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Biology of Human Tumors

Prognostic Significance of MajorHistocompatibility Complex Class II Expressionin Pediatric Adrenocortical Tumors: A St. Judeand Children's Oncology Group StudyEmilia Modolo Pinto1, Carlos Rodriguez-Galindo2, John Kim Choi1, Stanley Pounds3,Zhifa Liu3, Geoffrey Neale4, David Finkelstein5, John M. Hicks6, Alberto S. Pappo7,Bonald C. Figueiredo8, Raul C. Ribeiro7, and Gerard P. Zambetti1

Abstract

Purpose: Histologic markers that differentiate benign andmalignant pediatric adrenocortical tumors are lacking. Previousstudies have implicated an association of MHC class II expres-sion with adrenocortical tumor prognosis. Here, we determinedthe expression of MHC class II as well as the cell of origin ofthese immunologic markers in pediatric adrenocortical tumor.The impact of MHC class II gene expression on outcome wasdetermined in a cohort of uniformly treated children withadrenocortical carcinomas.

Experimental Design: We analyzed the expression of MHCclass II and a selected cluster of differentiation genes in 63pediatric adrenocortical tumors by Affymetrix Human U133 Plus2.0 or HT HG-U133þPM gene chip analyses. Cells expressingMHC class II were identified by morphologic and immunohis-tochemical assays.

Results: MHC class II expression was significantly greater inadrenocortical adenomas than in carcinomas (P ¼ 4.8 �10�6)and was associated with a higher progression-free survival (PFS)estimate (P ¼ 0.003). Specifically, HLA-DPA1 expression wasmost significantly associated with PFS after adjustment for tumorweight and stage. HLA-DPA1 was predominantly expressed byhematopoietic infiltrating cells and undetectable in tumor cells in23 of 26 cases (88%).

Conclusions: MHC class II expression, which is producedby tumor-infiltrating immune cells, is an indicator of diseaseaggressiveness in pediatric adrenocortical tumor. Our resultssuggest that immune responses modulate adrenocorticaltumorigenesis and may allow the refinement of risk strati-fication and treatment for this disease. Clin Cancer Res; 22(24);6247–55. �2016 AACR.

IntroductionAdrenocortical tumors are rare in children accounting for

approximately 0.2% of all pediatric malignancies (1). Cur-rently, prognosis is based on tumor size, surgical resectability,and metastasis at diagnosis (2–5). Adrenocortical adenomascarry a good prognosis; however, the outcome is variable forthe 80% of cases classified as carcinomas. Many small,

completely excised adrenocortical carcinomas later relapse,whereas others are cured (2–5).To determine the clinicalbehavior of pediatric resectability according to histologiccriteria is often challenging.

Previous studies have evaluated MHC class II expression as aprognostic marker for pediatric adrenocortical tumor (6–8).We previously demonstrated that the expression of specificMHC class II genes was higher in pediatric adrenocorticaladenomas compared with carcinomas (6). Consistent withthese findings, Leite and colleagues reported that lower expres-sion of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may con-tribute to more aggressive disease (7). However, Magro andcolleagues concluded that MHC class II expression did notdiscriminate between benign and malignant pediatric adreno-cortical tumors (8). Therefore, the relevance of MHC class II asan indicator of outcome for pediatric adrenocortical tumorremains to be established.

MHC class II markers are expressed mainly by hematopoi-etic cells of the immune system but have been observed innormal adult adrenal cortex cells with weak immunostainingin the inner fasciculate zone and strong staining in thereticularis zone (9). Conversely, fetal adrenocortical cells ofboth zones were invariably negative (9). Whether MHC classII gene expression in pediatric adrenocortical tumor arisesfrom the adrenocortical tumor or infiltrating immune cells isuncertain.

1Department of Pathology, St. Jude Children's Research Hospital, Memphis,Tennessee. 2International Outreach, St. Jude Children's Research Hospital,Memphis, Tennessee. 3Department of Biostatistics, St. Jude Children's ResearchHospital, Memphis, Tennessee. 4Hartwell Center, St. Jude Children's ResearchHospital, Memphis, Tennessee. 5Department of Computational Biology, St. JudeChildren's Research Hospital, Memphis, Tennessee. 6Department of Pathology,Texas Children's Hospital, Houston, Texas. 7Department of Oncology, St. JudeChildren's Research Hospital, Memphis, Tennessee. 8Instituto de Pesquisa Pel�ePequeno Príncipe and Faculdades Pequeno Príncipe, Curitiba, Brazil.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Gerard P. Zambetti and Raul C. Ribeiro, St. JudeChildren's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105.Phone: 901-595-3429; Fax: 901-525-8025; E-mail: [email protected];Raul C. Ribeiro, [email protected]

doi: 10.1158/1078-0432.CCR-15-2738

�2016 American Association for Cancer Research.

