intraclonal complexity in chronic lymphocytic leukemia ......1374 | calissano et al. | mol med...

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INTRODUCTIONChronic lymphocytic leukemia (CLL)

is a relatively frequent, incurable B-cellmalignancy (1,2). Even though some pa-

tients live for long periods with the dis-ease, many undergo progressive decline,leading to demise. Progression to a moreaggressive disease is often associated

with genomic changes (3), suggestingthat clonal evolution is a key factor in thedisease.

We previously found that CLL clonesare composed of subpopulations of cellsthat proliferate at different rates (4), asmeasured by in vivo deuterium (2H)- incorporation into newly synthesizedDNA of dividing cells (5,6). The mostproliferative fraction of a cancer clone isof major interest for several reasons.

Intraclonal Complexity in Chronic Lymphocytic Leukemia:Fractions Enriched in Recently Born/Divided andOlder/Quiescent Cells

Carlo Calissano,1 Rajendra N Damle,1,2 Sonia Marsilio,1 Xiao-Jie Yan,1 Sophia Yancopoulos,1 Gregory Hayes,3

Claire Emson,3 Elizabeth J Murphy,3,4 Marc K Hellerstein,3 Cristina Sison,5 Matthew S Kaufman,6

Jonathan E Kolitz,1,2 Steven L Allen,1,2 Kanti R Rai,1,6 Ivana Ivanovic,7 Igor M Dozmorov,7 Sergio Roa,8

Matthew D Scharff,8 Wentian Li,1 and Nicholas Chiorazzi1,2,9

1The Feinstein Institute for Medical Research, North Shore–LIJ Health System, Manhasset, New York, United States of America;2Department of Medicine, North Shore University Hospital, North Shore–LIJ Health System, Manhasset, and Hofstra North Shore–LIJSchool of Medicine, Hempstead, New York, United States of America; 3KineMed, Inc., Emeryville, California, United States ofAmerica; 4Department of Medicine, University of California, San Francisco, California, United States of America; 5Biostatistics Unit,The Feinstein Institute for Medical Research, North Shore–LIJ Health System, Manhasset, New York, United States of America;6Department of Medicine, Long Island Jewish Medical Center, North Shore–LIJ Health System, New Hyde Park, and Hofstra NorthShore–LIJ School of Medicine, Hempstead, New York, United States of America; 7Oklahoma Medical Research Foundation, OklahomaCity, Oklahoma, United States of America; 8Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York,United States of America; and 9Department of Molecular Medicine, Hofstra North Shore–LIJ School of Medicine, Hempstead, NewYork, United States of America

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with moredangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lym-phocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cellthat had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) andcluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA andhence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of twocell cycle–associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisonsof global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-prolifer-ation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunopheno-type was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analysessuggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, en-riched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cellsthat need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two pop-ulations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more ro-bust understanding of the development and clonal evolution of this currently incurable disease.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2011.00360

Address correspondence and reprint requests to Nicholas Chiorazzi, The Feinstein Institute

for Medical Research, 350 Community Drive, Manhasset, NY 11030. Phone: 516-562-1090;

Fax: 516-562-1011; E-mail: [email protected].

Submitted September 22, 2011; Accepted for publication September 22, 2011; Epub

(www.molmed.org) ahead of print September 23, 2011.

R E S E A R C H A R T I C L E

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First, the “proliferative compartment”may contain cells that developed newstructural DNA abnormalities leading tomore lethal disease. Furthermore, themost recently born fraction may be prog-eny of putative leukemic stem cells. Fi-nally, such cells would be potential tar-gets for therapies to abort clonalevolution.

We therefore studied the kinetic com-plexity of individual CLL clones to deci-pher fundamental insights about thepathophysiology of the disease. In partic-ular, we focused on further characteriz-ing the proliferative and resting compart-ments using differences in the densitiesof a surface membrane molecule upregu-lated after normal B-cell activation (clus-ter designation 5 [CD5]) and another in-volved in maintaining B-cell contact withstromal elements of solid lymphoid tis-sues (chemokine [C-X-C motif] receptor 4[CXCR4]). Using samples from patientsfor which CLL cells had been labeled invivo with 2H, we divided clones into sub-fractions enriched in the most prolifera-tive and most quiescent compartments.These fractions were then further charac-terized by comparing expression ofgenes encoding molecules usually upreg-ulated in dividing or resting populations.Finally, to provide a robust membranemap of these compartments that mightbe used for further characterization andtherapeutic targeting in patients, an ex-tended surface phenotype was definedwith a larger patient cohort.

