cancer research serologic markers of effective tumor ......microenvironment and immunology serologic...

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Published OnlineFirst February 2, 2010; DOI: 10.1158/0008-5472.CAN-09-3143

Microenvironment and Immunology
Cancer
Research

Serologic Markers of Effective Tumor Immunity againstChronic Lymphocytic Leukemia Include NonmutatedB-Cell Antigens

Ovidiu Marina1,7, Ursula Hainz1,2, Melinda A. Biernacki2,8, Wandi Zhang1, Ann Cai4, Jonathan S. Duke-Cohan2,4,Fenglong Liu3, Vladimir Brusic1, Donna Neuberg3, Jeffery L. Kutok4,5, Edwin P. Alyea2,4,6, Christine M. Canning1,2,Robert J. Soiffer2,4,6, Jerome Ritz1,2,4,6, and Catherine J. Wu1,2,4,6

Abstract

Authors' A2Medical ODana-Farbof 5PatholoMassachuProgram, Rof Medicine

Note: SupResearch O

PresentedMeeting ofAtlanta, Ge

CorresponHarvard InBoston, MAcwu@partn

doi: 10.115

©2010 Am

Cancer R

Downlo

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve du-rable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunityin eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL)responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody re-sponses developing in two DLI-treated patients who achieved long-term remission without graft-versus-hostdisease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantlyintracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma.Three antigens—C6orf130, MDS032, and ZFYVE19—were identified by both patients. Along with additionalcandidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein ex-pression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and theshared antigens do not represent minor histocompatibility antigens, as their sequences are identical in bothdonor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normalsubjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses wereserologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporallycorrelated with clinical disease regression. These B-cell antigens represent promising biomarkers of effectiveanti-CLL immunity. Cancer Res; 70(4); 1344–55. ©2010 AACR.

Introduction

Although chronic lymphocytic leukemia (CLL) is most of-ten controlled with combination chemotherapy (1), allogene-ic hematopoietic stem cell transplantation (HSCT) remainsthe only potentially curative treatment for this disease(2, 3). It has been long appreciated that these curative re-sponses rely on the ability of donor-derived immune effectorsto recognize and eliminate leukemic cells. The existence of

ffiliations: 1Cancer Vaccine Center and Departments ofncology and 3Biostatistics and Computational Biology,

er Cancer Institute; 4Harvard Medical School; Departmentsgy and 6Medicine, Brigham and Women's Hospital, Boston,setts; 7Will iam Beaumont Hospital, Transitional Yearoyal Oak, Michigan; and 8University of Connecticut School, Farmington, Connecticut

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

in part as a simultaneous oral session at the 49th Annualthe American Society of Hematology, December 9, 2007,orgia.

ding Author: Catherine J. Wu, Dana-Farber Cancer Institute,stitutes of Medicine, Room 416, 77 Avenue Louis Pasteur,02115. Phone: 617-632-5943; Fax: 617-632-3351; E-mail:

ers.org.

8/0008-5472.CAN-09-3143

erican Association for Cancer Research.

es; 70(4) February 15, 2010

on May 22, 202cancerres.aacrjournals.org aded from

the graft-versus-CLL response has been supported by studiesshowing that (a) myeloablative allogeneic, but not autolo-gous, HSCT consistently results in a plateau in survival after1 year and the development of undetectable minimal residualdisease (4); (b) disease remission is closely associated withimmunologic competence [i.e., following taper of immuno-suppression or development of graft-versus-host disease(GvHD); refs. 5, 6]; and finally (c) donor lymphocyte infusion(DLI) for posttransplant relapsed CLL effectively generatesdonor-derived tumor immunity against CLL (7). In this pro-cedure, infusion of donor lymphocytes without further che-motherapy or radiation therapy results in remission in 30%to 50% of patients with relapsed CLL (8–10).Because of its clear clinical efficacy in the absence of ad-

ditional therapy, DLI is a promising system for elucidatingthe mechanism of donor-derived immunity to CLL. Identi-fying and characterizing the antigenic targets of DLI mayprovide insight into the basis of the antitumor effect ofDLI. Although T cells clearly participate in graft-versus-leukemia (GvL) responses (11), several studies have impli-cated an important role for B-cell immunity in remissionof hematologic malignancies following transplant and DLI(12–14). Some B-cell–defined antigens (15) have beenshown to elicit T-cell responses, and effective tumor im-munity likely comprises a coordinated humoral and cellu-lar adaptive immune response.

1. © 2010 American Association for Cancer Research.

http://cancerres.aacrjournals.org/

Serologic Markers of Effective Immunity to CLL


To date, few CLL-specific target antigens have been identi-fied (16–19). In the current study, we identify several novelcandidate antigens through analysis of B-cell responses inCLL patients with clinically evident tumor immunity butwithout anti-host immune reactions following DLI. By usinghigh-density human protein microarrays probed with plasmaantibody from before and after DLI, we rapidly identified CLLantigens that elicit potent antibody responses in close tempo-ral association with clinical response to DLI. Potential uses ofthese antigens include immune monitoring of CLL followingtherapy and even as immunogens for CLL-specific vaccines.

