Leukemia (2002) 16, 2429–2437  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Expression profiling of B cell chronic lymphocytic leukemia suggests deficient CD1- mediated immunity, polarized cytokine response, altered adhesion and increased intracellular transport and processing of leukemic cells Z Zheng1, S Venkatapathy2, G Rao2 and CA Harrington2,3

1Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA; and 2Affymetrix, Inc, Santa Clara, CA, USA

We used oligonucleotide microarrays to profile the expression are thought to derive from CD5+ expressing B cells.2,4 CD5- of chronic lymphocytic leukemia (CLL) B cells from eight expressing normal B cells are a small population of B cells patients compared with CD5-expressing normal B cells from localized at the edge of the germinal center within the mantle four donors and with pooled normal circulating B cells. Of 6790 5 examined, we identified 87 genes that were differentially zone of the secondary lymphoid follicles. They are con- expressed at least two-fold between CLL and the normal B sidered a good candidate for the normal counterpart of CLL cells. CLL cells significantly down-regulated transcripts from cells because they share several characteristics with the malig- CD1c and CD1d genes, which encode known to nant CD5+ CLL cells. These include the propensity to produce present lipid antigen and mediate innate and adaptive immun- polyreactive, low-affinity IgM autoantibodies and the ity. The expression pattern was also consistent with reduced expression of the cross-reactive idiotypes in the antibody pro- signaling by interferon gamma but increased response to 2,6–8 interleukin 4 in leukemic cells. CLL cells increased the duced. Recently, human memory B cells have been pro- expression of several -associated extracellular matrix posed as the normal counterpart of CLL-B cells.9 However, and adhesion molecules, up-regulated many genes involved in this alternative model has difficulty accounting for the above intracellular protein transport and processing, while down- features of CLL-B, and the large percentage of CLL cases with regulating genes involved in proliferation and metabolism. leukemic cells having unmutated immunoglubulin Based on the expression pattern, we propose that CLL-B cells prolong their survival through increased interaction with sur- would argue against an origin of post-germinal center stage vival factors such as IL-4, and through various mechanisms of cells such as the memory cells. The ideal normal counterpart evading the immune response, such as turning off the of CLL will likely remain a subject of discussion. At present, expression of CD1c and CD1d, reducing immunogenic the CD5+ B cell remains a reasonable candidate as the normal response to interferon gamma, inactivating in B–T inter- counterpart of CLL and has been used as control cells in many action and increasing the expression of immunoglobulin recep- studies of CLL. tors which neutralize antibody-dependent cell-mediated cyto- toxicity. One approach to understanding the biological basis of CLL Leukemia (2002) 16, 2429–2437. doi:10.1038/sj.leu.2402711 pathophysiology would be to first correlate abnormal gene Keywords: CLL; microarray; expression profiling expression with the disease phenotypes. Recent advances in microarray technology allowfor comprehensive profiling of human cancer cells. This technique has been applied success- 10,11 Introduction fully in cancer classifications and diagnosis, and in gain- ing insights into functional pathways.12–14 Although B cell chronic leukemia is the most common leukemia in the expression profiling establishes only a correlative rather than western world, with approximately 10 000 new cases diag- causative role of identified genes in the biological process of nosed each year in the United States.1 It is characterized by the disease studied, experience has shown that the unique and the relentless accumulation in the blood, marrowand lymph- valuable global views of patterns, when oid tissues of mature monoclonal B lymphocytes that express coupled with available knowledge about gene functions, pro- the CD5 surface molecule. The leukemic CLL B cell accumu- vide the basis for formulating testable hypotheses about the 13,14 lation appears to be a result of defective apoptosis rather than disease pathways. Microarray profiling of hematological uncontrolled proliferation.2 CLL patients suffer immune malignancy for the purpose of exploring disease pathways deficiencies due to impaired humoral immunity characterized instead of disease classification has been reported recently in 15 by hypogammaglobulinemia. The leukemic B cells express a study of mantle cell (MCL). However, a hetero- very lowlevels of surface IgM or IgD, and upon antigen stimu- geneous population of cells was profiled, complicating the lation, are unable to undergo somatic hypermutation and iso- interpretation of results and reducing the sensitivity of the 15 type switching. In addition, CLL patients frequently develop study. CLL cells are especially appropriate for microarray autoimmune diseases, suggesting immune dysregulation.3 analysis because of the relative ease in obtaining a purified, Unlike many forms of chronic and acute leukemia, CLL developmentally homogenous leukemic cell population in lacks recurrent reciprocal chromosomal translocations and large quantities. Recently, oligonucleotide microarray has distinctive patterns of oncogene or tumor suppressor gene been used to compare the expression patterns of CLL-B cells 9 expression. As a result, it has been difficult to identify the mol- with several normal B cell populations. That study, however, ecular events that lead to the disease phenotypes. CLL-B cells emphasized sample classification based on the overall expression pattern comparison. It did not focus on analyzing the potential roles of differentially expressed genes in the pathogenesis of CLL. Here, we report the use of oligonucleo- Correspondence: Z Zheng at the present address: Amersham Biosci- tide microarrays to compare gene expression profiles between ences, 928 E. Arques Ave, Sunnyvale, CA 94085, USA; Fax: (408) purified CLL-B cells and CD5-expressing normal B cells, and 773-8343; e.mail: [email protected] 3Present address: Vaccine and Gene Therapy Institute, Oregon Health between CLL and normal circulating B cells, as a way to ident- and Science University, Portland, OR 97006, USA ify potential molecular deregulations that may contribute to Received 17 February 2002; accepted 26 June 2002 the pathophysiology of CLL. The results of our study revealed Expression profiling of CLL Z Zheng et al 2430 that in CLL there was significant down-modulation of genes netic beads coupled with goat anti-IgG (Dynal) were involved in both protein and lipid antigen presentation. We employed, exactly as in the protocol used for the isolation of also observed reduced RNA levels for genes involved in inter- CD5+ cells from human tonsils. Based on gene expression pat- feron gamma response but increased transcript levels for IL-4 tern comparisons, no obvious difference in gene expression response genes. The profile also indicated reduced cell pro- can be attributed to the additional positive selection (see liferation and metabolism but increased intracellular protein Results and discussion below). transport and processing, as well as increased production of For additional comparison, we isolated human peripheral extracellular matrix and adhesion molecules in CLL-B cells blood B cells directly from fresh buffy coats obtained from relative to normal CD5+ B cells. The significance of these healthy donors (Stanford Blood Center, Stanford, CA, USA) abnormal gene expression patterns to CLL pathobiology is dis- using CD19-Dynabeads (Dynal). Purified B cells on beads cussed. were lysed directly in Trizol for total RNA extraction. Total RNA from seven individual buffy coats was pooled for sub- sequent Poly(A) selection. Materials and methods The use of all patient and normal samples above were according to an IRB approved protocol. Cell isolation and RNA extraction

