Ida R. K. Bukholm

Re-expression of E-cadherin, α-catenin and β-catenin, but not of γ- catenin, in metastatic tissue from breast cancer patients

Tumour cell invasion and metastasis are the processes which kill most cancer patients. Tumour cells with the greatest invasive and metastatic capacity may be those with the highest number of genetic aberrations. The present study has analysed the expression of several tumour‐related proteins in both primary tumours and metastatic lesions from 34 breast cancer patients. Protein expression of p53, bcl‐2, p21, cyclin D1, E‐cadherin, α‐catenin, β‐catenin, and γ‐catenin was investigated by immunohistochemistry (IHC) using monoclonal antibodies. Metastatic tissue showed a different expression profile from the primary tumour in most patients. The most significant finding was the re‐expression of E‐cadherin, α‐catenin, and β‐catenin, and increased down‐regulation of γ‐catenin, in metastatic lesions. These results demonstrate that tumour cells, when released from the primary site and after regrowth elsewhere, are capable of re‐expression of adhesion molecules. γ‐catenin may play a different role in metastatic lesions than in primary tumours, since it is selectively down‐regulated in tumour tissue at the metastatic site. Copyright

Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch–repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGFβRII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGFβRII, in TIL and peripheral blood lymphocytes from patients with MSI+ tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI+ colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.

E-cadherin and α-, β-, and γ-catenin protein expression in relation to metastasis in human breast carcinoma

In the metastatic process, various cell–cell adhesion molecules seem to play an important role. E‐cadherin, a transmembrane protein with an extracellular and an intracellular domain, is one of the key players involved in cell–cell adhesion. The function of E‐cadherin in preventing metastasis in tumour development is believed to be dependent on intracellular catenins. In a previous study, the expression of E‐cadherin was examined in a series of human breast carcinomas. In that study, down‐regulation of E‐cadherin failed to correlate with lymph node and/or distant metastasis. In the present study, the expression of α‐, β‐, and γ‐catenins has been examined in a subset of the same tumours in order to evaluate their possible role in breast cancer metastasis. Tumour tissues from 90 primary breast carcinomas were immunostained for α‐, β‐, and γ‐catenins. Reduced or absent immunoreactivity in the tumour tissue was seen in 63 (70·0 per cent) for α‐catenin, in 50 (55·6 per cent) for β‐catenin, and in 50 (55·6 per cent) for γ‐catenin. Reduced expression of each of the catenins alone failed to correlate to metastasis. However, when all of the four proteins (E‐cadherin, α‐catenin, β‐catenin, and γ‐catenin) were analysed as one group, a significant association was seen between reduction in immunoreactivity of at least one of these four proteins and the presence of metastases. These results indicate that if one of these proteins is down‐regulated, the function of the others in suppressing metastasis is altered. A significant association was seen between lobular invasive tumours and β‐catenin expression.

Discovery and validation of breast cancer subtypes

BackgroundPrevious studies demonstrated breast cancer tumor tissue samples could be classified into different subtypes based upon DNA microarray profiles. The most recent study presented evidence for the existence of five different subtypes: normal breast-like, basal, luminal A, luminal B, and ERBB2+.ResultsBased upon the analysis of 599 microarrays (five separate cDNA microarray datasets) using a novel approach, we present evidence in support of the most consistently identifiable subtypes of breast cancer tumor tissue microarrays being: ESR1+/ERBB2-, ESR1-/ERBB2-, and ERBB2+ (collectively called the ESR1/ERBB2 subtypes). We validate all three subtypes statistically and show the subtype to which a sample belongs is a significant predictor of overall survival and distant-metastasis free probability.ConclusionAs a consequence of the statistical validation procedure we have a set of centroids which can be applied to any microarray (indexed by UniGene Cluster ID) to classify it to one of the ESR1/ERBB2 subtypes. Moreover, the method used to define the ESR1/ERBB2 subtypes is not specific to the disease. The method can be used to identify subtypes in any disease for which there are at least two independent microarray datasets of disease samples.

