Prognostic Significance of Interferon-γ and Its Signaling Pathway in Early Breast Cancer Depends on the Molecular Subtypes.

Interferons are crucial for adaptive immunity and play an important role in the immune landscape of breast cancer. Using microarray-based gene expression analysis, we examined the subtype-specific prognostic significance of interferon-γ (IFN-γ) as a single gene as well as an IFN-γ signature covering the signaling pathway in 461 breast cancer patients. Prognostic significance of IFN-γ, as well as the IFN-γ signature for metastasis-free survival (MFS), were examined using Kaplan-Meier as well as univariate and multivariate Cox regression analyses in the whole cohort and in different molecular subtypes. The independent prognostic significance of IFN-γ as a single gene was limited to basal-like breast cancer (hazard ratio (HR) 2.779, 95% confidence interval (95% CI) 1.117-6.919, p = 0.028). In contrast, the IFN-γ-associated gene signature was an independent prognostic factor in the whole cohort (HR 2.287, 95% CI 1.410-3.633, p < 0.001) as well as in the basal-like (HR 3.458, 95% CI 1.154-10.359, p = 0.027) and luminal B (HR 2.690, 95% CI 1.416-5.112, p = 0.003) molecular subtypes. These results underline the subtype-dependent prognostic influence of the immune system in early breast cancer.

Effects of definitive and salvage radiotherapy on the distribution of lymphocyte subpopulations in prostate cancer patients.

BACKGROUND: Radiotherapy (RT) is an established treatment for patients with primary and recurrent prostate cancer. Herein, the effects of definitive and salvage RT on the composition of lymphocyte subpopulations were investigated in patients with prostate cancer to study potential immune effects. PATIENTS AND METHODS: A total of 33 prostate cancer patients were treated with definitive (n = 10) or salvage RT (n = 23) after biochemical relapse. The absolute number of lymphocytes and the distribution of lymphocyte subpopulations were analyzed by multiparameter flow cytometry before RT, at the end of RT, and in the follow-up period. RESULTS: Absolute lymphocyte counts decreased significantly after RT in both patient groups and a significant drop was observed in the percentage of B cells directly after RT from 10.1 ± 1.3 to 6.0 ± 0.7% in patients with definitive RT and from 9.2 ± 0.8 to 5.8 ± 0.7% in patients with salvage RT. In contrast, the percentages of T and natural killer (NK) cells remained unaltered directly after RT in both patient groups. However, 1 year after RT, the percentage of CD3+ T cells was significantly lower in patients with definitive and salvage RT. The percentage of regulatory T cells was slightly upregulated in primary prostate cancer patients after definitive RT, but not after salvage RT. CONCLUSION: Definitive and salvage RT exert similar effects on the composition of lymphocyte subpopulations in prostate cancer patients. Total lymphocyte counts are lower in both patient groups compared to healthy controls and further decreased after RT. B cells are more sensitive to definitive and salvage RT than T and NK cells.

Overexpression of cytosolic, plasma membrane bound and extracellular heat shock protein 70 (Hsp70) in primary glioblastomas.

A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.

Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody.

Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed "TKD," comprising amino acids 450-461 (aa(450-461)) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70(+) mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.

Expression of aurora kinase A is associated with metastasis-free survival in node-negative breast cancer patients.

