b - cell chronic lymphocytic leukemia ( cll ) is the most common adult leukemia in the western world .
cll cells express 1 or more b cell associated antigens , generally cd19 , cd20 and cd23 with coexpression of cd5 , a t cell marker [ 1 , 2 ] .
fluorescent in situ hybridization ( fish ) for specific karyotypic markers   and polymerase chain reaction ( pcr ) for immunoglobulin heavy chain variable region ( igvh ) gene mutations [ 4 , 5 ] provide prognostic information .
the 70 kda zeta - associated protein ( zap-70 ) a cytoplasmic tyrosine kinase measured by flow cytometry [ 7 , 8 ] or by rt - pcr and immunohistochemistry ( ihc ) , appears to improve the prognostic significance of cytogenetics and igvh mutational status .
cll patients who are zap-70 positive irrespective of their clinical stage , cytogenetics and igvh mutational status have a shorter progression free survival .  
several groups have performed gene expression profiling ( gep ) on cll [ 1116 ] to establish a gene expression signature , prognostic groups , cell of origin , correlation with genomic defects and diagnostic markers .
these studies provide evidence that the cll gep is related to memory b - cells .
further , there appears to be a correlation between gep and genomic loss or gain suggesting that gene dosage may be important to cll pathogenesis [ 11 , 17 ] .
more recently microrna ( mirna ) expression profiles have been utilized to distinguish normal b - cells from cll cells and provide a signature that is associated with prognosis and progression .
the wnt / fzd canonical pathway has been proposed as a possible mechanism of oncogenesis in cll [ 1921 ] .
ror-2 , a close member of ror-1 , has been shown to bind wnt-5a via the cysteine rich domain ( crd ) and to activate the jnk signaling pathway .
current treatments can not cure cll and more effective therapies based on abrogating key oncogenic signaling pathways are needed . here , we pose several key objectives and goals to address this statement by utilizing gene expression and cytokine profiling of rai stage 0/i cll patients which include , ( a ) what are the differentially expressed genes in cll ?   previous studies used a microarray platform containing fewer genes , and therefore the current study with   > 10,000 novel genes would likely yield novel differentially expressed genes , ( b ) what oncogenic signaling pathway(s ) are likely to drive cll progression ?
, as they may help identify parallel cross - talking pathways , ( c ) what serum cytokines are associated with immune evasion and survival ?
,   that may activate cll and/or stromal cells , ( d ) what are   the six hallmarks of cancer for cll and can they be targeted rationally ? , ( e ) what is the status of p53 and zap-70   in low stage cll ? , since both are associated with a poor prognosis , ( f ) does ge change when cll cells are cultured for a given time ?
this would address the important question of why it is difficult to grow cll cells in culture and help identify differentially expressed genes after treatment with drugs ( e.g. , thalidomide , an immunomodulatory agent ) .
based on gep studies we identified over - expression of members of the non - canonical wnt / pcp - ror-1 signaling pathway genes that were validated by rt - pcr . two other pathways ( cxcl9/cxcr3 and flt3l - flt-3 rtk ) were identified where both ligand serum levels and cognate receptors , on cll cells , were elevated .  
we show that the majority of the patients differentially over - expressed zap-70 while p53 was down regulated .
the gep of cll cells at 0 hours and 24 hours in culture showed differential over - expression of the cytokine cxcl5 .
treatment of cll cells with thalidomide , a potent anti - angiogenic and immunomodulatory drug , had no effect on cell viability but led to the over - expression of cytokine   ccl5 .
together , these results provide novel biological insights and potential therapeutic targets for low - stage cll patients .
isolated and purified cll cells from patients were cultured in 10% human serum albumin ( gemini bioproducts , woodland , ca ) in rpmi-1640 media .
polyclonal antibodies to fzd3 ( r&d systems , mn ) , ror-1 ( r&d systems , mn ) and flt-3 ( santa cruz , ca ) were obtained .
secondary antibody at a 1 : 5000 dilution ( irdye   800 donkey anti - rabbit , rockland immunochemicals ) was used to visualize bands with a li - cor odyssey infrared imaging system .
