6796 Vol. 10, 6796–6806, October 15, 2004 Clinical Cancer Research Featured Article Variability in the Degree of Expression of Phosphorylated I␬B␣ in Chronic Lymphocytic Leukemia Cases With Nodal Involvement Antonia Rodrı´guez,1 Nerea Martı´nez,1 changes in the expression profile (mRNA and protein ex- Francisca I. Camacho,1 Elena Ruı´z-Ballesteros,2 pression) and clinical outcome in a series of CLL cases with 2 1 lymph node involvement. Activation of NF-␬B, as deter- Patrocinio Algara, Juan-Fernando Garcı´a, ␬ ␣ 3 5 mined by the expression of p-I B , was associated with the Javier Mena´rguez, Toma´s Alvaro, expression of a set of genes comprising key genes involved in 6 4 Manuel F. Fresno, Fernando Solano, the control of B-cell receptor signaling, signal transduction, Manuela Mollejo,2 Carmen Martin,1 and and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, Miguel A. Piris1 PIK3CD, and others. Cases with increased expression of ␬ ␣ 1Molecular Pathology Program, Centro Nacional de Investigaciones p-I B showed longer overall survival than cases with lower Oncolo´gicas, Madrid, Spain; 2Department of Genetics and Pathology, expression. A Cox regression model was derived to estimate 3 Hospital Virgen de la Salud, Toledo, Spain; Department of some parameters of prognostic interest: IgVH mutational Pathology, Hospital General Universitario Gregorio Maran˜o´n, ␬ ␣ 4 status, ZAP-70, and p-I B expression. The multivariate Madrid, Spain; Department of Hematology, Hospital Nuestra Sen˜ora analysis disclosed p-I␬B␣ and ZAP-70 expression as inde- del Prado, Talavera de la Reina, Toledo, Spain; 5Department of Pathology, Hospital Verge de la Cinta, Tortosa, Spain; pendent prognostic factors of survival. and 6Department of Pathology, Hospital Central de Asturias, Conclusions: A variable degree of activation of NF-␬B, Oviedo, Spain as determined by the expression of p-I␬B␣, is an identifiable event in CLL, and is correlated with changes in the expres- sion profile and overall survival. ABSTRACT Purpose: Based on previous preliminary observations, we hypothesize that the molecular and clinical variability of INTRODUCTION chronic lymphocytic leukemia (CLL) reflects differences in Chronic lymphocytic leukemia/small lymphocytic lym- the degree of nuclear factor (NF)-␬B activation, as deter- phoma (CLL/SLL) is a lymphoproliferative disorder character- mined by the expression of phosphorylated I␬B␣ (p-I␬B␣). ized by the slow proliferation and accumulation of monoclonal Experimental Design: The expression profile (mRNA mature CD5-positive B lymphocytes in peripheral blood, bone and protein expression) was analyzed with the Centro Na- marrow, lymph nodes, and other tissues. Ninety percent of cional de Investigaciones Oncolo´gicas Oncochip, a cDNA chronic lymphocytic leukemias are CLLs, and this is the most microarray containing 6386 cancer-related genes, and a tis- frequent adult leukemia in Western countries. sue microarray (TMA). The results were correlated with the Although CLL is a disease that is considered to be incur- IgVH mutational status, ZAP-70 expression, cytogenetic al- able with currently available therapy, its clinical course is het- terations, and clinical outcome. erogeneous; some patients have a more stable disease and die Results: We found correlations between the presence of after many years from unrelated causes, whereas others progress p-I␬B␣, a surrogate marker of NF-␬B activation, and very quickly and die within a few years. This variability has stimulated the search for prognostic markers with which to predict the outcome of patients and to allow treatments to be adapted to the specific risk. In addition to the Rai and Binet Received 4/19/04; revised 7/2/04; accepted 7/9/04. clinical staging systems (1, 2), specific cytogenetic [11q and 17p Grant support: This study was supported by grants from the Comu- deletions, 12 trisomy, 13q14 deletion (3)], molecular events nidad Auto´noma de Madrid (CAM 08.1/0011/2001.1), the Ministerio de [IgVH mutational index (4–6)], and the expression level of Sanidad y Consumo (FIS 01–0035), and the Ministerio de Ciencia y CD38 and ZAP70 (5–11) have been found to be associated with Tecnologı´a (SAF 2001–0060), Spain. F. Camacho is supported by an Olivia Roddom grant from the Spanish Association for Cancer Research different survival probabilities. (AECC). A. Rodrı´guez is supported by a grant from the Ministerio de The nuclear factor (NF)-␬B protein family, p105/p50, Sanidad y Consumo (FIS 02–9355). p100/p52, Rel A (p65), c-Rel, and Rel B play an important role The costs of publication of this article were defrayed in part by the in differentiation and survival in normal B cells and presumably payment of page charges. This article must therefore be hereby marked also in neoplastic B cells. NF-␬B proteins are present in the advertisement in accordance with 18 U.S.C. Section 1734 solely to ␬ ␣ indicate this fact. cytoplasm bound to I B proteins, which are inhibitory mole- Note: Supplementary data for this article can be found at Clinical cules that sequester NF-␬B dimers in the cytoplasm. After Cancer Research Online (http://clincancerres.aacrjournals.org) B-cell activation and subsequent phosphorylation, ubiquitina- Requests for reprints: Miguel Angel Piris, Molecular Pathology Pro- tion and proteasome degradation of I␬B␣, NF-␬B translocate to gram, Centro Nacional de Investigaciones Oncolo´gicas, C/Melchor Fer- na´ndez Almagro 3, E-28029 Madrid, Spain. Phone: 34-91-224-69-00; the nucleus, making possible the transcription of its target genes. Fax: 34-91-224-69-23; E-mail: [email protected]. NF-␬B activation after induction by CD40-CD154 ligation ©2004 American Association for Cancer Research. has been described in CLL cells (12) and is correlated with Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 6797 Bcl-2 expression (13). The presence of activated NF-␬B has feature in most lymphoid malignancies. Here we have investi- been shown to be activated by cytokines (14) and regulated by gated the correlation of the presence of phosphorylated I␬B␣ APRIL and BAFF genes (15). Electrophoretic mobility shift (p-I␬B␣), a marker of NF-␬B activation (17, 18), with changes analysis studies have confirmed the presence of NF-␬B activa- in the expression profile and clinical outcome of CLL cases. tion in a subset of CLL cases and have also revealed a subset of To achieve this, we used the Centro Nacional de Investi- cases lacking this (16). Nevertheless, the real incidence of this gaciones Oncolo´gicas (CNIO) Oncochip, a cDNA microarray, phenomenon and its precise features have yet to be fully eluci- containing 6386 cancer-related genes, and a tissue microarray dated. (TMA), containing 96 cores corresponding to 38 cases and Our previous preliminary observations led us to hypothe- controls. To facilitate simultaneous analysis using a cDNA size that the clinical variability of CLL reflects, at least partially, microarray and a TMA, we restricted the analysis to CLL cases differences in the degree of NF-␬B activation of CLL, a cardinal with nodal involvement, selected for being at Rai stage Ն 1. Fig. 1 Immunohistochemical staining in the TMA. The two columns on the left show nega- tive or low-level expression. Positive or strong expression for each marker is shown in the right-hand columns. A low- and high-power field is illustrated for each stain (original magni- fications of ϫ4 and ϫ40, re- spectively). Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. 6798 Differences in NF-␬B Activation in CLL Table 1 Description of the expression of the markers analyzed in mean survival-free disease time was 30 months (12–68 months). the TMA Progression with or without treatment occurred in 28 cases, with a Median Range Visual evaluation mean and median progression-free survival of 30 and 39 months, Syk 27.25 0.31–94.96 Strongly heterogeneous cytoplasmic respectively. The mean overall survival (OS) in this series was 52 expression. months (median, 46 months). This was shorter than that of other Lyn 63.05 20.46–82.61 Cytoplasmic expression, moderate series, perhaps because of the disease being more advanced at the variability. time of its diagnosis. Bcl2 54.86 17.35–77.89 Cytoplasmic, strong expression. p-I␬B␣ 2.63 0.01–65.43 Cytoplasmic and nuclear In most cases, CLL was considered the main cause of expression, highly variable. Two death, although four patients developed secondary gastrointes- major groups defined. tinal and breast carcinoma. Ki67 1.79 0.02–9.3 Nuclear expression, stronger in Tissue Microarray and Immunohistochemistry. A proliferation centers. c-Flip 0.02 0–1.24 Surface expression, low intensity. TMA block was constructed with a Tissue Arrayer device (Beecher IgM 51.76 0.26–82.63 Variable cytoplasmic-surface Instruments, MD) with two 1-mm-diameter cylinders from two expression. selected areas of each case and from appropriate controls. Sample cores were obtained from paraffin-embedded lymph node blocks corresponding to the initial diagnosis, which were available in 38 samples of the study. Paraffin sections were examined to select the involved area. The resulting TMA contained 96 cores from cases This meant that we could also use frozen, unmanipulated diag- and controls. Antibodies against Syk (Santa Cruz, CA), Lyn (Santa nostic lymph nodes, the NF-␬B activation status of which more Cruz Biotechnology), BclII (DAKO, Copenhagen, Denmark), closely reflects the in vivo status. These more advanced CLL p-I␬B␣ (Cell Signaling, Beverly, MA), Ki67 (DAKO), FLIP (Ab- cases are of additional interest because they require further cam, Cambridge, United Kingdom), and IgM (DAKO) were used clinical surveillance and early treatment and because auxiliary to determine protein expression. The immunohistochemical proto- prognostic variables could be identified.