MATERIALS AND METHODS

PatientsThe Institutional Review Board of the

North Shore–LIJ Health System approvedthese studies. After obtaining informedconsent in accordance with the Declara-tion of Helsinki, venous blood was col-lected from randomly chosen CLL pa-tients diagnosed by established criteria. Atotal of 15 subjects participating in the2H2O protocols were studied. Patientsdrank 2H2O for 6–12 weeks, dependingon the protocol, and cells were studied attwo time points during this period.

2H Measurements by GasChromatography/Mass Spectrometryand Calculation of the Fraction ofLabeled Cells

Peripheral blood mononuclear cellswere separated from heparinized venousblood and leukocyte-enriched fractionsby density gradient centrifugation usingFicoll-Paque (Pharmacia LKB Biotechnol-ogy, Piscataway, NJ, USA) and cryo -preserved until use. Calculation of thefraction of newly divided cells was per-formed after determination of 2H enrich-ment in plasma or saliva and of 2H en-richment in deoxyadenosine of genomicDNA as described (4).

Isolation of Cell Fractions on the Basisof Expression of CXCR4 and CD5

Peripheral blood mononuclear cellswere incubated with murine anti–humanmonoclonal antibodies (mAbs): CD5-FITC (fluorescein isothiocyanate),CXCR4-PE (phycoethrin), CD3–peridinin-chlorophyll-protein (CD3-PerCP) andCD19-allophycocyanin (CD19-APC) (allfrom BD Biosciences, San Jose, CA,USA). After gating on CD19+CD3–CD5+

events, cells were sorted with a BD FACSAria™ (Becton Dickinson Immuno-cytometry Systems, San Jose, CA, USA)on the basis of intensity of CXCR4 andCD5 (Figures 1A, B), and cell pelletswere stored at –80°C until performinggas chromato graphy/mass spectrometryor RNA extraction.

Gene Expression Profiling and GeneExpression Data Analyses

The quality of RNA extracted fromsorted cells using the RNeasy minikit(Qiagen, Valencia, CA, USA) was as-sessed using an Agilent RNA 6000 PicoKit (Agilent Technologies, ColoradoSprings, CO, USA). A total of 50 ng totalRNA was subjected to first- and second-strand reverse transcription followed bya single in vitro transcription amplifica-tion that incorporated biotin-labeled nu-cleotide to yield biotinylated antisenseRNA (a-RNA) using a TargetAmpNano-g Biotin-aRNA labeling kit (Epi-centre Biotechnologies, Madison, WI,

USA). Purified a-RNA was quantified,and the fragment size was ascertained ona Bioanalyzer (Agilent Technologies)using an Agilent RNA 6000 Nano Kit. Labeled a-RNA was hybridized to aHuman WG-6 v3.0 Expression BeadChipcontaining 48,804 probes and stainedusing streptavidin-Cy3 as per the manu-facturer’s instructions. The signal gener-ated was detected by high-performancelaser optics of the BeadArray Reader (Il-lumina, San Diego, CA, USA). Data werenormalized using quantile normalizationby BeadStudio software (Illumina). Todetermine differentially expressed genes,the CXCR4dimCD5bright versus CXCR4bright

CD5dim expression value ratio was com-puted for each patient and log-trans-formed, and a Student t test was per-formed using R (www.Rproject.org).Significant gene differences had 1.3 orgreater fold change and P < 0.01; for foldchange, we used the nth root of the prod-uct of all individual ratios, or the geo -metric mean of the ratio, for n samples.

