ESR1, PGR, ERBB2, and MKi67 mRNA expression in postmenopausal women with hormone receptor-positive early breast cancer: results from ABCSG Trial 6.

BACKGROUND: The purpose of this study was to assess the concordance of real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection of ESR1, PGR, ERBB2, and MKi67 messenger RNA (mRNA) in breast cancer tissues with central immunohistochemistry (IHC) in women treated within the prospective, randomized Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 6. PATIENTS AND METHODS: We evaluated ESR1, PGR, ERBB2, and MKi67 mRNA expression by Xpert® Breast Cancer STRAT4 (enables cartridge-based RT-qPCR detection of mRNA in formalin-fixed paraffin-embedded tissues) and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 protein expression by IHC [in situ hybridization (ISH) for HER2 IHC 2+] in 1115 surgical formalin-fixed paraffin-embedded specimens from patients of ABCSG Trial 6. Overall percent agreement (concordance), positive percent agreement (sensitivity), and negative percent agreement (specificity) between STRAT4 and IHC were determined for each marker. The primary objective of the study was concordance between STRAT4 mRNA measurements of ESR1, PGR, ERBB2, and MKi67 with central reference laboratory IHC (and ISH for HER2 IHC 2+ cases). Time to distant recurrence was analyzed by Cox models. RESULTS: All performance targets for ER, PR, and Ki67 were met. For HER2, the negative percent agreement target but not the positive percent agreement target was met. Concordance between STRAT4 and IHC was 98.9% for ER, 89.9% for PR, 98.2% for HER2, and 84.8% for Ki67 (excluding intermediate IHC 10%-20% staining). In univariable and multivariable Cox regression analyses, all four biomarkers tested by either STRAT4 RT-qPCR or by central IHC (ISH) had a comparable time to distant recurrence indicating similar prognostic value. CONCLUSIONS: With the exception of HER2, we demonstrate high concordance between centrally assessed IHC and mRNA measurements of ER, PR, and Ki67 as well as a high correlation of the two methods with clinical outcome. Thus, mRNA-based assessment by STRAT4 is a promising new tool for diagnostic and therapeutic decisions in breast cancer.

A clinical validation study of MammaPrint in hormone receptor-positive breast cancer from the Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG-8) biomarker cohort.

BACKGROUND: MammaPrint is a prognostic assay based on gene expression in tumors from patients with early breast cancer. MammaPrint has been extensively validated and Food and Drug Administration cleared in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue. We aimed to assess its prognostic performance in the biomarker cohort of the Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG-8) patient population, and to obtain a higher level of evidence with regard to its clinical validity after RNA extraction from FFPE biobank tissue. PATIENTS AND METHODS: A prespecified retrospective analysis to test the prognostic performance of the MammaPrint test to predict distant recurrence-free survival at 5 and 10 years as primary end point was carried out. MammaPrint risk, clinicopathological factors (after central pathological review), and clinical risk (using a modified version of Adjuvant! Online) were evaluated by Cox regression analyses. RESULTS: From 1347 available samples, 607 (45%) failed quality control after RNA extraction. In total, 658 (49%) patients were included in survival analyses: MammaPrint low risk versus high risk is a significant prognostic factor for distant recurrence-free survival at 5 years (94.0% versus 91.6%) with a significant risk reduction of 6.5% at 10 years (log-rank P value = 0.017, low risk 91.3% versus high risk 84.8%). The multivariable models suggest that hazard ratio (HR) is primarily driven by tumor stage (5-year HR 3.89; confidence interval 1.97-7.71) and nodal status (5-year HR 1.73; confidence interval 0.91-3.21). After adjustment for clinical risk groups, MammaPrint HRs remain stable with values just below 2.0 after the first 3 years. CONCLUSIONS: The MammaPrint test showed significant prognostic performance at 5 and 10 years of follow-up. In the particular cohort of ABCSG-8, the statistical independence from clinically assessed covariates remains unclear, and no conclusions concerning the clinical validity of the test can be drawn.

[Pericarditis as the initial manifestation of retroperitoneal fibrosis--a case report]. / Perikarditis als Erstmanifestation einer Retroperitonealfibrose--ein Fallbericht.

