PI3K Pathway Mutations and PTEN Levels in Primary and Metastatic Breast Cancer

The purpose of this work was to determine whether there are differences in PIK3CA mutation status and PTEN protein expression between primary and matched metastatic breast tumors as this could influence patient management. Fifty-micron paraffin sections were used for DNA extraction and 3-micron slides for immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH). ER, PR and HER2 IHC were repeated in a central laboratory for both primary and metastasis. PTEN levels were assessed by IHC and PI3K pathway mutations detected by a mass spectroscopy-based approach. Median age was 48 years (range, 30 to 83 years). Tumor subtype included 72% hormone receptor-positive/HER2-negative, 20% HER2-positive, and less than 7.8% triple receptor negative. Tissues were available for PTEN IHC in 46 primary tumors and 52 metastases. PTEN was lost in 14 (30%) primary tumors and 13 (25%) metastases. There were 5 cases of PTEN loss and eight cases of PTEN gain from primary to metastasis (26% discordance). Adequate DNA was obtained on 46 primary tumors and on 50 metastases for PIK3CA analysis. PIK3CA mutations were detected in 19 (40%) of primary tumors and 21 (42%) of metastases. There were five cases of PIK3CA mutation loss, and four cases of mutation gain (18% discordance). There was an increase of the level of PIK3CA mutations in four cases, and decrease in one from primary to metastasis. There is a high level of discordance in PTEN level, PIK3CA mutations, and receptor status between primary and metastatic disease that may influence patient selection and response to PI3K-targeted therapies

A novel AKT3 mutation in melanoma tumours and cell lines

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations

Dielectrophoresis has Broad Applicability to Marker-Free Isolation of Tumor Cells from Blood by Microfluidic Systems

The number of circulating tumor cells (CTCs) found in blood is known to be a prognostic marker for recurrence of primary tumors, however, most current methods for isolating CTCs rely on cell surface markers that are not universally expressed by CTCs. Dielectrophoresis (DEP) can discriminate and manipulate cancer cells in microfluidic systems and has been proposed as a molecular marker-independent approach for isolating CTCs from blood. To investigate the potential applicability of DEP to different cancer types, the dielectric and density properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic field-flow fractionation (DEP-FFF) and compared with like properties of the subpopulations of normal peripheral blood cells. We show that all of the NCI-60 cell types, regardless of tissue of origin, exhibit dielectric properties that facilitate their isolation from blood by DEP. Cell types derived from solid tumors that grew in adherent cultures exhibited dielectric properties that were strikingly different from those of peripheral blood cell subpopulations while leukemia-derived lines that grew in non-adherent cultures exhibited dielectric properties that were closer to those of peripheral blood cell types. Our results suggest that DEP methods have wide applicability for the surface-marker independent isolation of viable CTCs from blood as well as for the concentration of leukemia cells from blood. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4774307]Cancer Prevention and Research Institute of Texas (CPRIT) RP100934Kleberg Center for Molecular MarkersEntertainment Industry Foundation SU2C-AACR-DT0209NCI CA016672Biomedical Engineerin

Antibody-Independent Isolation of Circulating Tumor Cells by Continuous-Flow Dielectrophoresis

Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 106 nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4774304]Cancer Prevention and Research Institute of Texas (CPRIT) RP100934Kleberg Center for Molecular MarkersApoCell, IncEntertainment Industry Foundation SU2C-AACR-DT0209Imaging Research Cente

DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer

The majority of patients develop resistance against suppression of HER2-signaling mediated by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Using gene chip data of 88 HER2- positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further restricted to markers significantly associated with worse survival. Functional significance of the two strongest predictive markers was evaluated in vitro by gene silencing in HER2 overexpressing, trastuzumab resistant BC cell lines SKTR and JIMT-1. The strongest predictive MKPs were DUSP4/MKP-2 (AUC=0.75, p=0.0096) and DUSP6/MKP-3 (AUC=0.77, p=5.29E-05). Higher expression for these correlated to worse survival (DUSP4: HR=2.05, p=0.009 and DUSP6: HR=2, p=0.0015). Silencing of DUSP4 had significant sensitization effects – viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected controls (SKTR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Our findings suggest that DUSP4 may represent a new potential target to overcome trastuzumab resistance

A retrospective analysis of clinical outcome of patients with chemo-refractory metastatic breast cancer treated in a single institution phase I unit