BACKGROUND: Inhibitors targeting the cell cycle-regulated aurora kinase A (AURKA) are currently being developed. Here, we examine the prognostic impact of AURKA in node-negative breast cancer patients without adjuvant systemic therapy (n = 766). METHODS: AURKA was analyzed using microarray-based gene-expression data from three independent cohorts of node-negative breast cancer patients. In multivariate Cox analyses, the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER), and HER2 were considered. RESULTS: Patients with higher AURKA expression had a shorter metastasis-free survival (MFS) in the Mainz (HR 1.93; 95% CI 1.34 - 2.78; P < 0.001), Rotterdam (HR 1.95; 95% CI 1.45- 2.63; P<0.001) and Transbig (HR 1.52; 95% CI 1.14-2.04; P=0.005) cohorts. AURKA was also associated with MFS in the molecular subtype ER+/HER2- carcinomas (HR 2.10; 95% CI 1.70-2.59; P<0.001), but not in ER-/HER2- nor in HER2+ carcinomas. In the multivariate Cox regression adjusted to age, grade and tumor size, AURKA showed independent prognostic significance in the ER+/HER2- subtype (HR 1.73; 95% CI 1.24-2.42; P=0.001). Prognosis of patients in the highest quartile of AURKA expression was particularly poor. In addition, AURKA correlated with the proliferation metagene (R=0.880; P<0.001), showed a positive association with grade (P<0.001), tumor size (P<0.001) and HER2 (P<0.001), and was inversely associated with ER status (P<0.001). CONCLUSIONS: AURKA is associated with worse prognosis in estrogen receptor positive breast carcinomas. Patients with the highest AURKA expression (>75% percentile) have a particularly bad prognosis and may profit from therapy with AURKA inhibitors.

Prognostic Impact of LAG-3 mRNA Expression in Early Breast Cancer.

Background: Monoclonal antibodies against PD-1 or PD-L1 have been established in clinical practice for the treatment of both early and advanced/metastatic triple-negative breast cancer. Beyond the established immune checkpoints (ICPs) (PD-1 and CTLA-4), additional ICPs, such as lymphocyte activation gene-3 (LAG-3), are subject of current research. In the present retrospective gene-expression analysis, we evaluated the prognostic significance of LAG-3 in 461 patients with early breast cancer. In addition, we examined whether there was a correlation between the different ICP and CD8 expressions. Methods: Using microarray-based gene-expression analysis, we examined the prognostic significance of LAG-3 mRNA expression for metastasis-free survival (MFS) in the whole cohort of 461 breast cancer patients and among different molecular subtypes. Correlations were analyzed using Spearman's rho correlation coefficient. Results: In the whole cohort, LAG-3 expression had no significant impact on MFS (p = 0.712, log-rank). In the subgroup analyses, there was a trend that a higher LAG-3 expression was associated with a favorable outcome in the luminal B (p = 0.217), basal-like (p = 0.370) and HER2 (p = 0.089) subtypes, although significance was not reached. In contrast, in a multivariate Cox regression analysis, adjusted for age, tumor size, axillary nodal status, histological grade of differentiation and proliferation marker Ki-67, LAG-3 showed a significant influence on MFS (HR 0.574; 95% CI 0.369−0.894; p = 0.014). High LAG-3 significantly correlated with CD8 (ρ = 0.571; p < 0.001). Conclusions: LAG-3 expression had an independent impact on MFS. In addition to PD-1 and PD-L1, further immune checkpoints, such as LAG-3, could serve as therapeutic targets in breast cancer.

Long-term prognostic significance of HER2-low and HER2-zero in node-negative breast cancer.

BACKGROUND: Recently, novel antibody--drug conjugates (ADCs) showed clinical activity in a subset of advanced human epidermal growth factor receptor 2 (HER2)-negative patients. We investigated the prognostic significance of HER2-low and HER2-zero tumours. PATIENTS AND METHODS: The retrospective cohort study included 410 consecutive node-negative breast cancer patients without adjuvant systemic therapy treated between 1985 and 2000 (median follow-up: 16.73 [IQR 8.58-23.45] years). 351 (85.6%) were HER-2 negative and subdivided into HER2-zero (immunohistochemistry [IHC] score 0) and HER2-low (IHC score 1+ or 2+/in situ hybridisation [ISH]-negative). HER2 gene expression was available in 170 (48.4%) patients. Differences in HER2 status for immunohistochemistry, gene expression and clinico-pathologic parameters were assessed using Fisher's exact test, Pearson's correlation and Mann-Whitney test. Prognosis was investigated using the Kaplan-Meier method and Cox regression analyses. RESULTS: Of the 351 HER2-negative patients, 198 (56.4%) had HER2-low tumours and 153 (43.6%) were HER2-zero. Significant differences between HER2-zero and HER2-low tumours were found in histologic grading (P = 0.001), Ki-67 (P = 0.013) and HER2 gene expression (P = 0.002). HER2-low patients had significantly longer disease-free survival (DFS) (15-year rate: 67.5% [95% CI 61.0-74.7] vs. 47.3% [95% CI 39.9-56.1], P < 0.001) and overall survival (OS) (15-year rate: 75.4% [95% CI 69.4-81.9] vs. 66.8% [95% CI 59.5-74.9], P = 0.009). The OS difference was observed in hormone receptor (HR)-positive (P = 0.039) but not HR-negative (P = 0.086) tumours. The results of multivariable analyses confirmed the independent prognostic significance of HER2 status (DFS: HR, 0.546; 95% CI, 0.402-0.743; P < 0.001; OS: HR, 0.653; 95% CI, 0.458-0.932; P = 0.019). CONCLUSION: HER2-low patients had a better survival than HER2-zero patients.