HISTORY AND CLINICAL FINDINGS: A 70-year-old patient with invasive ductal breast cancer underwent conserving surgery of the right breast and right axillary dissection as well as postoperative irradiation therapy. Five months later, she presented with dyspnoea and progressive weakness. INVESTIGATIONS: Clinically, the patient showed anasarca and petechial hemorrhages, laboratory tests revealed thrombopenia, hepatic dysfunction, radiologic investigations showed enlargement of the liver and spleen, effusions of the pleura and pericardium, and ascite. Echocardiography showed pericardial effusion without cardiac tamponade. TREATMENT AND COURSE: Despite supportive therapy the patient's performance status deteriorated significantly, the diagnosis of the underlying disease could not be established, the patient died with the clinical signs of cardiovascular failure. Autopsy revealed progressive retroperitoneal fibrosis with systemic involvement of pleura, pericardium, epicardium, myocardium, lungs, and kidneys and pericarditis. Retrospectively clinical symptoms were interpreted as right heart insufficiency due to pericardial effusion. CONCLUSION: This case report reminds of occurrence of manyfold clinic manifestations of retroperitoneal fibrosis in dependence of particular organic involvement and that retroperitoneal fibrosis represents a differential diagnosis.

Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression.

The transactivation/transformation-domain associated protein (TRRAP) belongs to the Ataxia-telangiectasia mutated (ATM) super-family and has been identified as a cofactor for c-MYC-mediated oncogenic transformation. TRRAP and its yeast homolog (Tra1p) are components of histone acetyltransferase (HAT) complexes, SAGA (refs. 2,4,5), PCAF (ref. 3) and NuA4 (ref. 6), which are important for the regulation of transcription and cell cycle progression and also have a role in cell viability. Yet the biological function of this molecule and how it controls proliferation are still unclear. Here we show that null mutation of Trrap in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts. We use an inducible Cre-loxP system to show that loss of Trrap blocks cell proliferation because of aberrant mitotic exit accompanied by cytokinesis failure and endoreduplication. Trrap-deficient cells fail to sustain mitotic arrest despite chromosome missegregation and disrupted spindles, and display compromised cdk1 activity. Trrap is therefore essential for early development and required for the mitotic checkpoint and normal cell cycle progression.

Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures.

We investigated the effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on paracellular intestinal Ca2+ absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and bidirectional transepithelial 45Ca2+ fluxes in confluent Caco-2 cell cultures. The rise of TEER to steady-state levels of approximately 2,000 omega.cm2 was significantly attenuated by 1,25(OH)2D3 (by up to 50%) in a dose-dependent fashion between 10(-11) and 10(-8) M. Synthetic analogs of 1,25(OH)2D3, namely, 1 alpha,25-dihydroxy-16-ene,23-yne-vitamin D3 and 1 alpha,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency, whereas their genomically inactive 1-deoxy congeners were only marginally effective. Enhancement of transepithelial conductance of Caco-2 cell monolayers by vitamin D was accompanied by a significant increase in bidirectional transepithelial 45Ca2+ fluxes. Although 1,25(OH)2D3 also induced cellular 45Ca2+ uptake from the apical aspect of Caco-2 cell layers and upregulated the expression of calbindin-9kDa mRNA, no significant contribution of the Ca(2+)-adenosinetriphosphatase-mediated transcellular pathway to transepithelial Ca2+ transport could be detected. Therefore stimulation of Ca2+ fluxes across confluent Caco-2 cells very likely results from a genomic effect of vitamin D sterols on assembly and permeability of tight-junctional complexes.

Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of receptor expression.

The human colon adenocarcinoma-derived cell line Caco-2 was used as a model system to study the interaction of epidermal growth factors (EGF) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in control of colorectal cancer cell growth. The mitogenic stimulus of EGF was rapidly transduced via apical and basal membrane receptors alike into elevation of c-myc expression, causing a shift of Caco-2 cells from the G0/G1 into the S phase of the cell cycle. The stimulatory effect of EGF on cell division was effectively counteracted by 1,25(OH)2D3: the presence of the steroid hormone prevents the negative effect of EGF on vitamin D receptor abundance and concurrently minimises ligand-occupied EGF receptor numbers on both sides of Caco-2 cell monolayers. Our data suggest that EGF and 1,25-(OH)2D3 actions on mutual receptor levels represent a specific feature of the potent antimitogenic effect of the steroid hormone on colon cancer cells.

TNFalpha-mediated cell death is independent of cdc25A.

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.

Phenotypic heterogeneity of osteoblast-like MC3T3-E1 cells: changes of bradykinin-induced prostaglandin E2 production during osteoblast maturation.

We have examined clonal murine calvarial MC3T3-E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin-induced production of PGE2). It was found that the sublines investigated showed large variation of the above-mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above-mentioned agents. The most striking difference was observed with bradykinin-induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of 3H-bradykinin to its receptor and also correlated well with the bradykinin-induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast-like cell line MC3T3-E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Genes related to growth and invasiveness are repressed by sodium butyrate in ovarian carcinoma cells.