thalidomide powder ( sigma ) was freshly dissolved in dimethyl sulphoxide ( dmso ) at a 5 mm stock solution and diluted into rpmi-1640 media with 10% human serum immediately prior to use .
cd19 + normal pb b - cell rna from 4 normal volunteers was purchased from allcells , ca .
all patients studied had a diagnosis of cll stage 0 or i. patient blood samples ( n = 26 ) were collected after obtaining informed consent on an institutional review board approved protocol .
mononuclear cells were separated and either stored frozen at 80c or maintained in rpmi-1640 medium with 10% human serum ( gemini bioproducts , ca ) at 37c in 5% co2 .
separated serum was stored in 0.5  ml aliquots at 80c until further studies were performed .
mononuclear cells were separated from peripheral blood samples by ficoll - hypaque density centrifugation   and phenotype was determined by immunofluorescent flow cytometry analysis .
the cll cells were then maintained in culture in rpmi-1640 at 37c for further studies .
total rna was extracted using the rneasy mini kit ( qiagen , ca ) from 8 patient cll cells at collection .
further , rna was extracted from 4 patient cll cells in culture for 24 hours and after treatment with thalidomide ( 1  m ) for 24 hours .
the amount of total rna isolated from the cells was quantified using spectrophotometric od260 measurements with yields25  g / sample .
control rna was obtained from normal peripheral ( pb ) b - cells from 4 volunteers ( allcells , ca ) . 5  g of mrna was used to generate first - strand cdna by using a t7-linked oligo(dt ) primer .
after second - strand synthesis , in vitro transcription ( ambion ) was performed with biotinylated utp and ctp ( enzo diagnostics ) , resulting in 40- to 80-fold linear amplification of rna .
40  g of biotinylated rna was fragmented to 50- to 150-nt size before overnight hybridization at 45c to hg - u133a 2.0 affymetrix array comprising ~18,400 transcripts and 22,000 probe sets ( santa clara , ca ) .
after washing , arrays were stained with streptavidin  phycoerythrin ( molecular probes ) and scanned on a hewlett - packard scanner .
intensity for each feature of the array was captured by using genechip software ( affymetrix , ca ) , and a single raw expression level for each gene was derived from the 10 - 20 probe pairs representing each gene by using a trimmed mean algorithm .
intensity values were scaled such that overall intensity for each chip of the same type was equivalent .
the arrays were first inspected visually for artifacts and b2 oligo performance per manufacturer 's instructions ( affymetrix , inc .
chip cel files were then imported into r - bioconductor ( v. 1.7 ) ( http://genomebiology.com/2004/5/10/r80 ) .  
mas5 present / absent calls were generated with the  simpleaffy  ( http://bioinformatics.picr.man.ac.uk/simpleaffy/ ) package using a target scaling factor of 100 .
the scale factors were within 3-fold of each other as recommended in the guidelines provided in the affymetrix gene chip expression analysis : data analysis fundamentals manual .  
the quality control metrics were generated using the  qc  function provided in the  simpleaffy  package .  
all chips performed as follows :   the average backgrounds fell between 20 and 100 , hybridization controls ( biob , bioc , biod and cre ) were all called present with increasing signal intensities and the 3/5 ratios for the internal controls ( gapdh and -actin ) were less than 1.25 and 3 respectively .
further quality control was performed using the  affy  plm  package provided in the bioconductor suite .  
the microarray data were normalized by robust multi - chip analysis ( rma ) using the  fitplm  function ( default values ) .  
chip pseudo - images of the weights and residuals , normalized unscaled standard error ( nuse ) and relative log expression ( rle ) values were used for chip - level quality assessment .   
chip pseudo - images displayed no apparent artifacts that warranted excluding a chip from further analysis .  
log - fold changes were calculated from the normalized log2 expression values . for each cll sample at collection , in culture for 24 hours or treated with thalidomide for 24 hours a comparison to the control samples ( normal b - cells ) and lists of  robust increasers ' or  robust decreasers ' was generated utilizing the affymetrix data analysis program ( affymetrix mas 5.0 ) .