Normal CD5+ B cells were purified from four fresh human GeneChiparray expressionanalysis + tonsils collected from routine tonsillectomy procedures. CD5 + B cells like these were routinely used in most laboratories as Poly(A) RNA was selected using Oligotex mRNA midi kits (Qiagen, Valencia, CA, USA). Double-stranded cDNA was the normal controls in the studies of CLL-B cells. The tonsil + tissues were disrupted by gentle mechanical means, and the synthesized from poly(A) RNA with the SuperScript Choice resulting cell suspension was centrifuged through Ficoll– (Gibco) and oligo(dT) primers that contained a T7 RNA Ј Hypaque to obtain mononuclear cells. T cells were removed polymerase recognition sequence at the 5 end. Approxi- ␮ by two rounds of rosetting with an excess amount of fresh, mately 1 g of cDNA was subjected to in vitro transcription in neuroaminidase-treated sheep red blood cells (SRBC). Less the presence of biotinylated UTP and CTP (Enzo Diagnostic, than 1% of the remaining cells were CD2+ after rosetting. The Farmingdale, NY, USA) using the MegaScript T7 kit (Ambion, cells were then incubated with anti-CD5 MAb (Caltag, Bur- Austin, TX, USA). The labeled cRNA was fragmented and lingame, CA, USA) for 3–5 h at 4ºC, washed thoroughly in hybridized to a set of four oligonucleotide arrays (GeneChip PBS and incubated for 12 h at 4ºC with 20-fold excess (relative Hu6000 array set; Affymetrix) overnight. The arrays were then to target cells) of the magnetic beads coated with goat anti- washed, stained with phycoerythrin–streptavidin and scanned IgG (Dynal, Lake Success, NY, USA). CD5+ B cells attached according to the manufacturer’s instructions. The array con- to the beads were then magnetically separated from the tains 6790 probe sets for 6416 genes (5223 known human remaining cell suspension. After isolation, the CD5+ B cells genes and 1193 unnamed ESTs). Expression data were ana- on beads were directly lysed with Trizol (Gibco, Carlsbad, CA, lyzed using GeneChip 3.0 software (Affymetrix, Santa Clara, USA) for total RNA extraction. CA, USA). Global scaling of intensity values was used. The Heparinized venous blood samples from eight CLL patients average intensity over all genes on a chip was adjusted to with Rai stages varying from 0 to 2 (Table 1) were obtained 116 for all arrays to compensate for array-related variations in after written informed consent. None of the CLL patients hybridization efficiency and signal acquisition. Fluorescence received chemotherapy treatment during the 6 months prior intensity levels of less than 20 units (after scaling) were con- to the blood drawn for this study. Mononuclear cells were verted to 20, since discrimination of expression belowthis level could not be performed with confidence. For each gene, obtained after centrifugation over Ficoll–Hypaque, and CLL + cells from all but two samples were negatively selected by the CLL/TB5 expression ratio is calculated from the absolute depleting T cells using two rounds of anti-CD2 coated mag- expression value of one of the eight CLL samples and one of the four tonsilar CD5+ B cell samples. The average expression netic beads (Dynal). More than 96% of the purified CLL cells + + + ratio (Avg(CLL/TB5 )) was calculated by averaging the 32 were CD19 and CD5 in the CLL samples studies. Freshly + isolated CLL cells were immediately lysed in Trizol for total possible CLL/TB5 expression ratios. RNA extraction. For two of the CLL samples (CM and C72), additional steps Northern blot analysis of anti-CD5 coating followed by positive selection using mag- One microgram of polyA+ RNA from purified tonsillar, periph- eral blood, and CLL B cells was separated on a 1% agarose Table 1 Clinical features of enrolled CLL patients gel containing 0.6 M formaldehyde, transferred on to a nylon membrane (Nytran; Schleicher and Schuell, Keene, NH, USA), Sample Age WBC Hb Platelets Rai sIg 32 (109/l) (g/dl) (109/l) stage and hybridized with DNA probes randomly primed with P- dCTP. Hybridization signals were detected using a Phospho- C725 66 17.8 13.1 159 1 kappa Imager (Molecular Dynamics, Sunnyvale, CA, USA). C1010 75 98.2 15 222 1 kappa CH 34 198 11.7 142 2 lambda C72 46 19.1 15.3 198 0 kappa Results and discussion CM 71 54 11.6 99 2 kappa C101 50 42.7 15.5 155 0 NA Quality control of samples C117 45 28.4 13 262 0 kappa C828 81 10.8 12.4 125 1 NA To investigate the molecular basis underlying the different phenotypes of normal and CLL-B cells, we profiled the