Genes were assigned to specific func-tional categories using Ingenuity Path-way Analysis (IPA, www.ingenuity.com),Panther Classification System (Panther,www.pantherdb.org), DAVID Bioinfor-matics Resources 6.7, National Institute ofAllergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH)(DAVID, http://david.abcc.ncifcrf.gov/home.jsp) and GeneCards Batch Queries (GeneALaCart, www.genecards.org/BatchQueries/index.php). Significantlydifferent gene expression values wereclustered hierarchically on the basis of av-erage linkage on a correlation-based dis-tance (defined at http://arxiv.org/abs/cs/ 0402061). Heatmaps of gene expres-sion values for identified relevant func-tional categories were generated usingheatmap.2 in the gplots package of R.

Measurements of Surface andIntracellular Antigens by FlowCytometry

Peripheral blood mononuclear cellswere incubated with different combina-tions of murine anti–human mAbs di-rectly conjugated with the indicated fluo-


I N T R A C L O N A L C O M P L E X I T Y I N C H R O N I C L Y M P H O C Y T I C L E U K E M I A

rochrome: anti–CD5-FITC or -APC;CXCR4-PE, -APC, or -PECy7; CD3-PerCP; CD19-PerCP, -APC, -Pacific blueor -AmCyan; CD23, CD27, CD52, CD38,CCR7, CXCR3, BAFF-R, CTLA4, CD11a,CD11c, DR5, CD49d, CD62L and FcγRIIb,all in PE; and CD20-FITC (all from BDBiosciences). Unconjugated rabbit IgGantibody to receptor tyrosine kinase-likeorphan receptor 1 (ROR-1) was providedby Christoph Rader (National Cancer Institute/NIH, Bethesda, MD, USA). Sec-ondary staining was performed with PE-conjugated goat anti–rabbit IgG antibod-ies (Southern Biotech, Birmingham, AL,USA).

For surface membrane immunofluo-rescence, cells (2 × 105) in FACS buffer

(PBS + 10% fetal bovine serum + 1%sodium azide) were incubated with pri-mary antibody for 30 min at 4°C andthen exposed to secondary antibody for 25 min at 4°C, followed by fixationwith 0.1% formaldehyde in PBS. For intracellular detection of Ki-67 andminichromosome maintenance protein 6(MCM6), after membrane staining withanti–CXCR4-PE, –CD3-PerCP, –CD5-APCand –CD19- Pacific Blue (BD Biosciences),cells were fixed and permeabilized(Cytofix/ Cytoperm, BD Biosciences) andincubated with murine anti–Ki-67-FITCor anti–MCM6-FITC mAbs (BD Bio-sciences). Data were acquired with a BDLSRII flow cytometer or a FACS calibur(both Becton Dickinson Immunocytome-

try systems) and analyzed by FlowJov7.2.4 version. Within each CLL clone,relative expression in terms of percentpositive cells and mean fluorescent inten-sity of each surface or intracellularmarker was determined and compared.

All supplementary materials are availableonline at www.molmed.org.

RESULTSCLL clones can be divided into distinct

fractions on the basis of inverse surfaceexpression of CXCR4 and CD5. Byscreening a panel of chemokine receptorsexpressed on clones from a series of CLLpatients (4), we found an inverse rela-tionship between CXCR4 and CD5 densi-ties, with differing shapes among pa-tients (Figures 1A–D). The abundance ofcells representing the extremes of thesetwo populations (CXCR4dimCD5bright ver-sus CXCR4brightCD5dim) was relativelysmall and varied between patients(1–8%, not shown), with the majority ofcells (>90%) falling into an intermediatecategory (CXCR4intCD5int; Figures 1A–D).

We reasoned that high CD5 densitywould reflect cellular activation as in nor-mal human B cells (7), and low CXCR4levels would identify cells that internal-ized the receptor because of an activationevent and thereby passaged from a lym-phoid tissue to the periphery (8). As de-tailed elsewhere (5), incorporation of 2Hinto cellular DNA in patients given 2H2Ois a direct measure of newly synthesizedDNA and hence cell division, thereby al-lowing study of birth rates of CLL cells invivo (6,9,10). Therefore, we sorted CLLcells from nine patients consuming 2H2O(Figure 1E) into three fractions and quan-tified 2H-labeled deoxyadenosine in eachfraction by gas chromatography/massspectrometry. This step indicated that at21 d, the CXCR4dimCD5bright fraction wasmarkedly enriched in divided cells com-pared with the remainder of the clone,and this result lasted through 42 d (Fig-ure 1F); this disparity was reflected bysignificantly different CXCR4dimCD5bright

to CXCR4brightCD5dim enrichment ratios atthe two time points (12.6 at 21 d and 10.9