fundamentals ' guide was used to import these lists into genespring ( version 5.0 ) and obtain the intersection of robust increasers or decreasers across all patients .
increasers or decreasers common to cll patients were compared to normal pb b - cells . in order to assess quality and reproducibility of our data ,
gep for each patient was further classified according to the hallmarks of cancer , to determine cll specific  tumor profile signatures ' .
intersection of the gene lists for each target with the commonly up - regulated and down - regulated genes across patient samples was obtained .
this was also performed on the intersects of the up- and down - regulated gene lists with the genespring
one hundred nanograms of total rna were used for reverse transcriptase ( rt ) reactions ( 20 ml total volume ) carried out using superscript   iii platinum reverse transcriptase ( invitrogen , carlsbad , ca ) .  
reactions were incubated at 42c for 50 minutes followed by incubation at 37c with rnase h for 20 minutes .  
an opticon dna engine ( mj research , reno , nv ) was used to perform real - time fluorescence detection pcr .
1  l cdna was added to 12.5  l of platinum sybr green qpcr supermix - udg ( invitrogen , carlsbad , ca ) , 1  l of gene - specific or -actin specific primer pair ( see primer design ) , and 10.5 l of dh2o ( final volume of 25 l ) .  
amplification ( 95c for 15  s , 55c for 30  s , and 72c for 30  s ) was repeated for 44 cycles . threshold cycle value ( ct ) for each sample indicated the cycle at which a statistically significant increase in fluorescence was first detected .
these data were then normalized to -actin , which served as an unaffected control gene , for each data point and compared to a normal b - cell control to determine relative expression ratios .
pcr primers were designed using macvector ( accelrys , san diego , ca ) to produce amplicons with lengths ranging from 80 - 250 bp to optimize the efficiency of qpcr .  
-actin primers from quantumrna-actin internal standards ( ambion , austin , tx ) were used to normalize the qpcr data .
the following genes were analyzed by real time quantitative rt - pcr : axin1 , blk , csn1a1 , ctnnb1 , dvl1 , frat1 , fzd3 , fzd6 , flt-3 , gsk3   , itk , jak2 , jun , lef-1 , mapk10 , mapk8 , mapk9 , prkcb1 , ror-1 , ror-2 , syk , tcf4 , p53 , wnt-3a , wnt-5a and zap70 . stored ( 80c ) or fresh patient serum samples were utilized ( n = 26 patients ) .
array slides were thawed at room temperature as serum was diluted 3.5-fold with 1x blocking buffer ( cytokine array protocol , raybiotech , inc # aah - cyt - g1000 ) .
slides were blocked with blocking buffer and 50  l of each sample was hybridized with the slide for 2 hours ; 1  l internal controls were also added .  
after washing , provided biotin - conjugated anti - cytokine antibodies were diluted with 300  l of blocking buffer , added to the slide , and incubated for 2 hours at room temperature .  
washing steps were repeated , followed by addition of a 1:1500 dilution of alexa flour 555-conjugated streptavidin and incubated overnight at 4c .  
slides were washed multiple times and read on the axon genepix using the cy3 channel .
500  g clarified lysate per sample was incubated at 4c overnight with anti - ror-1 polyclonal antibodies followed by the addition of protein a agarose ( upstate biotechnology ) for a further 2 hours .
after page and transfer to immobilon membranes ( millipore , ma ) , immunoblotting was performed using the anti - ror-1 antibody . alternatively , 50100  g of protein from cell extracts was separated by page and immunoblotted with protein - specific antibodies .
protein bands were visualized utilizing the secondary antibody at a 1:5000 dilution ( irdye 800 donkey anti - rabbit , rockland immunochemicals ) with a li - cor odyssey infrared imaging system .
the purity of the cll samples estimated from the percentage of cd19 cells was at a mean of 89% ( range 8095% ) ( supplementary figure 1 ) which is very similar to that described by stratowa et al .   .
a gep was conducted on 8 low stage cll patients , 4 normal volunteers without age - matched cd19 + b - cells ( allcells , ca ) utilizing the affymetrix platform ( 22,000 genes ) with > 10,000 genes than previously published gep studies .