Materials and Methods

Preparation of cell and plasma samples. Serum or plas-ma samples were obtained from normal donors and patientsenrolled on clinical research protocols that were approvedby the Human Subjects Protection Committee at the Dana-Farber Cancer Institute (DFCI). Plasma was removed follow-ing brief centrifugation of whole blood and cryopreserved at−80°C until the time of analysis. Peripheral blood mononucle-ar cells (PBMC) from normal donors and patients were iso-lated by Ficoll/Hypaque density gradient centrifugation,cryopreserved with 10% DMSO, and stored in vapor-phaseliquid nitrogen until the time of analysis.Processing and analysis of protein microarrays. Protein

microarrays (Human ProtoArray v3, Invitrogen) from oneprinting lot were probed with patient plasma diluted 1:150and processed according to the manufacturer's recommen-dations, as previously described (20). These microarrays con-tain ∼5,000 NH2-terminal glutathione S-transferase (GST)fusion human proteins generated in an insect cell line, withproteins spotted in adjacent duplicates on nitrocellulose-coated glass slides. Binding of plasma immunoglobulin toprotein features on the array was detected using an AlexaFluor 647–conjugated anti-human IgG (H and L chain;1:2,000 dilution; Invitrogen). Protein microarrays werescanned and analyzed with correction for protein concentra-tion, as previously described (20). Significant signal changewas defined using the pretreatment and posttreatment Zscores, Zpre and Zpost, based both on a change in magnitudeZdelta = Zpost − max(0, Zpre) and in ratio Zmult = Zpost/max(1,Zpre) greater than a cutoff n. The cutoff n was set at 5 forsignificant interactions and 3 for borderline interactions.Both replicate spots were required to pass the test individu-ally for an interaction to be considered significant. Candidateantigens were ranked based on the largest cutoff n thatwould identify them as significantly changed. Microarray da-ta and calculated significance values are in the Gene Expres-sion Omnibus repository under accession GSE11564.Sequencing of candidate antigens from tumor and do-

nor tissue. The genes encoding the candidate antigens weredirectly sequenced using the M13 forward and M13 reverseprimers after gene-specific PCR amplification of genomicDNA and TA TOPO cloning (Invitrogen). Genomic DNAwas isolated from patient tumor and donor-generated EBVcell lines using a Wizard kit (Promega), following the manu-facturer's instructions. Flow cytometric analysis of patient

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tumor revealed all CD19+ cells to coexpress CD5, so tumorcells were immunomagnetically purified to >95% purity usingCD19+ microbeads (Miltenyi). Sequences were aligned usingSequencher (Gene Codes Corp.).Gene expression microarray data analysis. To compare

gene expression of our collection of target antigens betweennormal B cells and CLL cells, raw data files (.CEL) generatedon Affymetrix U95v2 microarrays by Klein and colleagues(21) were collected and normalized using Robust MultiarrayAveraging. We used Resourcerer (22) to map gene identifiersof the panel of target antigens to Affymetrix probe set IDs.For those targets that mapped to multiple probe set IDs,the median of the expression values was used. Raw data fileswere also processed using Affymetrix MAS 5.0, and geneswere defined as detectable if the detection call was “present”in ≥40% of samples for each cell type. The Fisher's exact testwas used for comparison between groups.Measurement of antigen-specific gene expression by

quantitative PCR. To evaluate the gene expression of thecandidate antigens, RNA was extracted (RNeasy kit, Qiagen)from normal PBMCs, immunomagnetically purified B cellsfrom normal PBMCs (CD19+ microbeads; Miltenyi), and B-CLLtumor cells. CLL RNA was extracted from cryopreserved sam-ples of peripheral blood or marrow that were known to con-tain >85% tumor cells. First-strand cDNA was generated from2 μg of total RNA by using random hexamers (Pharmacia LKBBiotechnology) and reverse transcriptase (SuperScript, LifeTechnologies) according to the manufacturer's instructions.Antigen-specific gene expression was measured by quantita-tive real-time PCR (qRT-PCR; Taqman Gene Expression As-says, Applied Biosystems) using a 7500 Fast Real-Time PCRcycler (Applied Biosystems). Transcript expression was mea-sured relative to glyceraldehyde-3-phosphate dehydrogenase(GAPDH). The significance of the difference in gene expressionof target antigens between cell populations was evaluated us-ing the exact Wilcoxon rank-sum test.Generation of cell lysates and Western blotting. Whole-

cell lysate was generated from patient samples by lysis withradioimmunoprecipitation assay buffer [1% NP40, 0.5% deox-ycholate, 0.1% SDS, 125 mmol/L sodium chloride, 50 mmol/LHEPES (pH 7.4)] in the presence of protease and phosphataseinhibitors. Lysates (20 μg of total protein per lane) weresubjected to gel electrophoresis using 4% to 15% gradientSDS-PAGE gels in Tris-glycine buffer and transferred ontonitrocellulose filters in Tris-glycine buffer containing 20%methanol. Antibodies specific to DAPK3 (1:500; Epitomics),MDS032 (1:1,000; Aviva Systems), SERBP1 (1:500; Abnova),ZFYVE19 (1:400; Abnova), and OGFOD1 (1:100; Abcam)were used to detect protein expression of the respectiveantigens by Western blot followed by incubation withhorseradish peroxidase (HRP)–linked secondary antibodies.Antibody to β-actin (1:5,000; Sigma-Aldrich) was used as acontrol to ensure equal loading of lanes.Plasmid acquisition and in vitro translation of candi-

date antigens. To facilitate protein expression for target val-idation and characterization, DNA sequences encodinga subset of the candidate antigens were either acquired inor cloned into Gateway (Invitrogen) expression vectors.