Figure 1. Intraclonal kinetic differences in CLL fractions defined by CXCR4 and CD5.(A–D) Contour plots of CLL cells on the basis of CXCR4 and CD5 levels. An inverse rela-tionship giving a crescent shape is clear in ~35% of analyses (A), evident in ~50% (B), sug-gested in ~10% (C) and absent in a few patients (D). (E) Representative example of 2H2Oenrichment in plasma (CLL875) of patients drinking 2H2O. CLL cells were flow-sorted on thebasis of CXCR4 and CD5 densities at time points indicated by arrows. The vertical dashedline indicates end of 2H2O consumption. (F) Average fraction of 2H-labeled cells in thethree fractions defined by CXCR4/CD5 densities. *CXCR4dimCD5bright fraction contains sig-nificantly higher levels of 2H-labeled DNA compared with CXCR4intCD5int andCXCR4brightCD5dim fractions (P < 0.01 for both). (G) Plot indicates the ratio of 2H-labeledcells in CXCR4dimCD5bright over CXCR4brightCD5dim fractions, indicating that CXCR4dim

CD5bright cells proliferated ~11 times more than CXCR4brightCD5dim cells (*P < 0.0001).(H, I) CXCR4dimCD5bright fractions are significantly enriched in cells expressing Ki-67 (H) andMCM6 (I) compared with CXCR4intCD5int and CXCR4brightCD5dim fractions (*P < 0.01 for both).



at 42 d, Figure 1G; P < 0.0001). See Sup-plementary Figure 1 for data describingthe kinetics of each fraction for each patient.

These findings were verified by ana-lyzing ex vivo, in the CXCR4/CD5 frac-tions, two cell cycle–related molecules,Ki-67 and MCM6, both expressed fromthe G1 to M phase. Comparisons ofCXCR4dimCD5bright versus CXCR4intCD5int

versus CXCR4brightCD5dim fractions in anexpanded cohort of CLL patients weresignificantly different (P < 0.01) for bothKi-67 and MCM6 (Figures 1H–I).

Collectively, these in vivo and ex vivofindings indicate that the CXCR4dim

CD5bright and CXCR4brightCD5dim fractionsof CLL clones are highly enriched com-pared with the total circulating leukemicload, in recently divided/young andmore resting/older members, respec-tively. For simplicity, we refer to these asthe “proliferative” and “resting” com-partments going forward, even thoughthe dominant intermediate populationcontributes cells with similar phenotypesto a significant extent.

Proliferative and Resting FractionsExhibit Gene Expression DifferencesConsistent with Their PutativeContrasting Proliferative Histories

Global gene expression profiling (GEP)was performed on isolated fractions fromthe same patients. Using Illumina Hu-manWG-6 v3.0 expression arrays to mea-sure the relative expression of 25,440genes and selecting genes for ≥1.3-folddifference in expression and P ≤ 0.01, wedefined 1,299 genes differentially ex-pressed between the two compartments;715 genes were more highly expressed inthe proliferative and 584 in the restingfraction (Supplementary Table 1). Thesegenes were then segregated into cate-gories that would support their differencein time from cellular activation and divi-sion and in migratory capacities using anumber of bioinformatic programs anddatabases (see Materials and Methods).

Genes involved in cell proliferation.We determined the extent that the twofractions differed in expression of genes

Figure 2. Heatmaps illustrating genes typically expressed differentially between the prolifer-ative versus resting fractions of CLL clones. (A) Pro- and antiproliferation genes up- ordownregulated in the proliferative versus resting fractions of the nine CLL clones analyzed.(B) Antiapoptotic genes. (C) Genes related to oxidative injury. (D) Trafficking-related genes.(A–D) Genes presented are significantly differentially expressed (Supplementary Table 1)and were assigned to categories using Ingenuity Pathway Analysis, Panther ClassificationSystem, DAVID Bioinformatics Resources 6.7 and GeneCards Batch Queries. Significantly dif-ferentially expressed gene expression values for each set were clustered using hierarchicalclustering on the basis of the average linkage on a correlation-based distance.



involved in normal or abnormal cell proliferation. Consistent with theCXCR4dim CD5bright fraction being enrichedin divided cells, 20 pro-proliferationgenes were found in this compartment(Figure 2A, top) and 10 in the CXCR4brightCD5dim fraction. In contrast, alarger number of genes with an antipro-liferation function were found in theCXCR4brightCD5dim fraction than theCXCR4dimCD5bright fraction (12 genes versus 7; Figure 2A, bottom).