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Here, we evaluated: (i) the expression of MHC class II genes;(ii) the cell of origin expressing MHC class II; and (iii) theimpact of MHC class II expression on clinical outcome in alarge group of uniformly treated pediatric adrenocorticaltumors. High MHC class II expression, in particular HLA-DPA1,was found associated with tumor-infiltrating immune cells anda more favorable progression-free survival (PFS).

Materials and MethodsParticipants and samples

The study included 52 participants in the St. Jude Children'sResearch Hospital (Memphis, TN) International Pediatric Adre-nocortical Tumor Registry (IPACTR; ClinicalTrials.gov, ID num-ber NCT00700414; ref. 2). The tumors were of any histology(adenomas, carcinomas, and undefined histology) and werenot uniformly treated. The IPACTR patients typically under-went surgery and chemotherapy for advanced stage disease(2–4). A second group, comprised of 34 subjects with histo-logically defined adrenocortical carcinomas, was uniformlytreated on the Children's Oncology Group (COG) ARAR0332protocol (ClinicalTrials.gov, ID number NCT00304070; ref. 4),which is a nonrandomized, single-arm, prospective study.Classification of adenoma, carcinoma, or tumors of undeter-mined histology in the first cohort (IPACTR) was based on themodified criteria of Weiss and colleagues (10–12) by twoindependent pathologists. In the COG cohort, only cases withdiagnosis of carcinoma, centrally reviewed by one of theauthors (J.M. Hicks), were enrolled on this study. The criteriafor carcinoma were based on a malignancy scoring system of 9gross and histopathologic features, including tumor weight(>400 g), tumor size (>10.5 cm), extension into periadrenalsoft tissue and/or adjacent organs, vena cava invasion, venousinvasion, capsular invasion, mitotic index (>15 per 20 highpower fields), and atypical mitotic figures, as previously definedby Wieneke and colleagues (13). Each item present is given ascore of 1. The identification of 4 or more of the above featuresis considered to be malignant. If there is metastatic disease to

lymph nodes or distant sites, the tumor is considered malignantas well. Written informed consent was obtained from parents orlegal guardians in all cases. This study was approved by the St.Jude Children's Research Hospital Institutional Review Boardand by the Cancer Therapy Evaluation Program of the NCI(Rockville, MD). Selected demographic and clinical features ofthe 86 patients are summarized in Table 1; detailed informa-tion is provided in Supplementary Table S1.

Gene expression data for pediatric adrenocortical tumors havebeen deposited in the National Center for Biotechnology Infor-mation, GEO profiles under accession code (COG cases,GSE76019; IPACTR cases, GSE76021).

Microarray analysisTotal RNA from tumor samples was used for RNA probe

preparation and hybridization to Affymetrix Human GenomeU133 Plus 2.0 or HT HG-U133þPM gene chip arrays accordingto the manufacturer's guidelines (Affymetrix; SupplementaryData, Section 1). Expressions of MHC class II and selected cellsurface marker genes (Supplementary Table S2) were evaluated inthe two cohorts of pediatric adrenocortical tumors (Supplemen-tary Table S1).

IHCFormalin-fixed, paraffin-embedded tumor sections (4 mm)

were stained for HLA-DPA1, HLA-DRA, and cell-surface mar-kers (CD3, CD4, CD8, CD68, and CD163) using BenchMark XT

Translational Relevance

Current prognostic stratification of pediatric adrenocorticaltumors is imprecise. Large tumors, incomplete surgical resec-tion, and regional and distant metastasis are associated withpoor prognosis. However, a substantial number of caseswithout these features relapse and eventually die from pro-gressive disease. Developing newmolecular prognostic criteriawould assist pathologists and oncologists to refine the histo-pathologic classification and clinical management of pediatricadrenocortical tumor. Here, we report that the expression ofMHC class II genes is significantly associated with prognosis,hence a potential biomarker to assist in differentiating benignfrom malignant adrenocortical tumors. We further identifiedtumor-infiltrating dendritic cells andmacrophages, but not thetumor cells, as the source of MHC class II antigen expression.Collectively, thesefindings provide anopportunity to improvethe risk stratification and outcome for pediatric adrenocorticaltumor.