we compared the most significantly over - expressed and down - regulated genes of cll versus normal b - cells in our gep to that of klein et al .
ten distinct genes represented by 15 probe sets were differentially expressed in all three studies ( table 1 ) .
hence , our data validate and add to the current knowledge on cll gep studies .
an analysis of differentially expressed genes by fold change between cll versus normal pb b - cells was performed in a gene - by - gene analysis for differences between the 2 groups .
genes that are  robustly  highly over - expressed or down regulated that are common to all 8 cll patients were compared to normal pb b - cells . for brevity only the 20 top over - expressed ( table 2(a ) ) or down regulated ( table 2(b ) ) genes are shown . of the over - expressed genes ( table 2(a ) ) , 5 are listed in table 1 ( abca6 , fibromodulin , lef-1 , igfbp4 , rasgrf1 ) confirming the data from klein et al . ,
novel over - expressed genes from this study are hba , hbb , pdgf - dd , ror-1 , fry , selp , hla - dqa1 and vipr1 all of which are implicated in human malignancies including leukemia , lymphoma and solid tumors .
it appears that the comparison of cll patients to normal pb b - cells demonstrates that several of the differentially genes are represented within the 6 hallmarks of cancer .
for cll they are tumor invasion ( col9a2 , rapgef3 , spock2 , wnt3 , coro1b , ccl5 , il2rb , map2k6 ) , anti - apoptosis and survival ( lef1 , igfbp4 , ror-1 , mybl1 , pim-1 , lck , flt-3 ) , cell cycle deregulation ( pftk1 , plk3 ) and angiogenesis ( pdgf - dd , fgf2 ) . consistent with cll several of the ig gene family members ( light chain variable and heavy chain constant ) are the most highly down regulated .
other highly down regulated genes of significance to tumor biology include ccr6 , bcl7a , myc , mafb , casp2 and hsp70b .  
serum samples from 26 cll patients and 4 normal volunteers were analyzed utilizing the cytokine array g-1000 platform consisting of 120 cytokines / chemokines / growth factors ( raybio , ga ) .
fold change was calculated for each patient , corrected for absolute lymphocyte count and a mean  sd obtained for each cytokine for all patients .
our cytokine profile identified all of the published elevated cytokines associated with cll cell survival ( il-1 , il-2 , il-4 , il-6 , il-8 , il-10 , tnf , ifn or  , g - csf or gm - csf ) or inhibition ( il-5 , tgf ) . in our cytokine profile
the 1st ranked is ifn ( 14.46 fold ) which is well known to prevent apoptosis of cll cells .
the 2nd ranked is igfbp-4 ( 14.22 fold ) and of significance is that it is also over - expressed in the gep indicating that serum levels reflect cll production of this cytokine .
the function(s ) igfbp-4 are not well characterized except for binding igf - i and igf - ii . in most human malignancies igfbp-4
the 3rd ranked is g - csf ( 10.89 fold ) which is known to decreases cll cell apoptosis .
the 4th   ranked is il-3 ( 8.45 fold ) which provides a proliferative signal to cll cells especially during the lymphoblastoid phase .
other significantly increased serum cytokines are flt-3 ligand ( 7.82 fold ) , scf ( 6.76 fold ) and sdf-1 ( 6.54 fold ) . utilizing both the gep and scp , an analysis of ligand - receptor ( l - r ) pairs of the cytokine - cytokine receptor or growth factor - growth factor receptor ( pdgf , tnf , tgf , wnt ) that are simultaneously elevated in our cohort of cll patients
table 3b highlights several of these l - r pairs with their respective fold changes and known function(s ) .
we identified 3 major oncogenic signaling pathways : gpcr cxcl9/cxcr3 pathway ; and the rtk pathways of wnt / fdz3-ror-1 pcp and flt-3l / flt-3 .
previously , it has been shown that cxcr3 is expressed on cll cells   and may play a role in chemotaxis .
moreover , the cxcr3 ligand , cxcl9 , is coexpressed in cll cells   implicating an autocrine mechanism of activation .