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DNA sequences in Gateway vectors were obtained from Plas-mID (Harvard Institute of Proteomics, Cambridge, MA;ref. 23), the Ultimate ORF collection (Invitrogen), or the Hu-man Orfeome Collection (generous gift of Dr. Mark Vidal,Center for Cancer Systems Biology, DFCI, Boston, MA;ref. 24). Additional DNA sequences were purchased fromthe American Type Culture Collection, Open Biosystems, orthe Resource Center of the German Human Genome Projector were directly PCR amplified from CLL tumor cell cDNAand cloned into the pDONR221 Gateway vector (Invitrogen).The majority of acquired cDNA clones originated from theI.M.A.G.E. Consortium (Lawrence Livermore National Lab-oratory; Supplementary Table S1; ref. 25). The inserts of allsequences in Gateway vectors were verified by sequencingand then shuttled into a Gateway-converted mammalian ex-pression vector containing a T7 promoter and a COOH-terminal GST tag (gift of Wagner Montor, Harvard Instituteof Proteomics) using LR Clonase (Invitrogen). Candidateantigens were expressed in vitro with rabbit reticulocyte ly-sate (TNT T7 Quick Coupled Transcription/Translation, Pro-mega) using biotinylated lysine tRNA (Transcend tRNA,Promega). Reactions (50 μL) using 1 to 2 μg of templateDNA and 2.5 μL of Transcend tRNA were incubated at 30°Cfor 90 min on a tabletop shaker at 950 rpm. A reaction using1.5 μg of DNA encoding luciferase was performed with eachsynthesis batch as a positive control.Immunoprecipitation of candidate antigens using pa-

tient plasma. Protein was immunoprecipitated using patientplasma, as previously described (20). In brief, 5 μL of reticu-locyte lysate product were immunoprecipitated with 5 μL ofserum or plasma at 4°C. To mirror the short incubation timeperiod used for the protein array probing, and to differentiatestrong antibody interactions from nonspecific cross-reactivity,we limited the immunoprecipitation reaction to 1 h. Immu-noprecipitated products were isolated using 50% protein A–Sepharose CL-4B beads (GE Healthcare) and visualized byimmunoblot. Protein was detected using immunoperoxidase-streptavidin (1:5,000 dilution; MP Biomedicals). The blotswere developed with SuperSignal West Femto detection sub-strate (Pierce Biotechnology) and imaged with BioMax Lightfilm (Kodak).For some experiments, immunoprecipitation of a single

candidate antigen against up to 48 serum or plasma sampleswas performed simultaneously to directly compare antigen-antibody affinity between plasma samples. Immunoprecipita-tion against GST was used as comparison for backgroundserum reactivity. As a control and for comparing protein bandsacross immunoblots, a 1:440 dilution of one batch of luciferasereticulocyte lysate product was loaded onto one to two lanes oneach immunoblot. Serum or plasma sample reactivity was com-pared against the range of reactivity evidenced by the 12 normalsubjects, taking into account the difference in the strength ofthe control band on each Western blot.

Results

DLI for CLL is associated with potent humoral immuneresponses.We identified two patients with relapsed CLL who

Cancer Res; 70(4) February 15, 2010


remained in continuous remission more than 8 years afterreceiving CD8-depleted DLI (26). As shown in Table 1, bothpatients A and B failed multiple regimens of conventionalchemotherapy before receiving myeloablative T-cell–depletedHSCT from HLA-matched sibling donors. Both relapsed withclinical disease within 5 years of transplantation and receivedDLI without any further chemotherapy or radiotherapy. Fol-lowing DLI, both patients had a clear GvL response withoutacute or chronic GvHD, showing rapid conversion to full do-nor hematopoiesis and gradual disappearance of histologi-cally evident marrow disease over 6 to 16 months.To identify the B-cell targets associated with CLL tumor

regression in these two patients, we used plasma collectedat 0 to 1 months before and 9 to 12 months after DLI assources of antibody for probing high-density protein micro-arrays. The vast majority of array proteins were bound atcomparable levels by pre- and post-DLI plasma antibodiesbased on raw fluorescence intensity. Using a concentration-dependent analysis, significant antigen-antibody interactionswere identified, shown as black diamonds in Fig. 1 (20).These candidate antigens comprised <1% of spotted proteins.No proteins had significantly decreased antibody reactivityfollowing immunotherapy.Targets of the DLI-induced humoral immune response

in patients with CLL. Analysis of the protein microarray datayielded 16 candidate DLI-associated antigens for patient Aand 21 for patient B. These antigens were ranked based onthe largest cutoff n that would identify them as significantlychanged. Although most candidate antigens were identifiedin only one patient, the two proteins encoded by genesZFYVE19 and C6orf130 were recognized by both patients. Athird protein, MDS032, was identified as a candidate antigenfor patient A and elicited borderline (n ≥ 3) antibody reactiv-ity for patient B. The candidate antigens and the shared an-tigen MDS032, only borderline significant for patient B, arelisted in Table 2. The candidate antigens as well as 26 inter-actions of borderline significance for patient A and 31 for pa-tient B are further described in Supplementary Table S1.We queried publicly available databases, including Entrez,

UniGene, Harvester, and SymAtlas (27–29), for informationon the function of the 35 identified candidate antigens, sum-marized in Table 2. Of the 25 known candidate antigens, 24are intracellular and 18 are reported as tumor associated(27). Eight have DNA or RNA binding activity, four are partof the ubiquitination pathway, two are tumor suppressors,and one each is lymphocyte associated or functions in thecell cycle, spermatogenesis, or apoptosis.Validation of serologic reactivity to candidate antigens.