Table 1. Clinical characteristics of 86 pediatric patients with adrenocorticaltumors

Characteristic All cases

Nonuniformlytreatedcohort 1(n ¼ 52)

Uniformlytreatedcohort 2(n ¼ 34)

GenderFemale 63 41 22Male 23 11 12

Age at presentationMean (mo) 68.47 68.74 68.06

Endocrine signsVirilization 47 27 20Mixed 16 12 4Nonfunctioning 14 7 7Other 9 6 3

Tumor histologyAdenoma 13 13 0Carcinoma 66 32 34Undefined 7 7 0

Disease stageI 28 18 10II 18 11 7III 28 18 10IV 12 5 7

Tumor weight (g)<200 49 28 21>200 32 22 10

Relapse(n) 24 12 12

Death(n) 21 13 8

Survival timeMean (mo) 44.38 50.77 34.6

Abbreviations: COG, Children's Oncology Group (uniformly treated cohort);IPACTR, International Pediatric Adrenocortical Tumor Registry (nonuniformlytreated cohort).

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(Ventana Medical Systems) or BOND-MAX (Leica Microsys-tems) automated stainers and reagents as listed in Supplemen-tary Table S3 according to the manufacturer's instructions.

Statistical analysisFor each microarray, we used the Bioconductor affy software

(www.bioconductor.org) to compute the mean values of robustmultiarray average–normalized log2 scale signals across allprobe sets annotated to MHC class II genes (14, 15) forcomparison of total MHC class II gene expression with histol-ogy and prognosis. The positive quantile transformation (16)was used to further normalize microarray data for comparisonof individual probe set values with histology and prognosis. Weused a discrimination gap statistic, as described in Supplemen-tary Data (Section 2), to select five individual probe sets in theIPACTR cohort, whose expression pattern most clearly distin-guished adenomas versus carcinomas, for further evaluation inthe COG cohort. For each of the five selected probe sets, asingle-predictor Cox regression analysis was used to evaluatethe univariate association of expression level with the estimateof PFS in the COG samples. These single-predictor Cox regres-sion models were used to perform a one-sided test of

the association of expression with PFS estimate at the P ¼0.05/5 ¼ 0.01 level (Bonferroni adjustment for multiple com-parisons). Comparisons that showed a significant association(with the Bonferroni correction) were used in a two-predictorCox regression model to compare expression with the PFSestimate after adjustment for tumor stage or tumor weight.PFS was defined as the time elapsed between protocol enroll-ment and the date of disease progression, death, or most recentfollow-up. The Kaplan–Meier method was used to estimatePFS probability as a function of time. All statistical analyseswere performed using R software (www.r-project.org), and allstatistical tests are two sided except where otherwise stated.

ResultsPatients

The age at diagnosis of the 63 females and 23 males was 3.1to 207.6 months (median, 38 months). Most patients presentedwith virilization (n ¼ 47), Cushing syndrome (n ¼ 7), aldo-steronism (n ¼ 2), or mixed hormonal secretion (n ¼ 16).Fourteen patients had nonfunctional tumors. Disease wasstaged according to the COG ARAR0332 protocol (4): stage I

Figure 1.

Gene expression profiling of MHC classII in the IPACTR cohort. Heatmap of38 probe sets annotated to MHC class IIand cluster of differentiation (CD)cell-surface marker genes for 29nonuniformly treated pediatricadrenocortical tumors. Red circles,adenomas; green circles, undeterminedhistology; blue circles, carcinomas.Probe sets and corresponding genesare identified at right and left,respectively.

MHC Class II Expression in Pediatric ACT

www.aacrjournals.org Clin Cancer Res; 22(24) December 15, 2016 6249




(n ¼ 28), II (n ¼ 18), III (n ¼ 28), and IV (n ¼ 12). Primarytumor samples were obtained in all cases, with the exception of3 biopsy samples (ACT080, ACT084, and ACT086) from COGpatients who had stage IV disease, as the tumors were consid-

ered inoperable. The median follow-up time among progres-sion-free patients was 52.3 months (range, 0.1–197.8 months).Clinical data for all patients are summarized in Table 1 andSupplementary Table S1.