our data confirm these individual observations , extend the finding that low stage cll patients express both cxcl9 and cxcr3 that may promote immune mediated cell homing in addition to chemotaxis .   the expression and activation of the classical wnt / fzd signaling cascade has been implicated in cll .
lu et al . , 2004 , quantified the gep of the wnt family and their cognate fzd receptors in cll cells , and determined the role of wnt signaling in promoting cll cell survival .
our gep identified ror-1 rtk to be over - expressed , which is a member of the non - canonical wnt / pcp pathway .
the coreceptor of ror-1 , fzd3 and fzd6 are also over - expressed implicating the activation of the wnt non - canonical pathway in cll ( table 3(b ) ) .
hence , we analyzed 20 representative members of the canonical and non - canonical pathway from the cell membrane to the nucleus by quantitative real time rt - pcr to ascertain which pathway was active in cll .
the members of the canonical pathway are down regulated ( dvl1 , axin , gsk-3 , tcf-1 ) except for lef-1 ( figure 1 ) , while members of the non - canonical pathway are over - expressed ( ror-1 , fzd3 , jak2 , jnk2 , itk ) ( figure 2 ) .
gep identified over - expression of several members of the non - canonical pathway : wnt3 ( 8-fold ) , wnt16 ( 2-fold ) , fzd3 ( 3-fold ) , fzd6 ( 2-fold ) and pck ( 7-fold ) to be over - expressed .
western blotting analyses performed on 4 representative cll patient cells for ror-1 confirmed expression at the protein level ( supplementary figure 2 ) .
these studies provide evidence for importance of the wnt - ror-1 non - canonical pathway in cll .  
the scp   and gep identified flt-3l to be elevated ( serum ) and flt-3 to be over - expressed ( microarray ) in all 8 cll patients .
rt - pcr of 20 cll patients for flt-3 showed that 8 ( 40% ) had over - expression while 12 ( 60% ) had no change   in expression ( supplementary figure 3 ) please see in supplementary materials avilable online at doi:10.1155/2007/453634 .
we next searched for downstream signaling molecules associated with flt-3 activation and identified over - expression of pim-1 ( 8-fold ) ( proviral insertional mutagenesis-1 serine / threonine kinase ) and c - myb ( 4-fold ) proto - oncogenes .
this novel finding implicates activation of the flt-3 rtk pathway in a subset of cll that may contribute to cell survival . in acute myeloid leukemia ( aml ) ,
flt-3 signaling up - regulates pim-1 expression and activation   but such a role has not been previously described for cll . hence , our finding of coexpression of flt-3l and flt-3 l - r pair is novel and requires further investigation . both zap-70 and p53
have been demonstrated to be important prognostic factors in cll . in a subset of cll patients with 17p deletion where p53 is lost an aggressive chemo - resistant clinical course ensues .
we conducted quantitative real time rt - pcr on 21 low stage cll patients for zap-70 and p53 expression ( supplementary figure 4 ) .
the results show that p53 is down - regulated in almost all patients , while zap-70 is over - expressed in 18 of 21 patients .
it is possible that a small fraction of contaminating normal t - cells in the purified cll samples may affect the zap-70 rt - pcr data , however the differential expression observed by gep suggests this is minimal .
the down regulation of p53 expression in low   stage cll appears to be an early event in the oncogenic program .
it is an enigma as to why cll cells purified from patients are not amenable to medium and long term cell culture .
these predominantly mature malignant b - cells are resistant to apoptosis but once in cell culture lose viability .
a key goal of the study was to identify factor(s ) that may contribute to loss of viability and differential gene expression by gep was utilized to investigate this phenomenon .
a gep study of 4 cll patients at 0 hours and at 24 hours in culture showed several genes to be differentially expressed   at 24 hours ( supplementary table 1 ) .
the chemokine cxcl5 was ~120 fold over - expressed followed by several other chemokines , ccl ( 2 , 3 and 23 ) and cxcl ( 1 , 2 and 3 ) subtypes .
interleukins-1 (  and  ) , il-6 and il-8 were also over - expressed .