BACKGROUND AND METHODS: Novel approaches to treat chemo-refractory metastatic breast cancer (MBC) are currently under investigation. This retrospective series reviews the outcome of 70 MBC patients who have participated in 30 phase I trials at the Royal Marsden Hospital from 2002 to 2009. RESULTS: The median treatment lines before phase I trial entry for MBC was 5 (range: 1-12 lines). The overall response rate was 11.4% (95% CI: 4.0-18.9%) and the clinical benefit rate at 4 months was 20% (95% CI: 10.6-29.3). The median time to progression was 7.0 weeks (95% CI: 6.4-7.5) and median overall survival was 8.7 months (95% CI: 7.6-9.8) from start of first phase I treatment. No patients discontinued trial because of treatment-related toxicities. Abnormal lactate dehydrogenase, serum albumin <35 mg per 100 ml, >or=5 previous treatment lines, liver metastases and Eastern Cooperative Group performance status >or=2 at study entry were significantly associated with poor overall survival in multivariate analysis. CONCLUSION: This retrospective analysis provides evidence that patients with MBC tolerate phase I clinical trials and a significant proportion of patients with chemo-refractory disease, particularly those with triple-negative or Her2-positive breast cancer, may benefit from treatment

Gene Expression Profiling Reveals New Aspects of PIK3CA Mutation in ERalpha-Positive Breast Cancer: Major Implication of the Wnt Signaling Pathway

BACKGROUND: The PI3K/AKT pathway plays a pivotal role in breast cancer development and maintenance. PIK3CA, encoding the PI3K catalytic subunit, is the oncogene exhibiting a high frequency of gain-of-function mutations leading to PI3K/AKT pathway activation in breast cancer. PIK3CA mutations have been observed in 30% to 40% of ERα-positive breast tumors. However the physiopathological role of PIK3CA mutations in breast tumorigenesis remains largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: To identify relevant downstream target genes and signaling activated by aberrant PI3K/AKT pathway in breast tumors, we first analyzed gene expression with a pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations. Genes of interest were then investigated in 249 ERα-positive breast tumors by real-time quantitative RT-PCR. A robust collection of 19 genes was found to be differently expressed in PIK3CA-mutated tumors. PIK3CA mutations were associated with over-expression of several genes involved in the Wnt signaling pathway (WNT5A, TCF7L2, MSX2, TNFRSF11B), regulation of gene transcription (SEC14L2, MSX2, TFAP2B, NRIP3) and metal ion binding (CYP4Z1, CYP4Z2P, SLC40A1, LTF, LIMCH1). CONCLUSION/SIGNIFICANCE: This new gene set should help to understand the behavior of PIK3CA-mutated cancers and detailed knowledge of Wnt signaling activation could lead to novel therapeutic strategies

An integrative genomic and proteomic analysis of PIK3CA, PTEN and AKT mutations in breast cancer

Phosphatidylinositol-3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT and PTEN mutations, and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in-vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor positive (33.8%) and HER2-positive (24.6%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers with PTEN protein levels also being significantly lower in hormone receptor-positive cancers. Unlike AKT1 mutations, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant impact on outcome in 166 hormone receptor-positive breast cancer patients after adjuvant tamoxifen. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and indeed inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines, and PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss but not PIK3CA mutations rendered cells sensitive to growth inhibition by the PI3K inhibitor LY294002. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer

Phosphatidylinositol 3-kinase pathway activation in breast cancer brain metastases

Activation status of the phosphatidylinositol 3-kinase (PI3K) pathway in breast cancer brain metastases (BCBMs) is largely unknown. We examined expression of phospho(p)-AKT, p-S6, and phosphatase and tensin homologue (PTEN) in BCBMs and their implications for overall survival (OS) and survival after BCBMs. Secondary analyses included PI3K pathway activation status and associations with time to distant recurrence (TTDR) and time to BCBMs. Similar analyses were also conducted among the subset of patients with triple-negative BCBMs. METHODS: p-AKT, p-S6, and PTEN expression was assessed with immunohistochemistry in 52 BCBMs and 12 matched primary BCs. Subtypes were defined as hormone receptor (HR)+/HER2-, HER2+, and triple-negative (TNBC). Survival analyses were performed by using a Cox model, and survival curves were estimated with the Kaplan-Meier method. RESULTS: Expression of p-AKT and p-S6 and lack of PTEN (PTEN-) was observed in 75%, 69%, and 25% of BCBMs. Concordance between primary BCs and matched BCBMs was 67% for p-AKT, 58% for p-S6, and 83% for PTEN. PTEN- was more common in TNBC compared with HR+/HER2- and HER2+. Expression of p-AKT, p-S6, and PTEN- was not associated with OS or survival after BCBMs (all, P > 0.06). Interestingly, among all patients, PTEN- correlated with shorter time to distant and brain recurrence. Among patients with TNBC, PTEN- in BCBMs was associated with poorer overall survival. CONCLUSIONS: The PI3K pathway is active in most BCBMs regardless of subtype. Inhibition of this pathway represents a promising therapeutic strategy for patients with BCBMs, a group of patients with poor prognosis and limited systemic therapeutic options. Although expression of the PI3K pathway did not correlate with OS and survival after BCBM, PTEN- association with time to recurrence and OS (among patients with TNBC) is worthy of further study