# Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, Catherine de Micco, Antoine Daunay, David Taieb, Peter van Loo, Celine Besse, et al.

► To cite this version:

Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, et al. Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors. Endocrine-Related Cancer, 2015, 22 (6), pp.953 - 967. 10.1530/erc-15-0086. cea-04598809

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# Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, Catherine de Micco, Antoine Daunay, David Taieb, Peter van Loo, Celine Besse, et al.

► To cite this version:

Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, et al.. Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors. Endocrine-Related Cancer, 2015, 22 (6), pp.953 - 967. 10.1530/erc-15-0086 . cea-04598809

HAL Id: cea-04598809

https://cea.hal.science/cea-04598809v1

Submitted on 3 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Copy number variations alter methylation and parallel *IGF2* overexpression in adrenal tumors

Helene Myrtue Nielsen<fnref n="1" />,<fnref n="2" />, Alexandre How-Kit<fnref n="1" />, Carole Guerin<fnref n="3" />, Frederic Castinetti<fnref n="4" />,
Hans Kristian Moen Vollan<fnref n="5" />,<fnref n="6" />,<fnref n="7" />, Catherine De Micco<fnref n="8" />, Antoine Daunay<fnref n="1" />, David Taieb<fnref n="9" />,
Peter Van Loo<fnref n="10" />,<fnref n="11" />, Celine Besse<fnref n="12" />, Vessela N Kristensen<fnref n="5" />,<fnref n="7" />,<fnref n="13" />, Lise Lotte Hansen<fnref n="2" />,
Anne Barlier<fnref n="14" />, Frederic Sebag<fnref n="3" /> and Jörg Tost<fnref n="15" />

Correspondence
should be addressed to J Tost
**Email**
tost@cng.fr

## Abstract

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the *IGF2/H19* locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying *IGF2* overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, *n* = 12), pheochromocytomas (PCC, *n* = 10), adrenocortical benign tumors (ACBT, *n* = 20), and adrenocortical carcinomas (ACC, *n* = 20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the *IGF2/H19* locus including the *H19* imprinting control region were integratively analyzed. *IGF2* overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the *H19* ICR suggesting

## Key Words

▶ adrenocortical tumors

▶ pheochromocytomas

▶ IGF2

▶ H19

▶ cancer

▶ imprinting

▶ DNA methylation

▶ copy number analysis

▶ tetraploidy

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹Laboratory for Functional Genomics, Fondation Jean Dausset - Centre d'Etude du Polymorphisme Humain (CEPH), Paris, France</footnote>

<footnote n="2">²Institute of Biomedicine, Aarhus University, Aarhus, Denmark</footnote>

<footnote n="3">³Endocrine and Metabolic Surgery Department, AP-HM La Conception, Marseille, France</footnote>

<footnote n="4">⁴Department of Endocrinology, AP-HM La Timone, Marseille, France</footnote>

<footnote n="5">⁵Department of Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway</footnote>

<footnote n="6">⁶Division of Surgery, Transplantation and Cancer Medicine, Department of Oncology, Oslo University Hospital, Oslo, Norway</footnote>

<footnote n="7">⁷The K G Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway</footnote>

<footnote n="8">⁸Pathology Department, AP-HM La Timone, Marseille, France</footnote>

<footnote n="9">⁹Nuclear Endocrine Imaging and Treatment Department, AP-HM La Timone, Marseille, France</footnote>

<footnote n="10">¹⁰Cancer Research UK, London Research Institute, London, UK</footnote>

<footnote n="11">¹¹Department of Human Genetics, University of Leuven, Leuven, Belgium</footnote>

<footnote n="12">¹²Genotyping Facilities, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France</footnote>

<footnote n="13">¹³Department of Clinical Molecular Biology (EpiGen), University of Oslo, Ahus, Lokerod, Norway</footnote>

<footnote n="14">¹⁴Laboratory of Molecular Biology, AP-HM La Conception and CRN2M, Aix-Marseille University, Marseille, France</footnote>

<footnote n="15">¹⁵Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France</footnote>
# Copy number variations alter methylation and parallel *IGF2* overexpression in adrenal tumors

Helene Myrtue Nielsen<fnref n="1" />,<fnref n="2" />, Alexandre How-Kit<fnref n="1" />, Carole Guerin<fnref n="3" />, Frederic Castinetti<fnref n="4" />,
Hans Kristian Moen Vollan<fnref n="5" />,<fnref n="6" />,<fnref n="7" />, Catherine De Micco<fnref n="8" />, Antoine Daunay<fnref n="1" />, David Taieb<fnref n="9" />,
Peter Van Loo<fnref n="10" />,<fnref n="11" />, Celine Besse<fnref n="12" />, Vessela N Kristensen<fnref n="5" />,<fnref n="7" />,<fnref n="13" />, Lise Lotte Hansen<fnref n="2" />,
Anne Barlier<fnref n="14" />, Frederic Sebag<fnref n="3" /> and Jörg Tost<fnref n="15" />

Correspondence
should be addressed to J Tost
**Email**
tost@cng.fr

## Abstract

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n = 12), pheochromocytomas (PCC, n = 10), adrenocortical benign tumors (ACBT, n = 20), and adrenocortical carcinomas (ACC, n = 20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting

## Key Words

▶ adrenocortical tumors

▶ pheochromocytomas

▶ IGF2

▶ H19

▶ cancer

▶ imprinting

▶ DNA methylation

▶ copy number analysis

▶ tetraploidy

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹Laboratory for Functional Genomics, Fondation Jean Dausset - Centre d'Etude du Polymorphisme Humain (CEPH), Paris, France</footnote>

<footnote n="2">²Institute of Biomedicine, Aarhus University, Aarhus, Denmark</footnote>

<footnote n="3">³Endocrine and Metabolic Surgery Department, AP-HM La Conception, Marseille, France</footnote>

<footnote n="4">⁴Department of Endocrinology, AP-HM La Timone, Marseille, France</footnote>

<footnote n="5">⁵Department of Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway</footnote>

<footnote n="6">⁶Division of Surgery, Transplantation and Cancer Medicine, Department of Oncology, Oslo University Hospital, Oslo, Norway</footnote>

<footnote n="7">⁷The K G Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway</footnote>

<footnote n="8">⁸Pathology Department, AP-HM La Timone, Marseille, France</footnote>

<footnote n="9">⁹Nuclear Endocrine Imaging and Treatment Department, AP-HM La Timone, Marseille, France</footnote>

<footnote n="10">¹⁰Cancer Research UK, London Research Institute, London, UK</footnote>

<footnote n="11">¹¹Department of Human Genetics, University of Leuven, Leuven, Belgium</footnote>

<footnote n="12">¹²Genotyping Facilities, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France</footnote>

<footnote n="13">¹³Department of Clinical Molecular Biology (EpiGen), University of Oslo, Ahus, Lokerod, Norway</footnote>

<footnote n="14">¹⁴Laboratory of Molecular Biology, AP-HM La Conception and CRN2M, Aix-Marseille University, Marseille, France</footnote>

<footnote n="15">¹⁵Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France</footnote>
that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with *IGF2* overexpression in adrenal tumors and that hypermethylation of the *H19* ICR is a consequence thereof.

Endocrine-Related Cancer
(2015) **22**, 953–967

Introduction

Tumors developing in the adrenal gland include a heterogeneous group of adrenocortical and adrenomedullary malignancies (Brunt & Moley 2001, Grumbach et al. 2003). Most adrenal tumors are benign and non-hormone producing. However, adrenocortical masses require attention as hypersecretion of steroid hormones and metabolic changes may be present, which might lead to hypertension, abdominal pain, gender disorders, weight gain, or increased blood sugar levels depending on which adrenal hormone is dysregulated (Low & Sahi 2012, Hodin et al. 2014). Adrenal tumors with increased aldosterone secretion lead to Conn's syndrome (CA), whereas pheochromocytomas (PCCs) secrete excess amounts of catecholamines (Low & Sahi 2012, Hodin et al. 2014). Adrenocortical carcinomas (ACCs) can present as either non-functional or functional tumors and represent the most aggressive group of adrenal tumors. ACCs have often metastasized at the time of diagnosis and therefore are associated with poor prognosis (Volante et al. 2008).