the imids thalidomide and lenalidomide are currently under evaluation as potential novel agents in phase i trials in cll ( 24 ) .
we investigated the effect of thalidomide on the differential gene expression of 4 cll patient cells treated in culture for 24 hours .
since there were gene expression changes at 24 hours in culture without thalidomide therapy , these changes were taken into account when analyzing the effects of thalidomide on changes in gene expression .
cell viability studies indicated thalidomide was not cytotoxic to cll cells ( data not shown ) .
thalidomide induced modest differential gene expression changes ( supplementary table 2 ) , with ccl5 ~2.5 fold over - expressed .
our study also analyzed potential therapeutic targets that may be amenable to targeting either with small molecular inhibitors and/or monoclonal antibodies .
for patients with cll the following are novel targets : small molecular inhibitors to protein kinases ( ror-1 , flt-3 , zap-70 , pim-1 , lck pftk1 , plk-3 ) , monoclonal antibodies to circulating ligands ( cxcl9 , igfbp4 , flt-3 ligand , pdgf - dd , fgf2 ) and monoclonal antibodies to cell surface receptor ( fzd3 , flt-3 , ror-1 , cxcr3 ) .
we undertook a gene expression and serum cytokine profile study of low stage cll patients to glean biological insights into disease pathogenesis .
first , our data confirmed the key gep findings of klein et al . , 2001 and jelinek et al .
second , we identified several significantly over - expressed novel genes that enhance further differentiation of cll from normal circulating pb b - cells .
this has allowed an extension of the molecular signature profile based on the six hallmarks of cancer   for cll .
third , a serum cytokine profile of 26 cll patients show several elevated cytokines / growth factors that are novel and help identify ligand - receptor pairs that may be pertinent to cll biology .
finally , quantitative real time rt - pcr and western blotting analyses of key genes and proteins respectively were utilized to validate and confirm the gep .    the wnt / fdz3-ror-1 pcp non - canonical pathway is over - expressed in cll cells .
the pcp signaling pathway controls tissue polarity and cell movement through activation of rhoa , c - jun n - terminal kinase ( jnk ) , and nemo - like kinase ( nlk ) .
human wnt5a , wnt5b , and wnt11 are non - canonical wnt ligands that transduce pcp signals through fzd3 or fzd6 receptors , and coreceptors ror-1 , ror-2 or ptk7 . the aberrant activation of wnt / pcp signaling pathway leads to a malignant phenotype with associated abnormal tissue polarity , invasion , and metastasis .
previously , lu et al . , 2004 , provided evidence for the wnt / fzd canonical pathway as a possible mechanism of oncogenesis and cell survival in cll .
rt - pcr of both the canonical and non - canonical pathway members , confirmed down regulation of the wnt / fdz canonical pathway ( dlv1 , axin , frat-1 , gsk-3 , -catenin , tcf ) except for lef-1 which was over - expressed in all patients ( figure 1 ) . over - expression of lef-1 in cll cells
has been described [ 12 , 17 , 19 ] and appears to play a role in pro - b cell development but not in mature b - cells .
in contrast , we found that fzd3 to be over - expressed and fzd6 to be down - regulated , while their coreceptor ror-1 and jnk-2 of the wnt / pcp pathway are over - expressed ( figure 2 ) .
ror-1 is a neural developmental kinase ( 937 amino acids )   but its cellular role has not been fully characterized .
a truncated ror-1 is found to be expressed in human leukemia and lymphoma cell lines and in several human neuroectodermal malignancies .
a mechanism is proposed for activation of the fzd3-ror-1 pcp pathway ( figure 3 ) via wnt binding to the ror-1 crd .
thus a switch from wnt / fzd canonical to wnt / pcp non - canonical signaling in cll is proposed .  
although key members of the wnt / fzd canonical pathway are down regulated in cll , lef-1 is over - expressed .
this discrepancy may be due to expression and activation of lef-1 via an alternative signaling pathway(s ) .
evidence points to the tgf pathway , where tgf-1 and -3 are elevated in the serum cytokine profile while tgf- receptor iii ( tgf- riii ,   -glycan ) and smad2 are over - expressed in the gep .