Significant microarray signals may result from true interac-tions, erroneous measurement, or interactions with misiden-tified proteins, such as due to improper synthesis orcoprecipitation of additional proteins. To confirm that theserologic targets identified by microarray analysis representtrue antibody-antigen interactions, we independently vali-dated antibody reactivity against our screened candidateantigens. Figure 2A schematically summarizes our validationstrategy. We expressed our candidate antigens as COOH-terminal GST fusion biotinylated proteins in a cell-free

Cancer Research





mammalian expression system, a different method of proteinmanufacture than used on the protein microarrays. We thenimmunoprecipitated the recombinant proteins with patientplasma and visualized immunoprecipitated proteins by im-munoblot. In this manner, interactions between serum anti-bodies and properly folded and posttranslationally modifiedantigen were detected, and nonspecific interactions were ex-cluded on the basis of size on immunoblot.Using this assay, we successfully validated 22 of the 30 test-

ed candidate antigen interactions, listed in Table 2. Eight in-teractions failed validation, and eight remained untested dueto limited patient sample. Figure 2B shows representative re-sults from these studies. Consistent with our bioinformaticanalysis, post-DLI plasma from both patients A and B immu-noprecipitated greater amounts of the shared antigensC6orf130, MDS032, and ZFYVE19 compared with pretreat-ment plasma. For SERBP1 and DAPK3, identified by patientA, and FAM122A and OGFOD1, identified by patient B, great-er immunoprecipitated antigen was evidenced only by thepost-DLI sample from the patient who identified the interac-tion. Equivalently high immunoprecipitation reactivityagainst the control EBNA1 construct was seen for all plasmasamples tested. Interactions of higher significance wereslightly more likely to be validated, with 88% of tested inter-actions with rank <10 validated, whereas only 57% of testedinteractions with rank ≥10 were validated (Table 2).A subset of candidate antigens is highly expressed in B

lineage cells. To broadly survey the gene expression of our col-lection of antigens, we first compared the gene expression ofour candidate antigens in primary CLL cells and normal B-cellsubsets using existing gene expression microarray datagenerated on the Affymetrix U95v2 array (21). Probe sets for18 of the 35 candidate antigenswere present on thismicroarray.In these data, 5 of the 18 matched candidate antigens—LCP2,PDCD4, MKNK1, SRPK2, and CHD2—showed higher expressionin CLL compared with normal B cells (P < 0.01) but none at thelevel of differential expression that was found for the most sig-nificantly upregulated 33 genes reported in that study.Because many of the candidate antigens were not repre-

sented on the gene expression microarray, we next directlyquantified antigen-specific gene expression in normal and



malignant B cells by using qRT-PCR. We performed gene ex-pression studies for nine candidate antigens in three tissuesources: normal PBMC, normal B cells, and CD19+CD5+

CLL tumor cells. No significant difference in gene expressionwas observed among the three tissue groups for three of theantigens (PRKAA1, POLR3D, and SPZ1). We observed in-creased expression in normal and malignant B cells com-pared with PBMCs for the three shared antigens C6orf130,MDS032, and ZFVYE19 as well as for OGFOD1, whereas athird pattern, in which antigen-specific gene expression is

Table 1. Clinical characteristics of patients A and B

Patient
Age atBMT (y)
Patient/donorsex

HSCTtype

Treatmentbefore BMT

Time torelapse(mo)

1. © 2

Pre-DLItreatment

010 Americ

Donor chimerismconversion/

pathologic response(mo after DLI)

Cancer Res; 70

an Association for C

Post-DLI

GvHDfo

(4) Febru

ancer R

Lastllow-up(y)

A
51 M/F(sister)
MM
yeloablativeRD TCD
Leukeran +
prednisone; 6 cyclesCHOP; fludarabine
60
None 6/16 None 10
B
49 M/F(sister)
MM
yeloablativeRD TCD
6 cycles CHOP;dexamethasone +BEAM

50
None 2/6 None 8
Abbreviations: BMT, bone marrow transplantation; MRD, matched related donor; TCD, T-cell depleted.

Figure 1. Post-DLI plasma contains greater antigen reactivity thanpre-DLI plasma. A, plots of the pre-DLI versus post-DLI medianbackground-subtracted fluorescence signal for all microarray proteins forpatients A and B. Each point represents the average across the tworeplicate protein spots on the microarray. B, calculated significance of themeasured pre- and post-DLI fluorescence, corrected for proteinconcentration, for each microarray protein (20). The black line denotesthe cutoff function used to determine significance. ⧫, significantinteraction; •, other protein.

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Table 2. Candidate antigens showing increased antibody reactivity after compared with before DLI forpatients A and B

Accession

ncer Res; 7

Downloade

Gene P

0(4) Februa

cand from

atient

ry 15, 20

cerres.