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody.

The major stress-inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1-fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near-infrared fluorescence imaging revealed an enrichment of Cy5.5-conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype-matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane-bound Hsp70, the fluorescence-labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour-selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumours. The anti-tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody-dependent cytotoxicity.

Gene expression profiling of luminal B breast cancers reveals NHERF1 as a new marker of endocrine resistance.

The luminal B subtype represents a group of high proliferating estrogen receptor positive breast cancers which are associated with a poor prognosis. Genes exclusively expressed in this subtype should help to better understand these tumors. In a finding cohort of 171 breast cancers luminal B specific genes were identified displaying strong expression in highly proliferating Ki-67 positive/ER positive tumors but no expression either in Ki-67 negative/ER positive or in Ki-67 positive/ER negative samples. The clinical relevance of the scaffold protein NHERF1 identified by this strategy was assessed in a total of 3,030 breast cancers. NHERF1 expression was associated with the luminal B subtype both in the finding and validation cohort. A positive correlation of NHERF1 expression with tumor size (P < 0.001), grade (P < 0.001), and HER2 status (P = 0.033) was observed. NHERF1 expression was associated with a worse survival in ER positive breast cancer (P < 0.001) and retained its prognostic value in multivariate analysis. For ER positive samples with low NHERF1 expression a benefit of endocrine therapy was detected (P = 0.007). In contrast no differences in disease free survival were found for high NHERF1 expressing breast cancers which were either treated with endocrine therapy or no systemic therapy. Our data indicate that NHERF1 expressing breast cancers seem to have a greater risk to develop resistance to endocrine therapy. However, based on previous findings of NHERF1 functioning in PI3K signalling from basic research, these tumors might be appropriate candidates for a targeted therapy of the PI3K/Akt pathway.

Ep-CAM RNA expression predicts metastasis-free survival in three cohorts of untreated node-negative breast cancer.

Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention as a prognostic factor in breast cancer. We aimed to validate the influence of Ep-CAM RNA expression in untreated node-negative breast cancer. Ep-CAM RNA expression was evaluated utilizing microarray-based gene-expression profiling in 194 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. The prognostic significance of Ep-CAM RNA expression for disease-free survival (DFS), metastasis-free survival (MFS), and breast cancer-specific overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for age, grading, pTstage, ER as well as PR receptor and HER-2 status. Additionally, Ep-CAM RNA expression was compared with immunohistochemistry (IHC) for Ep-CAM in 194 patients. The prognostic impact of Ep-CAM gene expression was validated in further 588 node-negative breast cancer patients. Levels of Ep-CAM RNA expression showed a significant correlation with IHC (P = 0.001) and predicted in univariate analysis DFS (P = 0.001, HR = 2.4), MFS (P = 0.003, HR = 2.5), and OS (P = 0.002, HR = 3.1) accurately. The prognostic influence of Ep-CAM RNA was significant also in multivariate analysis for DFS (P = 0.017, HR = 2.0), MFS (P = 0.049, HR = 1.9), and OS (P = 0.042, HR = 2.3), respectively. The association with MFS was confirmed in an independent validation cohort in univariate (P = 0.006, HR = 1.9) and multivariate (P = 0.035, HR = 1.7) analysis. Ep-CAM RNA correlated with the proliferation metagene (P < 0.001, R=0.425) Nevertheless, in multivariate analysis, Ep-CAM was associated with MFS independent from the proliferation metagene (P = 0.030, HR = 1.8). In conclusion, Ep-CAM RNA expression is associated with poor MFS in three cohorts of untreated node-negative breast cancer.