the tgf pathway is known to cross - talk with the wnt signaling pathway via smads [ 35 , 36 ] .
tgf can also induce novel lef-1 splice variants through a smad - independent signaling pathway .
therefore , in cll the expression and activation of lef-1 may occur through tgf  -smad dependent or independent mechanisms ( figure 3 ) .
moreover , cll cells respond abnormally to tgf stimulation   and over - expression of tgf-riii has been linked to over - expression of mutant k - ras .
hence , it is conceivable that over - expression of rasgrf1 in cll ( table 1 )   may activate the map kinase pathway leading to lef-1 activation .
mikels and nusse ( 2006 ) have shown that when ror-2 is expressed , wnt5a   and more recently igfbp-4   have been shown to inhibit canonical signaling downstream of -catenin stabilization at the level of tcf driven transcription .   a similar mechanism may operate in cll where ror-1 is expressed and an alternative pathway activates lef-1 .  
the study identified cxcl9-cxcr3 l - r pair where cxcl9 levels are elevated in the serum and cxcr3 was over - expressed in the gep ( table 3(b ) ) .
cxcr3 , a g - protein coupled chemokine receptor is normally expressed on th1 cells and its expression is associated with peripheral inflammation .
th1 cells express cxcr3 during activation and differentiation implying effector functions in both central and peripheral lymphoid tissue .
cll cells highly express cxcr3 , which promotes efficient migration in response to cxcl9 in comparison to normal pb b - cells .
cxcl9 is coexpressed with cxcr3 in cll   implicating an autocrine mechanism of malignant b - cell activation and promotion of migration ( figure 3 ) .
interferon- ( ifn ) the 1st ranked elevated cytokine ( table 3(a ) ) in the cytokine profile is known to induce cxcl9 required for t - cell recruitment and effector functions .
flt-3l is elevated in the serum cytokine profile and flt-3 is over - expressed by gep .
rt - pcr analysis of 20 cll patients confirmed over - expression of flt-3 ( 6 - 40 fold ) in a subset of cases ( 40% ) ( supplementary figure 3 ) .
flt-3 is constitutively activated due to internal tandem duplication ( it d ) mutations in the juxta - membrane domain in a majority of patients with acute myeloid leukemia ( aml ) and acute b - cell leukemia ( all ) .
2005 , demonstrated that pim-1 , a proto - oncogene with serine / threonine kinase activity is significantly down - regulated when flt-3 is inhibited with cep-701 , an flt-3 tyrosine kinase inhibitor .
a similar inhibitory effect with cep-701 was also shown for bad ( bcl-2 antagonist of cell death ) phosphorylation . in our gep , pim-1 is over - expressed while its substrates c - myb , bcl11b , stat4 and cdc25b are also over - expressed .
pim-1 is known to be up - regulated by stat 5 , which is also a downstream target of flt-3 signaling .
we propose that activated flt-3 up - regulates and activates pim-1 kinase providing proliferative and anti - apoptotic signals to some cll cells .  
hemoglobin ( hb ) a and b are over - expressed in cll patients ( table 2(a ) ) . alpha and beta - globin rna
have been detected in erythroid cells ( fetal liver and reticulocytes ) and non - erythroid tissues ( adult brain and liver , and cultured lymphoma , untransformed , and transformed fibroblast cell lines ) .
reactive oxygen species ( ros ) at physiological concentrations are required for normal cell function but excessive production of ros is detrimental to cells .
it has been shown that treatment of ht-29 colon cancer cells with hb enhanced proliferation via reactive oxygen species ( ros ) production and significantly blunts the cytotoxic effects of 5-fu and 5-dfur .
papillary thyroid carcinomas ( ptc ) express hbb at similar levels to normal thyroid gland but over - expression of hbb in kta2 cells ( anaplastic thyroid cancer cell line ) , significantly suppressed their growth .
in cll , there is progressive accumulation of well differentiated malignant cells that are arrested in g0/g1 and generation of excessive ros in this setting would be detrimental .