Over‐expression of cyclin a is highly associated with early relapse and reduced survival in patients with primary breast carcinomas

Progression through the mammalian cell cycle is facilitated by cyclin–cyclin‐dependent kinase (cdk) complexes, which are activated at specific points during the cell cycle. Alteration in cyclin–cdk complexess may lead to altered cell cycle and tumorigenesis. In this study, we analyzed expression of cyclins A, D1, D3 and E in tumor tissue from 170 patients with primary invasive breast carcinomas. Immunohistochemical methods were used to detect protein expression of these cyclins. We detected positive immunoreactivity in 55 (32%), 22 (13%), 38 (22%) and 37 (21.8%) of the samples for cyclins A, D1, D3 and E, respectively. A highly statistically significant association was observed between expression of cyclin A and early relapse (p = 0.001 univariate analysis, p = 0.006 multivariate analysis) as well as cancer‐specific death (p < 0.0001) during the follow‐up time. No association was observed between cyclin D1 or cyclin E, respectively, and relapse of disease or survival, while cyclin D3 over‐expression was associated with development of metastases during follow‐up (p = 0.005 univariate analysis, p = 0.01 multivariate analysis). However, cyclin D3 did not show any statistically significant association when cancer‐specific death was examined in a multivariate analysis (Cox regression for survival function).

DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response

BackgroundBreast cancer is the most frequent cancer in women and consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics. In this study, we aimed to identify DNA methylation based biomarkers to distinguish patients with locally advanced breast cancer who may benefit from neoadjuvant doxorubicin treatment.ResultsWe investigated quantitatively the methylation patterns in the promoter regions of 14 genes (ABCB1, ATM, BRCA1, CDH3, CDKN2A, CXCR4, ESR1, FBXW7, FOXC 1, GSTP1, IGF2, HMLH1, PPP2R2B, and PTEN) in 75 well-described pre-treatment samples from locally advanced breast cancer and correlated the results to the available clinical and molecular parameters. Six normal breast tissues were used as controls and 163 unselected breast cancer cases were used to validate associations with histopathological and clinical parameters.Aberrant methylation was detected in 9 out of the 14 genes including the discovery of methylation at the FOXC1 promoter. Absence of methylation at the ABCB1 promoter correlated with progressive disease during doxorubicin treatment. Most importantly, the DNA methylation status at the promoters of GSTP1, FOXC1 and ABCB1 correlated with survival, whereby the combination of methylated genes improved the subdivision with respect to the survival of the patients. In multivariate analysis GSTP1 and FOXC1 methylation status proved to be independent prognostic markers associated with survival.ConclusionsQuantitative DNA methylation profiling is a powerful tool to identify molecular changes associated with specific phenotypes. Methylation at the ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness. FOXC1 proved to be general prognostic factor, while ABCB1 and GSTP1 might be predictive factors for the response to and efficacy of doxorubicin treatment. Pharmacoepigenetic effects such as the reported associations in this study provide molecular explanations for differential responses to chemotherapy and it might prove valuable to take the methylation status of selected genes into account for patient management and treatment decisions.

Deregulation of cancer-related miRNAs is a common event in both benign and malignant human breast tumors

MicroRNAs (miRNAs) are endogenous non-coding RNAs, which play an essential role in the regulation of gene expression during carcinogenesis. The role of miRNAs in breast cancer has been thoroughly investigated, and although many miRNAs are identified as cancer related, little is known about their involvement in benign tumors. In this study, we investigated miRNA expression profiles in the two most common types of human benign tumors (fibroadenoma/fibroadenomatosis) and in malignant breast tumors and explored their role as oncomirs and tumor suppressor miRNAs. Here, we identified 33 miRNAs with similar deregulated expression in both benign and malignant tumors compared with the expression levels of those in normal tissue, including breast cancer-related miRNAs such as let-7, miR-21 and miR-155. Additionally, messenger RNA (mRNA) expression profiles were obtained for some of the same samples. Using integrated mRNA/miRNA expression analysis, we observed that overexpression of certain miRNAs co-occurred with a significant downregulation of their candidate target mRNAs in both benign and malignant tumors. In support of these findings, in vitro functional screening of the downregulated miRNAs in non-malignant and breast cancer cell lines identified several possible tumor suppressor miRNAs, including miR-193b, miR-193a-3p, miR-126, miR-134, miR-132, miR-486-5p, miR-886-3p, miR-195 and miR-497, showing reduced growth when re-expressed in cancer cells. The finding of deregulated expression of oncomirs and tumor suppressor miRNAs in benign breast tumors is intriguing, indicating that they may play a role in proliferation. A role of cancer-related miRNAs in the early phases of carcinogenesis and malignant transformation can, therefore, not be ruled out.