Rank*

10

aacrjo

Reproduced†

onurnals.org

Size(aminoacids)

C

May 2

hromosome

2, 2021. © 20

Protein

10 American Association

Function

NM_001348
DAPK3 A 1 Y 454 19p13.3 D eath-associatedprotein kinase 3
P
ositive regulator ofapoptosis
BC013778 S
LC7A6OS A 2 Y 309 16q22.1 S olute carrier family7 member 6 oppositestrand
U
ncharacterized
NM_015640
SERBP1 A 4 Y 387 1p31 S ERPINE1 mRNAbinding protein 1
R
egulation ofSERPINE1 mRNAstability
BC012596
ARPC4 A 6 — 168 3p25.3 A ctin related protein 2/3complex, subunit 4
A
ctin filamentpolymerization
NM_182692
SRPK2 A 8 — 688 7q22-q31.1 S FRS protein kinase 2 S pliceosome assemblyand splicing factortrafficking
BC002755
MKNK1 A 9 Y 465 1p33 M APK-interactingserine/threoninekinase 1
M
APK signalintegration
NM_018070
SSBP3 A 10 — 368 1p32.3 s sDNA binding protein 3 Tra nscription regulation BC038105 MPP7 A 11 — 576 10p11.23 M embrane protein,
palmitoylated 7L
ocalization of proteins
to tight junctions
BC027729 N/A A 12 — 130 3p25.1 N /A U ncharacterized NM_004252 S LC9A3R1 A 13 — 358 17q25.1 S olute carrier family 9,
isoform 3 regulator 1P
lasma scaffold protein
BC007347
CHD2 A 14 N 501 15q26 C hromodomainhelicaseDNA binding protein 2
E
ukaryotic nucleusorganization
NM_032567
SPZ1 A 15 Y 430 5q14.1 S permatogenic leucinezipper protein 1
C
ytokinesis duringdevelopment;spermatogenesis
BC016848
FAM131C A 16 Y 280 1p36.13 F amily with sequencesimilarity 131, member C
Hy
pothetical
BC021092
ZFYVE19 A,B 3,18 Y,Y 328 15q15.1 Z inc finger FYVEdomain-containingprotein 19
U
biquitin-proteinligase
NM_145063
C6orf130 A,B 5,19 Y,Y 152 6p21.1 H ypothetical U ncharacterized BC006005 MDS032 A,B 7,35 Y,Y 146 19p13.11 H ematopoietic stem/
progenitor cell proteinU
ncharacterized
BC036910 L
OC388882 B 1 Y 240 22q11.23 N /A‡ P ossibly nonsense-mediated mRNA decay
NM_138333
FAM122A B 2 Y 287 9q21.11 F amily with sequencesimilarity 122A
U
ncharacterized
BC031650
SH3RF2 B 3 N 220 5q32 S H3 domain containingring finger 2
U
biquitin conjugationpathway
BC037547
CDC20B B 4 Y 192 5q11.2 C ell division cycle20 homologue B
R
egulates theanaphase-promotingcomplex/cyclosome
BC001426
UQCRH B 5 Y 91 1p33 U biquinol-cytochrome creductase hinge protein
Mi
tochondrial
BC034401
N/A B 6 N 71 16 N /A U ncharacterized
(Continued on the following page)

Cancer Research

for Cancer Research.




significantly increased in CLL cells compared with normal Bcells, was observed for DAPK3 and SERBP1 (Fig. 3A). Consis-tent with the notion that GvL-associated antigens may be B-cell lineage genes, the three shared antigens C6orf130,MDS032, and ZFYVE19 showed significantly higher expres-sion levels in normal and malignant CD19+ B cells comparedwith PBMCs (P < 0.02). Western blotting of lysates from CLLB cells and normal B cells compared with PBMC confirmedthese results (Fig. 3B). These results indicate that a subset ofcandidate antigens is both immunogenic and highly ex-pressed in CLL.



Candidate antigens are not minor histocompatibilityantigens. The immunogenicity of candidate antigens canarise from sequence differences between donor, host, and tu-mor or differential presentation of antigen epitopes by tumorcells. Sequence differences may exist between donor and hostdue to gene polymorphisms, resulting in minor histocompat-ibility antigens (mHA). Direct sequencing of 6 to 10 indepen-dent copies of both tumor and donor cell templates revealedthe absence of sequence differences for ZFVYE19, MDS032,C6orf130, and DAPK3 between donor and CLL tumor cells.Thus, the selected subset of the identified antigens is not

Table 2. Candidate antigens showing increased antibody reactivity after compared with before DLI forpatients A and B (Cont'd)

Accession
Gene P atient Rank* Reproduced† Size(aminoacids)
C
hromosome
1. © 20

Protein

Cancer R

10 American Association

Function

NM_005522
HOXA1 B 7 Y 335 7p15.3 H omeobox A1 D evelopmenttranscription regulation
NM_005565
LCP2 B 8 Y 533 5q33.1-qter L ymphocyte cytosolicprotein 2
T
-cell antigen receptor-mediated signaling
BC032919
OGFOD1 B 9 Y 542 16q13 2 -Oxoglutarate andiron-dependentoxygenase domaincontaining 1
P
rotein metabolism
NM_001722
POLR3D B 10 Y 398 8q21 D NA-directed RNApolymerase IIIpolypeptide D
R
NA polymerase
NM_004401
DFFA B 11 N 331 1 p36.3-p36.2 D NA fragmentationfactor 45-kDa αpolypeptide
p
53-independent rolein chromosomestability
NM_016520
C9orf78 B 12 N 289 9q34.11 C hromosome 9 openreading frame 78
U
ncharacterized
NM_006251
PRKAA1 B 13 — 550 5p12 A MP-activated proteinkinase catalyticsubunit α-1
C
holesterol and fattyacid biosynthesis
NM_006002
UCHL3 B 14 N 230 13q22.2 U biquitin COOH-terminal esterase L3
P
rocessing of ubiquitinand ubiquitinatedproteins
BC026104
PDCD4 B 15 — 469 10q24 P rogrammed celldeath 4
I
nhibits neoplastictransformation
NM_001219
CALU B 16 N 315 7q32 C alumenin E ndoplasmic reticulumprotein folding/sorting
BC000108
WWP2 B 17 Y 335 16q22.1 W W domain-containingE3 ubiquitin proteinligase 2
U
biquitination
BC031228
N/A B 20 Y 79 11 N /A U ncharacterized BC047536 SCEL B 21 N 668 13q22 S ciellin P recursor to keratinocyte
cornified envelope

