Immunohistochemical analysis in a case of follicular lymphoma and its transformed counterpart.

<p>Sections from a follicular lymphoma (a) that subsequently acquired a <i>c-myc</i> translocation and transformed into a DLBCL (b) were analyzed for EBI3 expression by immunohistochemistry. The bar represents 50 µm.</p

Characteristics of mature aggressive B-cell lymphomas analyzed by immunohistochemistry.

<p>Characteristics of mature aggressive B-cell lymphomas analyzed by immunohistochemistry.</p

Immunohistochemical analysis of EBI3 expression in BL, BL/DLBCL and DLBCL.

<p>Sections from a case of BL (a), 2 cases of BL/DLBCL (b, c), and 2 cases of DLBCL (d, e) with or without <i>c-myc</i> translocation as indicated on each figure, were analyzed for EBI3 expression by immunohistochemistry. An inverse correlation between the presence of <i>c-myc</i> translocation and the positivity for EBI3 in tumoral cells was observed. Each picture is shown at the same magnification. The bar shown on (c) represents 50 µm.</p

<i>In vitro</i> effect of c-myc overexpression on <i>EBI3</i> expression level.

<p>Karpas 1106 cell line was transfected with a GFP reporter plasmid together with c-myc-ER expression vector or a control vector and cultured for various times in the absence or presence of 4-OHT. <i>EBI3</i> expression in GFP-positive cells isolated from vector control transfected cells (Col) or c-myc-ER transfected cells (c-myc-ER) was analyzed by RTqPCR. In (A), transfected cells were cultured for 20 hours in the presence of 4-OHT (200 nM). Data are expressed as mean ± SEM from 3 independent transfections. p value is indicated. In (B), cells were cultured for 20 hours in the absence or presence of 4-OHT (200 nM). In (C), cells were cultured with increasing concentrations of 4-OHT. The expression of <i>EBI3</i> in c-myc-ER transfected cells relative to that measured in control tranfected cells is represented. The percentage of decrease is indicated. A higher concentration of 4-OHT (1 µM) did not result in higher repression of <i>EBI3</i> expression (not shown). In (D), 4-OHT was added to the transfected cells for the last 16, 24 or 40 h of the culture. In (B)–(D), a representative experiment is shown.</p

Analysis of <i>EBI3</i> gene expression according to the presence or absence of <i>c-myc</i> translocation.

<p>Individual data for <i>EBI3</i> and <i>c-myc</i> were retrieved from GSE4475 and GSE10172 series <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024617#pone.0024617-Hummel1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024617#pone.0024617-Klapper1" target="_blank">[21]</a>. On (A), the expression of <i>EBI3</i> and of <i>c-myc</i> was analyzed according to the presence or not of <i>c-myc</i> translocations involving an Ig locus (Ig-myc) or not (non-Ig-myc). « others » designated lymphomas other than mBL, ie lymphomas classifed as non-mBL and lymphomas that could not be classified as mBL or non-mBL. On (B), <i>EBI3</i> expression among all lymphomas other than mBL is represented according to that of <i>c-myc</i>. On (C), <i>EBI3</i> expression among lymphomas that had initially received a pathological diagnosis of BL/DLBCL is shown according to that of <i>c-myc</i>. p values are indicated. NS: not significant.</p

Immunohistochemistry labeling results.

<p>A. 2+ score: weak-to-moderate complete membrane staining in more than 10% of tumor cells (objX40). B. 3+ score: strong complete membrane staining in more than 10% of tumour cells (objX40). C. Heterogeneous staining of a primary ovarian tumour (objX20). D: Heterogeneous staining of a metastasis (objX20) E. FISH: heterogeneous amplification of <i>HER2</i> in a tumor showing a cluster of tumor cells with amplification (white arrow, left part) and a cluster of non amplified tumor cells (orange arrow, right part). F: Clusters of red spots (<i>HER2</i> amplification) together with two green spots (centromere 17).</p

<i>HER2</i> in major published studies.

<p>A. Overexpression Review of selected articles evaluating <i>HER2</i> protein expression in large series of patients (including more than 50 tumour samples) published in international journals after 1994. Boxes represent % of <i>HER2</i> overexpression (scored as 2+ or 3+) and error bars show ±2 standard errors for each study. B. IHC and FISH status Review of selected articles evaluating <i>HER2</i> gene amplification (FISH or CISH) and/or <i>HER2</i> protein expression in large series of patients (including more than 50 tumour samples) published in international journals after 1994.<i>In situ</i> hybridisation represents FISH (fluorescence <i>in situ</i> hybridisation) and CISH (chromogenic <i>In situ</i> hybridisation) results. Mean <i>HER2</i> overexpression/amplification across studies is represented; IHC = Immunohistochemistry.</p

<i>HER2</i> gene amplification repartition according to <i>HER2</i> protein status.

<p>Perfect concordance in protein expression and gene amplification has been observed for samples with 3+ or 0/1+ IHC staining. In our study, 25% of equivocal samples (IHC 2+ staining) were amplified for <i>HER2</i> gene. Based on the HER2 reference scoring algorithm, 21 samples were considered as positive (15 scored 3+ by IHC and 6 scored 2+ validated by FISH). Three samples could not be compared by FISH due to fixation (one 3+ and two 2+ IHC scored). IHC = immunohistochemistry; FISH = fluorescence <i>in situ</i> hybridization.</p

Univariate analysis for progression-free survival and overall survival of biological and clinical parameters

<p>HR = Hazard ratio, CI = Confidence interval, * = P-value <0.05</p

Multivariate analysis of clinical parameters for progression-free survival and overall survival

<p>HR = Hazard ratio, CI = Confidence interval, * = P-value <0.05</p