Down-regulation of oncogene expression is one of the hallmarks of the process whereby transformed cells are forced into differentiation and/or growth arrest by potent inducers and therefore can represent an interim end point in cancer treatment. The differentiation inducer sodium butyrate (NaB) arrested growth of N.1 ovarian carcinoma cells and repressed expression of cyclin D1/prad1 and the invasiveness-related protease plasminogen activator-urokinase (plau). This was accompanied by the acquisition of a differentiated morphology, all of which characteristics were maintained as long as N.1 cells were exposed to the inducer. In accordance with a differentiated phenotype was the finding that fibronectin expression was increased significantly. Recently, it was shown that NaB represses the transcription factor c-myc by blocking Ca2+ signals and modulating serine threonine kinase activity. We wanted to investigate NaB-mediated interference on signals contributing to the expression on prad1, plau and growth arrest-specific 6 (gas6). Protein kinase A (PKA) inactivation de-repressed prad1 and plau transcript levels. NaB had onlygeneral but no specific influence on PKA-modulated prad1 and plau expression however. Protein kinase C activation up-regulated plau transcript levels, but not that of prad1. Prad1 expression seemed to depend on Ca2+-triggered signals. Constitutive plau expression was insensitive to additional Ca2+-mediated signals, but it became responsive upon NaB treatment.

IL-1 and TNF-alpha but no IL-2 expression is found in squamous cell carcinomas of the head and neck by RT-PCR.

We studied the expression of IL-1 (Interleukin 1), IL-2, IL-4, IL-10, GM-CSF (granulocyte/macrophage colony stimulating factor), TNF-alpha (tumor necrosis factor alpha) and IFN-gamma (Interferon gamma) in tumor specimens and adjacent mucosa from 12 patients with squamous cell carcinoma of the head and neck (SCCHN) by reverse-transcribed polymerase chain reaction (RT-PCR). No IL-2 and IL-4 expression was detected in any of the tumor specimens. High level expression of IL-1 and TNF-alpha was found in all 12 samples analyzed. Expression of GM-CSF was detected in 7 (58%) of 12 tumors, IL-10 was found in 3 (25%) and IFN-gamma in 6 (50%) of the samples. No qualitative differences in the cyotokine expression pattern were found between normal and tumor tissue. Our data indicate that there is a lack of cytokines typical for a specific, T-cell mediated immune response in SCCHN. A number of cytokines which are required for the initial steps of immune response however are produced. These results might have implications for future immuno-chemotherapeutical strategies.

Growth control of human colon-adenocarcinoma-derived Caco-2 cells by vitamin-D compounds and extracellular calcium in vitro: relation to c-myc-oncogene and vitamin-D-receptor expression.

The human colon-cancer cell line Caco-2, though of malignant origin, is still able to express the c-myc proto-oncogene in a regulable fashion. Transition from the logarithmic growth phase into the quiescent, i.e., confluent state, is accompanied by a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a concomitant reduction of c-myc mRNA and of nuclear association of c-myc protein. Conversely, growth stimulation by lowering extracellular [Ca++]0 to 0.25 mM results in up-regulation of c-myc expression levels and consequently inhibition of re-entry of Caco-2 cells into the G0/G1 phase. In contrast, regulation of c-myc in Caco-2 cells is completely resistant to vitamin-D sterols, since the anti-mitogenic action of I alpha, 25-dihdroxyvitamin D3 (I alpha, 25(OH)2D3) and of 2 synthetic analogs, I alpha, 25(OH)2-16-ene-23-yne-D3 and I alpha, 25(OH)2-26, 27-F6-16-ene-23-yne-D3, occurred independently of any change in c-myc mRNA and nuclear protein levels. Although the antiproliferative effect of the vitamin-D sterols requires high-affinity binding to the cytoplasmic vitamin-D receptor (VDR), vitamin-D sterols have no effect on VDR mRNA levels in Caco-2 cells. However, VDR mRNA expression changed in an antiparallel fashion to c-myc regulation upon transition between different growth states. This suggests that VDR mRNA abundance could nevertheless be important for vitamin-D-related c-myc-independent growth control in Caco-2 cells.

Growth regulation of human colon adenocarcinoma-derived cells by calcium, vitamin D and epidermal growth factor.

The Caco-2 cell line was utilized to analyze the role of nutrient factors such as calcium, vitamin D and epidermal growth factor (EGF) in epigenetic control of human colon carcinoma cell growth. Proliferative signals from either low extracellular calcium or EGF, respectively, are transduced in Caco-2 cells via an increase in c-myc proto-oncogene mRNA and nuclear protein expression levels. Activation of the EGF receptor is associated also with down-regulation of the cytoplasmic high-affinity vitamin D receptor (VDR). This would allow colon carcinoma cells to escape from the VDR-mediated anti-mitogenic action of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). However, Caco-2 cells have the unique property to synthesize the vitamin D hormone from 25-hydroxyvitamin D3. 1 alpha,25(OH)2D3, in turn, counteracts the negative effect of EGF on VDR abundancy and slow down tumor cell proliferation through a c-myc-independent pathway.

