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Histologic grading of breast cancer defines morphologic subtypes informative of metastatic potential, although not without considerable interobserver disagreement and clinical heterogeneity particularly among the moderately differentiated grade 2 (G2) tumors. We posited that a gene expression signature capable of discerning tumors of grade 1 (G1) and grade 3 (G3) histology might provide a more objective measure of grade with prognostic benefit for patients with G2 disease. To this end, we studied the expression profiles of 347 primary invasive breast tumors analyzed on Affymetrix microarrays. Using class prediction algorithms, we identified 264 robust grade-associated markers, six of which could accurately classify G1 and G3 tumors, and separate G2 tumors into two highly discriminant classes (termed G2a and G2b genetic grades) with patient survival outcomes highly similar to those with G1 and G3 histology, respectively. Statistical analysis of conventional clinical variables further distinguished G2a and G2b subtypes from each other, but also from histologic G1 and G3 tumors. In multivariate analyses, genetic grade was consistently found to be an independent prognostic indicator of disease recurrence comparable with that of lymph node status and tumor size. When incorporated into the Nottingham prognostic index, genetic grade enhanced detection of patients with less harmful tumors, likely to benefit little from adjuvant therapy. Our findings show that a genetic grade signature can improve prognosis and therapeutic planning for breast cancer patients, and support the view that low- and high-grade disease, as defined genetically, reflect independent pathobiological entities rather than a continuum of cancer progression. (Cancer Res 2006; 66(21): 10292-301)

Supplementary File 2 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

Supplementary File 3 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

Supplementary File 1 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

A comprehensive meeting on biological responses to DNA damage organized by Alan Lehmann, Deborah Barnes, Wouter Ferro, Robert Fuchs, Jan Hoeijmakers, Roland Kanaar, Leon Mullenders and Bert van Zeeland, was convened at Noordwijkerhout, The Netherlands, from April 2 to 7, 2006. This article summarizes information presented by speakers at the seven plenary sessions. Poster sessions with organized discussions constituted a fundamental aspect of the meeting—as did a marvelous evening of musical entertainment by a talented group of conferees.

Cyclin E is one of the key regulators of the G(1)/S transition in the cell cycle. Overexpression of cyclin E has been observed in several malignancies and is associated with high proliferation, aberrant expression of other cell cycle regulators and chromosomal instability in vitro. To explore potential associations between cyclin E deregulation and inactivation of the p53 tumor suppressor gene in human breast cancer, we investigated the immunohistochemical expression of cyclin E in paraffin embedded breast cancers from 270 women with known p53 status by cDNA based sequencing of the p53 gene. The breast cancers were divided into three subgroups according to the percentage of cyclin E-positive cells. One hundred and seventy-one patients (63%) had low cyclin E, 72 (27%) medium and 27 (10%) had high cyclin E content. Fifty-six percent (15/27) of the breast cancers with high cyclin E had p53 gene mutations, compared with 14% (24/171) of those with low cyclin E content (P < 0.0001). In p53 mutated breast cancers high cyclin E content was associated with insertions, deletions and nonsense point mutations in the p53 tumor suppressor gene, whereas low cyclin E was linked to p53 missense point mutations. We also observed statistically significant associations between a high cyclin E content and aneuploidy, high S phase, larger tumor size, estrogen receptor negativity, presence of axillary node metastases and high tumor grade. High cyclin E content was associated with poor overall survival in univariate and multivariate analysis (hazard ratio 2.4, 95% confidence interval 1.3-4.5). In summary, our findings demonstrate that overexpression of cyclin E is associated with an aggressive tumor phenotype and specific types of p53 mutations.

Background: Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMDTM) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53. Methods: The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products were analyzed and scored using an ALFTM automated DNA sequencer and ALFwin Fragment Analyzer software (Ver. 1.02). Results: The EMD technique had sensitivities of 45% and 64% and specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47). Conclusions: EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.

