Microtubule-associated protein tau correlates with estrogen receptor status but not with in vitro paclitaxel sensitivity in primary breast cancer.

BACKGROUND: Factors or signatures predicting response to chemotherapeutic agents are of great interest for breast cancer patient care. There is conflicting data regarding microtubule-associated protein tau as predictive marker of paclitaxel sensitivity. Paclitaxel plays an important role in the adjuvant and metastatic therapy of breast cancer. However, a substantial proportion of patients treated with paclitaxel do not derive benefit from this therapy. Therefore, evaluating potential predictive factors is increasingly important. The authors attempted to validate these findings in vitro utilizing the ATP tumorchemosensitivity assay (ATP-TCA). MATERIALS AND METHODS: The in vitro drug sensitivity to paclitaxel was evaluated in 48 fresh primary breast cancer specimens using the ATP-TCA. ATP-TCA results were analysed using the area under the curve (AUC) of growth inhibition. These results were correlated with the expression of tau mRNA measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Tau was also compared between patients with progesterone receptor (PgR) positive and negative and estrogen receptor (ER) positive and negative breast cancer, respectively. RESULTS: The correlation of tau with the AUC for paclitaxel was weak, Spearman Rho was -0.267 with a p-value of 0.064. As described before, multiple regression analysis confirmed T-stage (p = 0.01) and PR status (p = 0.01) as independent predictors of paclitaxel chemosensitivity. Using multiple regression analysis and defining tau mRNA expression as dependent variable estrogen receptor status as measured by immunohistochemistry was a highly significant predictor for tau mRNA expression (p < 0.001). Grade (p = 0.002) as well as PgR expression (p < 0.001) were also found to be predictors of tau mRNA expression. CON- CLUSIONS: In the present data set the authors were not able to show that MAP-tau mRNA could predict benefit from the addition of a taxane to adjuvant chemotherapy. They found that ER expression is associated with tau protein expression. Estrogen gene transcription is reported to carry weak predictive significance for endocrine sensitivity, therefore it might be worth pursuing whether, tau mRNA could possibly be a predictor for endocrine therapy response.

The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2- breast cancer patients.

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.

EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer.

BACKGROUND: In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. PATIENTS AND METHODS: We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan-Meier survival analysis. RESULTS: After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%-61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. CONCLUSION: The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer.

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.

New prognostic and predictive factors in breast cancer.

There are two major questions regarding systemic therapy of breast cancer: Firstly, which patients should be treated, and secondly, how should these patients be treated? Prognostic factors aim to foresee the outcome of patients irrespective of treatment while predictive factors intend to assess the outcome of patients receiving a certain systemic therapy and thus are intimately associated with sensitivity or resistance to therapy. Ideally, a predictive factor is also a therapeutic target as it is the case with estrogen receptor (ER) or HER-2. In order to avoid over- as well as under-treatment, it is advisable to select the appropriate treatment strategy on the basis of a careful risk assessment for each individual patient. Additionally to time-honoured clinicopathological factors additional prognostic factors like urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor 1 (PAI-1) or multiparameter gene-expression analyses have shown promising results especially in node-negative breast cancer. These multigene profiles offer new insights in breast cancer biology, like the important role of the tumor-associated immune system. ER, HER-2 and potentially newer prognostic factors like epithelial cell adhesion molecule (Ep-CAM) bridge the gap from prognosis to prediction and serve as therapeutic targets. This should allow us to quantify the risk of progression in each individual patient and tailor treatment accordingly, leading to a more personalized treatment recommendation.

Long-term outcome prediction by clinicopathological risk classification algorithms in node-negative breast cancer--comparison between Adjuvant!, St Gallen, and a novel risk algorithm used in the prospective randomized Node-Negative-Breast Cancer-3 (NNBC-3) trial.