Among the pro-proliferation genes up-regulated in the CXCR4dimCD5bright frac-tion were two canonical molecules in-volved in cell cycle progression: Cyclin E1(CCNE1) and cyclin D2 (CCND2).CCND2 plus Ki-67 and MCM6, whichwere enriched in this fraction on thebasis of flow cytometry (Figure 3), havebeen found higher in CLL lymph nodesthan peripheral blood (11). Notably, ofthe pro-proliferation genes that werehigher in resting cells (Figure 2A, top),myocyte-specific enhancer factor 2C(MEF2C) and midkine (MDK; neuritegrowth-promoting factor 2) promotegrowth of cell types that are often in aresting state: hematopoietic progenitors(12) and embryonic stem cells (13), respectively.

Of the antiproliferative genes higher inthe CXCR4brightCD5dim fraction (Figure 2A,bottom), BCL6, BACH2 and RGS2 are of in-terest because they control checkpoints inB-cell activation and maturation (14–16).

Genes involved in cell survival. Re-cently born/divided cells would alsolikely receive prosurvival/antiapoptoticsignals, whereas cells of the resting com-partment would be less likely to receivethese, being temporally further from anactivation signal and trophic support oftissue microenvironments. A total of 25antiapoptotic genes are higher in the pro-liferative versus 8 in the resting fraction(Figure 2B, bottom). Of note, MEF2C ap-pears necessary for B-cell proliferationand survival after in vitro B-cell receptor(BCR) stimulation (17). Several genes up-regulated in the resting fraction are of in-terest. TNF receptor superfamily 13C(TNFRSF13C) is the receptor for the

B-cell activating factor that mediates sur-vival in normal and non-Hodgkin lym-phoma B lymphocytes (18,19). TNFSF15codes a ligand for TNFRSF25 and TNFRSF6B that is not known to be ex-pressed in B cells and that inhibitsgrowth in other cells (20). Finally, inter-leukin (IL)-4R, which promotes normalB-cell and CLL cell survival (21) and pro-liferation (22) and is expressed by CLLcells (23), is upregulated in this fraction.

Prodeath genes are slightly more abun-dant in the resting fraction, suggestingthat this compartment contains older,less robust cells (Figure 2B, top). For ex-ample, harakiri (HRK), forkhead box O3(FOXO3) and leptin (LEP) (24–26) stimu-late apoptosis in various cell types, andtransmembrane 123 (TMEM123) encodes amucinlike gene that leads to oncotic celldeath upon cross-linking (27).

Genes involved in oxidative injury.Cells of the proliferative fraction upregu-lated more genes involved in the genera-tion or repair of oxidative injury than inresting cells (27 genes versus 6; Fig-ure 2C), likely because of increased meta-bolic activity in the dividing/recently di-vided cells. In addition, oxidative injuryis a known feature of CLL cells (28).

Products of catalase (CAT), glucose-6- phosphate dehydrogenase (G6PD), glu-tathione peroxidase 1 (GPX1), heme oxyge-nase (decycling) 1 (HMOX1) andthioredoxin (TXN) have a role in scaveng-ing free radicals.