Histologic grading of breast cancer defines morphologic subtypes informative of metastatic potential, although not without considerable interobserver disagreement and clinical heterogeneity particularly among the moderately differentiated grade 2 (G2) tumors. We posited that a gene expression signature capable of discerning tumors of grade 1 (G1) and grade 3 (G3) histology might provide a more objective measure of grade with prognostic benefit for patients with G2 disease. To this end, we studied the expression profiles of 347 primary invasive breast tumors analyzed on Affymetrix microarrays. Using class prediction algorithms, we identified 264 robust grade-associated markers, six of which could accurately classify G1 and G3 tumors, and separate G2 tumors into two highly discriminant classes (termed G2a and G2b genetic grades) with patient survival outcomes highly similar to those with G1 and G3 histology, respectively. Statistical analysis of conventional clinical variables further distinguished G2a and G2b subtypes from each other, but also from histologic G1 and G3 tumors. In multivariate analyses, genetic grade was consistently found to be an independent prognostic indicator of disease recurrence comparable with that of lymph node status and tumor size. When incorporated into the Nottingham prognostic index, genetic grade enhanced detection of patients with less harmful tumors, likely to benefit little from adjuvant therapy. Our findings show that a genetic grade signature can improve prognosis and therapeutic planning for breast cancer patients, and support the view that low- and high-grade disease, as defined genetically, reflect independent pathobiological entities rather than a continuum of cancer progression. (Cancer Res 2006; 66(21): 10292-301)

The aim of the present study was to study the expression and relationship of potential angiogenic factors. Paraffin-embedded tumour sections from 261 breast cancer patients were stained immunohistochemically for thrombospondin (TSP-1) expression. p53 status was previously determined by cDNA-based sequencing, and vascular endothelial growth factor (VEGF) protein expression had been previously analysed using an immunoassay. 241 cancers (92%) had detectable levels of TSP-1. No associations between TSP-1 and p53 status or VEGF were found. No correlations between TSP-1 and relapse-free (P = 0.3), breast cancer-corrected (P = 0.2) or overall survival (P = 0.5) were found. A correlation was found for patients with p53 mutations, but negative p53 expression, with higher VEGF levels (P = 0.009), but there was no correlation between this p53 group and those with low TSP-1 levels (P = 0.2). In conclusion, TSP-1 expression was not prognostic and was not associated with neither p53-status or VEGF expression.

Covalent alterations of DNA bases, which may have promutagenic or cytotoxic effects, are major consequences of endogenous DNA damage caused by hydrolysis, reactive oxygen species, and several metabolites and coenzymes. A common strategy for initiation of DNA base excision repair (BER) involves a DNA glycosylase that binds the altered deoxynucleoside in an extrahelical position and catalyzes cleavage of the base-sugar bond. Subsequently, an AP endonuclease or AP lyase activity incises the abasic site, followed by short-patch gap-filling, excision of the base-free sugar-phosphate residue, and ligation. The initial work that resulted in the discovery of DNA glycosylases and AP endonucleases is briefly reviewed. In recent years, it has been shown that the latter steps of the BER pathway differ greatly between mammalian cells and microorganisms such as yeast and bacteria. Three distinct subpathways of BER occur in mammalian cells, and these have been individually reconstituted with purified enzymes. Gene knockout mice are now revealing specific roles and backup mechanisms for repair functions in murine cells, and the results in general are also applicable to human cells. Future developments in the field of base excision repair include definition by proteomics of all factors involved in handling many different types of DNA lesions, clarification of mechanisms of repair of chromatin at a high level of accuracy, manifestation of repair proteins as drug targets for cellular sensitization to ionizing radiation and anticancer medicines, and elucidation of cross-talk between the base excision repair factors and other cellular proteins involved in a variety of stress responses.

Genomic instability is a major threat to living organisms. To counteract the damaging effects posed by endogenous and environmental agents, such as chemicals or radiation, micro-organisms devote several percent of their genome to encode proteins that function in the repair and recombination of DNA. For many years, a relatively small group of scientists have carefully delineated the molecular mechanisms of these repair processes, using the simplest model systems available, namely Escherichia coli and Saccharomyces cerevisiae . These studies, which until recently had only moderate impact outside of the field, now provide the cornerstone for exciting new research into analogous processes in hum an cells. The reason for this is the revelation that the biochemical pathways for the accurate replication, repair and recombination of DNA have been conserved through evolution.

Double-stranded DNA containing 7-methylguanine residues whose imidazole rings have been opened, i.e. 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine residues, may be prepared by treatment of DNA with dimethyl sulfate followed by prolonged incubation at pH 11.4. These substituted formamidopyrimidine residues are actively removed from DNA by a DNA glycosylase present in E. coli cell extracts. The enz;me shows no apparent cofactor requirement and has a molecular weight of about 30 000. The release of ring-opened 7-methyl-guanine residues is due to a previously unrecognized activity, different from the three known E. coli DNA glycosylases that release uracil, 3-methyladenine, and hypoxanthine from DNA. This enzyme may serve to repair a major secondary alkylation product in DNA. In addition, it may remove nonmethylated purines, whose imidazole rings have been opened, from X-irradiated DNA.