Figure 2.

Gene expression profiling of MHC classII and outcome in the COG cohort. A,Heatmap of 38 probe sets annotatedto MHC class II and clusterdifferentiation (CD) genes for 34uniformly treated pediatricadrenocortical tumors (COG cohort).Probe sets and corresponding genesare identified at right and left,respectively. B, Kaplan–Meierestimates of PFS of subjects withtumor expression of MHC II genes lessthan versus greater than the median inthe uniformly treated pediatricadrenocortical carcinomas in the COGcohort.

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Association of MHC class II gene expression to histologyand PFS

The median expression of 28 MHC class II probe sets wassignificantly higher in adenomas than in carcinomas (P ¼ 4.8 �10�6, Wilcoxon rank-sum test) in the IPACTR cohort (Fig. 1). Inaddition, greater MHC class II expression (Fig. 2A) was associated

with better PFS in the uniformly treated pediatric adrenocorticalcarcinomas in the COG cohort (Fig. 2B; P¼ 0.003, Cox regression).The 3-year PFS of subjects with less than median MHC class IIexpression was 47.1% [95% confidence interval (CI), 28.4%–

77.9%)], whereas that of subjects with greater than median MHCclass II expression was 88.2% (95% CI, 74.2%–100%; Fig. 2B).

Figure 3.

Identification of MHC class II genes associated with PFS of pediatric adrenocortical tumor. The five MHC class II probe sets (A–E) most differentiallyexpressed in adenomas versus carcinomas (IPACTR cohort) were used to assess PFS in the uniformly treated COG group of pediatric adrenocorticalcarcinomas (n ¼ 34).






Expression of specific MHC class II genes is associated withhistology and PFS

To identify the individual MHC class II probe sets associatedwith histology and PFS, we computed a discriminating gapsize (DGS) criterion (16) for differential gene expression inadenomas and carcinomas in the IPACTR cohort. The DGScriterion can also identify a gap in the expression distributionthat may distinguish between two distinct biological processes(see Supplementary Data, Section 2, for details). The probe setswere ranked according to the value of this DGS criterion, andthe top 5 were selected (HLA-DPA1, HLA-DQA1/2, HLA-DRA,HLA-DRB1, and HLA-DQB1; Supplementary Fig. S1).

Expression (< vs. �median) of each of the five probe sets withgreatest DGS was then compared with the PFS in the COG cohort(Fig. 3). We performed one-sided statistical tests to determinewhether greater expression of each probe set was associated withimproved PFS, with the Bonferroni adjustment for multiple tests.Greater expression of four of the five probe sets was significantlyassociated with greater PFS (one-sided raw P < 0.01 or 0.05/5;Supplementary Table S4).

In particular, expression ofHLA-DPA1 (probe set 211991_s_at)was significantly associated with tumor weight (P < 0.0001) anddisease stage (P ¼ 0.032) but not with age (P ¼ 0.397), gender(P ¼ 0.179), endocrine syndrome (P ¼ 0.071), surgery (P ¼0.864), or chemotherapy (P ¼ 0.106; Supplementary Table S5).

Association ofHLA-DPA1 expression with PFS after adjustmentfor disease stage

The prognostic value of HLA-DPA1 (probe set 211991_s_at)expression after adjustment for disease stage in the uniformlytreated COG cohort was evaluated. As expected, advanced stagewas associated with poor PFS. Five of 7 stage IV patients hadprogressive disease within one year and a sixth patient haddisease progression within two years (Fig. 4A). To date, none ofthe 15 stage I–III patients with greater than median expressionof HLA-DPA1 have died or experienced progressive disease,whereas the 12 stage I–III patients with less than medianexpression of HLA-DPA1 had a 3-year PFS of 58.3% (95% CI,36.2%–94.1%; Fig. 4B).

