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Published in final edited form as: Clin Cancer Res. 2016 December 15; 22(24): 6247-6255. doi:10.1158/1078-0432.CCR-15-2738.
Prognostic Significance of Major Histocompatibility Complex Class II Expression in Pediatric Adrenocortical Tumors: A St. Jude and Children’s Oncology Group Study
Emilia Modolo Pinto1, Carlos Rodriguez-Galindo2, John Kim Choi1, Stanley Pounds3, Zhifa Liu3, Geoff Neale4, David Finkelstein5, John Hicks7, Alberto S. Pappo6, Bonald Figueiredo8, Raul C. Ribeiro6,*, and Gerard P. Zambetti1,*
1Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
2Department of International Outreach, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
3Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
4Department of Hartwell Center, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
5Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
6Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, 38105, USA
7Department of Pathology, Texas Children’s Hospital, Houston 77030, USA
8Instituto de Pesquisa Pelé Pequeno Príncipe and Faculdades Pequeno Príncipe, Curitiba 80-250-200, Brazil
Abstract
Purpose-Histological markers that differentiate benign and malignant pediatric adrenocortical tumors (ACTs) are lacking. Previous studies have implicated an association of major histocompatibility complex (MHC) class II expression with ACT prognosis. Here we determined
`Corresponding authors: Raul C. Ribeiro, MD (raul.ribeiro@stjude.org) and Gerard P. Zambetti, PhD (gerard.zambetti@st.jude.org). Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed
Authors’ Contributions Conception and design: Emilia Modolo Pinto, Raul C. Ribeiro and Gerard P. Zambetti.
Provision of study materials or patients: Carlos Rodriguez-Galindo, Bonald Figueiredo, Raul C. Ribeiro.
Collection and assembly of data: Emilia Modolo Pinto, Stanley Pounds, John Kim Choi, Zhifa Liu, Geoff Neale, David Finkelstein, Raul C. Ribeiro, Gerard P. Zambetti
Data analysis and interpretation: Emilia Modolo Pinto, Stanley Pounds, John Kim Choi, John Hicks, Alberto Pappo, Zhifa Liu, Raul C. Ribeiro, Gerard P. Zambetti
Writing, critical review, and/or revision of the manuscript: all authors.
Additional information Gene expression data for pediatric adrenocortical tumors have been deposited in the National Center for Biotechnology Information (NCBI), GEO Profiles under accession code (COG cases, GSE76019; IPACTR cases, GSE76021).
the expression of MHC class II as well as the cell of origin of these immunological markers in pediatric ACT. The impact of MHC class II gene expression on outcome was determined in a cohort of uniformly-treated children with adrenocortical carcinomas.
Experimental Design-We analyzed the expression of MHC class II and a selected cluster of differentiation genes in 63 pediatric ACTs by Affymetrix Human U133 Plus 2.0 or HT HG- U133+PM gene chip analyses. Cells expressing MHC class II were identified by morphologic and immunohistochemical assays.
Results-MHC class II expression was significantly greater in adrenocortical adenomas than in carcinomas (P=4.8 × 10-6) and was associated with a higher progression-free survival (PFS) estimate (P=0.003). Specifically, HLA-DPA1 expression was most significantly associated with PFS after adjustment for tumor weight and stage. HLA-DPA1 was predominantly expressed by hematopoietic infiltrating cells and undetectable in tumor cells in 23 of 26 cases (88%).
Conclusion-MHC class II expression, which is produced by tumor- infiltrating immune cells, is an indicator of disease aggressiveness in pediatric ACT. Our results suggest that immune responses modulate adrenocortical tumorigenesis, and may allow the refinement of risk stratification and treatment for this disease.
Keywords
MHC class II; pediatric adrenocortical tumors; HLA-DPA1; dendritic cells
Introduction
Adrenocortical tumors (ACTs) are rare in children accounting for approximately 0.2% of all pediatric malignancies (1). Currently, prognosis is based on tumor size, surgical resectability, and metastasis at diagnosis (2-5). Adrenocortical adenomas carry a good prognosis; however, the outcome is variable for the 80% of cases classified as carcinomas. Many small, completely excised adrenocortical carcinomas later relapse, while others are cured (2-5). To determine the clinical behavior of pediatric ACT according to histologic criteria is often challenging.