Several genes and pathways have been implicated in the pathogenesis of ACCs including the WNT/β-Catenin pathway, and overexpression of the orphan nuclear receptor SF1 (Fassnacht et al. 2011, Simon & Hammer 2012). Recently, integrated large-scale analyses have identified alterations in driver genes including CTNNB1, TP53, CDKN2A, RB1, ZNRF3, and MEN1 (Barzon et al. 2001, Tissier et al. 2005, Assie et al. 2014, Juhlin et al. 2015). The insulin-like growth factor system has attracted much interest (Ribeiro & Latronico 2012) as the Insulin-like Growth Factor 2 (IGF2) is the most frequently over-expressed gene in ACCs (de Fraipont et al. 2005). High levels of IGF2 have been found to differentiate benign adrenocortical tumors from ACCs at both the RNA and the protein level (de Fraipont et al. 2005, Schmitt et al. 2006, Giordano et al. 2009, Ragazzon et al. 2011). Benign adrenal tumors express IGF2 and H19 at a level similar to normal adrenal tissue (Ilvesmaki et al. 1993a, Liu et al. 1995). In contrast, ACCs and PCCs show a downregulation of H19 expression and overexpression of IGF2 (Ilvesmaki et al. 1993a, Gicquel et al. 1997, Margetts et al. 2005, Slater et al.

2006, Soon et al. 2009, Meyer-Rochow et al. 2010, Ragazzon et al. 2011). Although IGF2 on its own is not sufficient for transformation, it has an active role in promoting ACC tumor growth (Guillaud-Bataille et al. 2014) and the IGF family plays an important role for the development of adrenal tumors, and has been proposed as therapeutic target (Ribeiro & Latronico 2012). *IGF2* and the neighboring *H19* are located on chromosome 11p15.5 and form the paradigmatic imprinted *IGF2/H19* locus (Supplementary Fig. 1, see section on supplementary data given at the end of this article). *IGF2* is expressed from the paternal allele only and due to its high expression levels in the fetal adrenal gland has been considered as a key mitogen for its early growth and development (Ilvesmaki et al. 1993b, Mesiano et al. 1993, O'Dell & Day 1998). After birth, *IGF2* expression levels decrease drastically and its expression is concentrated to the adrenal capsule and the periphery of the cortex (Baquedano et al. 2005). *H19* is maternally expressed and functions both as a long non-coding RNA involved in tumor suppression (Hao et al. 1993) and as a trans-regulator of a network of imprinted genes (Gabory et al. 2009).

Three differentially methylated regions (DMRs) are involved in the transcriptional regulation of the *IGF2/H19* locus (Supplementary Fig. 1, see section on supplementary data given at the end of this article). *IGF2* DMR0 and *IGF2* DMR2 are located between exons 2 and 3 and exons 8 and 9 respectively, while the *H19* DMR is located 4 kb upstream of the *H19* transcription start site. The *H19* DMR represents the imprinting control region (ICR) of the *IGF2/H19* locus and harbors seven binding sites for the methylation-sensitive insulator CTCF, a multifunctional protein involved in nuclear organization (Kanduri et al. 2000, Holmgren et al. 2001, Ohlsson et al. 2010), which brings downstream enhancers into physical proximity to either the *IGF2* or the *H19* promoter through parent-of-origin dependent methylation patterns of the *H19* ICR (Jinno et al. 1996, Frevel et al. 1999, Bell & Felsenfeld 2000, Kanduri et al. 2000).

The exact mechanisms underlying *IGF2* overexpression in adrenal tumors are poorly understood. DNA
that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with *IGF2* overexpression in adrenal tumors and that hypermethylation of the *H19* ICR is a consequence thereof.

Endocrine-Related Cancer
(2015) **22**, 953–967

Introduction

Tumors developing in the adrenal gland include a heterogeneous group of adrenocortical and adrenomedullary malignancies (Brunt & Moley 2001, Grumbach et al. 2003). Most adrenal tumors are benign and non-hormone producing. However, adrenocortical masses require attention as hypersecretion of steroid hormones and metabolic changes may be present, which might lead to hypertension, abdominal pain, gender disorders, weight gain, or increased blood sugar levels depending on which adrenal hormone is dysregulated (Low & Sahi 2012, Hodin et al. 2014). Adrenal tumors with increased aldosterone secretion lead to Conn's syndrome (CA), whereas pheochromocytomas (PCCs) secrete excess amounts of catecholamines (Low & Sahi 2012, Hodin et al. 2014). Adrenocortical carcinomas (ACCs) can present as either non-functional or functional tumors and represent the most aggressive group of adrenal tumors. ACCs have often metastasized at the time of diagnosis and therefore are associated with poor prognosis (Volante et al. 2008).

Several genes and pathways have been implicated in the pathogenesis of ACCs including the WNT/β-Catenin pathway, and overexpression of the orphan nuclear receptor SF1 (Fassnacht et al. 2011, Simon & Hammer 2012). Recently, integrated large-scale analyses have identified alterations in driver genes including CTNNB1, TP53, CDKN2A, RB1, ZNRF3, and MEN1 (Barzon et al. 2001, Tissier et al. 2005, Assie et al. 2014, Juhlin et al. 2015). The insulin-like growth factor system has attracted much interest (Ribeiro & Latronico 2012) as the Insulin-like Growth Factor 2 (IGF2) is the most frequently over-expressed gene in ACCs (de Fraipont et al. 2005). High levels of IGF2 have been found to differentiate benign adrenocortical tumors from ACCs at both the RNA and the protein level (de Fraipont et al. 2005, Schmitt et al. 2006, Giordano et al. 2009, Ragazzon et al. 2011). Benign adrenal tumors express IGF2 and H19 at a level similar to normal adrenal tissue (Ilvesmaki et al. 1993a, Liu et al. 1995). In contrast, ACCs and PCCs show a downregulation of H19 expression and overexpression of IGF2 (Ilvesmaki et al. 1993a, Gicquel et al. 1997, Margetts et al. 2005, Slater et al.

2006, Soon et al. 2009, Meyer-Rochow et al. 2010, Ragazzon et al. 2011). Although IGF2 on its own is not sufficient for transformation, it has an active role in promoting ACC tumor growth (Guillaud-Bataille et al. 2014) and the IGF family plays an important role for the development of adrenal tumors, and has been proposed as therapeutic target (Ribeiro & Latronico 2012). *IGF2* and the neighboring *H19* are located on chromosome 11p15.5 and form the paradigmatic imprinted *IGF2/H19* locus (Supplementary Fig. 1, see section on supplementary data given at the end of this article). *IGF2* is expressed from the paternal allele only and due to its high expression levels in the fetal adrenal gland has been considered as a key mitogen for its early growth and development (Ilvesmaki et al. 1993b, Mesiano et al. 1993, O'Dell & Day 1998). After birth, *IGF2* expression levels decrease drastically and its expression is concentrated to the adrenal capsule and the periphery of the cortex (Baquedano et al. 2005). *H19* is maternally expressed and functions both as a long non-coding RNA involved in tumor suppression (Hao et al. 1993) and as a trans-regulator of a network of imprinted genes (Gabory et al. 2009).

Three differentially methylated regions (DMRs) are involved in the transcriptional regulation of the *IGF2/H19* locus (Supplementary Fig. 1, see section on supplementary data given at the end of this article). *IGF2* DMR0 and *IGF2* DMR2 are located between exons 2 and 3 and exons 8 and 9 respectively, while the *H19* DMR is located 4 kb upstream of the *H19* transcription start site. The *H19* DMR represents the imprinting control region (ICR) of the *IGF2/H19* locus and harbors seven binding sites for the methylation-sensitive insulator CTCF, a multifunctional protein involved in nuclear organization (Kanduri et al. 2000, Holmgren et al. 2001, Ohlsson et al. 2010), which brings downstream enhancers into physical proximity to either the *IGF2* or the *H19* promoter through parent-of-origin dependent methylation patterns of the *H19* ICR (Jinno et al. 1996, Frevel et al. 1999, Bell & Felsenfeld 2000, Kanduri et al. 2000).

The exact mechanisms underlying *IGF2* overexpression in adrenal tumors are poorly understood. DNA
<table><tr><td>Research</td><td>H M Nielsen et al.</td><td>Mechanisms of IGF2 overexpression</td><td>22:6</td><td>955</td></tr></table>

hypermethylation of the *H19* promoter region has previously been associated with *IGF2* overexpression in ACCs (Gao *et al.* 2002) as well as somatic copy number changes, where loss of the maternal allele was accompanied by a duplication of the paternal allele (Gicquel *et al.* 1994, 1997). Maternal loss of chr11p15.5 has also been implicated in the *IGF2* overexpression observed in PCCs (Margetts *et al.* 2005).