Cells of origin of MHC class II expressionArchival tumor sections of 26 IPACTR cases (15 carcino-

mas, 6 adenomas, 5 of undetermined histology) were stainedfor hematoxylin and eosin (H&E) to confirm the pathologicdiagnosis. Eight adrenocortical tumors (ACT005, ACT008,ACT012, ACT030, ACT031, ACT032, ACT033, and ACT034)were stained with antibodies specific to HLA-DPA1 and HLA-DRA and markers of mononuclear hematopoietic cells (CD3,CD4, CD8, CD68, and CD163; Supplementary Table S3). H&Eand immunostaining results for a representative adenoma(ACT030, row a), early-stage carcinoma (ACT012, row b), andlate-stage carcinoma (ACT008, row c) are shown in Fig. 5A.HLA staining was predominantly cytoplasmic, with HLA-DRAbrighter than HLA-DPA1 (Fig. 5A, columns 2 and 3). MHCclass II staining was observed in two morphologic cell types,one that stained intensely for HLA-DRA and HLA-DPA1 andhad relatively short cytoplasmic processes and one that stainedless intensely and had longer cytoplasmic processes (Fig. 5Ainset, columns 2 and 3). The cells with short cytoplasmicprocesses were also strongly positive for CD68 (Fig. 5A, col-umn 4) and CD163 (not shown) but less positive for CD4

(Supplementary Fig. S2), suggesting tumor-infiltrating macro-phages. Other cells showing long cytoplasmic processes werenegative for CD68 (Fig. 5A, column 4) and CD163 but dimlypositive for CD4, consistent with dendritic cells (Supplemen-tary Fig. S2). Staining of sequential sections of the same tissueblock for CD3, CD4, and CD8 revealed both types of Tlymphocytes. Approximately 5% to 10% of the infiltratingcells were T lymphocytes (CD3þ). Moreover, many of thetumor-infiltrating cells dimly positive for CD4 in the adenomahad morphologic characteristic of histiocytes and dendriticcells (Supplementary Fig. S2).

Because of the strong association between HLA-DPA1gene expression and outcome, we analyzed the remaining18 IPACTR adrenocortical tumors (10 adrenocortical carcino-mas, 5 adrenocortical adenomas, and 3 of undetermined histo-logy) with anti-HLA-DPA1 antibody. HLA-DPA1 protein wasabsent or undetectable in the tumor cells in 15 of these cases(83%). However, three cases (ACT040, ACT047, and ACT048;17%) showed focal HLA-DPA1 immunostaining within theadrenocortical tumor cells (Fig. 5B; Supplementary Table S6).

DiscussionGreater expression of MHC class II genes, HLA-DPA1 in

particular, is associated with better prognosis for pediatric

Figure 4.

Association of stage disease and expression of HLA-DPA1 with PFS. A,Kaplan–Meier estimates of PFS according to stage disease only (stage I–III vs.IV). B, Kaplan–Meier estimates of PFS for cases with stage I–III diseaseaccording to expression of HLA-DPA1.

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adrenocortical tumors independent of histopathologic diagnosis.In most cases, MHC class II genes were expressed by tumor-infiltrating hematopoietic cells rather than adrenocortical tumorcells. These results suggest that immune surveillance, evident byelevated expression of MHC class II and the presence of tumor-associated macrophages and dendritic cells, may limit adreno-cortical progression, leading to less aggressive disease.

The histologic features of ACTs often do not allow unequiv-ocal classification as adenomas or carcinomas, and such casesare currently classified as tumors of indeterminate histology,undetermined malignant potential, or simply "adrenocorticalneoplasms" (13, 17, 18). In our IPACTR series, 7 of 52 tumors(13%) had undetermined histologic findings. Three of thesecases expressed high levels of MHC class II based on microarray

(ACT005 and ACT006) and IHC (ACT047), which is charac-teristic of pediatric adrenocortical adenomas. All three patientsexperienced tumor spillage during surgery but did not relapseand remained disease free at the time of the last follow-up(range, 20–56 months). Three of the remaining four adreno-cortical tumors of undetermined histology (ACT034, ACT039,and ACT046) expressed high levels ofHLA-DPA1 and are free ofdisease for more than 2 years. These clinical findings areconsistent with the observed high MHC class II expression andless aggressive disease.

Prognostic information is also lacking for pediatric adreno-cortical tumors classified as carcinomas, as their outcome variessubstantially after complete resection (3–5, 10–13). Tumorsize (weight or volume) is associated with prognosis, but small

Figure 5.

A, Immunohistochemical analysis ofMHC class II protein expression inadrenocortical tumors. Row a,adenoma; row b, stage I carcinoma;row c, stage III carcinoma. Note the highexpression of HLA-DRA and HLA-DPA1in the adenoma and low expression inthe two carcinomas. CD68 is acytoplasmic marker for cells ofthe macrophage lineage. B,Immunohistochemical analysis revealsHLA-DPA1 expression by adrenocorticaltumor cells in two pediatricadrenocortical tumors: ACT047 (a) andACT048 (b; magnification, 100�; inset,400�).






adrenocortical carcinomas too often behave aggressively (earlylocal and distant relapse), whereas large carcinomas mayhave an indolent course. We suggest that the expression ofMHC class II genes can be used to refine the risk classificationof adrenocortical tumors. Overexpression of MHC class IIgenes could more decisively identify patients with undeter-mined or carcinoma histology who are unlikely to requireadjuvant chemotherapy after complete surgical resection. Likechildhood adrenocortical tumors with a Weiss score of 0 or 1(10–13, 17), those with relatively large, completely excisedcarcinomas (stage II–III) with high expression of MHC class IIgenes could be spared intensive chemotherapy. Completelyresected carcinomas with low MHC class II gene expressioncould be treated with adjuvant chemotherapy irrespective oftheir size.

Overexpression of IGF2 and BUB1B is associated with poorprognosis in adult adrenocortical tumor (19, 20). In pediatricadrenocortical tumor, IGF2 is overexpressed in both adenomasand carcinomas (21) and therefore does not correlate with HLA-DPA1 levels or outcome (P > 0.10; Supplementary Table S5).BUBR1, the protein encoded by BUB1B, has a critical role inregulating the spindle assembly checkpoint and implicated inchromosome instability and cancer progression (22). Similar toadult adrenocortical tumor and other adult cancers (23, 24),overexpression of BUB1B inversely correlated with PFS in pedi-atric adrenocortical tumor from both cohorts (P ¼ 0.003; Sup-plementary Table S5).

MHC class II molecules are surface glycoproteins on anti-gen-presenting cells (e.g., dendritic cells, monocyte/macro-phages, and B lymphocytes) that presumably present tumorpeptides to CD4þ T cells (25). Previous studies of pediatricadrenocortical tumor have not definitively addressed the cellof origin expressing MHC class II, although it was suggested tooriginate from adrenocortical tumor cells and downregulatedconcurrently with tumor progression (6–8). However, MHCclass II is not expressed in the fetal adrenal cortex (26, 27), theputative origin of pediatric adrenocortical tumor (28), butrather, predominantly by tissue macrophages and a smallpopulation of CD45Rþ, IgMþ lymphoid cells (28). Our find-ings demonstrate that the cells expressing MHC class II genesin pediatric adrenocortical tumors, primarily adenomas, aremacrophages and dendritic cells. Dendritic cells are capable ofinitiating innate and adaptive tumor responses (29), suggest-ing a mechanism for suppressing pediatric adrenocorticaltumor progression. In contrast, three adrenocortical tumors(ACT40, ACT047, and ACT048) displayed focal tumor–cellexpression of HLA-DPA1 in addition to the infiltratinghematopoietic cells. Two of these patients (ACT047, ACT048)were more than 7 years of age at diagnosis and presented withvirilization and large tumors. Interestingly, these cases expe-rienced tumor spillage during surgery but did not relapseduring more than 20 months of follow-up. Whether tumor

cells expressing MHC class II are capable of participating inregulatory immune mechanisms and have prognostic implica-tions remains to be determined.

We have demonstrated that MHC class II gene expression inpediatric adrenocortical tumors originates from tumor-infiltrat-ing immune cells and is associated with more favorable prog-nosis. Our data support the inclusion of MHC class II expres-sion in addition to standard criteria (e.g., tumor size, mitoticactivity, atypical mitoses, and other histologic features) as atool for determining pediatric adrenocortical tumor diagnosis.Future studies will define the interaction of infiltrating hemato-poietic cells with the pediatric adrenocortical tumor microen-vironment, which may refine risk classification and facilitatethe development of immunotherapies for these tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed

Authors' ContributionsConception and design: E.M. Pinto, C. Rodriguez-Galindo, J.M. Hicks,R.C. Ribeiro, G.P. ZambettiDevelopment of methodology: E.M. Pinto, J.K. Choi, S. Pounds, J.M. Hicks,G.P. ZambettiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.M. Pinto, C. Rodriguez-Galindo, J.M. Hicks,B.C. Figueiredo, R.C. Ribeiro, G.P. ZambettiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.M. Pinto, J.K. Choi, S. Pounds, Z. Liu, G. Neale,D. Finkelstein, J.M. Hicks, A.S. Pappo, G.P. ZambettiWriting, review, and/or revision of the manuscript: E.M. Pinto, C. Rodriguez-Galindo, J.K. Choi, S. Pounds, J.M. Hicks, B.C. Figueiredo, R.C. Ribeiro,G.P. ZambettiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.M. Pinto, G.P. ZambettiStudy supervision: E.M. Pinto, R.C. Ribeiro, G.P. Zambetti

AcknowledgmentsWe thank the study participants and the clinicians and research staff at

participating institutions. We thank Jay Morris and Melanie Loyd for assis-tance with the expression arrays; Charlene Henry, Raven Childers, and theSt. Jude Children's Research Hospital anatomic pathology staff for assistancewith immunostaining studies. We thank Sharon Naron for editing themanuscript.

Grant SupportThis work was supported by a grant from Centocor, Inc., National Institutes

ofHealth Cancer Center support grant CA21765, and by the American LebaneseSyrian Associated Charities (ALSAC).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 16, 2015; revised April 28, 2016; accepted May 24, 2016;published OnlineFirst June 15, 2016.

References1. Altekruse SF, Kosary CL, Krapcho M, Neyman N, Aminou R, Waldron W,

et al. SEER Cancer Statistics Review, 1975–2007. Bethesda, MD:NationalCancer Institute; 2010. Available from: http://seer.cancer.gov/csr/1975_2007/.

2. Michalkiewicz E, Sandrini R, Figueiredo B, Miranda EC, Caran E,Oliveira-Filho AG, et al. Clinical and outcome characteristics of

children with adrenocortical tumors: a report from the Inter-national Pediatric Adrenocortical Tumor Registry. J Clin Oncol2004;22:838–45.

3. Ribeiro RC, Michalikiewicz E, Figueiredo BC, DeLacerda L, Sandrini F,PianovskyMD, et al. Adrenocortical tumors in children. Braz JMed Biol Res2000;33:1225–34.


Pinto et al.




4. Ribeiro RC, Pinto EM, Zambetti GP, Rodriguez-Galindo C. The Interna-tional Pediatric Adrenocortical Tumor Registry initiative: contributions toclinical, biological, and treatment advances in pediatric adrenocorticaltumors. Mol Cell Endocrinol 2012;351:37–43.

5. McAteer JP, Huaco JA, Gow KW. Predictors of survival in pediatric adre-nocortical carcinoma: a surveillance, epidemiology, and end results (SEER)program study. J Pediatr Surg 2013;48:1025–31.

6. West AN, Neale GA, Pounds S, Figueiredo B, Rodriguez-Galindo C,PianoviskiMA, et al. Gene expressionprofilingof childhood adrenocorticaltumors. Cancer Res 2007;67:600–8.

7. Leite FA, Lira RCP, Fedatto PF, Antonini SR, Martinelli CE Jr, de Castro M,et al. Low expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 is associ-atedwith poor prognosis in pediatric adrenocortical tumors (ACT). PediatrBlood Cancer 2014;6:1940–8.

8. MagroG, EspositoG, CecchettoG,Dall'lgna P,Marcato R,Gambini C, et al.Pediatric adrenocortical tumors: morphological diagnostic criteria andimmunohistochemical expression of matrix metalloproteinase type 2 andhuman leucocyte-associated antigen (HLA) class II antigens. Results fromthe Italian Pediatric Rare Tumor (TREP) Study project. Hum Pathol2012;43:31–9.

9. Khoury EL, Greenspan JS, Greenspan FS. Adrenocortical cells of the zonareticularis normally express HLA-DR antigenic determinants. Am J Pathol1987;127:580–91.

10. Weiss LM, Medeiros LJ, Vickery AL Jr. Pathologic features of prognosticsignificance in adrenocortical carcinoma. Am J Surg Pathol 1989;13:202–6.

11. Weiss LM. Comparative histologic study of 43 metastasizing and non-metastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163–9.

12. Bugg MF, Ribeiro RC, Roberson PK, Lloyd RV, Sandrini R, Silva JB, et al.Correlation of pathologic features with clinical outcome in pediatricadrenocortical neoplasia. A study of a Brazilian population. BrazilianGroup for Treatment of Childhood Adrenocortical Tumors. Am J ClinPathol 1994;101:625–9.

13. Wieneke J, Thompson L, Heffess C. Adrenal cortical neoplasms in thepediatric population: a clinicopathologic and immunophenotypic analysisof 83 patients. Am J Surg Pathol 2003;27:867–81.

14. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ,Scherf U, et al. Exploration, normalization, and summaries of highdensity oligonucleotide array probe level data. Biostatistics 2003;4:249–64.

15. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normal-ization methods for high density oligonucleotide array data based onvariance and bias. Bioinformatics 2003;19:185–93.

16. Pawlikowska I, Wu G, Edmonson M, Liu Z, Gruber T, Zhang J, et al. Themost informative spacing test effectively discovers biologically relevantoutliers ormultiplemodes in expression. Bioinformatics 2014;30:1400–8.

17. Chatterjee G, DasGupta S, Mukherjee G, Sengupta M, Roy P, Arun I, et al.Usefulness of Wieneke criteria in assessing morphologic characteristics ofadrenocortical tumors in children. Pediatr Surg Int 2015;31:563–71.

18. Sakoda A, Mushtaq I, Levitt G, Sebire NJ. Clinical and histopathologicalfeatures of adrenocortical neoplasms in children: retrospective review froma single specialist center. J Pediatr Surg 2014;49:410–15.

19. Drelon C, Berthon A, Val P. Adrenocortical cancer and IGF2: is the gameover or our experimental models limited? J Clin Endocrinol Metab2013;98:505–7.

20. de Reynies A, Assie G, Rickman DS, Tissier F, Groussin L, Rene-Corail F,et al. Gene expression profiling reveals a new classification of adrenocor-tical tumors and identifies molecular predictors of malignancy and sur-vival. J Clin Oncol 2009;27:1108–15.

21. Pinto EM, Chen X, Easton J, Finkelstein D, Liu Z, Pounds S, et al. Genomiclandscape of paediatric adrenocortical tumours. Nat Commun 2015;6:6302.

22. Baker DJ, Jin F, Jeganathan KB, van Deursen JM. Whole chromosomeinstability caused by Bub1 insufficiency drives tumorigenesis throughtumor suppressor gene loss of heterozygosity. Cancer Cell 2009;16:475–86.

23. Zhao Y, Ando K,Oki E, Ikawa-Yoshida A, Ida S, Kimura Y, et al. Aberrationsof BUBR1 and TP53 gene mutually associated with chromosomal insta-bility in human colorectal cancer. Anticancer Res 2014;34:5421–8.

24. Chen H, Lee J, Kljavin NM, Haley B, Daemen A, Johnson L, et al. Require-ment for BUB1B/BUBR1 in tumor progression of lung adenocarcinoma.Genes Cancer 2015;6:106–18.

25. Holling TM, Schooten E, van Den Elsen PJ. Function and regulation ofMHC class II molecules in T-lymphocytes: of mice and men. Hum Immu-nol 2004;65:282–90.

26. Marx C, Wolkersd€orfer GW, Brown JW, Scherbaum WA, Bornstein SR.MHC class II expression-a new tool to assess dignity in adrenocorticaltumours. J Clin Endocrinol Metab 1996;81:4488–91.

27. Oliver AM, Thomson AW, Sewell HF, Abramovich DR. Major histocom-patibility complex (MHC) class II antigen (HLA-DR, DQ, and DP) expres-sion in human fetal endocrine organs and gut. Scand J Immunol1988;27:731–7.

28. Coulter CL. Fetal adrenal development: insight gained from adrenaltumors. Trends Endocrinol Metab 2005;16:235–42.

29. Tel J, Anguille S, Waterborg CE, Smits EL, Figdor CG, de Vries IJ. Tumor-icidal activity of human dendritic cells. Trends Immunol 2014;35:38–46.






2016;22:6247-6255. Published OnlineFirst June 15, 2016.Clin Cancer Res Emilia Modolo Pinto, Carlos Rodriguez-Galindo, John Kim Choi, et al. Children's Oncology Group StudyII Expression in Pediatric Adrenocortical Tumors: A St. Jude and Prognostic Significance of Major Histocompatibility Complex Class

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