Purpose: The phosphatidylinositol 3 ¶-kinase/Akt pathway is frequently altered in breast cancer.PTEN, a phosphatase that opposes the effect of phosphatidylinositol 3 ¶-kinase, can be mutated or lost, whereas the PIK3CA gene is mutated. These have been proposed as alternative mechanisms, and their clinicalpathology significance is under discussion. In this study, we aimed to explore whether PIK3CA mutations and PTEN loss are mutually exclusive mechanisms, correlate with other known clinicopathologic markers, or have clinical implication in breast cancer. Experimental Design: Exons 9 and 20 of the PIK3CA gene were analyzed in 270 breast tumors, and mutations were detected by single-stranded conformational analysis followed by sequencing. The expression of PTEN was evaluated by immunohistochemistry in 201tumors. Results: PIK3CA mutations were found in 24% of the tumors and associated with estrogen receptor + status, small size, negative HER2 status, high Akt1, and high cyclin D1protein expression. PTEN was negative in 37% of the cases and PTEN loss was associated with PIK3CA mutations (P = 0.0024). Tumors presenting PTEN loss or both alterations were often estrogen receptor + , small in size, and HER2 -. PIK3CA mutations predicted for longer local recurrence-free survival. Moreover, PTEN loss by itself or combined with mutated PIK3CA tended to confer radiosensitivity. In addition, the patients with high S-phase fraction had longer recurrence-free survival if they carried mutations in the PIK3CA gene and/or had lost PTEN, whereas the same alterations were associated with shorter recurrence-free survival among patients with low S-phase fraction. Conclusions: PIK3CA mutations and PTEN loss were not mutually exclusive events and associated with similar prognostic factors.

The regulation of cell proliferation and cell survival in breast cancer is a complex interplay between steroid hormones, growth factors and their receptors. The understanding of the signalling pathways involved in these processes may help us find predictive factors for tumour aggressiveness and therapy resistance. Already recognised is the importance of the oestrogen receptor (ER) status of the tumour for predicting the benefit from endocrine treatment [1].Although it has not yet been fully established from clinical materials, experimental studies suggest that overexpression of different growth factor receptors in breast cancer makes cells less sensitive to tamoxifen and other cytotoxic drugs. These receptors include insulin-like growth factor CI = confidence interval; CMF = cyclophosphamide-methotrexate-5-fluorouracil; ER = oestrogen receptor; FITC = fluorescein isothiocyanate; pAkt = phosphorylated Akt; PI3-K = phosphatidylinositol 3-kinase; RR = rate ratio. Stål et al., licensee BioMed Central Ltd (Print ISSN 1465-5411; Online ISSN 1465-542X). This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any non-commercial purpose, provided this notice is preserved along with the article's original URL. AbstractBackground: The serine/threonine kinase Akt, or protein kinase B, has recently been a focus of interest because of its activity to inhibit apoptosis. It mediates cell survival by acting as a transducer of signals from growth factor receptors that activate phosphatidylinositol 3-kinase.

Estrogens play a crucial role in the development of breast cancer. Estradiol can be produced in the breast tissue in situ, and one of the enzymes involved in this process is 17b-hydroxysteriod dehydrogenase (17b-HSD) type 1 that catalyzes the interconversion of estrone (E1) to the biologically more potent estradiol (E2). The gene coding for 17b-HSD type 1 (HSD17B1) is located at 17q12-21, close to the more studied ERBB2 and BRCA1. The aim of this study was to investigate if HSD17B1 shows an altered gene copy number in breast cancer. We used real-time PCR and examined 221 postmenopausal breast tumors for amplification of HSD17B1 and ERBB2. In all, 32 tumors (14.5%) showed amplification of HSD17B1 and 21% were amplified for ERBB2. Amplification of the two genes was correlated (P ¼ 0.00078) and in 14 tumors (44%) with amplification of HSD17B1, ERBB2 was co amplified. The patients with amplification in at least one of the genes had a significantly worse outcome than patients without (P ¼ 0.0059). For estrogen receptor (ER)-positive patients who received adjuvant tamoxifen, amplification of HSD17B1 was related to decreased breast cancer survival (P ¼ 0.017), whereas amplification of ERRB2 was not. Amplification of HSD17B1 might be an indicator of adverse prognosis among ER-positive patients, and possibly a mechanism for decreased benefit from tamoxifen treatment.

Aim: The Mammalian Target of Rapamycin (mTOR) and its substrates S6K1 and S6K2 regulate cell growth, proliferation and metabolism through translational control. RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are situated in the commonly amplified 17q21-23 and 11q13regions. S6K1 amplification and protein overexpression have earlier been associated with a worse outcome in breast cancer, but information regarding S6K2 is scarce. The aim of this study was to evaluate the prognostic and treatment predictive relevance of S6K1/S6K2 gene amplification, as well as S6K2 protein expression in breast cancer. Results: S6K1 amplification/gain was detected in 10.7%/21.4% and S6K2 amplification/gain in 4.3%/21.3% of the tumors. S6K2 protein was detected in the nucleus (38%) and cytoplasm (76%) of the tumor cells. S6K1 amplification was significantly associated with HER2 gene amplification and protein expression. S6K2 amplification correlated significantly with high S6K2 mRNA levels, ER+ status and CCND1 amplification. S6K1 and S6K2 gene amplification was associated with a worse prognosis independent of HER2 and CCND1. S6K2 gain and nuclear S6K2 expression was related to an improved benefit from tamoxifen among patients with ER+ respectively ER+/PgR+ tumors. In the ER+/PgR-subgroup, nuclear S6K2 rather indicated decreased tamoxifen responsiveness. S6K1 amplification predicted reduced benefit from radiotherapy.Conclusions: This is the first study showing that S6K2 amplification and overexpression, like S6K1 amplification, have prognostic and treatment predictive significance in breast cancer.

The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event associated with oestrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels.Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole genome screening of DNA copy number changes in 29 breast tumours. To identify amplicon cores at 11q13 and 8p12, Genomic Identification of Significant Targets in Cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons (RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1(4EBP1), PPAPDC1B and FGFR1 (8p12)) were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, SNP arrays have enabled mapping of the 11q13 amplicon in breast tumours with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.3