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

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

Beyond trastuzumab: novel therapeutic strategies in HER2-positive metastatic breast cancer

The use of trastuzumab, a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) alteration present in 25 to 30% of breast cancers, has been associated with improved survival outcomes in both the adjuvant and metastatic settings. However, despite the robust clinical efficacy of trastuzumab in HER2-positive metastatic breast cancer (MBC), primary and secondary resistance remains a clinical challenge. Although lapatinib has demonstrated modest activity in this setting, trials reported to date have yet to demonstrate improvements in overall survival with its use. Novel therapeutic strategies to circumvent trastuzumab resistance are warranted, and agents targeting the HER, vascular endothelial growth factor, heat shock protein 90, phosphoinositide 3 kinase/Akt/mammalian target of rapamycin, and insulin-like growth factor-1 receptor pathways represent rational approaches in the management of HER2-positive disease. In this review, early-phase and emerging trial data surrounding the use of these promising agents in HER2-positive MBC will be discussed

An integrative multi-dimensional genetic and epigenetic strategy to identify aberrant genes and pathways in cancer

<p>Abstract</p> <p>Background</p> <p>Genomics has substantially changed our approach to cancer research. Gene expression profiling, for example, has been utilized to delineate subtypes of cancer, and facilitated derivation of predictive and prognostic signatures. The emergence of technologies for the high resolution and genome-wide description of genetic and epigenetic features has enabled the identification of a multitude of causal DNA events in tumors. This has afforded the potential for large scale integration of genome and transcriptome data generated from a variety of technology platforms to acquire a better understanding of cancer.</p> <p>Results</p> <p>Here we show how multi-dimensional genomics data analysis would enable the deciphering of mechanisms that disrupt regulatory/signaling cascades and downstream effects. Since not all gene expression changes observed in a tumor are causal to cancer development, we demonstrate an approach based on multiple concerted disruption (MCD) analysis of genes that facilitates the rational deduction of aberrant genes and pathways, which otherwise would be overlooked in single genomic dimension investigations.</p> <p>Conclusions</p> <p>Notably, this is the first comprehensive study of breast cancer cells by parallel integrative genome wide analyses of DNA copy number, LOH, and DNA methylation status to interpret changes in gene expression pattern. Our findings demonstrate the power of a multi-dimensional approach to elucidate events which would escape conventional single dimensional analysis and as such, reduce the cohort sample size for cancer gene discovery.</p

Functional proteomics can define prognosis and predict pathologic complete response in patients with breast cancer

<p>Abstract</p> <p>Purpose</p> <p>To determine whether functional proteomics improves breast cancer classification and prognostication and can predict pathological complete response (pCR) in patients receiving neoadjuvant taxane and anthracycline-taxane-based systemic therapy (NST).</p> <p>Methods</p> <p>Reverse phase protein array (RPPA) using 146 antibodies to proteins relevant to breast cancer was applied to three independent tumor sets. Supervised clustering to identify subgroups and prognosis in surgical excision specimens from a training set (n = 712) was validated on a test set (n = 168) in two cohorts of patients with primary breast cancer. A score was constructed using ordinal logistic regression to quantify the probability of recurrence in the training set and tested in the test set. The score was then evaluated on 132 FNA biopsies of patients treated with NST to determine ability to predict pCR.</p> <p>Results</p> <p>Six breast cancer subgroups were identified by a 10-protein biomarker panel in the 712 tumor training set. They were associated with different recurrence-free survival (RFS) (log-rank p = 8.8 E-10). The structure and ability of the six subgroups to predict RFS was confirmed in the test set (log-rank p = 0.0013). A prognosis score constructed using the 10 proteins in the training set was associated with RFS in both training and test sets (p = 3.2E-13, for test set). There was a significant association between the prognostic score and likelihood of pCR to NST in the FNA set (p = 0.0021).</p> <p>Conclusion</p> <p>We developed a 10-protein biomarker panel that classifies breast cancer into prognostic groups that may have potential utility in the management of patients who receive anthracycline-taxane-based NST.</p

HER2-family signalling mechanisms, clinical implications and targeting in breast cancer.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC

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

MYC Cooperates with AKT in Prostate Tumorigenesis and Alters Sensitivity to mTOR Inhibitors

MYC and phosphoinositide 3-kinase (PI3K)-pathway deregulation are common in human prostate cancer. Through examination of 194 human prostate tumors, we observed statistically significant co-occurrence of MYC amplification and PI3K-pathway alteration, raising the possibility that these two lesions cooperate in prostate cancer progression. To investigate this, we generated bigenic mice in which both activated human AKT1 and human MYC are expressed in the prostate (MPAKT/Hi-MYC model). In contrast to mice expressing AKT1 alone (MPAKT model) or MYC alone (Hi-MYC model), the bigenic phenotype demonstrates accelerated progression of mouse prostate intraepithelial neoplasia (mPIN) to microinvasive disease with disruption of basement membrane, significant stromal remodeling and infiltration of macrophages, B- and T-lymphocytes, similar to inflammation observed in human prostate tumors. In contrast to the reversibility of mPIN lesions in young MPAKT mice after treatment with mTOR inhibitors, Hi-MYC and bigenic MPAKT/Hi-MYC mice were resistant. Additionally, older MPAKT mice showed reduced sensitivity to mTOR inhibition, suggesting that additional genetic events may dampen mTOR dependence. Since increased MYC expression is an early feature of many human prostate cancers, these data have implications for treatment of human prostate cancers with PI3K-pathway alterations using mTOR inhibitors