Integration of pathway knowledge into a reweighted recursive feature elimination approach for risk stratification of cancer patients.

MOTIVATION: One of the main goals of high-throughput gene-expression studies in cancer research is to identify prognostic gene signatures, which have the potential to predict the clinical outcome. It is common practice to investigate these questions using classification methods. However, standard methods merely rely on gene-expression data and assume the genes to be independent. Including pathway knowledge a priori into the classification process has recently been indicated as a promising way to increase classification accuracy as well as the interpretability and reproducibility of prognostic gene signatures. RESULTS: We propose a new method called Reweighted Recursive Feature Elimination. It is based on the hypothesis that a gene with a low fold-change should have an increased influence on the classifier if it is connected to differentially expressed genes. We used a modified version of Google's PageRank algorithm to alter the ranking criterion of the SVM-RFE algorithm. Evaluations of our method on an integrated breast cancer dataset comprising 788 samples showed an improvement of the area under the receiver operator characteristic curve as well as in the reproducibility and interpretability of selected genes. AVAILABILITY: The R code of the proposed algorithm is given in Supplementary Material.

Prognostic Impact of Immunoglobulin Kappa C (IGKC) in Early Breast Cancer.

We studied the prognostic impact of tumor immunoglobulin kappa C (IGKC) mRNA expression as a marker of the humoral immune system in the FinHer trial patient population, where 1010 patients with early breast cancer were randomly allocated to either docetaxel-containing or vinorelbine-containing adjuvant chemotherapy. HER2-positive patients were additionally allocated to either trastuzumab or no trastuzumab. Hormone receptor-positive patients received tamoxifen. IGKC was evaluated in 909 tumors using quantitative real-time polymerase chain reaction, and the influence on distant disease-free survival (DDFS) was examined using univariable and multivariable Cox regression and Kaplan-Meier estimates. Interactions were analyzed using Cox regression. IGKC expression, included as continuous variable, was independently associated with DDFS in a multivariable analysis also including age, molecular subtype, grade, and pT and pN stage (HR 0.930, 95% CI 0.870-0.995, p = 0.034). An independent association with DDFS was also found in a subset analysis of triple-negative breast cancers (TNBC) (HR 0.843, 95% CI 0.724-0.983, p = 0.029), but not in luminal (HR 0.957, 95% CI 0.867-1.056, p = 0.383) or HER2-positive (HR 0.933, 95% CI 0.826-1.055, p = 0.271) cancers. No significant interaction between IGKC and chemotherapy or trastuzumab administration was detected (Pinteraction = 0.855 and 0.684, respectively). These results show that humoral immunity beneficially influences the DDFS of patients with early TNBC.

Gene Expression-Based Prediction of Neoadjuvant Chemotherapy Response in Early Breast Cancer: Results of the Prospective Multicenter EXPRESSION Trial.

PURPOSE: Expression-based classifiers to predict pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) are not routinely used in the clinic. We aimed to build and validate a classifier for pCR after NACT. PATIENTS AND METHODS: We performed a prospective multicenter study (EXPRESSION) including 114 patients treated with anthracycline/taxane-based NACT. Pretreatment core needle biopsies from 91 patients were used for gene expression analysis and classifier construction, followed by validation in five external cohorts (n = 619). RESULTS: A 20-gene classifier established in the EXPRESSION cohort using a Youden index-based cut-off point predicted pCR in the validation cohorts with an accuracy, AUC, negative predictive value (NPV), positive predictive value, sensitivity, and specificity of 0.811, 0.768, 0.829, 0.587, 0.216, and 0.962, respectively. Alternatively, aiming for a high NPV by defining the cut-off point for classification based on the complete responder with the lowest predicted probability of pCR in the EXPRESSION cohort led to an NPV of 0.960 upon external validation. With this extreme-low cut-off point, a recommendation to not treat with anthracycline/taxane-based NACT would be possible for 121 of 619 unselected patients (19.5%) and 112 of 322 patients with luminal breast cancer (34.8%). The analysis of the molecular subtypes showed that the identification of patients who do not achieve a pCR by the 20-gene classifier was particularly relevant in luminal breast cancer. CONCLUSIONS: The novel 20-gene classifier reliably identifies patients who do not achieve a pCR in about one third of luminal breast cancers in both the EXPRESSION and combined validation cohorts.

