for periampullary tumors, the genetic characterization of which has lagged. Cancer Res; 76(17); 5092-102. ©2016 AACR.

The inframe MSH2 codon 596 deletion is linked with HNPCC and associated with lack of MSH2 protein in tumours

Hereditary nonpolyposis colorectal cancer (HNPCC) may be caused by germline truncating mutations in DNA mismatch repair (MMR) genes. Whether or not missense or inframe mutations are disease-associated has become a practical clinical problem, because predictive genetic testing is employed to select high-risk persons for clinical examinations. Clinical examinations may reveal polyps to be removed and prevent cancer. One large kindred applying for health care had a N596del mutation in the MSH2 gene. The aim of this study was to determine whether or not the inframe mutation in this family was associated with disease, and to examine the tumours for presence of the MSH2 protein by immunohistochemistry. We demonstrated that the mutation was linked to disease with lod score 5.7 in the family, and all examined, but one manifest cancer, lacked the MSH2 protein.

Generation and characterisation of novel pancreatic adenocarcinoma xenograft models and corresponding primary cell lines

Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection in vitro, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding in vitro cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease.

Abstract A50: KRAS mutational status and expressions of p53 and S100A4 proteins in pancreatic adenocarcinomas.

Introduction: Currently, there are no established molecular predictors of clinical or therapeutic outcome in pancreatic adenocarcinomas. The purpose of this study was to elucidate the relationship between three potential prognostic markers ( KRAS mutations (codons 12 and 13) and S100A4 and p53 protein expression) and differentiation grade and survival in adenocarcinomas of the pancreatobiliary type. S100A4 plays a role in metastatic progression and the TP53 mutations are associated with poor prognosis in many cancers, but their significance as prognostic markers in pancreatic adenocarcinomas are uncertain (1), (2). Pancreatic cancers have a high incidence of activating KRAS mutations but their roles as prognostic factors are not clarified (3). Description: We examined 72 adenocarcinomas of the pancreatobiliary type with origin in the pancreas, stated by light microscopy. The tumors were classified according to the WHO Classification 2010 (4) and graded as: G1 well-differentiated, G2 moderately differentiated, G3 poorly differentiated, and G4 undifferentiated. The tumors were examined for KRAS mutations (codons 12 and 13) and were subjected to semiquantitative evaluation by immunohistochemical (IHC) staining of p53 (p53 (DO-1): sc-126, Santa Cruz Biotechnology, Inc.) (5), and S100A4 (6). KRAS mutational status was obtained by the WE-ARMS method (7). Statistical analyses were performed using SPSS (v.18, Chicago, IL). Results: The well- and middle-differentiated tumors were grouped and we found that 31/39 (.80) had a positive nuclear score for S100A4, 32/39 (.82) had a positive cytoplasm score for S100A4, 30/38 (.79) had a positive score for p53 and 27/30 (.90) were KRAS mutated. The poorly- and undifferentiated tumors were grouped and we found 23/29 (.79) had a positive nuclear score for S100A4, 25/29 (.86) had a positive cytoplasm score for S100A4, 22/29 (.76) had a positive score for p53 and 23/28 (.82) were KRAS mutated. The number of cases analyzed differs from the total number of cases as the quality of the tissue was not adequate for all the analyses in all cases. Conclusions: There are no statistically significant differences between histological grade and IHC score of p53 (nuclear score), S100A4 (nuclear and cytoplasmic score) or KRAS mutational status. Survival analyses will be presented. References: 1. P. Singh, R. Srinivasan, J. D. Wig, Pancreas 40, 644 (2011). 2. K. Boye, J. M. Nesland, B. Sandstad, G. M. Maelandsmo, K. Flatmark, Eur. J. Cancer 46, 2919 (2010). 3. M. Oliveira-Cunha, K. D. Hadfield, A. K. Siriwardena, W. Newman, Pancreas 41, 428 (2012). 4. WHO Classifications of Tumours, Pathology and Genetics of Tumours of the Digestive System. Hamilton S R and Aaltonen L A. 2010. Lyon, IARC Press. 5. D. C. Allred et al., J. Natl. Cancer Inst. 85, 200 (1993). 6. K. Flatmark et al., Tumour. Biol. 25, 31 (2004). 7. J. Hamfjord et al., Diagn. Mol. Pathol. 20, 158 (2011). Citation Format: Inger Marie Bowitz Lothe, Tone Ikdahl, Trond Buanes, Knut Jorgen Labori, Ole Petter Clausen, Gunhild M. Maelandsmo, Kjetil Boye, Tor Jacob Eide, Elin Kure. KRAS mutational status and expressions of p53 and S100A4 proteins in pancreatic adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A50.