Genes involved in cell trafficking.Lastly, 33 trafficking-related moleculeswere expressed at higher levels in theproliferative and 8 in the resting frac-tions (Figure 2D). Consistent with cells ofthe proliferative compartment having re-cently exited lymphoid tissues is theheightened expression of integrin, beta 7(ITGB7) and CXCR3. ITGB7 remains onthe surface of cells after leaving solid tis-sues and is involved in passage of cellsinto mucosa-associated lymphoid tissues(29,30); CXCR3 is involved in CLL cellmigration along interferon (IFN)- inducible protein 10 and IFN-γ–inducedmonokine gradients (31). However, anumber of other genes do not readily fitthe profile of recent emigrants of lym-phoid tissues. In particular, overexpres-sion of adhesion molecules platelet en-dothelial cell adhesion molecule 1 (PECAM1),cluster designation 11A (lymphocyte function-associated antigen 1)/ integrin,alpha L (CD11a/ITGAL) and cluster

Figure 3. Extended immunophenotype of proliferative and resting CLL fractions. Surfacemarkers were analyzed by flow cytometry in CXCR4dimCD5bright (X) and CXCR4brightCD5dim

(G) fractions. All surface molecules are depicted but CCR7 and CTLA4 were greater inCXCR4dimCD5bright fractions, in terms of percent positive cells (A) or mean fluorescence in-tensity (B) or both. CCR7 and CTLA4 are higher in the CXCR4brightCD5dim fraction. *P < 0.01and §P < 0.05 (paired Student t test).



designation 11c/ integrin, alpha X (comple-ment component 3 receptor 4 subunit)(CD11c/ITGAX), the latter two of whichwere confirmed by quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR) (Supplementary Figure 2) andflow cytometry (Figure 3), seems para-doxical, since these are usually expressedon cells docking on the endothelium andexiting the circulation.

Cells of the resting compartment ex-pressed more pleckstrin homology domaincontaining, family A member 2 (PLEKHA2)mRNA (Figure 2D) and CCR7 andCTLA4 protein (Figure 3). CCR7 pro-motes homing of normal B lymphocytesand CLL cells to lymph nodes and medi-ates neoplastic B-cell homing in patientswith widespread nodal dissemination(32). Also, CCR7 and CXCR4 promotemetastasis of solid tumors (33).PLEKHA2 has been found to be highlyexpressed in poor outcome CLL cells(unmutated IGHV or high ZAP-70 levels)and to have a role in adhesion to fi-bronectin and laminin (34).

qRT-PCR Analyses of Selected GenesConfirm Their Overexpression inProliferative or Resting Fractions

We performed qRT-PCR for genes cod-ing molecules expressed on or at B-cellsurfaces to provide a guide for develop-ing an extended surface membrane phe-notype and to corroborate the GEP data.A total of 67.8% (38/56) of genes wereconfirmed as significantly different be-tween the two fractions (SupplementaryFigure 2). Expression levels of CD5 andCXCR4 served as internal controls.

Surface Molecules Further Distinguishthe Proliferative and RestingCompartments

Finally, to permit an even more precisedefinition of the two fractions in CLLand to define molecules that might bevaluable therapeutic targets for thesefractions, the CXCR4/CD5 subsets weresubjected to a detailed multiparameterphenotypic analysis (Figures 3A, B). Mol-ecules were studied because they aremarkers of B-cell activation, survival and

migration or are targets of mAbs in usein humans. The proliferative compart-ment contained significantly more cellsthat expressed, at higher densities, CD38,CD20, CD23, CD27, CD52, JML-1, DR5,CXCR3, CD49d, CD62L, CD11a andCD11c (Figures 3A, B). Only ROR-1,FcγRIIb and BAFF-R exhibited differ-ences solely on the basis of density. Twomolecules were higher in the restingcompartment: CCR7 (both in the percentof positive cells and intensity) andCTLA4 (intensity). It is noteworthy thatseveral of these molecules are targets ofmAbs or compounds already in clinicaluse (35–41) or are being tested in humansubjects (23,42–46).

DISCUSSIONThe aim of this study was to further

delineate and isolate cells with differentkinetics in CLL clones, focusing on cellsthat had recently divided or are resting.Using 2H-incorporation into newly syn-

thesized DNA of dividing cells as a mea-sure of proliferation, we previouslydemonstrated intraclonal kinetic com-plexity in CLL, defining a small popula-tion (~0.1–1% of the leukemic clone)that divided daily (6). Because the 2H- incorporation approach does not permitvisualization of individual cells thathave divided, we subsequently enrichedfor cells of the proliferative fractionusing surface expression of a candidatemolecule, CD38, that is expressed on ac-tivated human B cells (47) and is associ-ated with poor clinical outcome in CLL(48). These studies indicated that withineach CLL clone, the CD38+ fraction con-tained ~2.5 times more recently dividedcells than the CD38– fraction (4). In thepresent study, using differential surfacemembrane densities of CXCR4 and CD5,we isolated fractions differing by ~11-fold in the percentage of recently di-vided cells (Figure 1G). In line with ourprevious study, CD38 expression was