Comparison of scores for bimodality of gene expression distributions and genome-wide evaluation of the prognostic relevance of high-scoring genes.

BACKGROUND: A major goal of the analysis of high-dimensional RNA expression data from tumor tissue is to identify prognostic signatures for discriminating patient subgroups. For this purpose genome-wide identification of bimodally expressed genes from gene array data is relevant because distinguishability of high and low expression groups is easier compared to genes with unimodal expression distributions.Recently, several methods for the identification of genes with bimodal distributions have been introduced. A straightforward approach is to cluster the expression values and score the distance between the two distributions. Other scores directly measure properties of the distribution. The kurtosis, e.g., measures divergence from a normal distribution. An alternative is the outlier-sum statistic that identifies genes with extremely high or low expression values in a subset of the samples. RESULTS: We compare and discuss scores for bimodality for expression data. For the genome-wide identification of bimodal genes we apply all scores to expression data from 194 patients with node-negative breast cancer. Further, we present the first comprehensive genome-wide evaluation of the prognostic relevance of bimodal genes. We first rank genes according to bimodality scores and define two patient subgroups based on expression values. Then we assess the prognostic significance of the top ranking bimodal genes by comparing the survival functions of the two patient subgroups. We also evaluate the global association between the bimodal shape of expression distributions and survival times with an enrichment type analysis.Various cluster-based methods lead to a significant overrepresentation of prognostic genes. A striking result is obtained with the outlier-sum statistic (p < 10-12). Many genes with heavy tails generate subgroups of patients with different prognosis. CONCLUSIONS: Genes with high bimodality scores are promising candidates for defining prognostic patient subgroups from expression data. We discuss advantages and disadvantages of the different scores for prognostic purposes. The outlier-sum statistic may be particularly valuable for the identification of genes to be included in prognostic signatures. Among the genes identified as bimodal in the breast cancer data set several have not yet previously been recognized to be prognostic and bimodally expressed in breast cancer.

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer.

INTRODUCTION: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. METHODS: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. RESULTS: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. CONCLUSIONS: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner.

Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression.

BACKGROUND: There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS: We copurified both RNA and DNA from a single 10-µm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS: In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%. CONCLUSIONS: This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

Improved prognostic classification of breast cancer defined by antagonistic activation patterns of immune response pathway modules.

BACKGROUND: Elucidating the activation pattern of molecular pathways across a given tumour type is a key challenge necessary for understanding the heterogeneity in clinical response and for developing novel more effective therapies. Gene expression signatures of molecular pathway activation derived from perturbation experiments in model systems as well as structural models of molecular interactions ("model signatures") constitute an important resource for estimating corresponding activation levels in tumours. However, relatively few strategies for estimating pathway activity from such model signatures exist and only few studies have used activation patterns of pathways to refine molecular classifications of cancer. METHODS: Here we propose a novel network-based method for estimating pathway activation in tumours from model signatures. We find that although the pathway networks inferred from cancer expression data are highly consistent with the prior information contained in the model signatures, that they also exhibit a highly modular structure and that estimation of pathway activity is dependent on this modular structure. We apply our methodology to a panel of 438 estrogen receptor negative (ER-) and 785 estrogen receptor positive (ER+) breast cancers to infer activation patterns of important cancer related molecular pathways. RESULTS: We show that in ER negative basal and HER2+ breast cancer, gene expression modules reflecting T-cell helper-1 (Th1) and T-cell helper-2 (Th2) mediated immune responses play antagonistic roles as major risk factors for distant metastasis. Using Boolean interaction Cox-regression models to identify non-linear pathway combinations associated with clinical outcome, we show that simultaneous high activation of Th1 and low activation of a TGF-beta pathway module defines a subtype of particularly good prognosis and that this classification provides a better prognostic model than those based on the individual pathways. In ER+ breast cancer, we find that simultaneous high MYC and RAS activity confers significantly worse prognosis than either high MYC or high RAS activity alone. We further validate these novel prognostic classifications in independent sets of 173 ER- and 567 ER+ breast cancers. CONCLUSION: We have proposed a novel method for pathway activity estimation in tumours and have shown that pathway modules antagonize or synergize to delineate novel prognostic subtypes. Specifically, our results suggest that simultaneous modulation of T-helper differentiation and TGF-beta pathways may improve clinical outcome of hormone insensitive breast cancers over treatments that target only one of these pathways.

Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells.

Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/2 > or = 7/3 < 5/5 < 4/6 < 3/7 < 2/8). In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC(50) of Hsp70=85 microg/ml) and hypoxic (EC(50) of Hsp70=55 microg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 microg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.

Exercise-induced extracellular 72 kDa heat shock protein (Hsp72) stimulates neutrophil phagocytic and fungicidal capacities via TLR-2.

This study evaluated the role of toll like receptor 2 (TLR-2) in the interaction of 72 kDa extracellular heat shock protein (Hsp72, a stress-inducible protein) with neutrophils and the participation on TLR-2 in the stimulation of neutrophil phagocytic and fungicidal capacities by post-exercise physiological concentrations of Hsp72. Human peripheral blood neutrophils were incubated with fluorescein isothiocyanate-conjugated Hsp72, and were analyzed by immunofluorescence microscopy and flow cytometry. Both methods revealed an interaction of Hsp72 with neutrophils. In addition, when neutrophils were pre-incubated with an anti-TLR-2 antibody this interaction was clearly decreased. Post-exercise circulating concentration of Hsp72 (8.6 ng/ml) stimulated the phagocytic and fungicidal capacities of neutrophils and this effect could be also blocked using an antibody against TLR-2. Phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK) and the nuclear transcription factor kappa beta (NF-kappabeta) were found to be involved in the signaling process, confirming the participation of TLR-2 in the stimulation of neutrophil function by Hsp72. In conclusion, TLR-2 is involved at least in part, in the stimulation of neutrophil phagocytic and fungicidal capacities induced by post-exercise physiological concentrations of Hsp72.

Anti-tumor activity of patient-derived NK cells after cell-based immunotherapy--a case report.

BACKGROUND: Membrane-bound heat shock protein 70 (Hsp70) serves as a tumor-specific recognition structure for Hsp70-peptide (TKD) plus IL-2 activated NK cells. A phase I clinical trial has shown that repeated re-infusions of ex vivo TKD/IL-2-activated, autologous leukapheresis product is safe. This study investigated the maintenance of the cytolytic activity of NK cells against K562 cells and autologous tumor after 6 plus 3 infusions of TKD/IL-2-activated effector cells. METHODS: A stable tumor cell line was generated from the resected anastomotic relapse of a patient with colon carcinoma (pT3, N2, M0, G2). Two months after surgery, the patient received the first monthly i.v. infusion of his ex vivo TKD/IL-2-activated peripheral blood mononuclear cells (PBMNC). After 6 infusions and a pause of 3 months, the patient received another 3 cell infusions. The phenotypic characteristics and activation status of tumor and effector cells were determined immediately before and at times after each infusion. RESULTS: The NK cell ligands Hsp70, MICA/B, and ULBP-1,2,3 were expressed on the patient's anastomotic relapse. An increased density of activatory NK cell receptors following ex vivo stimulation correlated with an enhanced anti-tumoricidal activity. After 4 re-infusion cycles, the intrinsic cytolytic activity of non-stimulated PBMNC was significantly elevated and this heightened responsiveness persisted for up to 3 months after the last infusion. Another 2 re-stimulations with TKD/IL-2 restored the cytolytic activity after the therapeutic pause. CONCLUSION: In a patient with colon carcinoma, repeated infusions of ex vivo TKD/IL-2-activated PBMNC initiate an intrinsic NK cell-mediated cytolytic activity against autologous tumor cells.