Leukemia Expression profiling of CLL Z Zheng et al 2431 expression of 6500 independent genes in freshly isolated leu- samples), using the program GeneCluster as previously kemic B cell samples from eight CLL patients (Table 1), as well described.13 The SOM algorithm, like other clustering algor- as purified control B cell samples. The control B cell samples ithms, serves to expose patterns in the gene expression data + included four tonsillar CD5 B cell samples and one pooled without using prior knowledge. The expression levels of the + peripheral blood CD19 B cell sample. 6790 probe sets represented on the GeneChip arrays were Some genes with previously characterized expression in used in the analysis. The default variation filter in the program CLL were included in the array and were used as internal con- was used to eliminate genes that did not change significantly trols. The expression levels of these genes are summarized in across samples. SOM was used to organize the remaining + Figure 1. As expected, tonsil CD5 cells and CLL cells genes into 18 clusters (Figure 2). Although these clusters were expressed high levels of the mRNA for the IgM heavy chain. generated without presumptions, they corresponded to clear Indicative of the monoclonal nature of the disease, there was biological relevance. For example cluster c7 identified 113 predominance in CLL cells of one of the two Ig chains genes that were consistently up-regulated in CLL-B cells and (lambda or kappa) expression that was consistent with avail- cluster c16 identified 148 genes consistently down-regulated able clinical phenotyping results (data not shown). CD4 in CLL. Most of the remaining clusters represent genes that expression was not detectable (mean expression intensity + were preferentially expressed in only one of the samples (same below): 22 for CLL vs 24 for tonsil CD5 cells), indicat- examined, thus capturing the unique signature pattern of the ing little or no contamination with T cells in the preparations. sample (eg cluster c1, c4 and c8). The lack of clusters rep- Expressions of CD3 and CD14 mRNA were low with similar resenting genes differentially expressed in only those two posi- levels between normal and CLL samples (CD3: 63 for CLL vs + tively selected CLL samples (C72 and CM) indicated that 75 for tonsil CD5 cells; CD14: 59 for CLL vs 55 for tonsil + under our experimental conditions, additional positive selec- CD5 cells). CD23, a molecule commonly associated with CLL,16 was highly expressed in CLL samples, while on aver- tion based on CD5 ligation of CLL cells had little effect on the age, CD23 expression in tonsillar CD5+ B cells was lower overall gene expression pattern in CLL. (2318 for CLL vs 1059 for tonsil CD5+ cells). As reported pre- viously,2 there was an increase in the level of bcl-2 mRNA, but no abnormal expression of p53 or Rb in CLL relative to normal CD5+ cells (Figure 1). The results described above indicate little degradation or contamination of our mRNA samples and demonstrate the robustness of the sample purification procedure.

