Ida Bukholm

TP53 mutation status and gene expression profiles are powerful prognostic markers of breast cancer

IntroductionGene expression profiling of breast carcinomas has increased our understanding of the heterogeneous biology of this disease and promises to impact clinical care. The aim of this study was to evaluate the prognostic value of gene expression-based classification along with established prognostic markers and mutation status of the TP53 gene (tumour protein p53) in a group of breast cancer patients with long-term (12 to 16 years) follow-up.MethodsThe clinical and histopathological parameters of 200 breast cancer patients were studied for their effects on clinical outcome using univariate/multivariate Cox regression. The prognostic impact of mutations in the TP53 gene, identified using temporal temperature gradient gel electrophoresis and sequencing, was also evaluated. Eighty of the samples were analyzed for gene expression using 42 K cDNA microarrays and the patients were assigned to five previously defined molecular expression groups. The strength of the gene expression based classification versus standard markers was evaluated by adding this variable to the Cox regression model used to analyze all samples.ResultsBoth univariate and multivariate analysis showed that TP53 mutation status, tumor size and lymph node status were the strongest predictors of breast cancer survival for the whole group of patients. Analyses of the patients with gene expression data showed that TP53 mutation status, gene expression based classification, tumor size and lymph node status were significant predictors of survival. Breast cancer cases in the basal-like and ERBB2+ gene expression subgroups had a very high mortality the first two years, while the highly proliferating luminal cases developed the disease more slowly, showing highest mortality after 5 to 8 years. The TP53 mutation status showed strong association with the basal-like and ERBB2+ subgroups, and tumors with mutation had a characteristic gene expression pattern.ConclusionTP53 mutation status and gene-expression based groups are important survival markers of breast cancer, and these molecular markers may provide prognostic information that complements clinical variables. The study adds experience and knowledge to an ongoing characterization and classification of the disease.

Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.MethodsQuantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.ResultsAberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.ConclusionsQuantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.

Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan‐related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N‐linked and O‐linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell–cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan‐related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cells glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly‐developing field of glycobiology.

Methylation profiling with a panel of cancer related genes: Association with estrogen receptor, TP53 mutation status and expression subtypes in sporadic breast cancer

Breast cancer is a heterogeneous disease that can be divided in subtypes based on histology, gene expression profiles as well as differences in genomic aberrations. Distinct global DNA methylation profiles have been reported in normal breast epithelial cells as well as in breast tumors. However, the influence of the tumor methylome on the previously described subgroups of breast cancer is not fully understood. Here we report the DNA methylation profiles of 80 breast tumors using a panel of 807 cancer related genes interrogating 1505 CpG sites. We identified three major clusters based on the methylation profiles; one consisting of mainly tumors of myoepithelial origin and two other clusters with tumors of predominantly luminal epithelial origin. The clusters were different with respect to estrogen receptor status, TP53 status, ErbB2 status and grade. The most significantly differentially methylated genes including HDAC1, TFF1, OGG1, BMP3, FZD9 and HOXA11 were confirmed by pyrosequencing. Gene Ontology analysis revealed enrichment for genes involved in developmental processes including homeobox domain genes (HOXA9, HOXA11, PAX6, MYBL2, ISL1 and IPF1) and (ETS1, HDAC1, CREBBP, GAS7, SPI1 and TBX1). Extensive correlation to mRNA expression was observed. Pathway analyses identified a significant association with canonical (curated) pathways such as hepatic fibrosis including genes like EGF, NGFR and TNF, dendritic cell maturation and the NF‐κB signaling pathway. Our results show that breast tumor expression subtypes harbor major epigenetic differences and tumors with similar gene expression profiles might belong to epigenetically different subtypes. Some of the transcription factors identified, with key roles in differentiation and development might play a role in inducing and maintaining the different phenotypes.

Short term results of complete (D3) vs. standard (D2) mesenteric excision in colon cancer shows improved outcome of complete mesenteric excision in patients with TNM stages I-II