BACKGROUND: Defining risk categories in breast cancer is of considerable clinical significance. We have developed a novel risk classification algorithm and compared its prognostic utility to the Web-based tool Adjuvant! and to the St Gallen risk classification. PATIENTS AND METHODS: After a median follow-up of 10 years, we retrospectively analyzed 410 consecutive node-negative breast cancer patients who had not received adjuvant systemic therapy. High risk was defined by any of the following criteria: (i) age <35 years, (ii) grade 3, (iii) human epithelial growth factor receptor-2 positivity, (iv) vascular invasion, (v) progesterone receptor negativity, (vi) grade 2 tumors >2 cm. All patients were also characterized using Adjuvant! and the St Gallen 2007 risk categories. We analyzed disease-free survival (DFS) and overall survival (OS). RESULTS: The Node-Negative-Breast Cancer-3 (NNBC-3) algorithm enlarged the low-risk group to 37% as compared with Adjuvant! (17%) and St Gallen (18%), respectively. In multivariate analysis, both Adjuvant! [P = 0.027, hazard ratio (HR) 3.81, 96% confidence interval (CI) 1.16-12.47] and the NNBC-3 risk classification (P = 0.049, HR 1.95, 95% CI 1.00-3.81) significantly predicted OS, but only the NNBC-3 algorithm retained its prognostic significance in multivariate analysis for DFS (P < 0.0005). CONCLUSION: The novel NNBC-3 risk algorithm is the only clinicopathological risk classification algorithm significantly predicting DFS as well as OS.

Gene expression of topoisomerase II alpha (TOP2A) by microarray analysis is highly prognostic in estrogen receptor (ER) positive breast cancer.

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 (chi(2)-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.

[Predictive and prognostic markers of breast cancer. Molecular biological analysis of fixed tumor tissue]. / Prädiktive und prognostische Brustkrebsmarker. Molekularbiologische Analyse von fixiertem Tumorgewebe als Basis.

A multitude of prognostic and predictive multiparameter algorithms based on the analysis of mRNA have been published in recent years. Many of the algorithms require fresh or fresh frozen tissue as a source of the mRNA. However, practical considerations suggest formalin-fixed paraffin-embedded tissue (FFPE tissue) to be a more suitable starting material for routine diagnostic applications. Therefore, Siemens Healthcare Diagnostics is developing a fully automated method to extract mRNA and DNA from FFPE tissue for use in pathology laboratories. Initially, the method will be used as part of a prognosis assay to predict the likelihood of distant metastasis and death for node-negative breast cancer patients.

Role of the progesterone receptor for paclitaxel resistance in primary breast cancer.

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 microg 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.

Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy.

Stress or heat shock proteins (HSPs) are the most conserved proteins present in both prokaryotes and eukaryotes. Their expression is induced in response to a wide variety of physiological and environmental insults. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and preventing their aggregation. HSPs have a dual function depending on their intracellular or extracellular location. Intracellular HSPs have a protective function. They allow the cells to survive lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several HSPs have also been demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream and downstream of the mitochondrial events. On the other hand, extracellular located or membrane-bound HSPs mediate immunological functions. They can elicit an immune response modulated either by the adaptive or innate immune system. This review will focus on HSP27, HSP70, and HSP90. We will discuss the dual role of these HSPs, protective vs. immunogenic properties, making a special emphasis in their utility as targets in cancer therapy.

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells.

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 gamma-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 gamma-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.

Inhibition of tumor growth by plasminogen-related protein-B.

BACKGROUND: Various fragments of the fibrinolytic protein plasminogen can act as antiangiogenic factors and inhibit the growth of primary and metastatic tumors in mice. Plasminogen-related gene-B encodes a putative 9 kDa protein virtually identical to the plasminogen N-terminal activation peptide, a 77-amino acid motif that is liberated from the parent plasminogen molecule during conversion to the serine proteinase plasmin. Previous data have documented enhanced transcription of plasminogen-related gene-B in neoplastic tissues. MATERIALS AND METHODS: We have tested the effects of recombinant versions of plasminogen-related protein-B and the plasminogen N-terminal activation peptide on the growth of tumors in mice, employing murine tumor cell lines implanted subcutaneously. RESULTS: The recombinant plasminogen-related protein-B significantly inhibited the growth of primary tumors in mice, while recombinant plasminogen N-terminal activation peptide elicited only a slight inhibition of tumor growth. CONCLUSION: These data suggest that plasminogen-related protein-B may have utility as a novel cancer therapeutic.

A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity.

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 (pep-scan) 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.

Homologous plasminogen N-terminal and plasminogen-related gene A and B peptides. Characterization of cDNAs and recombinant fusion proteins.