Global pattern of gene expression

To identify patterns of gene expression in CLL that differed from those in normal control CD5+ B cells, we first employed a self-organizing map (SOM) algorithm to analyze the 12 samples (four tonsillar CD5+ normal control and eight CLL

Figure 2 SOM analysis of gene expression in normal and CLL-B cells. Using GeneCluster,13 the 1958 genes that passed the default variation filter were grouped into 18 clusters. Each cluster is rep- resented by the average expression pattern for genes in the cluster. Normalized averaged expression levels are shown on the y-axis, with the two lines delineating 1 s.d. off the average. The 12 samples are represented on the x-axis, with the order being (from left to right) T1, T2, T3, T62, C101, C1010, C117, C72, C725, C828, CH and CM. The first four samples from the left are the tonsillar CD5+ B cells and the remaining eight CLL samples. The number of genes in each cluster is indicated at the top center of each cluster. Note that the cluster c7 Figure 1 Expression of control genes. Expression levels of selected represents 113 genes that showed up-regulation in all CLL samples genes in four tonsilar CD5+ B samples (triangles) and eight CLL-B and cluster c16 consists of 148 genes that were down-regulated in all samples (circles) studied. Expression levels of less than 20 are con- CLL samples. Most other clusters, such as c8, consist of genes that verted to 20. were preferentially expressed in only one of the samples examined.

Leukemia Expression profiling of CLL Z Zheng et al 2432 Differentially expressed genes between CLL and compared to normal CD5+ B cells. The complete list of these normal CD5+ B cells genes is shown in Table 2. The average expression levels together with their standard deviations in tonsil and CLL To represent quantitatively the gene expression differences samples, as well as the average CLL/TB5+ ratios, are listed in between normal and CLL cells, for each gene we obtained an Table 2. Their expressions in the pooled normal B cell sample average expression ratio in each CLL sample, ie average of are also included for reference. These genes could be grouped the four possible CLL/TB5+ ratio values from the CLL and four into different functional categories (Table 2). Overall, about TB5+ expression values. We first selected for genes that one-third of differentially expressed genes appeared to be showed two-fold or more differential expression between nor- related to immunoregulation, suggesting that the immune mal and CLL cells in at least six of the eight CLL samples. deregulation was the most prominent abnormality of CLL. We then used a hierarchical clustering program (Cluster17)to Some of the genes showing the most prominent differences cluster these differentially expressed genes. Among the 248 are described below. genes used in hierarchical clustering, 56 were excluded for further analysis due to lowsignal intensity (average of CLL and average of TB5+ both Ͻ100) or inconsistent expression CD1 gene family pattern (coefficient of variation for CLL/TB5+ ratios more than 100%), leaving 192 differentially expressed genes. Northern CLL cells display the characteristics of anergic B cells that are analysis of 10 randomly selected genes from the 192 genes defective in presenting antigens and mounting antibody all confirmed the results from the GeneChip hybridization responses to antigen.18,19 We observed down-regulation of B (two shown in Figure 3). Among these 192 genes, about half cell surface molecules that are believed to be critical in T-B (104 genes) were expressed in the normal peripheral blood B cell interaction, such as CD40 and CD83.20 The costimulatory cells at a level not significantly different (less than two-fold) molecule CD27, whose expression in B cells is suppressed by to the average expression level in the eight CLL samples. CD40,21 was up-regulated as expected. These 104 genes, although differentially expressed between Surprisingly, the two most down-regulated genes (25-fold tonsillar CD5+ B cells and circulating CLL cells, may contain and five-fold suppression, respectively) were CD1c and CD1d, genes related to the different anatomical locations of the two two members of the CD1 family of MHC-like molecules lymphocyte populations (circulating vs non-circulating) and responsible for presenting lipid rather than peptide antigen to were thus excluded for further consideration. Some of these T cells.22 In CLL-B cells these two CD1 mRNAs were virtually 104 genes, for example a cluster of 19 down-regulated genes undetectable (Table 2). Unique among MHC and MHC-like known to be involved in various aspects of cell proliferation, molecules, CD1 is recognized by the antigen receptors of two may account for some of the phenotypes of CLL-B (eg cell prominent T cell subset with innate immune function: the CD cycle arrest in CLL). They were nonetheless excluded, due to 1d-restricted human Valpha24 and the a similar expression pattern in the normal peripheral B cells, CD1c-specific human V␦1 gamma delta T cells.22 Thus, our which were also non-cycling. The remaining 88 genes are data indicate for the first time that the innate immunity likely to be an underestimate of genes differentially expressed mediated by the activation of CD1-restricted T cells may be between normal CD5+ B cells and CLL cells. compromised in CLL. The age distributions of our tonsil, normal blood, CLL blood The activation of NK T cells results in engagement of NK T samples are non-overlapping, with average age increasing in cell TCR and the lysis of microbial-infected antigen-presenting that order. There are 19 genes in the list of 88 with expression cells. Given that leukemic B cells are the major antigen level in normal blood falling between average levels in tonsil presenting cells in CLL, lack of CD1 expression on CLL-B may and in CLL. Among them, one gene (interferon receptor ifnar2- explain the increased chance of microbial infection in CLL 1) showed significant correlation between expression level in patients. However, more importantly, activation of NK T cells CLL patient and patient age (absolute value of correlation in vivo also induces a series of cellular activation events that coefficient Ͼ0.7) and is excluded from the final list. leads to the activation of innate cells and adaptive cells such The remaining 87 genes included 51 genes that were up- as B cells and T cells.22,23 Thus, defective CD1-restricted T regulated and 36 genes that were down-regulated in CLL cells cell activation conceivably can also lead to reduced adaptive response in CLL. Consistent with this hypothesis, CD69, which upon CD1-restricted T cell activation is induced on normal B cells,23 was expressed five-fold lower in CLL-B cells than in normal cells (Table 2). Given that CD1-restricted T cells play critical roles not only in microbial immunity, but also in autoimmune regulation24 and tumor prevention,25 it would be of great interest to study the status of T cells that recognize the CD1 molecules in CLL patients.