Figure 4. Hypothetical model of the lifecycle of a CLL B cell. Part 1: CLL cells rest on thestroma because of at least CXCR4-CXCL12 interactions. When stimulated, cells are acti-vated and divide, upregulating CD5, internalizing CXCR4 and detaching from stroma. Theprocess could be ligand-induced (for example, BCR or toll-like [TLR] or other pathways) orspontaneous. Low CXCR4 levels increase the chances of recently divided CLL cells(CXCR4dimCD5bright phenotype) exiting solid tissue and reaching peripheral blood. Part 2:Recently born/divided CLL cells reach peripheral blood as members of theCXCR4dimCD5bright fraction. Over time, possibly because of a lack of trophic input from thesolid tissue microenvironment, cells begin to reexpress CXCR4 to trek back to nutrient-richniches. This leads to expression of a CXCR4intCD5int and then CXCR4brightCD5dim mem-brane phenotype. The model considers the three fractions to be linked as a continuum.Part 3: CXCR4brightCD5dim CLL cells have the greatest chance of detecting and followinga CXCL12/SDF1 gradient, thereby reentering lymphoid solid tissue and receiving prosur-vival stimuli. Those that do not reenter die by exhaustion.



significantly higher in the CXCR4dim

CD5bright proliferative subset (Figure 3A).Finding increased numbers of cells ex-pressing Ki-67 and MCM6 in the samefraction confirmed the accuracy of thisapproach (Figures 1H, I). Finally, globalGEP on the same CXCR4/CD5 subsetsfurther validated that our approachidentified subsets of CLL clones differ-ing in proliferative histories, since theCXCR4dim CD5bright fraction overex-pressed more genes that support cell division (Figure 2A), block apoptosis(Figure 2B) and induce/repair oxidativedamage (Fig ure 2C) than the restingfraction, which contained more genesthat inhibit cell division (Figure 2A) andsurvival (Figure 2B). Nevertheless, thesefractions remain heterogeneous, likelycontaining cells from the intermediatefraction with characteristics distinctfrom those we are describing. Thus, thesituation is complex, and a more expan-sive collection and analysis of such datawill better elucidate unique features ofthe two compartments. Still, the detec-tion of gene (Figure 2) and protein (Figures 1 and 3) expression differencesconsistent with recently divided andresting cells, combined with the directin vivo demonstration of significant en-richment in cycled cells in the prolifera-tive fraction and a markedly reducedabundance in the resting fraction (Fig -ure 1), confirm that using CXCR4/CD5densities on CD19+ cells is a major im-provement in the delineation of thesetwo compartments.

The data conjure a lifecycle for indi-vidual CLL cells representing a contin-uum between the CXCR4dimCD5bright,CXCR4intCD5int and CXCR4 brightCD5dim

fractions (Figure 4). At one extreme isthe proliferative fraction, highly en-riched in young, vital cells that recentlyleft a solid lymphoid tissue where acti-vation and proliferation occurred. At theother end is the resting compartment,containing older, less robust cells thatmay have been circulating in the periph-ery longer and are attempting throughhigh CXCR4 levels to migrate into asolid tissue niche to avoid death. In this

model, the intermediate fraction, whichis the bulk of the clone, links the ex-tremes and is the fraction from whichmost of our current knowledge on circu-lating CLL cells is derived. This relation-ship is supported by the fact that BCRstimulation leads to downregulation ofCXCR4 expression (49), as depicted inFigure 4, part 1. Although our data areconsistent with this model, the processremains hypothetical because we haveonly studied cells in the peripheral cir-culation. Notably, it was recently shownby comparative GEP of bone marrowand lymph node cells that lymph nodesmay host most of the CLL proliferativeevents (11). In our study, cells with lowCXCR4 and high CD5 levels could in-clude emigrants from any site, and onlyone of the differentially expressed mole-cules involved in trafficking that we de-fined is site specific: ITGB7 suggests spe-cific passage to mucosa-associatedlymphoid tissues. Considering that atleast some CLL cells may be derivedfrom a human B-1 cell equivalent (50,51),the potential of migration to such tis-sues, which is favored for murine B-1cells (52), is provocative.