Abbreviations: MAPK, mitogen-activated protein kinase; N/A, not applicable.*Rank order by the significance of change between pre- and post-DLI reactivity.†Reactivity reproducible by immunoprecipitation assay. Y, successful; N, unsuccessful, —, not attempted.‡There is support for the transcript, as a nonsense-mediated mRNA decay candidate, but not for the protein (27).

es; 70(4) February 15, 2010 1349

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mHA but represents overexpressed CLL-associated or B-cell–associated antigens.Clinical response to DLI is temporally associated with

antibody responses to candidate antigens. After DLI thera-py, both patients A and B showed normalization of peripher-al blood lymphocytosis and disappearance of tumor cellsfrom the marrow space (Fig. 4A). Leukemia cells occupied25% of patient A's marrow intertrabecular space at 2 monthsafter DLI but decreased to undetectable levels within 24months. Similarly, CLL cells occupied 45% of the marrow in-tertrabecular space of patient B at 3 months after DLI butdecreased to <5% tumor involvement by 9 months after DLI.The development of humoral immunity was strongly cor-

related temporally with clinical response against CLL(Fig. 4A). To determine whether the kinetics of the humoralimmune response against the candidate antigens correlateswith the clinical response to DLI, we compared the diseaseburden with plasma reactivity against candidate antigensat various time points following DLI. We examined antigen-specific antibody reactivity over time by immunoblot follow-ing immunoprecipitation against DAPK3 and ZFYVE19 forpatient A and against OGFOD1 and ZFYVE19 for patient B.Increasing antibody reactivity against the tested antigenscorrelated with decreasing tumor involvement in marrow,whereas comparable reactivity against the control EBNA1 an-tigen was observed for all plasma samples. For patient A, theantibody reactivity to ZFYVE19 was constant from beforeDLI to 4 months after DLI and then increased, peaking at



17 months and decreasing at 24 months. Antibody reactivityto DAPK3 for the same patient followed a similar pattern,increasing from its pre-DLI level from 4 months to 17 monthsbefore decreasing at 24 months. For patient B, the antibodyreactivity to ZFYVE19 and OGFOD1 both increased at 6 and9 months relative to the pre-DLI level, with samples for fur-ther follow-up not available.The candidate antigens are broadly immunogenic in

CLL patients with GvL. We examined patterns of serologicreactivity against GST fusion proteins of DAPK3, OGFOD1,and ZFYVE19 by testing 84 plasma samples collected from72 patients with hematologic malignancies or normal volun-teers (Fig. 4B). Five of 12 patients with long-term remissionwith minimal or no GvHD after HSCT developed new anti-body reactivity after treatment against at least one of thethree antigens. In addition to reactivity against DAPK3 andZFYVE19 by patient A and OGFOD1 and ZFYVE19 by patientB, three additional patients (subjects 8–10) who underwentHSCT developed reactivity against one or more of the threeantigens (Fig. 4B, first two panels, and C). Patients 8 and 9underwent nonmyeloablative allogeneic HSCT, whereas pa-tient 10 underwent myeloablative allogeneic HSCT. Post-treatment samples were selected at 1 year followingtransplant when immunosuppressive medications had beentapered and GvHD was not noted.In contrast, we observed only occasional instances of reac-

tivity for these three antigens among normal donors or othercontrol groups. Only 1 of 12 normal volunteers was reactive

Figure 2. Validation studies confirm greater antigen-specific antibody reactivity in post-DLI compared with pre-DLI plasma. A, schematic representationof the immunoprecipation-based validation assay. B, validation of serologic reactivity generated by protein microarray analysis using immunoprecipitationfollowed by Western blot. Antigens were generated as biotinylated GST fusion proteins by in vitro transcription and translation using rabbit reticulocytelysate, immunoprecipitated with pre- and post-DLI plasma from patients A and B, and immunoblotted with streptavidin-HRP. Shown are ZFYVE19 (64 kDa),MDS032 (44.5 kDa), C6orf130 (45 kDa), SERPB1 (70 kDa), DAPK3 (80 kDa), FAM122A (58 kDa), OGFOD1 (91 kDa), and EBNA1 (48 kDa), with sizesincluding the antigen sequence on the microarray and attached GST tag. Sample full-length blots are presented in Supplementary Fig. S1.