Abnormal expression of genes involved in cytokine response

In the category of cytokine signaling, the receptor for interleu- kin 4, IL-4R, is up-regulated by more than five-fold. IL-4 has been shown to protect CLL-B cells from spontaneous26–28 and Figure 3 Verification of array hybridization results. Representative 29 Northern analysis of CCR7 and M-phase inducer phosphotase2 mRNA Fas-mediated apoptosis in vitro. The potential increase in IL- 30–32 expressions in purified normal peripheral blood B cells (BB), tonsillar 4 levels in T cells from CLL patients would further exacer- CD5+ B cells (TB) and CLL-B cells (CLL). bate the IL-4 induced response of CLL cells. Increased IL-4

Leukemia Expression profiling of CLL Z Zheng et al

+ 2433 Table 2 Differentially expressed genes between CLL-B cells and normal CD5 B cells

Accession Name Avg TB5+ (s.d.) AvgCLL (s.d.) BB Avg(CLL/TB5+)

Antigen presentation M63928 CD27 392 (188) 1340 (430) 241 3.9 R44494 CD83 116 (75) 37 (18) 160 0.49 R49884 CD40 506 (63) 219 (55) 109 0.44 X60592 CD40 243 (108) 86 (55) 20 0.42 Z11697 CD83 774 (301) 232 (100) 720 0.34 X14974 CD1 R3 gene for MHC-related antigen 198 (48) 43 (37) 124 0.23 (CD1d) Z22576 CD69 649 (338) 118 (104) 415 0.23 M28827 antigen CD1c 470 (69) 20 (0) 104 0.04

Cytokine signaling X52425 IL-4 receptor 503 (154) 2474 (1153) 863 5.3 X02875 (2Ј–5Ј) Oligo A synthetase E 130 (22) 405 (236) 152 3.2 M26383 IL-8 119 (67) 43 (8) 3681 0.48 J03143 Interferon-gamma receptor alpha chain 267 (51) 107 (33) 501 0.41 R34698 Interferon-inducible protein 9–27 522 (102) 191 (85) 807 0.38 R73660 Gamma-interferon-inducible protein IP-30 1493 (268) 489 (209) 1841 0.34

Immunoglobulin receptors U12255 IgG receptor FcRn 163 (44) 883 (179) 1789 5.7 M14766 Fc-Epsilon receptor CD23 1059 (650) 2318 (383) 839 2.8 M28696 Low-affinity IgG Fc receptor (FcGR2B) 146 (30) 343 (68) 154 2.4

Lymphocyte trafficking and adhesion/ECM/cytoskeleton U05291 Fibromodulin 54 (17) 1062 (359) 50 21.5 L31584 Chemokine receptor CCR7 177 (78) 1342 (371) 272 8.8 U25956 P- ligand 23 (6) 151 (60) 441 6.8 T70046 Filamin B 30 (12) 170 (157) 39 6.3 H14384 Agrin 44 (13) 240 (97) 27 6.0 M95610 Human alpha 2 type IX collagen (COL9A2) 20 (0) 115 (44) 20 5.7 L23823 Integrin beta 7 159 (42) 667 (289) 297 4.4 X69490 Titin 68 (7) 280 (144) 51 4.1 T41204 92 kDa type V collagenase 153 (67) 58 (23) 152 0.44 D44497 Coronin, actin-binding protein, 1A 3207 (687) 984 (479) 250 0.32 X59350 CD22 1312 (196) 272 (99) 618 0.21 M15395 LFA-1 339 (99) 37 (38) 354 0.12