Previous studies have evaluated Major Histocompatibility Complex (MHC) class II expression as a prognostic marker for pediatric ACT (6-8). We previously demonstrated that the expression of specific MHC class II genes was higher in pediatric adrenocortical adenomas compared to carcinomas (6). Consistent with these findings, Leite et al reported that lower expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may contribute to more aggressive disease (7). However, Magro and colleagues concluded that MHC class II expression did not discriminate between benign and malignant pediatric ACTs (8). Therefore, the relevance of MHC class II as an indicator of outcome for pediatric ACT remains to be established.
MHC class II markers are expressed mainly by hematopoietic cells of the immune system, but have been observed in normal adult adrenal cortex cells with weak immunostaining in the inner fasciculate zone and strong staining in the reticularis zone (9). Conversely, fetal adrenocortical cells of both zones were invariably negative (9). Whether MHC class II gene
expression in pediatric ACT arises from the adrenocortical tumor or infiltrating immune cells is uncertain.
Here we evaluated: 1) the expression of MHC class II genes; 2) the cell of origin expressing MHC class II; and 3) the impact of MHC class II expression on clinical outcome in a large group of uniformly-treated pediatric ACTs. High MHC class II expression, in particular HLA-DPA1, was found associated with tumor-infiltrating immune cells and a more favorable progression-free survival.
METHODS
Participants and Samples
The study included 52 participants in the St. Jude Children’s Research Hospital (St. Jude) International Pediatric Adrenocortical Tumor Registry (IPACTR) (clinicaltrials.gov, ID number NCT00700414) (2). The tumors were of any histology (adenomas, carcinomas and undefined histology) and were not uniformly treated. The IPACTR patients typically underwent surgery, and chemotherapy for advanced stage disease (2-4). A second group comprised of 34 subjects with histologically-defined adrenocortical carcinomas were uniformly treated on the Children’s Oncology Group (COG) ARAR0332 protocol (clinicaltrials.gov, ID number NCT00304070) (4), which is a non- randomized, single-arm, prospective study. Classification of adenoma, carcinoma or tumors of undetermined histology in the first cohort (IPACTR) was based on the modified criteria of Weiss and colleagues (10-12) by two independent pathologists. In the COG cohort, only cases with diagnosis of carcinoma, centrally reviewed by one of the authors (JH), were enrolled on this study. The criteria for carcinoma were based on a malignancy scoring system of nine gross and histopathological features, including tumor weight (>400 g), tumor size (>10.5 cm), extension into periadrenal soft tissue and/or adjacent organs, vena cava invasion, venous invasion, capsular invasion, mitotic index (>15 per 20 high power fields), and atypical mitotic figures, as previously defined by Wienecke (13). Each item present is given a score of 1. The identification of 4 or more of the above features is considered to be malignant. If there is metastatic disease to lymph nodes or distant sites, the tumor is considered malignant, as well. Written informed consent was obtained from parents or legal guardians in all cases. This study was approved by the St. Jude Institutional Review Board and by the Cancer Therapy Evaluation Program of the National Cancer Institute. Selected demographic and clinical features of the 86 patients are summarized in Table 1; detailed information is provided in Supplementary Table 1.
Microarray Analysis
Total RNA from tumor samples were used for RNA probe preparation and hybridization to Affymetrix Human Genome U133 Plus 2.0 or HT HG-U133+PM gene chip arrays according to the manufacturer’s guidelines (Affymetrix, Santa Clara, CA) (Data Supplement, section 1). Expression of MHC class II and selected cell surface marker genes (Supplementary Table 2) were evaluated in the two cohorts of pediatric adrenocortical tumors (Supplementary Table 1).
Immunohistochemistry
Formalin-fixed, paraffin-embedded tumor sections (4-um) were stained for HLA-DPA1, HLA-DRA and cell surface markers (CD3, CD4, CD8, CD68 and CD163) using Benchmark XT (Ventana Medical, Tucson, AZ) or BondMax (Leica Microsystems, Bannockburn, IL) automated stainers and reagents as listed in Supplementary Table 3 according to the manufacturer’s instructions.
Statistical Analysis
For each microarray, we used the Bioconductor affy software (www.bioconductor.org) to compute the mean values of robust multi-array average (RMA)-normalized log2-scale signals across all probe-sets annotated to MHC class II genes (14-15) for comparison of total MHC class II gene expression with histology and prognosis. The positive quantile transformation (PQT) (16) was used to further normalize microarray data for comparison of individual probe-set values with histology and prognosis. We used a discrimination gap statistic, as described in Data Supplement section 2, to select five individual probe-sets in the IPACTR cohort whose expression pattern most clearly distinguished adenomas vs. carcinomas, for further evaluation in the COG cohort. For each of the five selected probe- sets, a single-predictor Cox regression analysis was used to evaluate the univariate association of expression level with the estimate of progression-free survival (PFS) in the COG samples. These single-predictor Cox regression models were used to perform a one- sided test of the association of expression with PFS estimate at the P= 0.05/5 = 0.01 level (Bonferroni adjustment for multiple comparisons). Comparisons that showed a significant association (with the Bonferroni correction) were used in a two-predictor Cox regression model to compare expression with the PFS estimate after adjustment for tumor stage or tumor weight. PFS was defined as the time elapsed between protocol enrollment and the date of disease progression, death, or most recent follow-up. The Kaplan-Meier method was used to estimate PFS probability as a function of time. All statistical analyses were performed using R software (www.r-project.org) and all statistical tests are two-sided except where otherwise stated.
RESULTS
Patients
The age at diagnosis of the 63 females and 23 males was 3.1 to 207.6 months (median, 38 months). Most patients presented with virilization (n=47), Cushing syndrome (n=7), aldosteronism (n=2), or mixed hormonal secretion (n=16). Fourteen patients had non- functional tumors. Disease was staged according to the COG ARAR0332 protocol (4): stage I (n=28), II (n=18), III (n=28) and IV (n=12). Primary tumor samples were obtained in all cases, with the exception of three biopsy samples (ACT080, ACT084 and ACT086) from COG patients who had stage IV disease, as the tumors were considered inoperable. The median follow-up time among progression-free patients was 52.3 months (range, 0.1 - 197.8 months). Clinical data for all patients are summarized in Table 1 and Supplementary Table 1.
Association of MHC Class II Gene Expression to Histology and Progression-free Survival
The median expression of 28 MHC class II probe sets was significantly higher in adenomas than in carcinomas (P=4.8 x 10-6, Wilcoxon rank-sum test) in the IPACTR cohort (Fig 1). Additionally, greater MHC class II expression (Fig 2A) was associated with better progression-free survival (PFS) in the uniformly-treated pediatric adrenocortical carcinomas in the COG cohort (Fig 2B; P= 0.003, Cox regression). The three-year PFS of subjects with less than median MHC class II expression was 47.1% (95% CI: 28.4 - 77.9%) while that of subjects with greater than median MHC class II expression was 88.2% (95% CI: 74.2 - 100%) (Fig 2B).
Expression of Specific MHC Class II Genes is Associated with Histology and Progression- free Survival
To identify the individual MHC class II probe sets associated with histology and PFS, we computed a discriminating gap size (DGS) criterion (16) for differential gene expression in adenomas and carcinomas in the IPACTR cohort. The DGS criterion can also identify a gap in the expression distribution that may distinguish between two distinct biological processes (see Data Supplement section 2 for details). The probe sets were ranked according to the value of this DGS criterion, and the top 5 were selected (HLA-DPA1, HLA-DQA1/2, HLA- DRA, HLA-DRB1 and HLA-DQB1) (Supplementary Fig 1).
Expression (< vs. ≥ median) of each of the five probe-sets with greatest DGS was then compared to the PFS in the COG cohort (Fig 3). We performed one-sided statistical tests to determine whether greater expression of each probe set was associated with improved PFS, with the Bonferroni adjustment for multiple tests. Greater expression of four of the five probe-sets was significantly associated with greater PFS (one-sided raw p-values <. 01, or 0.05/5) (Supplementary Table 4).
In particular, expression of HLA-DPA1 (probe-set 211991_s_at) was significantly associated with tumor weight (P< 0.0001) and disease stage (P=0.032) but not with age (P=0.397), gender (P=0.179), endocrine syndrome (P= 0.071), surgery (P= 0.864) or chemotherapy (P=0.106) (Supplementary Table 5).
Association of HLA-DPA1 Expression with Progression-free Survival after Adjustment for Disease Stage
The prognostic value of HLA-DPA1 (probe-set 211991_s_at) expression after adjustment for disease stage in the uniformly-treated COG cohort was evaluated. As expected, advanced stage was associated with poor progression-free survival (PFS). Five of seven stage IV patients had progressive disease within one year and a sixth patient had disease progression within two years (Fig 4A). To date, none of the 15 stage I-III patients with greater than median expression of HLA-DPA1 have died or experienced progressive disease while the 12 stage I-III patients with less than median expression of HLA-DPA1 had a three-year PFS of 58.3% (95% CI: 36.2-94.1%; Fig 4B).
Cells of Origin of MHC Class II Expression
Archival tumor sections of 26 IPACTR cases (15 carcinomas, 6 adenomas, 5 of undetermined histology) were stained for hematoxylin and eosin (H&E) to confirm the pathological diagnosis. Eight ACTs (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 3). H&E and immunostaining results for a representative adenoma (ACT030, row a), early-stage carcinoma (ACT012, row b) and late-stage carcinoma (ACT008, row c) are shown in Figure 5A. HLA staining was predominantly cytoplasmic, with HLA-DRA brighter than HLA-DPA1 (Fig 5A, columns 2 and 3). MHC class II staining was observed in two morphologic cell types, one that stained intensely for HLA-DRA and HLA-DPA1 and had relatively short cytoplasmic processes and one that stained less intensely and had longer cytoplasmic processes (Fig 5A inset, columns 2 and 3). The cells with short cytoplasmic processes were also strongly positive for CD68 (Fig 5A, column 4) and CD163 (not shown) but less positive for CD4 (Supplementary Fig 2), suggesting tumor-infiltrating macrophages. Other cells showing long cytoplasmic processes were negative for CD68 (Fig 5A, column 4) and CD163 but dimly positive for CD4, consistent with dendritic cells (Supplementary Fig 2). Staining of sequential sections of the same tissue block for CD3, CD4 and CD8 revealed both types of T lymphocytes. Approximately 5-10% of the infiltrating cells were T- lymphocytes (CD3-positive). Moreover, many of the tumor-infiltrating cells dimly positive for CD4 in the adenoma had morphologic characteristic of histiocytes and dendritic cells (Supplementary Fig 2).
Because of the strong association between HLA-DPA1 gene expression and outcome, we analyzed the remaining 18 IPACTR ACTs (10 ACC, 5 ACA and 3 of undetermined histology) with anti-HLA-DPA1 antibody. HLA-DPA1 protein was absent 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 the adrenocortical tumor cells (Fig 5B and Supplementary Table 6).
DISCUSSION
Greater expression of MHC class II genes, HLA-DPA1 in particular, is associated with better prognosis for pediatric adrenocortical tumors independent of histopathological diagnosis. In most cases, MHC class II genes were expressed by tumor-infiltrating hematopoietic cells rather than adrenocortical tumor cells. These results suggest that immune surveillance, evident by elevated expression of MHC class II and the presence of tumor-associated macrophages and dendritic cells, may limit adrenocortical progression leading to less aggressive disease.
The histological features of ACTs often do not allow unequivocal classification as adenomas or carcinomas, and such cases are currently classified as tumors of indeterminate histology, undetermined malignant potential, or simply “adrenocortical neoplasms” (13,17-18). In our IPACTR series, seven of 52 tumors (13%) had undetermined histological findings. Three of these cases expressed high levels of MHC class II based on microarray (ACT005 and ACT006) and IHC (ACT047), which is characteristic of pediatric adrenocortical adenomas.
Clin Cancer Res. Author manuscript; available in PMC 2017 December 15.
All three patients experienced tumor spillage during surgery, but did not relapse and remained disease-free at the time of the last follow up (range 20-56 months). Three of the remaining four ACTs of undetermined histology (ACT034, ACT039 and ACT046) expressed high levels of HLA-DPA1 and are free of disease for more than 2 years. These clinical findings are consistent with the observed high MHC class II expression and less aggressive disease.
Prognostic information is also lacking for pediatric adrenocortical tumors classified as carcinomas, as their outcome varies substantially after complete resection (3-5; 10-13). Tumor size (weight or volume) is associated with prognosis, but small adrenocortical carcinomas too often behave aggressively (early local and distant relapse), while large carcinomas may have an indolent course. We suggest that the expression of MHC class II genes can be used to refine the risk classification of ACTs. Overexpression of MHC class II genes could more decisively identify patients with undetermined or carcinoma histology who are unlikely to require adjuvant chemotherapy after complete surgical resection. Like childhood ACTs with a Weiss score of 0 or 1 (10-13, 17) those with relatively large, completely excised carcinomas (stage II-III) with high expression of MHC class II genes could be spared intensive chemotherapy. Completely resected carcinomas with low MHC class II gene expression could be treated with adjuvant chemotherapy irrespective of their size.
Overexpression of IGF2 and BUB1B is associated with poor prognosis in adult ACT (19- 20). In pediatric ACT, IGF2 is overexpressed in both adenomas and carcinomas (21) and therefore does not correlate with HLA-DPA1 levels or outcome (P > 0.10; Supplementary Table 5). BUBR1, the protein encoded by BUB1B, has a critical role in regulating the spindle-assembly checkpoint and implicated in chromosome instability and cancer progression (22). Similar to adult ACT and other adult cancers (23-24) overexpression of BUB1B inversely correlated with progression-free survival in pediatric ACT from both cohorts (P=0.003) (Supplementary Table 5).
MHC class II molecules are surface glycoproteins on antigen-presenting cells (e.g., dendritic cells, monocyte/macrophages, and B-lymphocytes) that presumably present tumor peptides to CD4+ T cells (25). Previous studies of pediatric ACT have not definitively addressed the cell of origin expressing MHC class II, although it was suggested to originate from adrenocortical tumor cells and downregulated concurrently with tumor progression (6-8). However, MHC class II is not expressed in the fetal adrenal cortex (26-27), the putative origin of pediatric ACT (28), but rather, predominantly by tissue macrophages and a small population of CD45R+, IgM+ lymphoid cells (28). Our findings demonstrate that the cells expressing MHC class II genes in pediatric ACTs, primarily adenomas, are macrophages and dendritic cells. Dendritic cells are capable of initiating innate and adaptive tumor responses (29), suggesting a mechanism for suppressing pediatric ACT progression. In contrast, three ACTs (ACT40, ACT047 and ACT048) displayed focal tumor-cell expression of HLA-DPA1 in addition to the infiltrating hematopoietic cells. Two of these patients (ACT047, ACT048) were more than 7 years of age at diagnosis and presented with virilization and large tumors. Interestingly, these cases experienced tumor spillage during surgery, but did not relapse during more than 20 months of follow up. Whether tumor cells
expressing MHC class II are capable of participating in regulatory immune mechanisms and have prognostic implications remains to be determined.
We have demonstrated that MHC class II gene expression in pediatric ACTs originates from tumor-infiltrating immune cells and is associated with more favorable prognosis. Our data supports the inclusion of MHC class II expression in addition to standard criteria (e.g., tumor size, mitotic activity, atypical mitoses and other histological features) as a tool for determining pediatric ACT diagnosis. Future studies will define the interaction of infiltrating hematopoietic cells with the pediatric adrenocortical tumor microenvironment, which may refine risk classification and facilitate the development of immunotherapies for these tumors.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank the study participants and the clinicians and research staff at participating institutions. We thank Jay Morris and Melanie Loyd for assistance with the expression arrays; Charlene Henry, Raven Childers and the St. Jude anatomic pathology staff for assistance with immunostaining studies. We thank Sharon Naron for editing the manuscript.
Grant Support This work was supported by a grant from Centocor, Inc., National Institutes of Health Cancer Center Support Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC).
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Translational Relevance
Current prognostic stratification of pediatric adrenocortical tumors (ACTs) is imprecise. Large tumors, incomplete surgical resection and regional and distant metastasis are associated with poor prognosis. However, a substantial number of cases without these features relapse and eventually die from progressive disease. Developing new molecular prognostic criteria would assist pathologists and oncologists to refine the histopathological classification and clinical management of pediatric ACT. Here we report that the expression of MHC class II genes is significantly associated with prognosis, hence a potential biomarker to assist in differentiating benign from malignant ACTs. We further identified tumor infiltrating dendritic cells and macrophages, but not the tumor cells, as the source of MHC class II antigen expression. Collectively, these findings provide an opportunity to improve the risk stratification and outcome for pediatric ACT.
HLA-DPB1
201137_s_at
HLA-DQA1
203290_at
HLA-DMB
203932_at
HLA-DRB1
204670_x_at
HLA-DOB
205671_s_at
HLA-DOA
206313_at
HLA-DRB3
208306_x_at
HLA-DRA
208894_at
HLA-DRB1
209312_x_at
HLA-DRB4
209728_at
HLA-DQB1
209823_x_at
HLA-DQB1
210747_at
HLA-DRA
210982_s_at
HLA-DOA
211142_x_at
HLA-DQB1
211654_x_at
HLA-DQB1
211656_x_at
HLA-DPA1
211990_at
HLA-DPA1
211991_s_at
HLA-DQA1/2
212671_s_at
HLA-DQB1
212998_x_at
HLA-DPA1
213537_at
HLA-DQA1
213831_at
HLA-DRB1
215193_x_at
HLA-DQB2
215536_at
HLA-DRB4
215666_at
HLA-DOA
217001_x_at
HLA-DRB6
217362_x_at
HLA-DMA
217478_s_at
CD3D
213539_at
CD3E
205456_at
CD3G
206804_at
CD4
203547_at
CD4
216424_at
CD8A
205758_at
CD68
203507_at
CD163
203645_s_at
CD163
215049_x_at
CD163
216233_at
ACT029
ACT018
ACT001
ACT003
ACT006
ACT020
ACT005
ACT019
ACT015
ACT021
ACT002
ACT013
ACT004
ACT023
ACT014
ACT028
ACT016
ACT008
ACT026 ACT011
ACT012
ACT007
ACT025
ACT017
ACT022
ACT009
ACT027
ACT024
ACT010
Case ID
4
Histology
4
HLA-DPB1
HLA-DQA1
HLA-DMB
HLA-DRB1
HLA-DOB
HLA-DOA
HLA-DRB3
HLA-DRA
HLA-DRB1
HLA-DRB4
HLA-DQB1
HLA-DQB1
HLA-DRA
HLA-DOA
HLA-DQB1
HLA-DQB1
HLA-DPA1
HLA-DPA1
HLA-DQA1/2
HLA-DQB1
HLA-DPA1
HLA-DQA1
HLA-DRB1
HLA-DQB2
HLA-DRB4
HLA-DOA
HLA-DRB6
HLA-DMA
CD3D
CD3E
CD3G
CD4
CD4
CD8A
CD68
CD163
CD163
CD163
201137_s_at
203290_at
203932_at
204670_x_at
205671_s_at
206313_at
208306_x_at
208894_at
209312_x_at
209728_at
209823_x_at
210747_at
210982_s_at
211142_x_at
211654_x_at
211656_x_at
211990_at
211991_s_at
212671_s_at
212998_x_at
213537_at
213831_at
215193_x_at
215536_at
215666_at
217001_x_at
217362_x_at
217478_s_at
213539_at
205456_at
206804_at
203547_at
216424_at
205758_at
203507_at
203645_s_at
215049_x_at
216233_at
Case ID
4
ACT079 ACT080
ACT059
ACT054
ACT084
ACT060
ACT058
ACT056
ACT053
ACT076
ACT072
ACT061
ACT062
ACT055
ACT064
ACT067
ACT074
ACT075
ACT066
ACT065
ACT085
ACT077
ACT081
ACT071
ACT057
ACT083
ACT070
ACT063
ACT068
ACT073
ACT086
ACT078
ACT069
ACT082
4
Progression-Free Survival
1.0
0.8
n = 17; 88.2%
0.6
0.4
0.2
n = 17; 47.1%
< median expression
0.0
≥ median expression
P = 0.003
0
1
2
3
4
5
6
7
years
A.
B.
Progression-Free Survival
1.0
Progression-Free Survival
1.0
T
0.8
n=17
0.8
I
n=17
0.6
0.6
0.4
+
0.4
0.2
n=17
n=17
HLA-DPA1
0.2
211991_s_at
HLA-DQA1/2
0.0
P = 0.0031
0.0
212671_s_at
P = 0.00074
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
years
years
C.
D.
Progression-Free Survival
1.0
Progression-Free Survival
1.0
i.
0.8
n=17
0.8
n=17
0.6
0.6
0.4
0.4
+
-+-
0.2
n=17
HLA-DRA 208894_at
n=17
0.2
HLA-DRB1
0.0
P = 0.0043
0.0
215193_x_at
P = 0.0032
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
years
years
E.
Progression-Free Survival
1.0
THE
0.8
n=17
0.6
HLA- < median
0.4
+
HLA- > median
n=17
0.2
HLA-DQB1
0.0
211654
at
P = 0.0063
0
1
2
3
4
5
6
7
years
A.
1.0
Progression-Free Survival
0.8
n=27; 79.9%
+
0.6
0.4
0.2
n=7: 14.3%
·I·
Stage I-III Stage IV
0.0
P= 0.0000368
0
1
2
3
4
5
6
7
years
B.
1.0
n=15; 100%
Progression-Free Survival
0.8
0.6
n=12; 58.3%
0.4
0.2
HLA-DPA1 < median; Stage I-III
HLA-DPA1 > median; Stage I-III
0.0
P= 0.00046
0
1
2
3
4
5
6
7
years
H&E
HLA-DRA
HLA-DPA1
CD68
a1
a2
a3
a4
b1
b2
b3
b4
c1
c2
c3
c4
a
b
| Characteristic | All cases | Non-uniformly treated cohort 1 (n = 52) | Uniformly treated cohort 2 (n = 34) |
|---|---|---|---|
| Gender | |||
| Female | 63 | 41 | 22 |
| Male | 23 | 11 | 12 |
| Age at presentation | |||
| Mean (mo) | 68.47 | 68.74 | 68.06 |
| Endocrine signs | |||
| Virilization | 47 | 27 | 20 |
| Mixed | 16 | 12 | 4 |
| Non-functioning | 14 | 7 | 7 |
| Other | 9 | 6 | 3 |
| Tumor histology | |||
| Adenoma | 13 | 13 | 0 |
| Carcinoma | 66 | 32 | 34 |
| Undefined | 7 | 7 | 0 |
| Disease stage | |||
| I | 28 | 18 | 10 |
| II | 18 | 11 | 7 |
| III | 28 | 18 | 10 |
| IV | 12 | 5 | 7 |
| Tumor weight (g) | |||
| < 200 | 49 | 28 | 21 |
| >200 | 32 | 22 | 10 |
| Relapse | |||
| (n) | 24 | 12 | 12 |
| Death | 8 | ||
| (n) | 21 | 13 | |
| Survival time | |||
| Mean (mo) | 44.38 | 50.77 | 34.6 |
Abbreviations: IPACTR, International Pediatric Adrenocortical Tumor Registry (non-uniformly treated cohort); COG, Children’s Oncology Group (uniformly-treated cohort).