Relationship between abnormal p53 protein and failure to express p21 protein in human breast carcinomas.

Alterations in the p53 protein are a common feature in most malignancies, including breast carcinomas. p53 protein alterations contribute to malignant transformation in several ways, through genomic instability and accumulation of additional genetic alterations in other genes, through alteration of the p53‐dependent apoptotic pathway, and through downregulation of downstream effector proteins such as p21 (WAF1/CIP1), necessary for cell‐cycle growth arrest. Cell‐cycle arrest is needed to allow DNA repair after injury. This study examines the relationship between abnormalities in p53 protein and expression of p21 protein in 70 cases selected from a series of 212 sporadic human breast carcinomas. Immunohistochemistry (IHC) was used for detection of p53 and p21 protein expression. Constant denaturant gel electrophoresis (CDGE) was used for detection of mutations in exons 5–8 of the TP53 gene. A highly significant association was found between abnormalities in p53, scored as protein accumulation and/or mutations, and lack of p21 expression. p21 was also shown to be downregulated in samples without p53 alterations, indicating that other mechanisms are also involved in turning off this gene.

Loss of heterozygosity at 11q23.1 in breast carcinomas: Indication for involvement of a gene distal and close to ATM

Previous reports have suggested that heterozygotes for ataxia‐telangiectasia (A‐T) have an increased risk of cancer, in particular breast cancer. The ATM gene, responsible for A‐T, was recently cloned. Loss of heterozygosity (LOH) in the chromosome band 11q23, where the ATM gene is located, has been reported in several types of tumours including breast carcinomas. Whether the ATM gene is the target, and the sole target, for the LOH seen in this region is not yet known. In this study, 169 primary breast carcinomas and 10 metastases were examined for allelic imbalance (AI) using 10 microsatellite markers mapping to 11q23.1. Nine of the markers reside within a 10 Mb region surrounding the ATM gene, whereas the tenth locus, APOC‐3, is located more than 12 Mb telomeric from this region. The highest frequencies of alteration were found for APOC‐3 (45%), and for two markers located approximately 200 and 900 kb telomeric from ATM, DIIS1294 (44%) and DIIS1818 (44%). The marker located within the ATM gene, DIIS2179, was altered in 37% of the informative tumours. The present deletion map indicates that three distinct regions at 11q23.1 may be involved in breast cancer development; one between the markers DIIS1294 and DIIS1818, a second close to APOC‐3, and a third that is possibly the ATM‐gene itself. Genes Chromosom. Cancer 18:175–180, 1997.

Expression and gene amplification of primary (A, B1, D1, D3, and E) and secondary (C and H) cyclins in colon adenocarcinomas and correlation with patient outcome

Background/Aims: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3, and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle progression and have not previously been evaluated in colon cancer. This study examined cyclin protein expression and gene amplification in colon adenocarcinoma and the correlation with patient outcome. Methods: Immunohistochemistry and real time quantitative polymerase chain reaction were used to determine cyclin expression and gene amplification in 219 tumours. The results were compared with clinical variables and patient outcomes. Results: Cyclin H was overexpressed in all tumours, cyclin C in 88%, cyclin B1 in 58%, cyclin A in 83%, cyclin D3 in 36%, cyclin E in 25%, and cyclin D1 in 11% of the tumours. Extra gene copies of cyclin A were seen in 6.2% of the tumours, cyclin B1 in 9%, cyclin C in 26.9%, cyclin D1 in 55%, cyclin D3 in 20.5%, cyclin E in 19.1%, and cyclin H in 5.1%. A significant correlation between protein overexpression and gene amplification was seen for cyclin C only. High expression of cyclin A was independently associated with improved survival. Amplification of cyclin C was independently associated with an unfavourable prognosis. Conclusions: Amplification of the cyclin C gene was related to an unfavourable prognosis and high protein expression of cyclin A was associated with a better outcome in colon adenocarcinoma.