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to OGFOD1, and none of 12 to the other two antigens. Only1 of 9 post-HSCT CLL nonresponder patients and 1 of 10 un-treated CLL patients were reactive to both DAPK3 andZFYVE19. Similarly, only 2 of 12 chemotherapy-treated CLLpatients displayed reactivity against DAPK3, and 1 of 12against ZFYVE19. No evidence of reactivity was observedamong nine chronic myelogenous leukemia patients success-fully treated with DLI or among eight allotransplanted CLLpatients who developed GvHD, suggesting that the antigensare broadly immunogenic in patients with CLL who have de-veloped GvL responses.

Discussion

Identification of target antigens remains a priority in can-cer immunology. Few CLL-associated antigens have beenpreviously described, and of these, few have been shown toconsistently generate cytotoxicity against primary CLL cells



(16, 17, 19, 21, 30). To identify CLL antigens associated withthe GvL effect, we have focused on naturally immunogenicantigens associated with long-lasting immunity to CLL in-duced by DLI. Antibody-based approaches for target antigendiscovery are attractive because of their ease of use and themounting evidence that antibody responses correlate withmeaningful clinical responses to treatment. For example,Miklos and colleagues (12) reported a highly significant cor-relation between relapse-free survival and the presence of an-tibody responses against Y-encoded minor histocompatibilityantigens. Other investigators have reported antigen-specificB-cell responses as reliable markers of clinical response fol-lowing vaccination (31). To this end, we dissected the im-mune responses of two patients who achieved long-termdisease remission without clinically apparent GvHD andidentified 35 unique antigens that elicited significant anti-body reactivity following, but not before, DLI.Although our candidate CLL-associated antigens are

predominantly intracellular proteins, they are potentially

Figure 3. Gene and protein expression of the shared candidate antigens in normal PBMC, normal CD19+ B cells, and CLL cells. A, gene expressionwas measured by gene-specific qRT-PCR and normalized to transcript levels of GAPDH. Black bars denote mean values. Significance of the differencebetween groups by the Wilcoxon rank-sum test is shown on each graph. The number of samples in each group is given below the figures. Candidateantigens with similar relative expression in both normal and CLL B cells (C6orf130, MDS032, ZFYVE19, and OGFOD1) or with increased expression in CLLcells compared with normal B cells (DAPK3 and SERBP1). B, protein expression of candidate antigens measured by Western blot of cell lysates forPBMC (n = 2), normal CD19+ B cells (n = 2), and CLL cells (n = 5). Samples presented are independent of the samples tested in A. Protein lysates wereloaded at 20 μg/lane. Separate Western blots were probed with commercial antibodies to DAPK3, SERBP1, OGFOD1, MDS032, ZFYVE19, and β-actin(as control).




Marina et al.

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reliable markers for T-cell targeting. Our recent studies showthat treatment-associated tumor antigens that elicit high-titer B-cell responses are also targets of CD8+ T-cell immunity(32). Intracellular antigen may complex with antibody forenhanced delivery of antigen through FcγR-mediated path-ways to antigen-presenting cells, thus augmenting T-cell re-sponses (33). For example, vaccination with the intracellulartumor antigen NY-ESO-1 consistently results in antigen-specific antibody production that participates in cross-priming of this antigen to T cells (34). Our findings thus



provide a rich source of candidate CLL antigens, and ourfuture studies will explore the extent to which antigen-specific antibody responses contribute to the developmentof coordinated T-cell responses against our most promisingtargets.Our candidate antigens were identified using high-density

protein microrarrays, a powerful emerging technology. Thisimmunoproteomic screening platform enables high-throughputscreening of thousands of proteins for interactions and mea-sures the molecular biomarker end points of disease more

Figure 4. Serologic reactivity against CLL candidate antigens. A, the antibody responses of patients A and B temporally correlate with the clinical antitumorresponse. DAPK3 (80 kDa), OGFOD1 (91 kDa), ZFYVE19 (64 kDa), and EBNA1 (48 kDa) were generated as biotinylated GST fusion proteins by in vitrotranscription and translation. These were immunoprecipitated using protein A beads with pre- and post-DLI plasma and detected with streptavidin-HRP.The bottom graphs depict the percent of marrow intertrabecular space filled with CLL cells following DLI and the WBC counts across time. B, plasmareactivity of multiple patient and normal subject groups against antigens DAPK3, OGFOD1, and ZFYVE19 measured using immunoprecipitation.Interactions were graded against that of normal subjects, with light gray denoting weak interactions and dark gray denoting strong interactions. CML,chronic myelogenous leukemia. GvL, denoted as present (+) or absent (−); GvHD, denoted as present (+) or absent (−). Treatments included DLI (*),nonmyeloablative allogeneic HSCT ( † ), and myeloablative allogeneic HSCT ( ‡ ). Numbers denote individual patients, with the first two groups havingmatched preimmunotherapy and postimmunotherapy samples. C, the antibody responses of patients 8, 9, and 10 against antigens DAPK3, OGFOD1,and ZFYVE19 developed over time after allogeneic HSCT. Antibody responses were measured as in A. The preparative regimen and salvage chemotherapyfor HSCT resulted in disease remission and precludes comparison of WBC and bone marrow time courses. Full-length blots are presented inSupplementary Fig. S2.

