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Background: ER+/HER2− breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy. Methods: A total of 1702 postmenopausal ER+/HER2− breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan–Meier method and Cox regression analysis were used in an early (0–5 years) and late time interval (>5 years post diagnosis). Results: EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up. Conclusion: The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.

Introduction Overexpression of Topoisomerase II alpha (TOP2A) has been implicated with gene amplification of the 17q21 amplicon and consecutively with ErbB2 overexpression and amplification. However, gene amplification does not necessarily correlate with RNA and protein expression. There is growing evidence that TOP2A protein expression is a strong prognostic and TOP2A gene amplification might be a predictive marker (particularly for the use of anthracyclines). Methods Large scale analysis was performed using Affymetrix microarray data from n = 1,681 breast cancer patients to evaluate TOP2A expression. Results TOP2A expression showed a strong correlation with tumor size (χ²-test, P < 0.001), grading (P < 0.001), ErbB2 (P < 0.001) and Ki67 expression (P < 0.001) as well as nodal status (P = 0.042). Survival analysis revealed a significant prognostic value in ER positive (n = 994; log rank P < 0.001), but not in ER negative breast cancer patients (n = 369, P = 0.35). The prognostic impact of TOP2A expression was independent of Ki67 expression in ER positive tumors (P = 0.002 and P = 0.007 for high and low Ki67, respectively). Moreover a worse prognosis of high TOP2A expressing tumors was found in the subgroup of ErbB2 negative tumors (P < 0.001) and a trend among ErbB2 positive tumors (P = 0.11). The prognostic value of TOP2A was independent of whether the patients were untreated or had received adjuvant therapy. In multivariate Cox regression analysis including standard parameters TOP2A emerged to be the top prognostic marker (HR 2.40, 95% CI 1.68-3.43, P < 0.001). Conclusion TOP2A expression is an independent prognostic factor in ER positive breast cancer and could be helpful for risk assessment in ER positive breast cancer patients.

CX+/CX− and Colo+/Colo− tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal γ -irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX−, Colo−). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to γ -irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX− and Colo−, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX− and Colo− tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX−, Colo+/Colo− nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.

Background: Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed. Methods: Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts ( n =86 and n =55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n =212, and GeparQuattro, n =383). Results: A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts ( P <0.0001; P <0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) ( n =61, P =0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels ( P =0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P =0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro. Conclusion: In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pepscan) analysis, the epitope of this mAb was mapped as the C-terminal–localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454–461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal–extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450–463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454–461), the 12-mer TKDNNLLGRFEL (aa 450–461), the 13-mer C-terminal–extended peptide NLLGRFELSGIPP (aa 454–466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450–463), Hsc70 TKDNNLLGKFELTG (aa 450–463), and DnaK AADNKSLGQFNLDG (aa 447–460) failed to activate NK activity.

Low-level flights over tundra wetlands in Alaska and Canada have been conducted during the Airborne Measurements of Methane Emissions (AirMeth) campaigns to measure turbulent methane fluxes in the atmosphere. In this paper we describe the instrumentation and new calibration procedures for the essential pressure parameters required for turbulence sensing by aircraft that exploit suitable regular measurement flight legs without the need for dedicated calibration patterns. We estimate the accuracy of the mean wind and the turbulence measurements. We show that airborne measurements of turbulent fluxes of methane and carbon dioxide using cavity ring-down spectroscopy trace gas analysers together with established turbulence equipment achieve a relative accuracy similar to that of measurements of sensible heat flux if applied during low-level flights over natural area sources. The inertial subrange of the trace gas fluctuations cannot be resolved due to insufficient high-frequency precision of the analyser, but, since this scatter is uncorrelated with the vertical wind velocity, the covariance and thus the flux are reproduced correctly. In the covariance spectra the -7/3 drop-off in the inertial subrange can be reproduced if sufficient data are available for averaging. For convective conditions and flight legs of several tens of kilometres we estimate the flux detection limit to be about 4 mg m−2 d−1 for w′CH4′‾, 1.4 g m−2 d−1 for w′CO2′‾ and 4.2 W m−2 for the sensible heat flux.

Paclitaxel plays an important role in the treatment of primary breast cancer. However, a substantial proportion of patients treated with paclitaxel does not appear to derive any benefit from this therapy. We performed a prospective study using tumour cells isolated from 50 primary breast carcinomas. Sensitivity of primary tumour cells to paclitaxel was determined in a clinically relevant range of concentrations (0.85–27.2  μ g ml −1 paclitaxel) using an ATP assay. Chemosensitivity data were used to study a possible association with immunohistochemically determined oestrogen and progesterone receptor (ER and PR) status, as well as histopathological parameters. Progesterone receptor (PR) mRNA expression was also determined by quantitative RT–PCR. We observed a clear association of the PR status with chemosensitivity to paclitaxel. Higher levels of immunohistochemically detected PR expression correlated with decreased chemosensitivity ( P =0.008). Similarly, high levels of PR mRNA expression were associated with decreased paclitaxel chemosensitivity ( P =0.007). Cells from carcinomas with T-stages 3 and 4 were less sensitive compared to stages 1 and 2 ( P =0.013). Multiple regression analysis identified PR receptor status and T-stage as independent predictors of paclitaxel chemosensitivity, whereas the ER, N-stage, grading and age were not influential. In conclusion, in vitro sensitivity to paclitaxel was higher for PR-negative compared with PR-positive breast carcinoma cells. Thus, PR status should be considered as a possible factor of influence when designing new trials and chemotherapy protocols.

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.

Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1-10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.

Purpose: Gene expression profiling has the potential to lead to a more accurate prognostic classification of breast cancer patients. Recently, a set of 70 genes has been identified that allowed risk classification of node negative patients. We analysed the prognostic utility of a subset of genes belonging to that list of 70 genes and compared them with established prognostic factors in a collective of 150 untreated node-negative breast cancer patients. Methods: cRNA of 150 untreated node-negative breast cancer patients was hybridized on the Affymetrix HG-U133A array and probe sets corresponding to the set of 70 genes were identified. A good and poor prognosis profile was generated for the expression of 52 genes. The prognostic utility of this gene-expression signature was then compared with established prognostic factors (i.e. histological grade, tumor size, steroid hormone receptor status, St. Gallen risk-category). The prognostic utility for disease-free survival (DFS) was evaluated using univariable and multivariable statistical analyses. Results: 33 (22%) of the tumors were assigned to the good and 117 (78%) to the poor prognosis group based on the gene-expression signature. This led to a sensitivity of 93% with a specificity of 26%. 5-year DFS was 94% in the good and 78% in the poor prognosis group (p = 0.167). Of the prognostic factors analysed only histological grade was associated with DFS (p<0,0005) in multivariate analysis. Conclusion: The histological grade had a higher prognostic utility than the gene-expression signature in our cohort of untreated nodenegative breast cancer patients.