therefore , over - expression of hba and hbb may counter excessive ros production and be protective to cll cells .   
we show that p53 expression is down regulated and zap-70 is over - expressed in low stage cll ( supplementary figure 4 ) .
when the white cell count or the absolute lymphocyte count ( alc ) was plotted against p53 or zap-70 level of expression , there is no significant correlation .
hence , the loss of p53 or gain of zap-70 function is independent of disease burden , but may be related to initiation and progression of the abnormal cll clone .  
cll cells are resistant to apoptosis but once in cell culture appear to lose viability .
several different approaches to maintain cll cells in culture have been investigated but none has been successfully implemented .
we hypothesize that a major reason for inability to grow these cells is due to the removal of a factor or factors from their natural ( plasma or serum ) environment .
we demonstrate that cll cells elaborate a chemokine - cytokine response to being in culture at 24 hours .
it is known that cll cells regulate the microenvironment through modulation of the cytokine milieu that recruit and activate immune cells ( t - cells ) . since standard culture conditions do not have access to accessory survival mechanisms , these cells undergo apoptosis over a period of 1 - 2 weeks .  
targeting of the tumor microenvironment with immunomodulatory ( imid ) agents such as thalidomide and lenalidomide has been evaluated in cll   with promising clinical activity .
thalidomide therapy in cll cells showed no cytotoxicity , and a similar result has been reported for lenalidomide in in vitro tumor models of cll .
we also evaluated differential gene expression between thalidomide untreated and treated cll cells in culture for 24 hours and showed very modest gene expression changes .
this is consistent with its proposed mechanism of action on the stroma and activation of the immune system rather than a direct effect on malignant cells .
the lack of a direct effect of thalidomide on cll cells in culture and activity in patients implies a role for imids in modulating the stromal microenvironment for their therapeutic efficacy .
our study identified 3 novel differentially expressed oncogenic pathways pertinent to low stage cll patients .
these pathways are implicated aberrant regulation of cell proliferation , survival and migration of cll cells .
further , in most of the patients p53 is down - regulated while zap70 is up - regulated suggesting a common mechanism of pathogenesis .
interferon , flt-3l and igfbp4 are the most prominent serum cytokines in low stage cll with the latter implicated in modulating the wnt signaling pathway .  
the 6 essential alterations in cell physiology that collectively dictate malignant growth ( cll ) are represented by 2530 up - regulated over - expressed cancer genes and down - regulated tumor suppressors which appear to determine a slow proliferator phenotype .
this cll phenotype has gained the ability to avoid immune attack by nk and t - cells by secreting factors that inhibit effector activity and appears to subvert the bone marrow stromal environment for survival .
our data provide a platform to extend these studies to high stage ( rai iii / iv ) cll patients .
a recent gep study of waldenstrom macroglobulinemia ( wm ) showed a phenotype similar to cll and pb b - cells than multiple myeloma   implicating a small set of differentially expressed genes which may distinguish between these lymphoproliferative diseases .
 gene expression profiling ( gep ) of 8 stage 0/i untreated chronic lymphocytic leukemia ( cll ) patients showed over - expression of frizzled 3 ( fzd3)/ror-1 receptor tyrosine kinase ( rtk ) , flt-3 rtk and cxcr3 g - protein coupled receptor ( gpcr ) . 
 rt - pcr of 24 genes in 21 patients of the wnt pathway corroborated the gep . 
 transforming growth factor , fibromodulin , tgfriii and smad2 are also over - expressed by gep . 
 serum cytokine profiling of 26 low stage patients showed elevation of ifn , csf3 , flt-3l and insulin - like growth factor binding protein 4 . in order to ascertain why cll cells grow poorly in culture , a gep of 4 cll patients cells at 0 hr and 24 hr in culture demonstrated over expression of cxcl5 , ccl2 and cxcl3 , that may recruit immune cells for survival . 
 treatment with thalidomide , an immunomodulatory agent , showed elevation of ccl5 by gep but was not cytotoxic to cll cells . 
 our data suggest an interplay of several oncogenic pathways , cytokines and immune cells that promote a survival program in cll . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion