Hemoglobin overexpression and splice signature as new features of inflammatory breast cancer?

INTRODUCTION: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. OBJECTIVES: The objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. METHODS: W e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. RESULTS: A 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p<10-7). This splice signature was associated with poor metastasis-free survival in hormone receptor-negative non-IBC (p=0.02), but had no prognostic value in IBC. PAM analysis of dysregulated genes in IBC compared to non-IBC identified a 10-gene signature highly predictive of IBC phenotype and conferring a poor prognosis in non-IBC. The genes most commonly upregulated in IBC were 3 hemoglobin genes able to reliably discriminate IBC from non-IBC (p<10-4). Hb protein expression in epithelial breast tumor cells was confirmed by immunohistochemistry. CONCLUSION: IBC has a specific spliced transcript profile and is characterized by hemoglobin gene overexpression that should be investigated in further functional studies.

Clinical value of R-spondins in triple-negative and metaplastic breast cancers.

BACKGROUND: RSPO ligands, activators of the Wnt/ß-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). METHODS: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT-PCR. The effect of RSPO on the Wnt/ß-catenin pathway activity was determined by luciferase assay, western blotting, and qRT-PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/ß-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. RESULTS: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10-4). RSPO2 and RSPO4 stimulate Wnt/ß-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. CONCLUSIONS: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Estrogen-Receptor, Progesterone-Receptor and HER2 Status Determination in Invasive Breast Cancer. Concordance between Immuno-Histochemistry and MapQuant™ Microarray Based Assay.

BACKGROUND: Hormone receptor status and HER2 status are of critical interest in determining the prognosis of breast cancer patients. Their status is routinely assessed by immunohistochemistry (IHC). However, it is subject to intra-laboratory and inter-laboratory variability. The aim of our study was to compare the estrogen receptor, progesterone receptor and HER2 status as determined by the MapQuant™ test to the routine immuno-histochemical tests in early stage invasive breast cancer in a large comprehensive cancer center. PATIENTS AND METHODS: We retrospectively studied 163 invasive early-stage breast carcinoma with standard IHC status. The genomic status was determined using the MapQuant™ test providing the genomic grade index. RESULTS: We found only 4 tumours out of 161 (2.5%) with discrepant IHC and genomic results concerning ER status. The concordance rate between the two methods was 97.5% and the Cohen's Kappa coefficient was 0.89. Comparison between the MapQuant™ PR status and the PR IHC status gave more discrepancies. The concordance rate between the two methods was 91.4% and the Cohen's Kappa coefficient was 0.74. The HER2 MapQuant™ test was classified as « undetermined ¼ in 2 out of 163 cases (1.2%). One HER2 IHC-negative tumour was found positive with a high HER2 MapQuant™ genomic score. The concordance rate between the two methods was 99.3% and the Cohen's Kappa coefficient was 0.86. CONCLUSION: Our results show that the MapQuant™ assay, based on mRNA expression assay, provides an objective and quantitative assessment of Estrogen receptor, Progesterone receptor and HER2 status in invasive breast cancer.

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.

Analysis of correlation between mitotic index, MIB1 score and S-phase fraction as proliferation markers in invasive breast carcinoma. Methodological aspects and prognostic value in a series of 257 cases.

BACKGROUND: The study was designed in order to evaluate the degree of correlation of mitotic index (MI), Ki67 (MIB1) score and S-phase fraction (SPF) as markers of cell proliferation and prognosis in breast cancer. MATERIALS AND METHODS: The series analysed corresponded to 257 consecutive invasive breast carcinoma, treated at the Institut Curie, France, in 1995. Nottingham histological grade and MIB1 semiquantitative and quantitative score were assessed on histological sections, whereas SPF was calculated using flow cytometry analysis of fine-needle aspiration products. Proliferation indices were compared to pathological data and to overall survival (OS) and disease-free survival (DFS) (minimum follow-up: 72 months). RESULTS: The median values for the proliferation markers were 9/10 HPF for MI, 32.4% for MIB1 and 3.7% for SPF. A high rate of correlation (r=0.96; p<0.001) was observed between semi-quantitative and quantitative MIBI evaluation. A positive correlation was found between the three markers (r ranging from 0.54 to 0.61;p<0.001). Univariate analysis of markers associated to disease outcome showed that MIB1, axillary node status (N) and progesterone receptor (PR) status were significantly associated with OS and that MIB1 and SPF were associated with DFS, together with node and hormone receptor status. In multivariate analysis, when proliferation markers were adjusted on the N and PR status, only MIB1 retained a prognostic value for OS (RR= 1.83) [1.00;3.35] and SPF for DFS (RR= 1.58) [1.02-2.44] (p=0.04). CONCLUSION: A good level of correlation was observed between the values of the three markers of tumour cell proliferation analysed. In this series of invasive breast cancers, MIB1 immunostaining was found to be a prognostic marker of both OS and DFS. The median (32.4%) was a valuable cut-off value for prognostic assessment. Semi-quantitative and quantitative evaluations provided very similar values. MIB1 can thus be considered as a reliable prognostic maker, usable in small size tissue specimens which are inappropriate for MI or SPF analysis. The impact of MIB1 compared to that of the other proliferative markers will be further assessed in a subgroup of T1N0M0 for which the prognostic assessment is of major interest.

Increased expression of transforming growth factor-beta after cerebral ischemia in the baboon: an endogenous marker of neuronal stress?

There has been an increasing interest in recent years in the evaluation of the neuronal and glial responses to ischemic insult. Some cytokines, including transforming growth factor-beta (TGF-beta), that are overexpressed after experimental stroke in rodents are thought to be implicated in the neuronal processes that lead to necrosis. Thus, such cytokines could predict tissue fate after stroke in humans, although data are currently sparse for gyrencephalic species. The current study addressed the expression pattern of TGF-beta1 in a nonhuman primate model of middle cerebral artery occlusion. Focal permanent ischemia was induced for 1 or 7 days in 6 baboons and the following investigations were undertaken: cerebral oxygen metabolism (CMRO2) positron emission tomography studies, magnetic resonance imaging, postmortem histology, and reverse transcription-polymerase chain reaction. The aim of the current study was to correlate the expression of TGF-beta1 to the underlying metabolic and histologic state of the threatened cerebral parenchyma. The authors evidenced increased TGF-beta1 mRNA levels (up to 25-fold) in those regions displaying a moderate (20% to 49%) reduction in CMRO2. The current findings suggest that the greatly enhanced expression of TGF-beta1 in the penumbral zones that surround tissue destined to infarction may represent a robust index of potentially salvageable brain. The current investigation, in the nonhuman primate, strengthens the authors' hypothesis, derived from rodent models, that TGF-beta1 may be involved in the physiopathology of human stroke.