Retinoic acid induced death of ovarian carcinoma cells correlates with c-myc stimulation.

In the ovarian adenocarcinoma subline N.I, all-trans retinoic acid (ATRA) induced substantial cell death. This response was elicited only at decreased serum levels. Exposure of N.I cells to increasing concentrations of ATRA was accompanied by a considerable up-regulation of c-myc transcript levels that correlated with the rate of cell killing, which itself was an active process as judged by sustained transcriptional expression. ATRA-triggered rounding and detaching of single cells from the substratum was accompanied by degradation of genomic DNA. We show that the N.I model cell line, which is otherwise highly ATRA-resistant, can undergo an ATRA-triggered suicide program when serum is limited. The accompanying c-myc up-regulation seems to be mediated by retinoic-acid-receptor-independent pathways involving membrane-associated phospholipases instead, because manoalide partly suppressed c-myc induction by ATRA but left constitutive c-myc expression unaffected.

Diversity of TCRAV and TCRBV sequences used by human T-cell clones specific for a minimal epitope of Bet v 1, the major birch pollen allergen.

T-cell clones (TCC) were raised from the peripheral blood of patients suffering from tree pollen allergy. All TCC were restricted by HLA-DR molecules. In order to investigate possible intervention targets in Type I allergic diseases, we examined T-cell receptor (TCR) alpha and beta chain nucleotide sequences of five allergen-reactive human CD4+ TCC specific for a C-terminal epitope (BV 144) of Bet v 1, the major birch pollen allergen. Proliferation assays using synthetic peptides revealed the 10-mer LRAVESYLLA as minimal epitope for three TCC; two TCC also displayed reactivity with the nonapeptide LRAVESYLL. Two TCC expressed TCRBV2S3, all other BV144-specific TCC used diverse TCRAV and TCRBV gene segments. Moreover, the junctional regions encoding the third complementary determining regions (CDR3) of the TCR showed a striking heterogeneity in length and amino acid composition. Nevertheless, all TCC showed an arginine residue in the N-terminal region of their TCRBV CDR3 loops. Therefore, therapeutical strategies aimed at the clonal deletion of allergen-specific T-cell clones, providing help for IgE synthesis, will not be feasible. Our results cast a doubt on the theory that the CDR3 exclusively provides the primary contact with the peptide bound in the major histocompatibility (MHC) groove, and suggest additional interaction with MHC class II.

Familial hemophagocytic lymphohistiocytosis associated with disseminated T-cell lymphoma: a report of two siblings.

Two siblings with evidence of disseminated T-cell lymphoma at the time of diagnosis of familial hemophagocytic lymphohistiocytosis (FHL) are reported, an association which has not been described previously. The first child with typical clinical and laboratory features of FHL died shortly after admission, before diagnosis could be established. Retrospective analysis of autoptic tissue revealed marked hemophagocytosis as well as morphological and immunohistochemical features suggestive of disseminated T-cell lymphoma. In the second child, FHL was diagnosed in time. Subsequent histologic investigation of bone marrow biopsies displayed a focal infiltration by T-cell lymphoma. DNA hybridization studies provided evidence of a monoclonal T-cell receptor beta chain gene rearrangement. Following conventional chemotherapeutic induction for FHL, the patient received an allogeneic bone marrow transplant (BMT) from a related healthy donor. Currently, 17 months after BMT, the boy is in unmaintained remission from FHL and T-cell lymphoma. The current pathogenetic concepts for FHL and a possible relationship between T-cell lymphoma and FHL are discussed.

cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and localization to chromosome 3q26.3-3q28: a free left Alu Arm is inserted in the 3' noncoding region.

Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one of the four enzymes of the peroxisomal beta-oxidation pathway. Here, we report the full-length human cDNA sequence and the localization of the corresponding gene on chromosome 3q26.3-3q28. The cDNA sequence spans 3779 nucleotides with an open reading frame of 2169 nucleotides. The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present. DNA sequence comparison of the coding region showed an 80% homology between human and rat bifunctional enzyme cDNA. The 3' noncoding sequence contains 117 nucleotides homologous to an Alu repeat. Based on sequence comparison, we propose that these nucleotides are a free left Alu arm with 86% homology to the Alu-J family. RNA analysis shows one band with highest intensity in liver and kidney. This cDNA will allow in-depth studies of molecular defects in patients with defective peroxisomal bifunctional enzyme. Moreover, it will also provide a means for studying the regulation of peroxisomal beta-oxidation in humans.