While genetic and epigenetic mechanisms are closely intertwined, it is currently unclear if DNA methylation and genetic aberrations are independent events leading to *IGF2* overexpression or if DNA methylation patterns reflect only changes at the genetic level. In the current study, we provide the first integrated analysis of gene expression, DNA methylation and genetic variation of the *IGF2/H19* locus in adrenal tumor subtypes using high-resolution molecular technologies to unravel the driving force behind *IGF2* overexpression in these tumors.

# Materials and methods

## Study group

Sixty-two adrenal tumor samples from the Timone Hospital (Marseille, France) were analyzed in this study. Clinical and pathological data are summarized in Table 1. Four groups of ACTs were included in the study: 12 aldosterone producing adenomas in the context of Conn's syndrome (CA), ten pheochromocytomas (PCCs), 20 adrenocortical carcinomas (ACCs) of which two samples (51 and 52) were derived from the two adrenal glands of the same patient, and 20 adrenocortical benign tumors (ACBT). ACBT were either non-secreting cortical tumors or cortisol secreting adrenal tumors with no evidence for malignancy. ACTH dependent bilateral adrenal hyperplasia and ACTH independent adrenal hyperplasia (primary pigmented nodular disease) were not included in this cohort. Adrenocortical tumors were staged using the Weiss score, PCCs were staged using the Pheochromocytoma of the Adrenal gland Scale Score (PASS), which both take histological features such as tumor size, presence of necrosis and mitotic activity including atypical mitoses into account (Weiss 1984, Thompson 2002, Lau & Weiss 2009). An adrenocortical tumor with a Weiss score of three or higher was classified as ACC and PCCs with a PASS score of four or higher were classified as malignant. All pheochromocytomas lacked a positive familial history indicating hereditary disease and had no mutations in the *SDHB*, *SDHD* and *VHL* genes as assessed by Sanger sequencing. Written informed consent was obtained

<table><caption>**Table 1** Clinical and pathological data on the adrenal tumors</caption><thead><tr><th>Patient subgroups</th><th>All</th><th>Percentage/range</th></tr></thead><tbody><tr><td>Conn's adenoma</td><td></td><td></td></tr><tr><td>Number of samples</td><td>12</td><td></td></tr><tr><td>Female</td><td>6</td><td>50%</td></tr><tr><td>Male</td><td>6</td><td>50%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>47</td><td>(31–68)</td></tr><tr><td>Tumor size in mm (median, range)</td><td>14.5</td><td>(10–22)</td></tr><tr><td>Adrenocortical benign tumors (ACBT)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>20</td><td></td></tr><tr><td>Female</td><td>16</td><td>80%</td></tr><tr><td>Male</td><td>4</td><td>20%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>55.5</td><td>(23–68)</td></tr><tr><td>Weiss score (mean, range)</td><td>0.35</td><td>0–2</td></tr><tr><td>Tumor size in mm (median, range)</td><td>40</td><td>(20–60)</td></tr><tr><td>Pheochromocytomas (PCC)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>10</td><td></td></tr><tr><td>Female</td><td>6</td><td>60%</td></tr><tr><td>Male</td><td>4</td><td>40%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>50.5</td><td>(41–67)</td></tr><tr><td>PASS score (mean, range)</td><td>2.7</td><td>(0–9)</td></tr><tr><td>Tumor size in mm (median, range)</td><td>40</td><td>(26–80)</td></tr><tr><td>Malignant carcinomas (ACC)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>20</td><td></td></tr><tr><td>Female</td><td>12</td><td>60%</td></tr><tr><td>Male</td><td>8</td><td>40%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>48</td><td>(28–78)</td></tr><tr><td>Weiss score (mean, range)</td><td>5.65</td><td>(3–6)</td></tr><tr><td>No. of patients with metastases</td><td>4</td><td></td></tr><tr><td>TNM stage I or II</td><td>7</td><td></td></tr><tr><td>TNM stage III or IV</td><td>11</td><td></td></tr><tr><td>Tumor size in mm (median, range)</td><td>50</td><td>(33–190)</td></tr></tbody></table>

from all patients and the study was approved by the local ethics committee.

## Isolation of DNA and RNA

All tumoral tissue samples were carefully evaluated by microscopy to ensure that sampling was from tumoral tissue and not adjacent normal tissue. Genomic DNA was isolated from 20 mg of fresh frozen tumors using the QIAamp DNA Mini Kit with a proteinase K treatment. Total RNA was extracted from 15 to 20 mg of the same tumor tissue using the RNeasy minikit (Qiagen) followed by DNAse treatment according to the manufacturer's instructions. DNA from whole blood samples of the corresponding patients were extracted using the MagAttract DNA Blood Midi M48 Kit (Qiagen).

## Gene expression analysis

According to the manufacturer's instructions, 500 ng of total RNA was reverse transcribed using Superscript III and oligo(dT) primers (Life Technologies). RNA concentrations
<table><tr><td>Research</td><td>H M Nielsen et al.</td><td>Mechanisms of IGF2 overexpression</td><td>22:6</td><td>955</td></tr></table>

hypermethylation of the *H19* promoter region has previously been associated with *IGF2* overexpression in ACCs (Gao *et al.* 2002) as well as somatic copy number changes, where loss of the maternal allele was accompanied by a duplication of the paternal allele (Gicquel *et al.* 1994, 1997). Maternal loss of chr11p15.5 has also been implicated in the *IGF2* overexpression observed in PCCs (Margetts *et al.* 2005).

While genetic and epigenetic mechanisms are closely intertwined, it is currently unclear if DNA methylation and genetic aberrations are independent events leading to *IGF2* overexpression or if DNA methylation patterns reflect only changes at the genetic level. In the current study, we provide the first integrated analysis of gene expression, DNA methylation and genetic variation of the *IGF2/H19* locus in adrenal tumor subtypes using high-resolution molecular technologies to unravel the driving force behind *IGF2* overexpression in these tumors.

# Materials and methods

## Study group

Sixty-two adrenal tumor samples from the Timone Hospital (Marseille, France) were analyzed in this study. Clinical and pathological data are summarized in Table 1. Four groups of ACTs were included in the study: 12 aldosterone producing adenomas in the context of Conn's syndrome (CA), ten pheochromocytomas (PCCs), 20 adrenocortical carcinomas (ACCs) of which two samples (51 and 52) were derived from the two adrenal glands of the same patient, and 20 adrenocortical benign tumors (ACBT). ACBT were either non-secreting cortical tumors or cortisol secreting adrenal tumors with no evidence for malignancy. ACTH dependent bilateral adrenal hyperplasia and ACTH independent adrenal hyperplasia (primary pigmented nodular disease) were not included in this cohort. Adrenocortical tumors were staged using the Weiss score, PCCs were staged using the Pheochromocytoma of the Adrenal gland Scale Score (PASS), which both take histological features such as tumor size, presence of necrosis and mitotic activity including atypical mitoses into account (Weiss 1984, Thompson 2002, Lau & Weiss 2009). An adrenocortical tumor with a Weiss score of three or higher was classified as ACC and PCCs with a PASS score of four or higher were classified as malignant. All pheochromocytomas lacked a positive familial history indicating hereditary disease and had no mutations in the *SDHB*, *SDHD* and *VHL* genes as assessed by Sanger sequencing. Written informed consent was obtained

<table><caption>**Table 1** Clinical and pathological data on the adrenal tumors</caption><thead><tr><th>Patient subgroups</th><th>All</th><th>Percentage/range</th></tr></thead><tbody><tr><td>Conn's adenoma</td><td></td><td></td></tr><tr><td>Number of samples</td><td>12</td><td></td></tr><tr><td>Female</td><td>6</td><td>50%</td></tr><tr><td>Male</td><td>6</td><td>50%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>47</td><td>(31–68)</td></tr><tr><td>Tumor size in mm (median, range)</td><td>14.5</td><td>(10–22)</td></tr><tr><td>Adrenocortical benign tumors (ACBT)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>20</td><td></td></tr><tr><td>Female</td><td>16</td><td>80%</td></tr><tr><td>Male</td><td>4</td><td>20%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>55.5</td><td>(23–68)</td></tr><tr><td>Weiss score (mean, range)</td><td>0.35</td><td>0–2</td></tr><tr><td>Tumor size in mm (median, range)</td><td>40</td><td>(20–60)</td></tr><tr><td>Pheochromocytomas (PCC)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>10</td><td></td></tr><tr><td>Female</td><td>6</td><td>60%</td></tr><tr><td>Male</td><td>4</td><td>40%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>50.5</td><td>(41–67)</td></tr><tr><td>PASS score (mean, range)</td><td>2.7</td><td>(0–9)</td></tr><tr><td>Tumor size in mm (median, range)</td><td>40</td><td>(26–80)</td></tr><tr><td>Malignant carcinomas (ACC)</td><td></td><td></td></tr><tr><td>Number of samples</td><td>20</td><td></td></tr><tr><td>Female</td><td>12</td><td>60%</td></tr><tr><td>Male</td><td>8</td><td>40%</td></tr><tr><td>Age at surgery, years (median, range)</td><td>48</td><td>(28–78)</td></tr><tr><td>Weiss score (mean, range)</td><td>5.65</td><td>(3–6)</td></tr><tr><td>No. of patients with metastases</td><td>4</td><td></td></tr><tr><td>TNM stage I or II</td><td>7</td><td></td></tr><tr><td>TNM stage III or IV</td><td>11</td><td></td></tr><tr><td>Tumor size in mm (median, range)</td><td>50</td><td>(33–190)</td></tr></tbody></table>

from all patients and the study was approved by the local ethics committee.

## Isolation of DNA and RNA

All tumoral tissue samples were carefully evaluated by microscopy to ensure that sampling was from tumoral tissue and not adjacent normal tissue. Genomic DNA was isolated from 20 mg of fresh frozen tumors using the QIAamp DNA Mini Kit with a proteinase K treatment. Total RNA was extracted from 15 to 20 mg of the same tumor tissue using the RNeasy minikit (Qiagen) followed by DNAse treatment according to the manufacturer's instructions. DNA from whole blood samples of the corresponding patients were extracted using the MagAttract DNA Blood Midi M48 Kit (Qiagen).

## Gene expression analysis

According to the manufacturer's instructions, 500 ng of total RNA was reverse transcribed using Superscript III and oligo(dT) primers (Life Technologies). RNA concentrations
were established using a NanoDrop 2000c spectro-
photometer (Thermo Scientific, Wilmington, DE, USA)
and the RNA integrity number (RIN) was assessed
using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Massy, France).

Expression analyses were performed following the
MIQE guidelines to ensure accurate quantification of
gene expression (Bustin *et al.* 2009). The *IGF2* and *H19*
expression assays used in this study are both spanning
exon-exon boundaries and have previously been published
(Dejeux *et al.* 2009, Koukoura *et al.* 2011) (Supplementary
Table 1, see section on supplementary data given at the end
of this article). Experiments were performed in duplicate
for each sample using a cut-off of 10% coefficient of
variance (CV). Two samples (9 and 16) were excluded
from the expression analysis due to a CV value above the
threshold and low RIN respectively. The average RIN
among the remaining 60 RNA samples was 8.5. A dilution
series of commercial cDNA (10–0.001 ng/µl) was prepared
to determine PCR efficiency (*E*). For each primer set,
*E* was >90%.

The geNorm reference gene selection kit (Primer-
Design, Southampton, UK) was used to identify the most
stably expressed reference genes across the different
adrenocortical tumors using the qbasePLUS Software
(Biogazelle, Zwijnaarde, Belgium). The expression of
*IGF2* and *H19* was normalized to the geometric mean of
the three reference genes (*SDHA*, *ATP5B*, and *CYC1*). The
final RT-qPCRs included 5 µl of cDNA (1 ng/µl), 5 µl primer
(300 nM), and 10 µl of SYBR Green I Master mix (Roche).
The real-time PCR program was initiated with 10 min
at 95 °C followed by 45 cycles of 15 s at 95 °C and 60 s at
60 °C using a LightCycler 480 II (Roche). Melting curve
analysis was performed after each run initiated with 5 s at
95 °C, 60 s at 65 °C and finally with a temperature ramping
from 65 °C to 95 °C (0.1 °C/s) to exclude amplification of
unspecific products.

DNA methylation analysis by pyrosequencing

Quantitative DNA methylation values were obtained
using pyrosequencing as previously described (Tost &
Gut 2007). Briefly, 500 ng of genomic DNA was treated
with sodium bisulfite using the EpiTect Bisulfite Kit
(Qiagen) according to the manufacturer's instructions.
The final PCR contained 10 ng of bisulfite treated DNA,
1×PCR buffer (Qiagen), assay specific MgCl₂ concen-
trations, 100 µM of each nucleotide, 200 nM of each
primer, and 2 U of HotStarTaq (Qiagen). The PCR program
was initiated with an initial denaturation step of 15 min

at 95 °C followed by 50 cycles of 30 s denaturation at
95 °C, 30 s at the assay specific annealing temperature
and 15 s elongation at 72 °C. The final extension was
performed for 5 min at 72 °C. Primer sequences and assay-
specific modifications to the general protocol are given in
Supplementary Table 1, see section on supplementary data
given at the end of this article.

Single nucleotide polymorphism analysis

Fifty-seven tumor samples had sufficient high-quality
material available for genome-wide single nucleotide
polymorphism (SNP) analysis using the Illumina-
Omni2.5M array (Illumina, Inc., San Diego, CA, USA)
(Gunderson et al. 2005, Peiffer et al. 2006)). Arrays were
imaged using iScan scanners and the BeadStudio software
(Illumina) was used to call genotypes and extract data for
downstream analysis.

The log-transformed ratio between measured and
expected SNP signal intensity (LogR) and B-allele frequen-
cies (BAF) were visualized using tools implemented in the
'copynumber' R library (Gentleman et al. 2004, Nilsen
et al. 2012). Inspection of raw data profiles demonstrated a
previously described asymmetry in the intensity of the two
alleles for each SNP remaining after the normalization
steps implemented in Illumina's Genome Studio Software
(Staaf et al. 2008). This affected both the allelic proportions
and the copy number estimates. Data were corrected for
this bias using a quantile normalization approach
implemented in the tQN method (Staaf et al. 2008). Bias
in the estimated copy number related to GC binding
artifacts was corrected for using the method by (Cheng
et al. 2011). Allele specific copy number estimates
corrected for ploidy and infiltration of normal cells were
identified using ASCAT (Van Loo et al. 2010). For the
correct estimation of the percentage of the tumor cell
fraction ASCAT is dependent on genomic aberrations.
Therefore ASCAT cannot estimate the tumor percentage of
diploid tumor samples without aberrations correctly. As
such, we have excluded the tumor percentage estimation
for diploid tumors without genetic aberrations in Table 2.
Copy number changes were verified by genotyping rs680,
which is located in *IGF2* (chr11:2,153,634) for paired
tumor and blood samples heterozygous for rs680.

Genotyping rs680

The PCR for genotyping contained 10 ng of genomic DNA,
1× PCR buffer (Qiagen), 1 mM MgSO₄, 100 µM of each
nucleotide, 200 nM of each primer, and 3 U of Platinum
were established using a NanoDrop 2000c spectro-
photometer (Thermo Scientific, Wilmington, DE, USA)
and the RNA integrity number (RIN) was assessed
using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Massy, France).

Expression analyses were performed following the
MIQE guidelines to ensure accurate quantification of
gene expression (Bustin *et al.* 2009). The *IGF2* and *H19*
expression assays used in this study are both spanning
exon-exon boundaries and have previously been published
(Dejeux *et al.* 2009, Koukoura *et al.* 2011) (Supplementary
Table 1, see section on supplementary data given at the end
of this article). Experiments were performed in duplicate
for each sample using a cut-off of 10% coefficient of
variance (CV). Two samples (9 and 16) were excluded
from the expression analysis due to a CV value above the
threshold and low RIN respectively. The average RIN
among the remaining 60 RNA samples was 8.5. A dilution
series of commercial cDNA (10–0.001 ng/µl) was prepared
to determine PCR efficiency (*E*). For each primer set,
*E* was >90%.

The geNorm reference gene selection kit (Primer-
Design, Southampton, UK) was used to identify the most
stably expressed reference genes across the different
adrenocortical tumors using the qbasePLUS Software
(Biogazelle, Zwijnaarde, Belgium). The expression of
*IGF2* and *H19* was normalized to the geometric mean of
the three reference genes (*SDHA*, *ATP5B*, and *CYC1*). The
final RT-qPCRs included 5 µl of cDNA (1 ng/µl), 5 µl primer
(300 nM), and 10 µl of SYBR Green I Master mix (Roche).
The real-time PCR program was initiated with 10 min
at 95 °C followed by 45 cycles of 15 s at 95 °C and 60 s at
60 °C using a LightCycler 480 II (Roche). Melting curve
analysis was performed after each run initiated with 5 s at
95 °C, 60 s at 65 °C and finally with a temperature ramping
from 65 °C to 95 °C (0.1 °C/s) to exclude amplification of
unspecific products.

DNA methylation analysis by pyrosequencing

Quantitative DNA methylation values were obtained
using pyrosequencing as previously described (Tost &
Gut 2007). Briefly, 500 ng of genomic DNA was treated
with sodium bisulfite using the EpiTect Bisulfite Kit
(Qiagen) according to the manufacturer's instructions.
The final PCR contained 10 ng of bisulfite treated DNA,
1×PCR buffer (Qiagen), assay specific MgCl₂ concen-
trations, 100 µM of each nucleotide, 200 nM of each
primer, and 2 U of HotStarTaq (Qiagen). The PCR program
was initiated with an initial denaturation step of 15 min

at 95 °C followed by 50 cycles of 30 s denaturation at
95 °C, 30 s at the assay specific annealing temperature
and 15 s elongation at 72 °C. The final extension was
performed for 5 min at 72 °C. Primer sequences and assay-
specific modifications to the general protocol are given in
Supplementary Table 1, see section on supplementary data
given at the end of this article.

Single nucleotide polymorphism analysis

Fifty-seven tumor samples had sufficient high-quality
material available for genome-wide single nucleotide
polymorphism (SNP) analysis using the Illumina-
Omni2.5M array (Illumina, Inc., San Diego, CA, USA)
(Gunderson et al. 2005, Peiffer et al. 2006)). Arrays were
imaged using iScan scanners and the BeadStudio software
(Illumina) was used to call genotypes and extract data for
downstream analysis.

The log-transformed ratio between measured and
expected SNP signal intensity (LogR) and B-allele frequen-
cies (BAF) were visualized using tools implemented in the
'copynumber' R library (Gentleman et al. 2004, Nilsen
et al. 2012). Inspection of raw data profiles demonstrated a
previously described asymmetry in the intensity of the two
alleles for each SNP remaining after the normalization
steps implemented in Illumina's Genome Studio Software
(Staaf et al. 2008). This affected both the allelic proportions
and the copy number estimates. Data were corrected for
this bias using a quantile normalization approach
implemented in the tQN method (Staaf et al. 2008). Bias
in the estimated copy number related to GC binding
artifacts was corrected for using the method by (Cheng
et al. 2011). Allele specific copy number estimates
corrected for ploidy and infiltration of normal cells were
identified using ASCAT (Van Loo et al. 2010). For the
correct estimation of the percentage of the tumor cell
fraction ASCAT is dependent on genomic aberrations.
Therefore ASCAT cannot estimate the tumor percentage of
diploid tumor samples without aberrations correctly. As
such, we have excluded the tumor percentage estimation
for diploid tumors without genetic aberrations in Table 2.
Copy number changes were verified by genotyping rs680,
which is located in *IGF2* (chr11:2,153,634) for paired
tumor and blood samples heterozygous for rs680.

Genotyping rs680

The PCR for genotyping contained 10 ng of genomic DNA,
1× PCR buffer (Qiagen), 1 mM MgSO₄, 100 µM of each
nucleotide, 200 nM of each primer, and 3 U of Platinum
<table><caption>**Table 2** IGF2 and H19 expression levels compared to DNA methylation and genetic status of 11p15.5</caption><thead><tr><th colspan="9">Conn's adenoma</th></tr><tr><th>ID</th><th>IGF2 exp.</th><th>H19 exp.</th><th>Genomic status of 11p15.5</th><th>Ploidy</th><th>H19 ICR meth.</th><th>DMR2 meth.</th><th>DMR0 meth.</th><th>Tumor cell in %</th></tr></thead><tbody><tr><td>1</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>2</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>3</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>4</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>5</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>6</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>7</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>8</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>9</td><td>NA</td><td>NA</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>10</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>11</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>NA</td><td>NA</td></tr><tr><td>12</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><th colspan="9">Adrenocortical Benign Tumours</th></tr><tr><th>ID</th><th>IGF2 exp.</th><th>H19 exp.</th><th>Genomic status of 11p15.5</th><th>Ploidy</th><th>H19 ICR meth.</th><th>DMR2 meth.</th><th>DMR0 meth.</th><th>Weiss</th><th>Tumor cell in %</th></tr><tr><td>13</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>14</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Hypermeth.</td><td>0</td><td>NA</td></tr><tr><td>15</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>16</td><td>NA</td><td>NA</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>17</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>18</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>19</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>20</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>21</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>22</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>23</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>24</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>1</td><td>NA</td></tr><tr><td>25</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>26</td><td>Normal</td><td>Normal</td><td>NA</td><td>NA</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>NA</td></tr><tr><td>27</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>2</td><td>NA</td></tr><tr><td>28</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>29</td><td>Normal</td><td>Normal</td><td>NA</td><td>NA</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>30</td><td>Overexp.</td><td>Normal</td><td>NA</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>0</td><td>NA</td></tr><tr><td>31</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><td>32</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>0</td><td>NA</td></tr><tr><th colspan="9">Pheochromocytomas</th></tr><tr><th>ID</th><th>IGF2 exp.</th><th>H19 exp.</th><th>Copy number status of 11p15.5</th><th>Ploidy</th><th>H19 ICR meth.</th><th>DMR2 meth.</th><th>DMR0 meth.</th><th>PASS</th><th>Tumor cell in %</th></tr><tr><td>33</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Normal</td><td>Normal</td><td>2</td><td>38%</td></tr><tr><td>34</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>6</td><td>53%</td></tr><tr><td>35</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>6</td><td>38%</td></tr><tr><td>36</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>1</td><td>53%</td></tr><tr><td>37</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>71%</td></tr><tr><td>38</td><td>Overexp.</td><td>Normal</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>9</td><td>60%</td></tr><tr><td>39</td><td>Overexp.</td><td>Downreg.</td><td>UPD</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>0</td><td>79%</td></tr><tr><td>40</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>65%</td></tr><tr><td>41</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>0</td><td>81%</td></tr><tr><td>42</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>3</td><td>NA</td></tr><tr><th colspan="9">Adrenocortical Carcinoma</th></tr><tr><th>ID</th><th>IGF2 exp.</th><th>H19 exp.</th><th>Copy number status of 11p15.5</th><th>Ploidy</th><th>H19 ICR meth.</th><th>DMR2 meth.</th><th>DMR0 meth.</th><th>Weiss</th><th>Tumor cell in %</th></tr><tr><td>43</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>3</td><td>54%</td></tr><tr><td>44</td><td>Overexp.</td><td>Downreg.</td><td>UPD genotype</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>7</td><td>72%</td></tr><tr><td>45</td><td>Overexp.</td><td>Downreg.</td><td>UPD</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>3</td><td>70%</td></tr><tr><td>46</td><td>Overexp.</td><td>Downreg.</td><td>UPD genotype</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>5</td><td>86%</td></tr><tr><td>47</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>4</td><td>84%</td></tr><tr><td>48</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>3</td><td>59%</td></tr></tbody></table>
<table><caption>**Table 2** IGF2 and H19 expression levels compared to DNA methylation and genetic status of 11p15.5</caption><thead><tr><th>Conn's adenoma</th><th>ID</th><th>IGF2 exp.</th><th>H19 exp.</th><th>Genomic status of 11p15.5</th><th>Ploidy</th><th>H19 ICR meth.</th><th>DMR2 meth.</th><th>DMR0 meth.</th><th>Tumor cell in %</th></tr></thead><tbody><tr><td>1</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>2</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>3</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>4</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>5</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>6</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>7</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>8</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>9</td><td>NA</td><td>NA</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>10</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>11</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>NA</td><td>NA</td></tr><tr><td>12</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td colspan="10"><strong>Adrenocortical Benign Tumours</strong></td></tr><tr><td>13</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>14</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>15</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>16</td><td>NA</td><td>NA</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>17</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>NA</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>18</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>19</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>20</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>21</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>22</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>23</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>24</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>25</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>26</td><td>Normal</td><td>Normal</td><td>NA</td><td>NA</td><td>NA</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td>27</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>28</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>29</td><td>Normal</td><td>Normal</td><td>NA</td><td>NA</td><td>NA</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>30</td><td>Overexp.</td><td>Normal</td><td>NA</td><td>NA</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>NA</td></tr><tr><td>31</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td>32</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>NA</td></tr><tr><td colspan="10"><strong>Pheochromocytomas</strong></td></tr><tr><td>33</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Normal</td><td>38%</td></tr><tr><td>34</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>53%</td></tr><tr><td>35</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Hypermeth.</td><td>38%</td></tr><tr><td>36</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>53%</td></tr><tr><td>37</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>71%</td></tr><tr><td>38</td><td>Overexp.</td><td>Normal</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>60%</td></tr><tr><td>39</td><td>Overexp.</td><td>Downreg.</td><td>UPD</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>79%</td></tr><tr><td>40</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>65%</td></tr><tr><td>41</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Hypometh.</td><td>Normal</td><td>81%</td></tr><tr><td>42</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>NA</td></tr><tr><td colspan="10"><strong>Adrenocortical Carcinoma</strong></td></tr><tr><td>43</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>54%</td></tr><tr><td>44</td><td>Overexp.</td><td>Downreg.</td><td>UPD genotype</td><td>Normal</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>72%</td></tr><tr><td>45</td><td>Overexp.</td><td>Downreg.</td><td>UPD</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>70%</td></tr><tr><td>46</td><td>Overexp.</td><td>Downreg.</td><td>UPD genotype</td><td>Normal</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>86%</td></tr><tr><td>47</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>Normal</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>84%</td></tr><tr><td>48</td><td>Overexp.</td><td>Normal</td><td>Normal</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>59%</td></tr></tbody></table>
<table><caption>Table 2 Continued</caption><tr><td>49</td><td>Overexp.</td><td>Normal</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>5</td><td>68%</td></tr><tr><td>50</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>8</td><td>NA</td></tr><tr><td>51</td><td>Overexp.</td><td>Downreg.</td><td>Triploid</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>81%</td></tr><tr><td>52</td><td>Overexp.</td><td>Downreg.</td><td>NA</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>NA</td></tr><tr><td>53</td><td>Overexp.</td><td>Downreg.</td><td>Triploid</td><td>4</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>6</td><td>32%</td></tr><tr><td>54</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>6</td><td>95%</td></tr><tr><td>55</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>88%</td></tr><tr><td>56</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>2</td><td>72%</td></tr><tr><td>57</td><td>Normal</td><td>Downreg.</td><td>UPD genotype</td><td>4</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>8</td><td>81%</td></tr><tr><td>58</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>7</td><td>39%</td></tr><tr><td>59</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>6</td><td>70%</td></tr><tr><td>60</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>7</td><td>67%</td></tr><tr><td>61</td><td>Overexp.</td><td>Downreg.</td><td>Loss of two alleles<br>(one of each)</td><td>4</td><td>Normal</td><td>Hypometh.</td><td>Hypermeth.</td><td>5</td><td>59%</td></tr><tr><td>62</td><td>Normal</td><td>Downreg.</td><td>Tetraploid</td><td>4</td><td>Normal</td><td>Hypometh.</td><td>Hypometh.</td><td>4</td><td>81%</td></tr></table>

NA, no aberrations; for correct estimation of the tumor percentage, ASCAT is dependent on genomic aberrations. For diploid tumors without aberrations, the tumor percentage can therefore not be calculated.

Taq DNA Polymerase High Fidelity (Life Technologies) in a final volume of 25 µl. The PCR program was initiated with a denaturation step of 4 min at 95 °C followed by 50 cycles of 30 s at 95 °C, an annealing temperature at 56 °C for 30 s, and elongation at 72 °C for 15 s. The final extension was performed for 4 min at 72 °C. Quantitative genotyping was performed on 10 µl of the PCR product using pyrosequencing. Primer sequences are given in Supplementary Table 1, see section on supplementary data given at the end of this article.

## Statistical analysis

The non-parametric Mann-Whitney test was used to assess potential differences between groups for the methylation and the expression analysis. A two-sided t-test was used to test for associations between categorized molecular alterations and categorical clinical parameters. Correlation analyses were made using the Spearman's rank correlation coefficient. A nominal *P* value <0.05 was considered significant.

## Results

To provide a comprehensive analysis of the mechanisms underlying *IGF2* overexpression in adrenal tumors, gene expression levels were integrated with copy number and ploidy analysis, and the DNA methylation status of three DMRs including the ICR upstream of *H19* (*H19* DMR) and two secondary DMRs (DMR0 and DMR2) within *IGF2* (Supplementary Fig. 1, see section on supplementary data given at the end of this article).

## Differential *IGF2* and *H19* expression characterize PCCs and ACCs

Gene expression changes of *IGF2* and *H19* were analyzed in 60 fresh frozen adrenal tumors (Fig. 1). *IGF2* was considered overexpressed when the expression level was three times above the s.d. of the mean *IGF2* expression value observed for the CAs, which express *IGF2* and *H19* at levels comparable to normal adrenal tissue (Ilvesmaki *et al.* 1993a, Liu *et al.* 1995). *H19* was likewise considered downregulated when the expression level of individual samples was below three times the s.d. of the mean *H19* expression level observed for CAs.

*IGF2* showed overexpression in 85% (17/20) of the ACCs and 100% (10/10) of the PCCs, whereas only 4/19 ACBTs and 3/11 CAs showed overexpression of *IGF2* (*P*≤0.0003 for both ACC and PCC vs CAs; Mann-Whitney test) (Fig. 1). Expression levels did not correlate with clinical parameters such as presence of metastases, TNM stage. Only a tendency for a positive correlation between *IGF2* expression and tumor size for the ACCs was observed, but did not reach statistical significance (*R*²=0.273, *P*=0.067, Spearman's correlation). Decreased expression levels of *H19* also characterized both ACCs (18/20) and PCCs (7/10) when compared to the CA samples (*P*=0.0001 and *P*=0.01 respectively; Mann-Whitney test). However, the level of *H19* down regulation was less pronounced for PCCs compared to ACCs (*P*=0.0053; Mann-Whitney test) (Fig. 1). Concomitant *IGF2* overexpression and *H19* downregulation characterized the majority of the ACCs (15/20), PCCs (7/10), and a single ACBT, but was not present in all cases (Table 2).
<table><caption>Table 2 Continued</caption><tr><td>49</td><td>Overexp.</td><td>Normal</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>5</td><td>68%</td></tr><tr><td>50</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>8</td><td>NA</td></tr><tr><td>51</td><td>Overexp.</td><td>Downreg.</td><td>Triploid</td><td>4</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>81%</td></tr><tr><td>52</td><td>Overexp.</td><td>Downreg.</td><td>NA</td><td>NA</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>NA</td></tr><tr><td>53</td><td>Overexp.</td><td>Downreg.</td><td>Triploid</td><td>4</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>6</td><td>32%</td></tr><tr><td>54</td><td>Overexp.</td><td>Downreg.</td><td>Normal</td><td>2</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>6</td><td>95%</td></tr><tr><td>55</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>8</td><td>88%</td></tr><tr><td>56</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Normal</td><td>2</td><td>72%</td></tr><tr><td>57</td><td>Normal</td><td>Downreg.</td><td>UPD genotype</td><td>4</td><td>High</td><td>Normal</td><td>Hypermeth.</td><td>8</td><td>81%</td></tr><tr><td>58</td><td>Normal</td><td>Downreg.</td><td>Normal</td><td>2</td><td>Normal</td><td>Normal</td><td>Normal</td><td>7</td><td>39%</td></tr><tr><td>59</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>6</td><td>70%</td></tr><tr><td>60</td><td>Overexp.</td><td>Downreg.</td><td>Loss of one allele</td><td>2</td><td>High</td><td>Hypometh.</td><td>Hypermeth.</td><td>7</td><td>67%</td></tr><tr><td>61</td><td>Overexp.</td><td>Downreg.</td><td>Loss of two alleles (one of each)</td><td>4</td><td>Normal</td><td>Hypometh.</td><td>Hypermeth.</td><td>5</td><td>59%</td></tr><tr><td>62</td><td>Normal</td><td>Downreg.</td><td>Tetraploid</td><td>4</td><td>Normal</td><td>Hypometh.</td><td>Hypometh.</td><td>4</td><td>81%</td></tr></table>

NA, no aberrations; for correct estimation of the tumor percentage, ASCAT is dependent on genomic aberrations. For diploid tumors without aberrations, the tumor percentage can therefore not be calculated.

Taq DNA Polymerase High Fidelity (Life Technologies) in a final volume of 25 µl. The PCR program was initiated with a denaturation step of 4 min at 95 °C followed by 50 cycles of 30 s at 95 °C, an annealing temperature at 56 °C for 30 s, and elongation at 72 °C for 15 s. The final extension was performed for 4 min at 72 °C. Quantitative genotyping was performed on 10 µl of the PCR product using pyrosequencing. Primer sequences are given in Supplementary Table 1, see section on supplementary data given at the end of this article.

## Statistical analysis

The non-parametric Mann-Whitney test was used to assess potential differences between groups for the methylation and the expression analysis. A two-sided t-test was used to test for associations between categorized molecular alterations and categorical clinical parameters. Correlation analyses were made using the Spearman's rank correlation coefficient. A nominal P value <0.05 was considered significant.

## Results

To provide a comprehensive analysis of the mechanisms underlying *IGF2* overexpression in adrenal tumors, gene expression levels were integrated with copy number and ploidy analysis, and the DNA methylation status of three DMRs including the ICR upstream of *H19* (*H19* DMR) and two secondary DMRs (DMR0 and DMR2) within *IGF2* (Supplementary Fig. 1, see section on supplementary data given at the end of this article).

## Differential *IGF2* and *H19* expression characterize PCCs and ACCs

Gene expression changes of *IGF2* and *H19* were analyzed in 60 fresh frozen adrenal tumors (Fig. 1). *IGF2* was considered overexpressed when the expression level was three times above the s.d. of the mean *IGF2* expression value observed for the CAs, which express *IGF2* and *H19* at levels comparable to normal adrenal tissue (Ilvesmaki *et al.* 1993a, Liu *et al.* 1995). *H19* was likewise considered downregulated when the expression level of individual samples was below three times the s.d. of the mean *H19* expression level observed for CAs.

*IGF2* showed overexpression in 85% (17/20) of the ACCs and 100% (10/10) of the PCCs, whereas only 4/19 ACBTs and 3/11 CAs showed overexpression of *IGF2* (*P*≤0.0003 for both ACC and PCC vs CAs; Mann-Whitney test) (Fig. 1). Expression levels did not correlate with clinical parameters such as presence of metastases, TNM stage. Only a tendency for a positive correlation between *IGF2* expression and tumor size for the ACCs was observed, but did not reach statistical significance (*R*²=0.273, *P*=0.067, Spearman's correlation). Decreased expression levels of *H19* also characterized both ACCs (18/20) and PCCs (7/10) when compared to the CA samples (*P*=0.0001 and *P*=0.01 respectively; Mann-Whitney test). However, the level of *H19* down regulation was less pronounced for PCCs compared to ACCs (*P*=0.0053; Mann-Whitney test) (Fig. 1). Concomitant *IGF2* overexpression and *H19* downregulation characterized the majority of the ACCs (15/20), PCCs (7/10), and a single ACBT, but was not present in all cases (Table 2).
<figure><img src="image_3.png" /><figcaption>**Figure 1**

Quantitative expression analysis of *IGF2* and *H19* in adrenocortical tumors. *IGF2* and *H19* expression values are normalized to the geometric mean of the three reference genes (*SDHA*, *ATP5B*, and *CYC1*). Data is presented on a logarithmic scale.</figcaption></figure>

Aberrant DNA methylation is found throughout the
IGF2/H19 locus

We further aimed to clarify the involvement of abnormal
DNA methylation patterns of the three DMRs at the
IGF2/H19 locus (IGF2 DMR0, IGF2 DMR2, and H19 DMR
(ICR)) in adrenocortical tumorigenesis. For each analyzed
region, a sample was scored as having aberrant DNA
methylation if its mean DNA methylation level of the total
number of CpG sites analyzed in each region differed by
more than 10% from the CA samples. The median DNA
methylation level for each CpG site analyzed is shown in
Fig. 2 for each subgroup. The imprinting control region,
H19 ICR, contains seven CTCF binding sites, of which
three were analyzed (CTCF2, CTCF3, and CTCF6),
whereby the sixth binding site has been reported
to correlate best with IGF2 expression (Takai *et al.* 2001).

The DNA methylation levels of the three CTCF binding
sites analyzed were highly correlated (ρ>0.75, P=2.2×
10⁻¹⁶, Spearman's correlation). PCCs and ACCs showed
hypermethylation of the H19 ICR compared to the
samples from patients with CA and ACBTs (P<0.0001
for both the ACCs and PCCs, Mann-Whitney test). The
DNA methylation level of the three CTCF binding sites
was not as high for PCC samples as for ACCs (P=0.022,
Mann-Whitney test) and this difference could neither be
explained by copy number changes nor content of tumor
cells in the samples. Of note, the H19 ICR DNA
methylation level correlated positively with the IGF2
expression for the PCCs (ρ=0.93, P=0.00013, Spearman's
correlation), while no correlation between IGF2
expression and H19 ICR methylation was observed for
the ACCs (ρ=0.34, P=0.14, Spearman's correlation).
Increased DNA methylation levels of the H19 ICR were
observed for four adenomas (samples 17, 19, 30, and 31).
However, their DNA methylation levels only exceeded our
chosen threshold by 1-5% and only one of these (sample
30) had concomitant IGF2 overexpression.

A high correlation was also observed for the DNA
methylation levels for the two regions analyzed within the
IGF2 DMR2 (DMR2a and DMR2b, ρ=0.63, P=1.43×10⁻⁷,
Spearman's correlation). A gradient of hypomethylation
of IGF2 DMR2 was observed with CA samples presenting
the highest DNA methylation level followed by the ACBTs
with a significant difference between the two groups
(P=0.0076, Mann-Whitney test). The PCCs and ACCs
had the lowest DNA methylation levels when compared to
CA samples (P=0.014 and P=0.019 respectively, Mann-
Whitney test) (Fig. 2). The observed hypomethylation of
the IGF2 DMR2 was independent of the somatic copy
number changes of the region (see the following section)
and associated with neither Weiss nor PASS score nor
IGF2 expression levels (Table 2). DNA methylation
changes did not correlate with any clinical parameters in
the tumor groups.

The ACCs further displayed a slight, though signi-
ficant, higher DNA methylation level for three CpG sites
analyzed in the IGF2 DMR0 compared to all other groups
(P=0.0067, Kruskal-Wallis test), which correlated
positively with the Weiss score (ρ=0.60, P=0.039,
Spearman's correlation).

Somatic copy number changes of chr11p15.5 are
abundant in ACCs and PCCs

Genome-wide SNP analysis was used to establish
the chromosomal status of chr11:1 986 296-2 223 233
<figure><img src="image_3.png" /><figcaption>**Figure 1**

Quantitative expression analysis of *IGF2* and *H19* in adrenocortical tumors. *IGF2* and *H19* expression values are normalized to the geometric mean of the three reference genes (*SDHA*, *ATP5B*, and *CYC1*). Data is presented on a logarithmic scale.</figcaption></figure>

Aberrant DNA methylation is found throughout the
IGF2/H19 locus

We further aimed to clarify the involvement of abnormal
DNA methylation patterns of the three DMRs at the
IGF2/H19 locus (IGF2 DMR0, IGF2 DMR2, and H19 DMR
(ICR)) in adrenocortical tumorigenesis. For each analyzed
region, a sample was scored as having aberrant DNA
methylation if its mean DNA methylation level of the total
number of CpG sites analyzed in each region differed by
more than 10% from the CA samples. The median DNA
methylation level for each CpG site analyzed is shown in
Fig. 2 for each subgroup. The imprinting control region,
H19 ICR, contains seven CTCF binding sites, of which
three were analyzed (CTCF2, CTCF3, and CTCF6),
whereby the sixth binding site has been reported
to correlate best with IGF2 expression (Takai *et al.* 2001).

The DNA methylation levels of the three CTCF binding
sites analyzed were highly correlated (ρ>0.75, P=2.2×
10⁻¹⁶, Spearman's correlation). PCCs and ACCs showed
hypermethylation of the H19 ICR compared to the
samples from patients with CA and ACBTs (P<0.0001
for both the ACCs and PCCs, Mann-Whitney test). The
DNA methylation level of the three CTCF binding sites
was not as high for PCC samples as for ACCs (P=0.022,
Mann-Whitney test) and this difference could neither be
explained by copy number changes nor content of tumor
cells in the samples. Of note, the H19 ICR DNA
methylation level correlated positively with the IGF2
expression for the PCCs (ρ=0.93, P=0.00013, Spearman's
correlation), while no correlation between IGF2
expression and H19 ICR methylation was observed for
the ACCs (ρ=0.34, P=0.14, Spearman's correlation).
Increased DNA methylation levels of the H19 ICR were
observed for four adenomas (samples 17, 19, 30, and 31).
However, their DNA methylation levels only exceeded our
chosen threshold by 1-5% and only one of these (sample
30) had concomitant IGF2 overexpression.

A high correlation was also observed for the DNA
methylation levels for the two regions analyzed within the
IGF2 DMR2 (DMR2a and DMR2b, ρ=0.63, P=1.43×10⁻⁷,
Spearman's correlation). A gradient of hypomethylation
of IGF2 DMR2 was observed with CA samples presenting
the highest DNA methylation level followed by the ACBTs
with a significant difference between the two groups
(P=0.0076, Mann-Whitney test). The PCCs and ACCs
had the lowest DNA methylation levels when compared to
CA samples (P=0.014 and P=0.019 respectively, Mann-
Whitney test) (Fig. 2). The observed hypomethylation of
the IGF2 DMR2 was independent of the somatic copy
number changes of the region (see the following section)
and associated with neither Weiss nor PASS score nor
IGF2 expression levels (Table 2). DNA methylation
changes did not correlate with any clinical parameters in
the tumor groups.