The cDNA corresponding to exons 2-4 of the processed human plasminogen (Pgn) gene, encoding the N-terminal peptide domain (NTP), has been cloned, expressed in Escherichia coli as a recombinant protein (r-NTP) containing a hexahistidine tag, and refolded to the native structure that contains two internal cystine bridges. RNA expression of the two Pgn-related genes, PRG A and PRG B, that potentially encode 9-kDa polypeptides having extensive similarity to the NTP has been investigated. Using RNA-based PCR with liver RNA as template, we demonstrate that PRG A encodes a detectable mRNA species. PRG A and PRG B have been found to be transcribed in the liver and yield virtually identical mRNAs. Neither of the PRGs are expressed in a variety of other normal tissues, as determined by Northern blot analysis. Factor-Xa digestion of the tagged r-NTP yields cleavage products which indicates that the expressed r-NTP domain of Pgn is endowed with a flexible conformation. Recombinant PRG B protein (r-PRG B) fused to a hexahistidine tag was purified and analyzed for structural integrity. Preliminary 1H-NMR spectroscopic data for r-NTP and r-PRG B indicate relatively fast amide 1H-2H exchange in 2H2O and close conformational characteristics for the two homologous polypeptides. Far ultraviolet-CD spectra for r-NTP and r-PRG B at pH 7.0 indicate similar defined secondary structure content for both domains, with 13-17% alpha-helix and 24-27% antiparallel beta-sheet. The fact that two transcriptionally active genes encode almost identical polypeptides supports the hypothesis that the Pgn NTP, together with the putative polypeptides encoded by the PRGs, may serve an important function, such as controlling the conformation of Pgn and thus its susceptibility to tissue activators.

Biochemical properties of recombinant human beta-glucuronidase synthesized in baby hamster kidney cells.

The cDNA sequence encoding human beta-glucuronidase [Oshima, Kyle, Miller, Hoffmann, Powell, Grubb, Sly, Troplak, Guise and Gravel (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 685-689] was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant in one step by use of immunoaffinity chromatography, the biochemical properties of the enzyme were examined. With a pH optimum of 4.0, a Km of 1.3 mM and thermal stability up to 68 degrees C, this protein has characteristics very similar to those described for beta-glucuronidase from human placenta [Brot, Bell and Sly (1978) Biochemistry 17, 385-391. However, the recombinant product has several structural properties not previously reported for beta-glucuronidase isolated from natural sources. First, recombinant beta-glucuronidase is synthesized as a tetramer consisting of two disulphide-linked dimers. As can be inferred from the cDNA sequence, the enzyme possesses five cysteine residues after cleavage of the signal peptide. By introducing a C-terminal truncation, we eliminated the last cysteine at position 644. In the mutant, covalent linkage between two monomers is no longer observed, indicating that Cys-644 is involved in intermolecular disulphide-bond formation. The functional role of the disulphide bond remains elusive, as it was shown that (i) intracellular transport of the mutant is not impaired and (ii) it is still able to form an enzymically active tetramer. A second feature that has not previously been observed for beta-glucuronidase from any origin is the existence of two enzymically active species for recombinant beta-glucuronidase, when examined by gel filtration on a TSK 3000 column. With apparent molecular masses of 380 kDa and 190 kDa we propose that they represent tetramers and dimers respectively. Partial N-terminal sequencing and electrophoresis under denaturing conditions revealed that the dimers consist of subunits that have been proteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall protein backbone as well as its activity are resistant to a number of proteases. Only the C-terminal portion is susceptible to protease action, and the disulphide-linked form is readily converted into non-disulphide-bonded subunits. Pulse-chase analysis shows that human beta-glucuronidase remaining intracellular in BHK cells after synthesis undergoes a similar proteolytic processing event, i.e. a reduction in mass of 3-4 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

The fate of the three subunits of major histocompatibility complex class I molecules.

At the surface of the murine T lymphoma cell line RMA-S, the expression of "empty" class I molecules can be dramatically enhanced by culture at 26 degrees C. These class I molecules are unstable following transfer to 37 degrees C unless they are loaded with exogenously added peptides. Class I heterodimers that have failed to bind peptide ("empty" class I molecules) dissociate and the class I heavy chains are degraded. Internalization, if it precedes breakdown, would be the rate-limiting step. Radioiodinated peptides (VSV NP 8-mer or Sendai NP 9-mer) dissociate from the class I molecules in the absence of exogenous peptide or beta 2 microglobulin and appear in the medium. Release of the iodinated peptides does not result in a reduction in the quantity of stable assembled class I molecules. This paradox may be explained by a more rapid off-rate for radioiodinated peptides, when compared with their unlabeled counterparts, which constitute about 99% of the total in our radiolabeled preparations. In the medium the peptide is rapidly modified by serum- and cell-derived proteases. The short half-life of empty class I molecules and of free ligand would effectively preclude sensitization of innocent bystanders for lysis by cytotoxic T lymphocytes.