Cancer Research





directly than DNA microarray technology (35). High-densityprotein microarrays offer several distinct advantages overconventional screening platforms. All proteins spotted onprotein microarrays are distinct open reading frames (ORF)expressed in a eukaryotic system that preserves potentiallyantigenic posttranslational modifications. Protein micro-arrays also offer a degree of standardization across expe-riments because the same proteins of comparableconcentration are printed across multiple microarrays. Onthe other hand, they are unlikely to contain antigens thatare specific to a tumor or to an individual, such as personalimmunogenic tumor-restricted mutations. Protein microar-rays are also not yet well established; we therefore devotedextensive efforts to developing a novel bioinformatic analysis(20) as well as a reliable method for independent validationof identified antigens. Because errors in the manufacture ofprotein microarrays have been described, which could leadto misidentified antigens on high-throughput screening(36), we developed an immunoprecipitation-based validationassay to sensitively detect the interaction of antibodies withproperly folded protein antigens with size determination viaimmunoblot.Overall, 22 of 30 (73%) tested antigen interactions were ex-

perimentally confirmed to elicit greater antibody reactivity inthe post-DLI compared with pre-DLI plasma. Characteriza-tion of several of the validated target antigens led us to theinsight that many of the identified targets of GvL are en-coded by nonmutated B-cell lineage genes. Increasing numb-ers of mHAs have been described over the years, includingsome with selective B-cell expression (37). Screens for GvL-associated antigens following HSCT have been biased toidentify mHAs because their primary criterion for selectionis differential cytolytic T-cell reactivity against host tissuecompared with donor cells. These studies show that alloim-munity is a fundamentally important mechanism underlyingGvL. However, they neglect the potential contribution of im-munity against other classes of tumor-associated antigensthat have been defined in other nontransplant tumor sys-tems, such as nonmutated differentiation, aberrantly ex-pressed antigens (38), or even self-antigens that areimmunogenic when presented by tumor but are normally se-questered from immune detection (39). Our current studiestherefore reveal that classes of antigen other than mHAs (i.e.,nonmutated tumor-associated antigens) comprise a compo-nent of effective GvL responses. These results are consistentwith recent studies by Nishida and colleagues (40) showingthat, in addition to T-cell responses against alloantigens,T-cell reactivity against CLL-specific antigens can be de-tected following nonmyeloablative HSCT in associationwith clinical response.The discovery that the targets of effective anti-CLL immu-

nity include nonmutated antigens that are highly expressedin CLL suggests that these antigens may have broader anti-CLL immunologic utility beyond the donor-host pairs fromwhich they were identified. Although patients A and B devel-oped reactivity against largely different panels of antigens, asubset of antigens elicited humoral immunity in both pa-tients. Moreover, close to half of our cohort of 12 CLL pa-



tients with GvL responses reacted against at least one ofthe three candidate antigens tested. These results suggestthat, as a collection, these antigens would be useful for mon-itoring immune responses against CLL after immune-basedtherapies. For example, several ongoing studies are examin-ing the effects of vaccination with whole autologous tumor inwhich the specific immunogens are unknown (41, 42).We selected for further characterization three antigens

identified by both patients' sera and three antigens foundto be highly expressed in CLL. Of the shared antigens,C6orf130 is a predicted ORF in chromosome 6. MDS032(USE1) is an uncharacterized hematopoietic stem/progenitorcell protein that localizes to the endoplasmic reticulum andfunctions in the formation of a SNARE complex (43).ZFYVE19 is a zinc finger FYVE domain-containing protein,which has been reported as an in-frame fusion partner tothe mixed-lineage leukemia gene (44). The FYVE domainmediates recruitment of proteins to membranes containingphosphatidylinositol 3-phosphate, particularly endosomes.Among the three nonshared antigens, DAPK3 (death-associatedprotein kinase 3) plays a role in apoptosis (45) and spermato-genesis (46). SERBP1 (SERPINE1 mRNA binding protein 1)is significantly overexpressed in ovarian epithelial cell tu-mors, with increased expression in advanced disease, sug-gesting a role in tumor invasion and metastasis (47).OGFOD1 (2-oxoglutarate and iron-dependent oxygenase do-main containing 1) has been reported in tumor cell pseudo-podial protrusions (48).In closing, our identification of nonmutated and common-

ly immunogenic CLL antigens reveals that immune re-sponses following effective immunotherapy are directed, atleast in part, to antigens present on CLL tumor cells.As the identified candidate antigens represent immunogenictumor-associated antigens and not alloantigens, these maybe attractive targets for immunotherapy directed at CLL out-side of the context of allogeneic transplantation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Roslyn Gerwin and Beth Witten for expert technical assistance,Drs. David Hill and Mark Vidal for providing plasmids, and the DFCItransplant team and Pasquarello Tissue Bank for providing clinical samples.

Grant Support

Howard Hughes Medical Institute Medical Research Training Fellowship(O. Marina) and Department of Defense grant W81XWH-07-1-0080, Milesand Eleanor Shore Award, National Cancer Institute grant 5R21CA115043-2,and Early Career Physician-Scientist Award of the Howard Hughes MedicalInstitute (C.J. Wu). C.J. Wu is a Damon-Runyon Clinical Investigator supported(in part) by the Damon-Runyon Cancer Research Foundation grant CI-38-07.

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

Received 8/25/09; revised 11/11/09; accepted 12/10/09; publishedOnlineFirst 2/2/10.




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2010;70:1344-1355. Published OnlineFirst February 2, 2010.Cancer Res Ovidiu Marina, Ursula Hainz, Melinda A. Biernacki, et al. AntigensChronic Lymphocytic Leukemia Include Nonmutated B-Cell Serologic Markers of Effective Tumor Immunity against

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