BackgroundThe aim of the present study was to investigate whether the new method of complete mesocolic excision (CME) with a high (apical) vascular tie (D3 resection) had an immediate effect compared with a conventional (standard) approach even in those patients without lymph node metastases.MethodsA cohort of 189 consecutive patients with tumour–nodal–metastasis (TNM) stages I–II and a mean age of 73xa0years were operated on in the period from January 2007 to December 2008 in three community teaching hospitals. The CME approach (nxa0=xa089), used in hospital A, was compared to the standard technique used (nxa0=xa0105) in two other hospitals, B and C. Lymph node yields from the specimens were used as a surrogate measure of radical resections. Outcome was analysed after a median follow-up of 50.2xa0months.ResultsIn-hospital mortality rate was 2.8xa0% in the CME group and 8.6xa0% in the standard group. The 3-year overall survival (OS) in the CME group was 88.1 versus 79.0xa0% (pxa0=xa00.003) in the standard group, and the corresponding disease-free survival (DFS) was 82.1 versus 74.3xa0% (pxa0=xa00.026). Cancer-specific survival was 95.2xa0% in the CME group versus 90.5xa0% in the standard group (pxa0=xa00.067). Age, operative technique, and T category were significant in multiple Cox regressions of OS and DFS.ConclusionsCompared with the standard (D2) approach, introduction of CME surgical management of colon cancer resulted in a significant immediate improvement of 3-year survival for patients with TNM stage I–II tumours as assessed by OS and DFS.

Overall survival after resection for colon cancer in a national cohort study was adversely affected by TNM stage, lymph node ratio, gender, and old age

BackgroundA national surveillance program of colon cancer treatment was introduced in 2007. We examined prognostic factors for colon cancer operated in 2000 with an aim of improving survival in the new program and a special focus on the merit of lymph node yield.MethodsA cohort of 269 patients, 152 women (56.5%), with a mean age of 71xa0years, was operated for colon cancer in 2000 at three teaching hospitals and followed up for 7xa0years.ResultsOverall 5-year survival was 58.0%, and overall hospital mortality was 5.2%, with 4.5% in elective cases and 12.5% after urgent surgery. In only 41.1% of the specimens were 12 or more lymph nodes retrieved, but this did not affect survival in the combined cohort, although one of the hospitals achieved a significantly better result with a harvest of 12 or more lymph nodes. In a multivariate analysis, old age, gender, a high lymph node ratio (LNR) at stage III, and tumor–node–metastasis stage were adverse factors for survival.ConclusionsThe operative mortality was high and should be reassessed. The lymph node count did not have a significant impact on outcome overall, whereas the LNR proved significant for stage III. A prospective protocol using overall lymph node yield as a surrogate measure for more radical surgery, nevertheless, seems warranted to improve the lymph node harvest according to international recommendations.

Improved Lymph Node Harvest from Resected Colon Cancer Specimens Did Not Cause Upstaging from TNM Stage II to III

BackgroundThe number of lymph nodes retrieved and examined from a resected colon cancer specimen may be crucial for correct staging. We examined if efforts to increase the lymph node harvest to more than 12 lymph nodes per specimen would upstage some patients from TNM stage II to III.MethodsThree hospitals compared results from 2000 with those of 2007 in 421 resected patients with stage II and III colon cancer. Hospital A endeavored to improve the surgical procedure while the pathologists enhanced the quality of lymph node sampling. Hospital B did not make any marked changes, while hospital C introduced the GEWF lymph node solvent (glacial acetic acid, ethanol, distilled water, and formaldehyde) in their pathology method.ResultsIn 2000, 12 or more lymph nodes were harvested in 39.6, 45.0, and 21.1% of the specimens from the three hospitals, while the figures for 2007 were 85.7, 42.0, and 90.3%, respectively. The significant increase in lymph node harvest in two of the hospitals in 2007 compared to 2000 (pxa0<xa00.001) did not affect the share of patients with stage III in 2007 (38.7%) compared to 2000 (44.1%) (pxa0=xa00.260). The number of positive lymph nodes and the lymph node ratio (LNR) decreased from 2000 to 2007. A lymph node yield of 12 or more was not associated with an increased probability of positive lymph nodes in a multivariable logistic regression analysis.ConclusionMore radical surgery and dedicated pathologists and the use of the GEWF solvent significantly increased the lymph node yield but did not upstage patients from TNM stage II to III.

Up-regulation of CLDN1 in gastric cancer is correlated with reduced survival

BackgroundThe genetic changes in gastric adenocarcinoma are extremely complex and reliable tumor markers have not yet been identified. There are also remarkable geographical differences in the distribution of this disease. Our aim was to identify the most differentially regulated genes in 20 gastric adenocarcinomas from a Norwegian selection, compared to matched normal mucosa, and we have related our findings to prognosis, survival and chronic Helicobacter pylori infection.MethodsBiopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 20 patients immediately following surgical resection of the tumor. Whole genome, cDNA microarray analysis was performed on the RNA isolated from the sample pairs to compare the gene expression profiles between the tumor against matched mucosa. The samples were microscopically examined to classify gastritis. The presence of H. pylori was examined using microscopy and immunohistochemistry.Results130 genes showed differential regulation above a predefined cut-off level. Interleukin-8 (IL-8) and Claudin-1 (CLDN1) were the most consistently up-regulated genes in the tumors. Very high CLDN1 expression in the tumor was identified as an independent and significant predictor gene of reduced post-operative survival. There were distinctly different expression profiles between the tumor group and the control mucosa group, and the histological subsets of mixed type, diffuse type and intestinal type cancer demonstrated further sub-clustering. Up-regulated genes were mapped to cell-adhesion, collagen-related processes and angiogenesis, whereas normal intestinal functions such as digestion and excretion were associated with down-regulated genes. We relate the current findings to our previous study on the gene response of gastric epithelial cells to H. pylori infection.ConclusionsCLDN1 was highly up-regulated in gastric cancer, and CLDN1 expression was independently associated with a poor post-operative prognosis, and may have important prognostic value. IL-8 and CLDN1 may represent central links between the gene response seen in acute H. pylori infection of gastric epithelial cells, and ultimately gastric cancer.

The 5p12 breast cancer susceptibility locus affects MRPS30 expression in estrogen-receptor positive tumors

Genome‐wide association studies have identified numerous loci linked to breast cancer susceptibility, but the mechanism by which variations at these loci influence susceptibility is usually unknown. Some variants are only associated with particular clinical subtypes of breast cancer. Understanding how and why these variants influence subtype‐specific cancer risk contributes to our understanding of cancer etiology. We conducted a genome‐wide expression Quantitative Trait Locus (eQTL) study in a discovery set of 287 breast tumors and 97 normal mammary tissue samples and a replication set of 235 breast tumors. We found that the risk‐associated allele of rs7716600 in the 5p12 estrogen receptor‐positive (ER‐positive) susceptibility locus was associated with elevated expression of the nearby gene MRPS30 exclusively in ER‐positive tumors. We replicated this finding in 235 independent tumors. Further, we showed the rs7716600 risk genotype was associated with decreased MRPS30 promoter methylation exclusively in ER‐positive breast tumors. In vitro studies in MCF‐7 cells carrying the protective genotype showed that estrogen stimulation decreased MRPS30 promoter chromatin availability and mRNA levels. In contrast, in 600MPE cells carrying the risk genotype, estrogen increased MRPS30 expression and did not affect promoter availability. Our data suggest the 5p12 risk allele affects MRPS30 expression in estrogen‐responsive tumor cells after tumor initiation by a mechanism affecting chromatin availability. These studies emphasize that the genetic architecture of breast cancer is context‐specific, and integrated analysis of gene expression and chromatin remodeling in normal and tumor tissues will be required to explain the mechanisms of risk alleles.

Expression of adhesion proteins E-cadherin, α-catenin, β-catenin and γ-catenin is different in T1 and T2 breast tumours

Background: Breast cancer is the most common malignancy in women. Although an increasing number of patients with breast cancer are being cured by surgery, a considerable number of patients suffer relapse in the form of metastases after surgery. E‐cadherin and catenins have documented roles in breast cancer progression. Mammography is supposed to decrease breast cancer mortality by detecting tumours while they are small and before they have reached a clinically detectable stage. Aim: In the present study, we wanted to evaluate whether there are differences in expression patterns of adhesion proteins, shown to be crucial in the metastatic process, between small tumours detected by mammography and clinically detected large tumours. Methods: Expression of E‐cadherin, &agr;‐catenin, &bgr;‐catenin and &ggr;‐catenin was analysed using immunohistochemistry methods in 86 invasive breast carcinomas detected by mammography and compared with 90 clinically palpable invasive breast carcinomas. Results: In the group of tumours detected by mammography (86 samples), reduced expression of E‐cadherin was observed in 12 (14%) samples. Reduced expression of &agr;‐catenin was observed in four (4.6%) samples, and three (3.5%) samples showed reduced expression of &bgr;‐catenin. All samples showed strong expression of &ggr;‐catenin. When expression patterns of these proteins were evaluated in 90 clinically detected tumours, we observed reduced expression of E‐cadherin in 58 (64.4%) samples, 12 (13.3%) samples showed reduced expression of &agr;‐catenin, while nine (10%) samples showed reduced expression of &bgr;‐catenin. Strong expression of &ggr;‐catenin was detected in all tumours also in this group. Statistical analyses revealed a highly significant difference in expression of E‐cadherin (p<0.001). However, no statistically significant differences were observed in expression of &agr;‐catenin (p = 0.081) and &bgr;‐catenin (p = 0.092) between the two groups of tumours. Conclusion: Results indicate that T1 breast tumours harbour less alterations in E‐cadherin‐catenin complexes and therefore are probably less likely to disseminate, and patients probably have a better prognosis than if tumours are diagnosed as T2.