This hypothetical lifecycle also sug-gests “stem-like” or “initiating” capabili-ties of CLL subclones (53). For instance,the CXCR4brightCD5dim cells could be nestled on stromal elements (Figure 4,part 1) before being released and givingrise to the dividing CXCR4dimCD5bright

cells of the proliferative and bulk frac-tions (Figure 4, part 2); therefore, theseCXCR4brightCD5dim resting cell fractionscould be a distinct self-renewing subsetfrom which all clonal members emanate.In a related scenario, these same cellscould be members of the CXCR4bright

CD5dim fraction of the resting compart-ment of the blood on their way back to asustaining niche in a lymphoid tissue(Figure 4, part 3), where they might di-vide again. The former possibility wouldbe consistent with a B-1–like cell, becausein mice these cells have self-renewal ca-pacity (54), implying that the ability tomigrate effectively would be the key fac-tor determining survival of the clone. If

the latter possibility is the case, a carefulanalysis of the resting compartmentmight yield a subset of cells with stem-like/initiating capacities.

Delineation of intratumor kinetic het-erogeneity may also have therapeuticimplications. Although most anticancertherapies are predicated on eliminatingthe entire neoplastic clone, preferen-tially targeting the proliferative andresting compartments might be effica-cious. Thus, preferentially eliminatingthe most recently born/divided cellswould limit the fount of clonal evolu-tion; preventing cells from reenteringsolid tissue would thwart the less ro-bust, apoptosis-prone resting cells fromreceiving survival signals. Collectively,these approaches would lock a cloneinto a steady state of genetic abnormali-ties and eventually lead to clonalshrinkage based on spontaneous celldeath (6,55) and death due to survivalsignal deprivation (56).

Although speculative, this approachcould be tested, since relevant com-pounds are already in clinical use orevaluation. For example, the proliferativecompartment expresses higher levels ofCD20, CD23, CD38, CD49d, CD52,CD11a and DR5 (Figure 3), all of whichcan be targeted by specific, availablemAbs (23,35–46). Other targets of poten-tial interest in the proliferative fractionare CXCR3 (GEP, qRT-PCR, flow), CD27(flow), ITGB7 (GEP and qRT-PCR), Ly96(GEP), CD62L (flow) and CD11c/ITGAX(flow and GEP). For the resting compart-ment, the most relevant molecules ap-pear to be CXCR4 and CCR7 (flow andGEP). Plerixaflor, a CXCL12 (CXCR4 lig-and) inhibitor used for stem cell mobi-lization, is being tested in CLL treatment(39). Although no compound is presentlyavailable to target CCR7 in vivo, an anti-CCR7 mAb kills CLL cells in vitro with-out affecting T cells that also expressCCR7 (57). Thus, an anti-CXCR4 and/oranti-CCR7 approach could limit the abil-ity of CXCR4bright and CCR7bright cells toreenter solid tissues, fostering restingcompartment apoptosis. Finally, target-ing IL-4R and CTLA4 is feasible because



a recombinant IL-4–Pseudomonas exo-toxin fusion protein that binds IL-4R (23)and overcomes apoptosis resistance ofCLL cells (58) is in clinical use, andmAbs reactive with CTLA4 (40,41) arecurrently being used to potentiate T-cellresponses against tumors (59).

ACKNOWLEDGMENTSThis work was supported in part by

the National Cancer Institute/NIH(CA81554) and by philanthropic contri-butions from The Karches Foundation,Prince Family Foundation, Marks Foun-dation, Jerome Levy Foundation, LeonLevy Foundation, Andrew and Mona Albert Fund Inc., and Joseph ElettoLeukemia Research Fund. We thankAarti Damle and The Feinstein Institute’smicroarray core facility for gene expres-sion analyses.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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intraclonal complexity in chronic lymphocytic leukemia ......1374 | calissano et al. | mol med...

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