Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez, A.
Right arrow Articles by Piris, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez, A.
Right arrow Articles by Piris, M. A.
Clinical Cancer Research Vol. 10, 6796-6806, October 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Variability in the Degree of Expression of Phosphorylated I{kappa}B{alpha} in Chronic Lymphocytic Leukemia Cases With Nodal Involvement

Antonia Rodríguez1, Nerea Martínez1, Francisca I. Camacho1, Elena Ruíz-Ballesteros2, Patrocinio Algara2, Juan-Fernando García1, Javier Menárguez3, Tomás Alvaro5, Manuel F. Fresno6, Fernando Solano4, Manuela Mollejo2, Carmen Martin1 and Miguel A. Piris1

1 Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; 2 Department of Genetics and Pathology, Hospital Virgen de la Salud, Toledo, Spain; 3 Department of Pathology, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 4 Department of Hematology, Hospital Nuestra Señora del Prado, Talavera de la Reina, Toledo, Spain; 5 Department of Pathology, Hospital Verge de la Cinta, Tortosa, Spain; and 6 Department of Pathology, Hospital Central de Asturias, Oviedo, Spain


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Based on previous preliminary observations, we hypothesize that the molecular and clinical variability of chronic lymphocytic leukemia (CLL) reflects differences in the degree of nuclear factor (NF)-{kappa}B activation, as determined by the expression of phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}).

Experimental Design: The expression profile (mRNA and protein expression) was analyzed with the Centro Nacional de Investigaciones Oncológicas Oncochip, a cDNA microarray containing 6386 cancer-related genes, and a tissue microarray (TMA). The results were correlated with the IgVH mutational status, ZAP-70 expression, cytogenetic alterations, and clinical outcome.

Results: We found correlations between the presence of p-I{kappa}B{alpha}, a surrogate marker of NF-{kappa}B activation, and changes in the expression profile (mRNA and protein expression) and clinical outcome in a series of CLL cases with lymph node involvement. Activation of NF-{kappa}B, as determined by the expression of p-I{kappa}B{alpha}, was associated with the expression of a set of genes comprising key genes involved in the control of B-cell receptor signaling, signal transduction, and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, PIK3CD, and others. Cases with increased expression of p-I{kappa}B{alpha} showed longer overall survival than cases with lower expression. A Cox regression model was derived to estimate some parameters of prognostic interest: IgVH mutational status, ZAP-70, and p-I{kappa}B{alpha} expression. The multivariate analysis disclosed p-I{kappa}B{alpha} and ZAP-70 expression as independent prognostic factors of survival.

Conclusions: A variable degree of activation of NF-{kappa}B, as determined by the expression of p-I{kappa}B{alpha}, is an identifiable event in CLL, and is correlated with changes in the expression profile and overall survival.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a lymphoproliferative disorder characterized by the slow proliferation and accumulation of monoclonal mature CD5-positive B lymphocytes in peripheral blood, bone marrow, lymph nodes, and other tissues. Ninety percent of chronic lymphocytic leukemias are CLLs, and this is the most frequent adult leukemia in Western countries.

Although CLL is a disease that is considered to be incurable with currently available therapy, its clinical course is heterogeneous; some patients have a more stable disease and die after many years from unrelated causes, whereas others progress 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 clinical staging systems (1 , 2) , specific cytogenetic [11q and 17p deletions, 12 trisomy, 13q14 deletion (3) ], molecular events [IgVH mutational index (4, 5, 6) ], and the expression level of CD38 and ZAP70 (5, 6, 7, 8, 9, 10, 11) have been found to be associated with different survival probabilities.

The nuclear factor (NF)-{kappa}B protein family, p105/p50, p100/p52, Rel A (p65), c-Rel, and Rel B play an important role in differentiation and survival in normal B cells and presumably also in neoplastic B cells. NF-{kappa}B proteins are present in the cytoplasm bound to I{kappa}B{alpha} proteins, which are inhibitory molecules that sequester NF-{kappa}B dimers in the cytoplasm. After B-cell activation and subsequent phosphorylation, ubiquitination and proteasome degradation of I{kappa}B{alpha}, NF-{kappa}B translocate to the nucleus, making possible the transcription of its target genes.

NF-{kappa}B activation after induction by CD40-CD154 ligation has been described in CLL cells (12) and is correlated with Bcl-2 expression (13) . The presence of activated NF-{kappa}B has been shown to be activated by cytokines (14) and regulated by APRIL and BAFF genes (15) . Electrophoretic mobility shift analysis studies have confirmed the presence of NF-{kappa}B activation in a subset of CLL cases and have also revealed a subset of cases lacking this (16) . Nevertheless, the real incidence of this phenomenon and its precise features have yet to be fully elucidated.

Our previous preliminary observations led us to hypothesize that the clinical variability of CLL reflects, at least partially, differences in the degree of NF-{kappa}B activation of CLL, a cardinal feature in most lymphoid malignancies. Here we have investigated the correlation of the presence of phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}), a marker of NF-{kappa}B activation (17 , 18) , with changes in the expression profile and clinical outcome of CLL cases.

To achieve this, we used the Centro Nacional de Investigaciones Oncológicas (CNIO) Oncochip, a cDNA microarray, containing 6386 cancer-related genes, and a tissue microarray (TMA), containing 96 cores corresponding to 38 cases and controls. To facilitate simultaneous analysis using a cDNA microarray and a TMA, we restricted the analysis to CLL cases with nodal involvement, selected for being at Rai stage ≥ 1. This meant that we could also use frozen, unmanipulated diagnostic lymph nodes, the NF-{kappa}B activation status of which more closely reflects the in vivo status. These more advanced CLL cases are of additional interest because they require further clinical surveillance and early treatment and because auxiliary prognostic variables could be identified.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
Cases were selected for study based on the availability of frozen lymph node tissue at the moment of diagnosis of CLL. Frozen samples were supplied by the Hospital Virgen de la Salud, Toledo; Hospital Gregorio Marañón, Madrid; Hospital Doce de Octubre, Madrid; Hospital Central de Asturias, Oviedo; Hospital Verge de la Cinta, Tortosa; and the Tumor Bank Network of the CNIO. Diagnosis of CLL was based on the National Cancer Institute-sponsored Working Group guidelines and the World Health Organization Classification criteria (19 , 20) .

Lymph node biopsies were done to assess CLL involvement at diagnosis. Lymph node tissue was frozen and paraffin-embedded following standard procedures. Expert pathologists did blind evaluations of morphology and immunohistochemistry previous to entering into the study. Frozen reactive lymph node samples were used as controls.

Forty-one cases were found to be suitable for study in accordance with the established criteria. Clinical data were obtained from medical records. The series comprised 25 males and 16 females. The mean age at diagnosis was 67 years, with only two patients under 50 (37 and 39) years of age. At the moment of diagnosis, all patients had lymph node involvement, in addition to peripheral blood lymphocytosis corresponding to a stage ≥1 on the Rai scoring system. According to the Binet classification, 10 cases (24%) were stage A, 23 (56%) cases were stage B, five (12%) cases were stage C, and three (7%) cases could not be assigned a category. The average follow-up after diagnosis was 52 months, with a maximum of 132 months. Criteria to consider progressive disease and to determine need for treatment were based on NCI guidelines (20) . First-line treatment was Chlorambucil ± Prednisone in 19 patients, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like regimen in seven patients and nucleotide analogous-based therapy in four patients. Their mean survival-free disease time was 30 months (12–68 months). Progression with or without treatment occurred in 28 cases, with a mean and median progression-free survival of 30 and 39 months, respectively. The mean overall survival (OS) in this series was 52 months (median, 46 months). This was shorter than that of other series, perhaps because of the disease being more advanced at the time of its diagnosis.

In most cases, CLL was considered the main cause of death, although four patients developed secondary gastrointestinal and breast carcinoma.

Tissue Microarray and Immunohistochemistry.
A TMA block was constructed with a Tissue Arrayer device (Beecher Instruments, MD) with two 1-mm-diameter cylinders from two 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 and controls. Antibodies against Syk (Santa Cruz, CA), Lyn (Santa Cruz Biotechnology), BclII (DAKO, Copenhagen, Denmark), p-I{kappa}B{alpha} (Cell Signaling, Beverly, MA), Ki67 (DAKO), FLIP (Abcam, Cambridge, United Kingdom), and IgM (DAKO) were used to determine protein expression. The immunohistochemical protocol was carried out as described previously (21) .

All hybridized TMAs were scanned with the BLISS system (Bacus Laboratories, Inc, Lombard, IL), which uses a three charge-coupled device chip for the three colors red, green, and blue sensor optically coupled to a microscope. Tissue microarrays were quantitatively scored on a scale of 0 to 100 with TMAscore v.1.0 image analysis software (Bacus Laboratories, Inc., Lombard, IL), which uses WebSlide v.1.0 (Bacus Laboratories, Inc.) virtual slides.

ZAP-70 expression (Cell Signaling) was classified as positive or negative after visual inspection and comparison with the level of expression of reactive T cells.

Microarray Assays.
RNA isolation and amplification for microarray experiments were performed as described previously (22) . Universal Human Reference RNA (Stratagene) was used as a reference. From each amplified RNA product, 2.5 µg was labeled with cyanine 3-conjugated dUTP (Cy3) in the case of the samples and with cyanine 5-conjugated dUTP (Cy5) in the case of the reference RNA.

Hybridizations were done in the CNIO OncoChip (22) . Hybridized slides were scanned with the Scanarray 5000 XL (GSI Lumonics Kanata, Ontario, Canada) or with the Agilent G2565AA Microarray Scanner System (Agilent, Palo Alto, CA), and images were analyzed with the GenePix 4.0 program (Axon Instruments Inc., Union City, CA).

To adjust for differences in the red and green labeling, Cy3/Cy5 ratios were normalized with the Diagnosis and Normalization of Microarray Data (DNMAD) tool based on standard print-tip loess.7

To organize patterns on a symmetrical scale, ratios were log-transformed (base 2), and duplicated spots on the OncoChip were averaged to the median. Only genes that had data present in >70% of cases were considered in the subsequent analysis. To obtain the expression profile of each tumor we referred the ratios of the tumors to the mean of ratios of reactive lymph nodes from four different patients.

Cluster analysis was done with the Gene Cluster program (23) . Student’s t test was used to identify genes differentially expressed in the classes defined by p-I{kappa}B{alpha} expression.8

Definition of Functional Signatures.
All genes printed on the CNIO Oncochip array were analyzed with the Gene Ontology-based application FATIGO, which is part of the GEPAS suite (24) .9 Genes that remained unclassified by FATIGO were sought in the GENECARDS database10 and biological functions assigned. Genes were grouped in different categories modified in accordance with published observations about pathogenesis of B-cell lymphoma as NF-{kappa}B, B-cell receptor (BCR) signaling and lymph node-homing categories. Functional signatures for cell growth and maintenance (806 clones), apoptosis (146 clones), NF-{kappa}B pathway (167 clones), BCR signaling (54 clones), cell communication (808 clones), lymph node-homing (13 clones) and metabolism (1409 clones) were included here.

VH Gene Analysis.
Rearranged IgVH genes were amplified with a semi-nested PCR method following standard published procedures (25) . A mixture of six framework 1 (FR1) VH family-specific primers and two consensus primers for the JH gene in the first round of PCR and JH internal primers in the second round were used.

The direct-sequencing procedure was done with an ABI PRISM 310 or 3700 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany) following the manufacturer’s procedure. Mutations were identified by comparison with the germline sequence (immunoglobulin BLAST11 and V BASE sequence directories12 ).

A sequence was considered mutated when there was <98% homology with the closest germline VH genes.

Fluorescence In situ Hybridization Analysis.
Fluorescence in situ hybridization analysis was done with LSI p53/LSI ATM and LSI D13S319/LSI 13q34/CEP 12 Multi-color Probe Sets, from Vysis (Downers Grove, IL). LSI p53 (17p13.1) covers the entire p53 gene, LSI D13S319, detects the 13q14.3 region, LSI 13q34 detects a region near the subtelomere of chromosome 13q, and CEP 12 detects the {alpha} satellite, centromeric region of chromosome 12. LSI ATM detects the 11q22.3 region of chromosome 11, which includes the entire ataxia telangiectasia-mutated gene (ATM).

Hybridizations on the tissue microarray were done as described in Moreno-Bueno et al. (26) . The cutoff value for deletions was taken as the mean plus three times the SD of the proportion of cells with one signal in the three normal non-tumoral samples. This value corresponded to 25% of cells. Tumor samples with >25% of cells with one signal were considered to be subject to genomic loss involving the targeted genomic region. The cutoff value for trisomy was established with the same three normal nontumoral samples. Gain or amplification was defined as the presence (in >5% of tumor cells) of three distinctive signals.

Statistical Analysis.
Relationships between the levels of expression of the different markers were explored with the Pearson correlation. The significance of associations between characteristics was determined with Fisher’s exact test.

The mean expression for each value was compared to identify genes discriminating between groups established on the basis of the presence or absence of a condition by Student’s t test.13 The associated P value is presented as the adjusted P value derived from procedures that control the false discovery rate. Genes with adjusted P values using False Discovery Rate (FDR)_indep <0.2 are shown.

Cases were divided into two groups with values of p-I{kappa}B{alpha} greater than and less than the median of the series, respectively.

Cox proportional hazard models were used to evaluate the possible associations between OS and a number of variables. OS curves were derived with the Kaplan-Meier method. Statistical significance of associations between individual variables and OS was determined with the log-rank test.

Statistical analyses were done with the SPSS (SPSS Inc., Chicago, IL) program and with tools for random permutation tests.14


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tissue Microarray Data.
Captured images of expression of Syk, Lyn, Bcl2, p-I{kappa}B{alpha}, Ki67, c-FLIP, and IgM are shown in Fig. 1Citation . Cases were divided into two groups with respect to the median value of protein detection in the whole series (Table 1)Citation . For p-I{kappa}B{alpha}, this threshold indeed separates two groups exhibiting distinct low and high levels of expression.



View larger version (128K):
[in this window]
[in a new window]
  		Fig. 1. Immunohistochemical staining in the TMA. The two columns on the left show negative 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 magnifications of x4 and x40, respectively).

 

View this table:
[in this window]
[in a new window]
  		Table 1 Description of the expression of the markers analyzed in the TMA

 
Levels of expression for the markers showed that p-I{kappa}B{alpha} expression was closely correlated with the level of expression of Syk, Lyn, and Bcl2. Details of the results are given in Table 2Citation . Other significant associations are also described in this table.


View this table:
[in this window]
[in a new window]
  		Table 2 Correlation between markers analyzed in the TMA

 
cDNA Microarray Data.
Hierarchical clustering (Stanford University Cluster) of 41 CLL samples and four reactive lymph nodes analyzed by cDNA microarrays revealed relative homogeneity of all of the CLL cases. Reactive lymph node cases were grouped together (Fig. 2)Citation . The reproducibility of the technique was confirmed by repeating the hybridization of four samples and finding that parallel hybridizations clustered together (supplementary information).



View larger version (75K):
[in this window]
[in a new window]
  		Fig. 2. Hierarchical clustering of the CLL series, showing the relative homogeneity of this series. Red indicates high-level expression, and green indicates low-level expression.

 
Two-fold over- or underexpressed clones with respect to reactive lymph nodes in at least 50% of CLL cases were considered to make up the CLL signature. This includes 213 genes, comprising 114 down-regulated and 99 up-regulated genes. Genes were grouped according to functional signatures, paying particular attention to the functional clusters of NF-{kappa}B, BCR signaling and lymph-node homing. The results of the expression profiling of these gene sets are detailed in Tables 3Citation and 4Citation .


View this table:
[in this window]
[in a new window]
  		Table 3 CLL signature genes divided into functional groups

 

View this table:
[in this window]
[in a new window]
  		Table 4 Expression-profiling genes upregulated in NF-{kappa}B and BCR groups

 
Relevant up-regulated genes and proteins related with BCR- and NF-{kappa}B-signaling included LYN, BTK, CCR7, BCL2, BAFF and other genes. CCR7, a well characterized NF-{kappa}B target (27) , is a major receptor providing the triggering signals for B-cell entry into lymph nodes (28) , which is consistent with the fact that the CLL cells analyzed here were derived from involved lymph nodes.

Lyn is a member of the Src-family protein tyrosine kinases that, along with Blk and Fyn, play an essential role in pre-B-cell receptor-mediated NF-{kappa}B activation and B-cell development (29) .

Btk has been shown to couple I{kappa}B kinase, I{kappa}B{alpha}, and NF-{kappa}B to the BCR, linking these key pathways (30) ; additionally, BTK directly regulates bcl-x expression by transcriptional control in response to BCR engagement (31) .

Increased Bcl2 expression was also observed in this series, confirming previous observations (32) that linked Bcl2 expression to NF-{kappa}B activation in CLL cells (33) .

BAFF, a member of the tumor necrosis factor superfamily is involved in normal and leukemic B-cell survival and differentiation. BAFF binds to B cells and through the B-cell co-receptor complex promotes NF-{kappa}B activation (15 , 34) .

Genes found to be up-regulated in the lymph node-homing group included CCR7 and SELL. SELL (L-selectin, CD62L) is a cell surface adhesion protein that mediates the adherence of lymphocytes to endothelial cells of high endothelial venules in peripheral lymph nodes, the expression of which was found to be increased in a subset of CLL patients (32) . Interestingly, loss of L-selectin from the cell surface inhibited the migration of activated lymphocytes to lymph nodes and resulted in their preferential migration to the spleen (35) .

ZAP-70 Expression.
ZAP-70 could be evaluated in 35 cases of the complete series. Expression was positive in 20 (57%) cases and negative in the remaining 15 (43%) cases. In the light of recent results suggesting that this expression is a surrogate of IgVH mutational status (8 , 10 , 36) , we investigated this correlation in our series. We found that 15 of the 20 positive cases, but only three of the15 negative cases, belonged to the unmutated group. This association is significant (P < 0.05).

Mutational Status.
DNA was obtained from 40 cases of the complete series. IgVH gene rearrangement was identified in 36 patients. Forty-seven sequences were amplified, with double or triple rearrangement in seven and two cases, respectively. Only four of the nine multiple rearrangements showed differences in mutational status with respect to VH family usage. The spectrum of VH family usage was as follows: VH 1 and VH 3 both in 16 cases, VH 4 in 11 cases, VH 2 in 2 cases, and VH 5 and VH 6 both in 1 case. Distribution of different subfamily usage was similar to the described, with a more frequent representation of VH1.69 (10 sequences) (37 , 38) . Taking a >2% variability with respect to the germline VH sequence, 18 IgVH sequences from 17 patients were considered as mutated, and 29 sequences from 19 patients had a low mutational index. Percentages of mutated (48%) and unmutated (52%) cases were calculated based on 36 rearrangements. All cases with VH1.69 subfamily usage were unmutated.

FISH Analysis.
To identify chromosomal abnormalities and assess their involvement in the findings in this series, we did FISH analysis on TMA cores, thereby investigating deletions in 11q22 (ATM), 17p13 (p53), 13q14 regions, and 12 trisomy. The distribution of chromosomal abnormalities was as follows: 11q22 deletion was present in five cases of 32 assessable cores (15%); only one case featured deletion of 17p13 out of 30 assessable cores (3%); 13q14 deletion was found in four of 28 assessable cores (14%); finally, only one of the 32 assessable cases was positive for trisomy of chromosome 12 (3%). Two cases had double chromosomal abnormalities, both with 11q22 and 13q14 deletions. All cases with 11q22 deletion were from the high-level p-I{kappa}B{alpha} expression group.

Correlation with p-I{kappa}B{alpha}.
CLL cases were divided into two groups according to the median value (2.63) of p-I{kappa}B{alpha} protein detection in the whole series. Employing previously defined functional signatures, Student’s t test was used to identify genes that were differentially expressed in these two groups. In total, 894 genes with the best adjusted P value (see Material and Methods) were differentially expressed (Fig. 3A and B)Citation Citation . The most characteristic genes of the high-level p-I{kappa}B{alpha} expression group are listed in Tables 5Citation and 6Citation .



View larger version (81K):
[in this window]
[in a new window]
  		Fig. 3. Student’s t test analysis. A, differentially expressed genes identified in cases with high versus low levels of expression of phosphorylated I{kappa}B{alpha}. B, detail showing the most closely associated genes with highest P value within each group. Red indicates high-level expression, green indicates low-level expression, and missing values are in gray.

 


View larger version (66K):
[in this window]
[in a new window]
  		Fig. 3A. Continued

 

View this table:
[in this window]
[in a new window]
  		Table 5 Differentially expressed genes in cases with high p-I{kappa}B{alpha} expression

 

View this table:
[in this window]
[in a new window]
  		Table 6 NF-{kappa}B genes associated with high level of p-I{kappa}B{alpha} expression

 
Among the genes to be deregulated in the cases with NF-{kappa}B activation, some of the most noticeable were PIK3CD, LYN, IRAK1, IGF1, FAF1, BAFF, RBBP2, HDAC6, CDC16, PMSCL2, PPAP2B, SELL, and MALT1.

PIK3CD codes for the p110{delta} catalytic subunit of the phosphoinositide 3-kinase (PI3K) family, preferentially expressed by leukocytes, which plays a role, after antigen-receptor triggering and SYK activation, in protein kinase C-ß- and AKT/PKB-dependent B-cell survival (39) .

IGF1 and its receptor, IGF1-R, are key regulators of cell growth and development. After activation, they induce a cascade of tyrosine phosphorylations, leading to the eventual activation of Akt and up-regulation of the antiapoptotic protein Bcl-xL (40) .

FAF1 is a member of the death-inducing signaling complex in Fas-mediated apoptosis and has also been shown to be able to play a role as an NF-{kappa}B activity suppressor through cytoplasmic retention of p65, via physical interaction (41) .

BAFF expression was greater in cases with NF-{kappa}B activation, thereby confirming the proposed role of BAFF as a promoter of NF-{kappa}B activation in leukemic B-cells (15 , 34) .

It is of particular note that the increased expression of RBBP2 and HDAC6, a binding partner for NF-{kappa}B p50 and p65 (42) , associates NF-{kappa}B activation in CLL with histone acetylation.

In contrast, cases with a low level of NF-{kappa}B activation express CUL3, PTH, CARD12, TRAF3, MADH4, GAD2, DNAI1, UBE2D3, GTF2B, HEAB, POL2RG, G3BP, and ARG1. These findings suggest abnormalities in the ubiquitination and protein degradation in this group of cases.

Associations of p-I{kappa}B{alpha} expression with VH mutational status and chromosomal abnormalities were sought using Fisher’s exact test. There was no substantial relationship between the level of p-I{kappa}B{alpha} expression and IgVH mutational index, although we observed that high levels of p-I{kappa}B{alpha} were associated with 11q22 (ATM) deletion (P < 0.05).

Survival Analysis.
Using the Kaplan-Meier method to assess the prognostic importance of p-I{kappa}B{alpha} in the OS of CLL, we found that cases with a high level of expression had better survival probability than cases with a low level of expression (P < 0.05) (Fig. 4)Citation . The five-year OS probability was 40 and 60% in the low- and high-level p-I{kappa}B{alpha} expression groups, respectively.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Kaplan-Meier curve showing association between p-I{kappa}B{alpha} expression and OS.

 
Kaplan-Meier analysis of IgVH mutational status revealed a trend toward longer survival in the mutated group. No relation was found between chromosomal abnormalities and OS in this series.

A Cox proportional hazards regression model was derived to estimate a number of variables of potential prognostic interest, such as IgVH mutational status, ZAP-70, and p-I{kappa}B{alpha} expression. This multivariate analysis showed ZAP-70 expression and p-I{kappa}B{alpha} to be independent prognostic factors of survival (P < 0.01; Table 7Citation ).


View this table:
[in this window]
[in a new window]
  		Table 7 Multivariate Cox hazard ratios of Zap-70 and p-I{kappa}B{alpha} expression

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results from our study show that activation of NF-{kappa}B, as determined by the expression of p-I{kappa}B{alpha}, is a relatively frequent phenomenon in CLL with lymph node involvement. The intensity of this expression parallels NF-{kappa}B activation and oscillates along the series. Additionally, the expression of p-I{kappa}B{alpha} is linked with the expression of a large set of key genes involved in the control of B-cell signaling, signal transduction and apoptosis.

CLL cells are capable of responding to external cell proliferation signals, such as microenvironmental stimuli fostering CD40 stimulation (43) , and anti-apoptotic signals after antigen-receptor triggering. BCR complex is formed by surface IgM that is non-covalently associated with CD79a/CD79b signal transduction units. Following sIg cross-linking, it activates the BCR signaling pathway downstream. In normal B-lymphocytes, this leads to proliferation, differentiation, apoptosis, survival, and immune tolerance, depending on the B-cell developmental stage and cell environment. Molecules downstream of BCR include members of the Src kinase family (Lyn, Fyn, and Blk; ref. 29 ), the Tec kinase family (Btk; ref. 44 ), Syk tyrosine kinase (45 , 46) , members of the protein kinase C family of serine-threonine kinases (29) , PI3K; ref. 39 ), the mitogen-activated protein kinase family (45) , the antiapoptotic NF-{kappa}B complex (29) , and ZAP-70 (47) in the case of CLL cells. Activation of B-cell co-receptor complex (CD21, CD19 and CD81) by the BAFF gene (34) and tumor necrosis factor receptor CD40-CD154 interactions amplify BCR signaling (48) .

This study, including the mRNA and protein expression level of a majority of these genes, suggests the possible significance of BCR signaling in the activation of NF-{kappa}B and highlights the expression of a selected number of genes playing a critical role in this process, including SYK, IRAK1, and PI3K. Interestingly, this phenomenon seems to involve preferentially a group of CLL cases, thus further increasing the biological heterogeneity of CLL.

IRAK1 is a key TRAF6-interacting protein that mediates NF-{kappa}B activation downstream of tumor necrosis factor and Toll/IL-1R-containing receptors (49) . In fact, in this CLL series, the expression of both IRAK1 and IL1 appears to be closely related to that of phosphorylated I{kappa}B{alpha}.

This study also underlines the relevance of the PI3K pathway in the molecular pathogenesis of CLL cells. Although the molecular details of the recruitment, activation, and signaling of PI3Ks by the BCR are not fully understood, it is clear that they are at least partially regulated by BCR- and Syk-dependent phosphorylation of CD19 and other B-cell PI3K adaptor molecules (39) . Here, the results highlight a simultaneous expression of a large set of the key molecules in this process in NF-{kappa}B activated CLL cases, including SYK, IRAK1, IL1, PIK3CD, and PIK3CB. Studies at the protein level have confirmed these findings; these observations also extend to the expression of BclII and Lyn.

In this study, Bcl2 protein expression level was closely related with p-I{kappa}B{alpha}, Syk, and other molecules involved in the BCR signaling that leads to NF-{kappa}B activation. Indeed, the high level of BclII protein expression is routinely observed in the diagnosis of small lymphocytic neoplasms and may be seen in bone marrow and lymph node examinations. Thus, increased Bcl2 protein expression is a common and substantial finding in the major types of small lymphocytic neoplasms, including mantle cell lymphoma, follicular lymphoma, and CLL. The routes leading to Bcl2 expression, nevertheless, differ in distinct neoplasms (21) .

Variation of NF-{kappa}B activation in CLL is not an entirely novel observation, because electrophoretic mobility shift analysis data showing variability in tumoral cells and cell lines have been already reported (16) . Phosphorylated I{kappa}B{alpha} has been used as a surrogate for the activation of NF-{kappa}B, because it constitutes a key step in the release of NF-{kappa}B subunits and eventual nuclear translocation (17 , 18) . Moreover, a variable response to BCR signaling in CLL cells has been reported previously (33 , 50 , 51) and linked to the presence of decreased levels of functionally relevant adhesion molecules, cell-signaling receptors, and 11q22-q23 deletion (16) . Gene expression profiling studies (52 , 53) have also showed the presence of an IgVH mutational status gene signature, composed of genes that have various expressions among mutated and unmutated cases, thus underlining the relevance of BCR signaling to the generation of the cellular expression profile.

The data from this study reveal the variable degree of expression of p-IkB{alpha}, a surrogate of NF-{kappa}B activation, at least in CLL cases with nodal involvement, linking this heterogeneity with the expression of genes involved in the BCR signaling and NF-{kappa}B activation. Whether this finding is also relevant in CLL cases with early clinical stages remains to be clarified.

If this finding is confirmed in larger CLL series with early or advanced stages, p-I{kappa}B{alpha} expression could be added to the list of biological variables predicting clinical behavior that already includes IgVH somatic mutation, ZAP-70 expression, and other cytogenetic alterations. Data here obtained do not provide an explanation for the more adverse clinical course in cases lacking NF-{kappa}B activation; a reason for this phenomenon would be given by the presence of alternative cytogenetic or molecular alterations. This contrasts with the situation observed in multiple myeloma, where an increased NF-{kappa}B activity is associated with enhanced tumor cell survival (54) .

The study has also identified a large set of molecules that could be functionally assayed in the future with the aim of identifying therapeutic targets.


   ACKNOWLEDGMENTS
 
We thank the CNIO Tumor Bank for providing most of the samples analyzed. The authors acknowledge the technical assistance of the Genomics and Histology Units and the Molecular Pathology Program. We also thank Juan C. Cigudosa for carrying out FISH analysis and Ramon Diaz for statistical advice.


   FOOTNOTES
 
Grant support: This study was supported by grants from the Comunidad Autónoma de Madrid (CAM 08.1/0011/2001.1), the Ministerio de Sanidad y Consumo (FIS 01–0035), and the Ministerio de Ciencia y Tecnología (SAF 2001–0060), Spain. F. Camacho is supported by an Olivia Roddom grant from the Spanish Association for Cancer Research (AECC). A. Rodríguez is supported by a grant from the Ministerio de Sanidad y Consumo (FIS 02–9355).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article can be found at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org)

Requests for reprints: Miguel Angel Piris, Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, C/Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Phone: 34-91-224-69-00; Fax: 34-91-224-69-23; E-mail: mapiris{at}cnio.es

7 http://dnmad.bioinfo.cnio.es. Back

8 http://bioinfo.cnio.es/cgi-bin/tools/multest/multest.cgi. Back

9 http://fatigo.bioinfo.cnio.es. Back

10 http://bioinformatics.weizmann.ac.il/cards. Back

11 http://www.ncbi.nlm.nih.gov/igblast. Back

12 http://www.mrc-cpe.cam.ac.uk/vbase-ok. Back

13 http://bioinfo.cnio.es/cgi-bin/tools/multest/multest.cgi. Back

14 http://bioinformatica.cnio.es/. Back

Received 4/19/04; revised 7/ 2/04; accepted 7/ 9/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Binet JL, Lepoprier M, Dighiero G, et al A clinical staging system for chronic lymphocytic leukemia: prognostic significance. Cancer (Phila) 1977;40:855-64.
  2. Rai KR, Montserrat E Prognostic factors in chronic lymphocytic leukemia. Semin Hematol 1987;24:252-6.[Medline]
  3. Dohner H, Stilgenbauer S, Benner A, et al Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000;343:1910-6.[Abstract/Free Full Text]
  4. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-54.[Abstract/Free Full Text]
  5. Hamblin TJ, Orchard JA, Ibbotson RE, et al CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99:1023-9.[Abstract/Free Full Text]
  6. Damle RN, Wasil T, Fais F, et al Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840-7.[Abstract/Free Full Text]
  7. Ibrahim S, Keating M, Do KA, et al CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001;98:181-6.[Abstract/Free Full Text]
  8. Wiestner A, Rosenwald A, Barry TS, et al ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 2003;101:4944-51.[Abstract/Free Full Text]
  9. Krober A, Seiler T, Benner A, et al V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002;100:1410-6.[Abstract/Free Full Text]
  10. Crespo M, Bosch F, Villamor N, et al ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-75.[Abstract/Free Full Text]
  11. Durig J, Nuckel H, Cremer M, et al ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003;17:2426-34.[CrossRef][Medline]
  12. Furman RR, Asgary Z, Mascarenhas JO, Liou HC, Schattner EJ Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells. J Immunol 2000;164:2200-6.[Abstract/Free Full Text]
  13. Viatour P, Bentires-Alj M, Chariot A, et al NF- kappa B2/p100 induces Bcl-2 expression. Leukemia 2003;17:1349-56.[CrossRef][Medline]
  14. Zaninoni A, Imperiali FG, Pasquini C, Zanella A, Barcellini W Cytokine modulation of nuclear factor-kappaB activity in B-chronic lymphocytic leukemia. Exp Hematol 2003;31:185-90.[CrossRef][Medline]
  15. Kern C, Cornuel JF, Billard C, et al Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. Blood 2004;103:679-88.[Abstract/Free Full Text]
  16. Sembries S, Pahl H, Stilgenbauer S, Dohner H, Schriever F Reduced expression of adhesion molecules and cell signaling receptors by chronic lymphocytic leukemia cells with 11q deletion. Blood 1999;93:624-31.[Abstract/Free Full Text]
  17. Sun Q, Matta H, Chaudhary PM The human herpes virus 8-encoded viral FLICE inhibitory protein protects against growth factor withdrawal-induced apoptosis via NF-kappa B activation. Blood 2003;101:1956-61.[Abstract/Free Full Text]
  18. Liu L, Eby MT, Rathore N, Sinha SK, Kumar A, Chaudhary PM The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex. J Biol Chem 2002;277:13745-51.[Abstract/Free Full Text]
  19. Jaffe ES, Harris NL, Stein H, Vardiman JW Pathology and genetics of tumours of haematopoietic and lymphoid tissues. World health organization classification of tumours 2001 IARC Press Lyon, Fance
  20. Cheson BD, Bennett JM, Grever M, et al National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:4990-7.[Free Full Text]
  21. Martinez N, Camacho FI, Algara P, et al The molecular signature of mantle cell lymphoma reveals multiple signals favoring cell survival. Cancer Res 2003;63:8226-32.[Abstract/Free Full Text]
  22. Tracey L, Villuendas R, Ortiz P, et al Identification of genes involved in resistance to interferon-alpha in cutaneous T-cell lymphoma. Am J Pathol 2002;161:1825-37.[Abstract/Free Full Text]
  23. Eisen MB, Spellman PT, Brown PO, Botstein D Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 1998;95:14863-8.[Abstract/Free Full Text]
  24. Herrero J, Al-Shahrour F, Diaz-Uriarte R, et al GEPAS: a web-based resource for microarray gene expression data analysis. Nucleic Acids Res 2003;31:3461-7.[Abstract/Free Full Text]
  25. Algara P, Mateo MS, Sanchez-Beato M, et al Analysis of the IgV(H) somatic mutations in splenic marginal zone lymphoma defines a group of unmutated cases with frequent 7q deletion and adverse clinical course. Blood 2002;99:1299-304.[Abstract/Free Full Text]
  26. Moreno-Bueno G, Sanchez-Estevez C, Cassia R, et al Differential gene expression profile in endometrioid and nonendometrioid endometrial carcinoma: STK15 is frequently overexpressed and amplified in nonendometrioid carcinomas. Cancer Res 2003;63:5697-702.[Abstract/Free Full Text]
  27. Mathas S, Lietz A, Janz M, et al Inhibition of NF-kappaB essentially contributes to arsenic-induced apoptosis. Blood 2003;102:1028-34.[Abstract/Free Full Text]
  28. Okada T, Ngo VN, Ekland EH, et al Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J Exp Med 2002;196:65-75.[Abstract/Free Full Text]
  29. Saijo K, Schmedt C, Su IH, et al Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development. Nat Immunol 2003;4:274-9.[CrossRef][Medline]
  30. Petro JB, Khan WN Phospholipase C-gamma 2 couples Bruton’s tyrosine kinase to the NF-kappaB signaling pathway in B lymphocytes. J Biol Chem 2001;276:1715-9.[Abstract/Free Full Text]
  31. Petro JB, Castro I, Lowe J, Khan WN Bruton’s tyrosine kinase targets NF-kappaB to the bcl-x promoter via a mechanism involving phospholipase C-gamma2 following B cell antigen receptor engagement. FEBS Lett 2002;532:57-60.[CrossRef][Medline]
  32. Korz C, Pscherer A, Benner A, et al Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes. Blood 2002;99:4554-61.[Abstract/Free Full Text]
  33. Bernal A Survival of leukemic B cells promoted by engagement of the antigen receptor. Blood 2001;98:3050-7.[Abstract/Free Full Text]
  34. Hase H, Kanno Y, Kojima M, et al BAFF/BLyS can potentiate B-cell selection with the B-cell co-receptor complex. Blood 2003;:
  35. Venturi GM, Tu L, Kadono T, et al Leukocyte migration is regulated by L-selectin endoproteolytic release. Immunity 2003;19:713-24.[CrossRef][Medline]
  36. Orchard JA, Ibbotson RE, Davis Z, et al ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet 2004;363:105-11.[CrossRef][Medline]
  37. Fais F, Ghiotto F, Hashimoto S, et al Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Investig 1998;102:1515-25.[Medline]
  38. Kipps TJ, Tomhave E, Pratt LF, Duffy S, Chen PP, Carson DA Developmentally restricted immunoglobulin heavy chain variable region gene expressed at high frequency in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 1989;86:5913-7.[Abstract/Free Full Text]
  39. Okkenhaug K, Vanhaesebroeck B PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol 2003;3:317-30.[CrossRef][Medline]
  40. Sekharam M, Zhao H, Sun M, et al Insulin-like growth factor 1 receptor enhances invasion and induces resistance to apoptosis of colon cancer cells through the Akt/Bcl-x(L) pathway. Cancer Res 2003;63:7708-16.[Abstract/Free Full Text]
  41. Park MY, Jang HD, Lee SY, Lee KJ, Kim E Fas associated factor 1 inhibits NF-kB activity by interfering with nuclear translocation of RelA (p65) subunit of NF-kB. J Biol Chem 2003;:
  42. Zhang W, Kone BC NF-kappaB inhibits transcription of the H(+)-K(+)-ATPase alpha(2)-subunit gene: role of histone deacetylases. Am J Physiol Renal Physiol 2002;283:F904-11.[Abstract/Free Full Text]
  43. Granziero L, Ghia P, Circosta P, et al Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001;97:2777-83.[Abstract/Free Full Text]
  44. Khan WN Regulation of B lymphocyte development and activation by Bruton’s tyrosine kinase. Immunol Res 2001;23:147-56.[CrossRef][Medline]
  45. Kawauchi K, Ogasawara T, Yasuyama M Activation of extracellular signal-regulated kinase through B-cell antigen receptor in B-cell chronic lymphocytic leukemia. Int J Hematol 2002;75:508-13.[Medline]
  46. Lanham S, Hamblin T, Oscier D, Ibbotson R, Stevenson F, Packham G Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia. Blood 2003;101:1087-93.[Abstract/Free Full Text]
  47. Chen L, Widhopf G, Huynh L, et al Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood 2002;100:4609-14.[Abstract/Free Full Text]
  48. Haxhinasto SA, Bishop GA Synergistic B cell activation by CD40 and the B cell antigen receptor: Role of BCR-mediated kinase activation and TRAF regulation. J Biol Chem 2003;:
  49. Suzuki N, Suzuki S, Duncan GS, et al Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature (Lond) 2002;416:750-6.[CrossRef][Medline]
  50. Zupo S, Cutrona G, Mangiola M, Ferrarini M Role of surface IgM and IgD on survival of the cells from B-cell chronic lymphocytic leukemia. Blood 2002;99:2277-8.[Free Full Text]
  51. Hivroz C, Geny B, Brouet JC, Grillot-Courvalin C Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C. J Immunol 1990;144:2351-8.[Abstract]
  52. Rosenwald A, Alizadeh AA, Widhopf G, et al Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med 2001;194:1639-47.[Abstract/Free Full Text]
  53. Klein U, Tu Y, Stolovitzky GA, et al Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 2001;194:1625-38.[Abstract/Free Full Text]
  54. Berenson JR, Ma HM, Vescio R The role of nuclear factor-kappaB in the biology and treatment of multiple myeloma. Semin Oncol 2001;28:626-33.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
K. Lin, M. A. Glenn, R. J. Harris, A. D. Duckworth, S. Dennett, J. C. Cawley, M. Zuzel, and J. R. Slupsky
c-Abl Expression in Chronic Lymphocytic Leukemia Cells: Clinical and Therapeutic Implications.
Cancer Res., August 1, 2006; 66(15): 7801 - 7809.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. Ruiz-Ballesteros, M. Mollejo, A. Rodriguez, F. I. Camacho, P. Algara, N. Martinez, M. Pollan, A. Sanchez-Aguilera, J. Menarguez, E. Campo, et al.
Splenic marginal zone lymphoma: proposal of new diagnostic and prognostic markers identified after tissue and cDNA microarray analysis
Blood, September 1, 2005; 106(5): 1831 - 1838.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez, A.
Right arrow Articles by Piris, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez, A.
Right arrow Articles by Piris, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research