Protein processing/metabolism X69910 P63 mRNA for transmembrane protein 20 (0) 959 (308) 313 47.9 T92055 ABC-1 36 (12) 316 (125) 40 9.9 M55621 N-acetylglucosaminyltransferase I 108 (114) 236 (61) 106 6.6 D16111 Phosphatidylethanolamine binding protein 294 (41) 1743 (402) 195 6.0 L41559 Pterin-4A-carbinolamine dehydratase 34 (21) 146 (58) 20 5.5 (PCBD) X62822 Beta-galactoside alpha-2,6-sialyltransferase 45 (35) 148 (72) 64 4.9 U26648 Syntaxin 5A 95 (61) 216 (42) 71 4.1 T48904 Heat shock 27 kDa protein 364 (118) 909 (451) 181 2.7 T96942 Mitochondrial short-chain enoyl-CoA 184 (92) 355 (153) 154 2.4 hydratase R08183 10 kDa heat shock protein, mitochondrial 410 (180) 134 (122) 42 0.41 X05309 C3B/C4B receptor (CR1) F allotype 138 (65) 40 (21) 113 0.39 K03001 Aldehyde dehydrogenase 2 199 (39) 63 (29) 287 0.33 X69433 Mitochondrial isocitrate dehydrogenase 184 (27) 59 (32) 117 0.32 M13792 Adenosine deaminase (ADA) 282 (43) 77 (61) 21 0.28 R32804 Glucose transporter type 3 189 (117) 43 (20) 686 0.27 X76648 Glutaredoxin 238 (77) 40 (17) 293 0.18

(continued on next page) signaling via increase in both IL-4R on the CLL-B cell and IL- plays a central role in tumor surveillance and immunity,33 and 4 from T cell would likely offer a survival advantage for leu- this function of IFN-gamma is achieved primarily through kemic CLL-B cells. direct action on the tumor cells to increase their immuno- In contrast, molecules involved in responding to TH1 cyto- genicity.34,35 The major consequence of reduced IFN-gamma kine IFN-gamma (IFN-gamma receptor and two IFN-gamma signaling in CLL may be decreased susceptibility of CLL cells regulated genes, IP-30 and 9-27 protein) are suppressed by to tumor immunity and thus increased CLL-B survival. two- to three-fold in CLL cells. The reduced response to IFN- gamma in CLL has not been reported before. IFN-gamma

Leukemia Expression profiling of CLL Z Zheng et al

2434 + Table 2 Differentially expressed genes between CLL-B cells and normal CD5 B cells (continued)

Accession Name Avg TB5+ (s.d.) AvgCLL (s.d.) BB Avg(CLL/TB5+)

Transcriptional and growth regulation M24069 DNA-binding protein A (dbpA) 37 (19) 533 (177) 164 18.0 T40507 dbpA 106 (26) 1143 (404) 409 11.3 M74718 TCF4 120 (20) 574 (214) 46 4.9 M81934 Human CDC25B 22 (4) 100 (70) 20 4.7 X66924 Heir-1 mRNA for helix–loop–helix protein 163 (37) 630 (402) 52 4.0 (ID3) H46136 Homeobox protein HOX-A10 123 (28) 53 (46) 20 0.4 X51345 JUN-B 2073 (1185) 689 (423) 4467 0.4 U00115 BCL-6 295 (90) 92 (27) 303 0.33 M22638 LYL-1 498 (128) 136 (93) 57 0.29 L25941 Integral nuclear envelope inner membrane 321 (149) 75 (26) 347 0.27 protein (LBR) H24033 C-MYC 245 (98) 44 (17) 178 0.20 R37053 P55-c-FOS Proto-oncogene 282 (239) 21 (1) 385 0.17 U22431 Hypoxia-inducible factor 1 alpha 169 (76) 25 (9) 218 0.17 R12588 G2/Mitotic-specific cyclin B2 254 (82) 37 (21) 91 0.16

Antiapoptosis M14745 BCL-2-alpha 39 (16) 230 (71) 98 6.8 D15057 Defender against apoptosis (DAD-1) 85 (34) 216 (49) 96 3.0 U27467 BCL-2 related mRNA (A1) 451 (100) 160 (69) 1081 0.4

Signaling M77349 TGF-beta induced gene product (BIGH3) 37 (33) 643 (169) 192 26.0 M80899 Human novel protein AHNAK 20 (0) 495 (377) 52 24.8 U20982 Insulin-like growth factor binding Protein-4 34 (18) 686 (311) 30 24.6 (IGFBP4) H24596 RAF kinase inhibitor protein 48 (56) 494 (281) 20 19.5 R70806 Diacylglycerol kinase 175 (107) 782 (307) 128 12.4 M97675 Human transmembrane receptor (ROR1) 20 (0) 182 (119) 20 9.1 M93425 Protein-tyrosine phosphatase G1 40 (5) 276 (134) 123 7.0 H50438 M-phase inducer phosphatase 2 62 (33) 302 (90) 106 6.0 L25081 Transforming protein RHOC 68 (34) 256 (120) 100 4.7 U02570 Human CDC42 GTPase-activating protein 35 (20) 101 (56) 35 3.6 X07109 PKC type beta II 196 (39) 592 (163) 222 3.1 R61874 PAK-interacting exchange factor alpha 324 (49) 148 (61) 311 0.5 H67367 RAN-specific GTPase-activating protein 162 (56) 59 (35) 20 0.4 H24635 Protein-tyrosine phosphatase PAC-1 156 (21) 50 (37) 173 0.3 R67358 MAP kinase phosphatase-1 764 (425) 182 (176) 1516 0.3

Miscellaneous X79204 Ataxin 1 24 (7) 179 (74) 53 8.0 T52999 Unknown 48 (8) 225 (84) 90 4.8 R56038 Unknown 30 (12) 124 (66) 39 4.6 M16405 M4 Muscarinic acetylcholine receptor 74 (77) 163 (84) 36 4.6 H82820 B 50 (42) 129 (70) 283 4.0 R40717 Unknown 35 (17) 109 (48) 47 3.8 T55709 Unknown 51 (36) 120 (58) 20 3.7 R81175 Glutamate receptor 7 47 (28) 104 (26) 28 3.0 R43911 Unknown 1131 (104) 2277 (575) 669 2.0 H40677 Unknown 156 (44) 37 (37) 110 0.3 R45299 Unknown 316 (212) 36 (18) 79 0.2 X04011 X-CGD gene involved in chronic 241 (50) 34 (21) 496 0.1 granulomatous disease

The genes are listed in putative functional categories and within each category are sorted by fold changes (Avd(CLL/TB5+)). BB, normal peripheral CD19+ cells pooled from seven individuals.

Increased expression of immunoglobulin receptors FcGR2B was recently shown to be the target for deregu- lation by chromosomal translocation in B cell follicular lym- We also observed induction of several immunoglobulin recep- phoma, and overexpression of FcGR2B may play a role in tor messages in CLL-B cells. These include the neonatal IgG tumor progression in follicular lymphoma.36 Increased receptor FcRn (5.7-fold increase), the Fc-epsilon receptor FcGR2B engagement may reduce antibody-dependent cell- CD23 (2.8-fold increase) and the lowaffinity IgG Fc receptor mediated cytotoxicity of antibodies against tumor cells in FcGR2B (2.4-fold increase). The overexpression of CD23 is vivo,37 therefore it is likely that increased FcGR2B also con- consistent with increased response to IL-4, as CD23 can be tributes to the survival of leukemic cells. The observation of induced by IL-4 in vitro in both normal B cells and CLL-B.26 increased FcGR2B expression in CLL should be relevant to the

Leukemia Expression profiling of CLL Z Zheng et al 2435 treatment of CLL as several antibody therapeutics, including deregulation. For example, several genes involved in cellular rituximab (anti-CD20) and CAMPATH (anti-CD52), are cur- proliferation pathways were also identified in the differentially rently in clinical trials for treatment of CLL. regulated set. Most of the identified proliferative proto- oncogenes (c-MYC, Jun-B, c-Fos, Hox-A10, BCL-6, G2-spe- cific Cyclin B2) were down-regulated, while inhibitor for pro- Increased expression of the extracellular matrix liferation (Id3) was over expressed. This pattern is expected proteins given the cell cycle arrest phenotype of CLL-B cells. Consist- ent with the quiescent state of CLL-B cells, many genes It is surprising that CLL cells strongly up-regulate several extra- involved in cellular metabolism (such as aldehyde dehydro- cellular matrix proteins. The most highly induced gene in this genase 2) were down-regulated (Table 2). category is fibromodulin, with 21.5-fold induction in CLL. Fib- It should be noted that although c-Myc is the primary tran- romodulin is a that interacts with the type I and scriptional factor that regulates proliferation in many tumor II collagen fibrils in the extracellular matrix and has a role in cell types, it is not clear whether its down-regulation is the the assembly of collagen fibrils in connective tissues.38 Also cause for the growth arrest in CLL. Many c-Myc target genes overexpressed in CLL-B cells is the type IX collagen, a fibril- have been identified51 and are represented in our array. How- associated collagen with similar function to fibromodulin.39 ever, they were not significantly down-regulated in CLL (data Corresponding to increased expression of these matrix pro- not shown). In addition, the c-Myc partner Max expression teins, there is decreased expression of collagenase V involved was not significantly decreased relative to CD5+ B cells in the degradation of extracellular matrix. These together with (average fold change = 0.70). the increased expression of P-selectin ligand, agrin, and inte- The transcriptional factor dbpA was highly induced in CLL grin beta7 suggest increased adhesiveness and reduced (average 15-fold) relative to CD5+ B cells. Moreover, the high mobility of CLL cells relative to normal counterparts. This is expressions were specific to CLL as they were not significantly particularly noteworthy given that the CLL-B cells are circulat- up-regulated in purified CD5+ B cells from a MCL patient ing whereas the CD5+ normal counterparts for this study are (unpublished observation). A closely related protein dbpB cells in tissue. Most of these molecules were not over- (YB1) was highly but not differentially expressed in CLL expressed in normal circulating B cells (Table 2). The contri- (average fold change = 0.76), indicating the specificity of bution of these phenotypic changes to the characteristics of dbpA overexpression. As a DNA binding protein containing CLL cell trafficking remains to be determined. There was no the cold-shock domain, dbpA is known to inhibit the induc- correlation, however, between the expression of these mol- tion of MHC molecules by IFN-gamma,52 but it has never ecules and the degree of leukemic infiltration of lymphoid been described in human B cells. It will be of interest to inves- organs in our patients. tigate the potential role of this transcriptional regulator in The interactions between cancer cells and tissue micro- pathogenesis of CLL. environment are critical for cancer survival and progression.40 Although there are at least 25 genes on our GeneChip with For example, interaction with fibronectin prevents CLL-B from known roles in apoptosis, we found only three anti-apoptotic apoptosis.41 It is not clear with what cell types the CLL-B cells molecules differentially expressed in CLL. In addition to Bcl2, may have increased contact. One possible candidate could the anti-apopototic molecule DAD-1 was overexpressed in be the bone marrowstromal cells, whichadhere to CLL cells CLL cells by about three-fold. DAD-1 does not belong to the but not normal B cells and provide survival factors for Bcl2 family and it protects cells from apoptosis by mech- CLL.42,43 The increased expression of chemokine receptor anisms distinct from those used by bcl-2.53 Paradoxically, the CCR7 (Figure 3) is noteworthy, as the role of this receptor on Bcl2 family antiapoptotic molecule A1 was decreased in CLL. the B cells is not primarily for B cell homing to lymph While the Bcl-2 family of proteins plays a major role in pro- organs,44,45 but rather for bringing T cells and activated B cells tecting CLL-B cells from apoptosis induced by therapeutic within lymph organs together for interaction.45 CLL-B cells agents,54 it is not clear whether they are critical in establishing express CCR7 at a level similar to activated normal B cells the pro-survival phenotype of CLL in vivo, since prior to ther- (unpublished observation). Being the major accessory cells in apy CLL cells do not encounter those therapeutic agents. In CLL, the leukemic cells may compete with competent, acti- fact, when cultured in vitro, CLL-B cells, even with over- vated B cells for T cell interaction.46 Moreover, once in con- expression of Bcl2, are more prone to spontaneous apoptosis tact, CLL-B cells quickly inactivate T cells by downregulating than normal peripheral blood B cells or CD5+ normal B CD40L and costimulatory molecules on the T cell surface,47,48 cells.42 This observation suggests the increased steady-state rendering T cells anergic. Further study is needed to elucidate level of antiapoptotic molecules, while important in determin- the role of CCR7 in CLL pathogenesis. The expression of agrin ing response to therapy, is not sufficient to explain the CLL in CLL-B cells, but not normal B cells, is also unusual. When pathogenesis. secreted from T cells agrin can promote the formation of the Finally, we found in CLL cells significant up-regulation of contact site (immunological synapse) between activated T genes for protein processing (N-acetylglucosaminyltransferase cells and antigen-presenting cells.49,50 It will be of interest to 1, PCBD, beta-galactoside alpha-2,6-sialyltransferase) and test whether agrin from CLL-B cells has a similar function in protein trafficking/transport (p63, ABC-1, phosphatidylethano- promoting immunological synapse formation, or in clustering lamine binding protein, syntaxin 5A). Among them, the p63 molecules on CLL-B cells to form contact site with other mRNA, for a transmembrane protein localized to the ER-Golgi cell types. intermediate compartment,55 shows a 48-fold increase, the highest in the entire list. Fewstudies have explored the role of intracellular protein processing and transport in the disease Changes in non-immune-related gene expression phenotype of CLL. Our results correlate the deregulation of this process with CLL. Some proteins encoded by genes ident- The comprehensive nature of microarray gene expression pro- ified here may modulate functions of immunoregulatory pro- filing allows one to gain valuable insights beyond immune teins. For example, beta-galactoside alpha 2,6-sialyltransfer-

Leukemia Expression profiling of CLL Z Zheng et al 2436 ase sialylates CD22 and can abrogate CD22-mediated B DA. Immunoglobulin V gene expression in CD5 B-cell malig- lymphocyte adhesion in vitro.56 Identifying specific protein nancies. Ann NY Acad Sci 1992; 651: 373–383. processing and trafficking pathways that are affected, and the 8 Kipps TJ, Duffy SF. Relationship of the CD5 B cell to human tonsil- lar lymphocytes that express autoantibody-associated cross- target molecules that they regulate, will help understand the reactive idiotypes. J Clin Invest 1991; 87: 2087–2096. pathogenesis of the disease. 9 Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, Husson CLL is a complex human disease characterized by abnormal H, Freedman A, Inghirami G, Cro L, Baldini L, Neri A, Califano cell death rather than increased proliferation. Our comprehen- A, Dalla-Favera R. 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