The ACCs further displayed a slight, though signif-
cant, higher DNA methylation level for three CpG sites
analyzed in the IGF2 DMR0 compared to all other groups
(P=0.0067, Kruskal-Wallis test), which correlated
positively with the Weiss score (ρ=0.60, P=0.039,
Spearman's correlation).

Somatic copy number changes of chr11p15.5 are
abundant in ACCs and PCCs

Genome-wide SNP analysis was used to establish
the chromosomal status of chr11:1 986 296-2 223 233
<figure><img src="image_4.png" /><figcaption>**Figure 2**</figcaption></figure>

DNA methylation levels for the three DMRs of the *IGF2/H19* locus for each subgroup of adrenocortical tumors. (A) Schematic presentation of the *IGF2/H19* locus. *IGF2* harbors 9 exons whereas *H19* harbors five exons, which are indicated by the black and white squares. Transcription start sites are indicated with arrows. The *IGF2/H19* locus has three differential methylated regions (DMRs) named *DMR0*, *DMR2* and *H19* DMR. The parental-specific (M=maternal allele, P=paternal allele) DNA methylation status is presented by black bars for each DMR (black=methylated,

harboring the *IGF2/H19* locus as this technology allows the detection of copy number changes as well as copy number neutral events. The chromosomal and allelic status was established using the BAF and Log R ratios, estimated by ASCAT (Van Loo *et al.* 2010), allowing discrimination between different forms of events: poly- ploidy, allelic loss and gain, and uniparental disomy (UPD). ASCAT results were confirmed by genotyping rs680 using pyrosequencing, which confirmed loss of hetero- zygosity for 8/8 informative samples (Supplementary Table 2, see section on supplementary data given at the end of this article).

Somatic copy number changes of the *IGF2/H19* locus were restricted to PCC and ACC samples (Table 2). PCC samples remained in the diploid state, while ACCs were often tetraploid (7/19 analyzed) (Table 2). Representative samples showing different somatic copy number changes of the chr11p15.5 locus are shown in Fig. 3. Figure 3A represents a diploid sample with no genetic alterations accounting for 36/57 of the samples analyzed within this study including 12/12 CA samples, 16/16 ACBTs, 4/10 PCCs

white = unmethylated). The *H19* DMR makes up the imprinting control region (ICR) of the locus containing seven CTCF binding sites. For each DMR a number of CpG sites were analyzed (*DMR0* = 3, *DMR2* = 26, *CTCF2* = 17, *CTCF3* = 11, and for *CTCF6* = 15) and the DNA methylation levels correlated highly between the three sites (*ρ* > 0.75, *P* = 2.2 × 10⁻¹⁶, Spearman's correlation). Each CpG site is presented as a filled circle. (B) The mean DNA methylation levels of single CpG sites are shown for each of the four adrenocortical tumor subtypes (Conn's adenoma, ACBTs, PCCs, and ACCs).

and 4/19 ACCs. Seven out of 12 diploid ACCs and half of the PCCs presented with loss of a single allele (Fig. 3B). Seven ACC samples were found to be tetraploid of which two samples (51 and 53) had lost a single allele of chr11p15.5 resulting in triploidy of this region (Fig. 3C). Another three tetraploid ACC samples (44, 46, and 57) presented with an UPD-like genotype as a consequence of loss of two alleles of the same parental origin (Fig. 3D). Of the two remaining tetraploid samples, ACC sample 61 had lost one of each allele, thus having normal allelic dosage, whereas the other (sample 62) did not show any loss of chr11p15.5. UPD is characterized by copy number neutral variation and occurred in a single ACC sample (45) and a single PCC sample (39), both of which were diploid (Fig. 3E).

IGF2 overexpression in PCCs and ACCs correlates with
somatic copy number changes of chr11p15.5

Thirty-six diploid samples did not show any copy number
changes at chr11p15.5 (Fig. 4), of which 13 (36%)
overexpressed *IGF2* (Table 2). Of these 13 diploid samples,
<figure><img src="image_4.png" /><figcaption>**Figure 2**</figcaption></figure>

DNA methylation levels for the three DMRs of the *IGF2/H19* locus for each subgroup of adrenocortical tumors. (A) Schematic presentation of the *IGF2/H19* locus. *IGF2* harbors 9 exons whereas *H19* harbors five exons, which are indicated by the black and white squares. Transcription start sites are indicated with arrows. The *IGF2/H19* locus has three differential methylated regions (DMRs) named *DMR0*, *DMR2* and *H19* DMR. The parental-specific (M=maternal allele, P=paternal allele) DNA methylation status is presented by black bars for each DMR (black=methylated,

harboring the *IGF2/H19* locus as this technology allows the detection of copy number changes as well as copy number neutral events. The chromosomal and allelic status was established using the BAF and Log R ratios, estimated by ASCAT (Van Loo *et al.* 2010), allowing discrimination between different forms of events: poly- ploidy, allelic loss and gain, and uniparental disomy (UPD). ASCAT results were confirmed by genotyping rs680 using pyrosequencing, which confirmed loss of hetero- zygosity for 8/8 informative samples (Supplementary Table 2, see section on supplementary data given at the end of this article).

Somatic copy number changes of the *IGF2/H19* locus were restricted to PCC and ACC samples (Table 2). PCC samples remained in the diploid state, while ACCs were often tetraploid (7/19 analyzed) (Table 2). Representative samples showing different somatic copy number changes of the chr11p15.5 locus are shown in Fig. 3. Figure 3A represents a diploid sample with no genetic alterations accounting for 36/57 of the samples analyzed within this study including 12/12 CA samples, 16/16 ACBTs, 4/10 PCCs

white = unmethylated). The *H19* DMR makes up the imprinting control region (ICR) of the locus containing seven CTCF binding sites. For each DMR a number of CpG sites were analyzed (*DMR0* = 3, *DMR2* = 26, *CTCF2* = 17, *CTCF3* = 11, and for *CTCF6* = 15) and the DNA methylation levels correlated highly between the three sites (*ρ* > 0.75, *P* = 2.2 × 10⁻¹⁶, Spearman's correlation). Each CpG site is presented as a filled circle. (B) The mean DNA methylation levels of single CpG sites are shown for each of the four adrenocortical tumor subtypes (Conn's adenoma, ACBTs, PCCs, and ACCs).

and 4/19 ACCs. Seven out of 12 diploid ACCs and half of the PCCs presented with loss of a single allele (Fig. 3B). Seven ACC samples were found to be tetraploid of which two samples (51 and 53) had lost a single allele of chr11p15.5 resulting in triploidy of this region (Fig. 3C). Another three tetraploid ACC samples (44, 46, and 57) presented with an UPD-like genotype as a consequence of loss of two alleles of the same parental origin (Fig. 3D). Of the two remaining tetraploid samples, ACC sample 61 had lost one of each allele, thus having normal allelic dosage, whereas the other (sample 62) did not show any loss of chr11p15.5. UPD is characterized by copy number neutral variation and occurred in a single ACC sample (45) and a single PCC sample (39), both of which were diploid (Fig. 3E).

IGF2 overexpression in PCCs and ACCs correlates with somatic copy number changes of chr11p15.5

Thirty-six diploid samples did not show any copy number changes at chr11p15.5 (Fig. 4), of which 13 (36%) overexpressed *IGF2* (Table 2). Of these 13 diploid samples,
<figure><img src="image_5.png" /><figcaption>**Figure 3**</figcaption></figure>

ASCAT profiles of adrenal tumors. (A) A Conn's adenoma sample (1) being diploid and with no somatic copy number changes at chr11p15.5 thus presenting a normal sample. (B) A tetraploid ACC sample (51), for which loss of a single allele resulted in triploidy of chr11p15. (C) An UPD-like

genotype (57) being tetraploid with loss of two alleles of the same parental origin. (D) A diploid sample (39) with a copy number neutral variation presenting an UPD. (E) The PCC 33 represents samples being diploid and with loss of a single allele of chr11p15.
<figure><img src="image_5.png" /><figcaption>**Figure 3**</figcaption></figure>

ASCAT profiles of adrenal tumors. (A) A Conn's adenoma sample (1) being diploid and with no somatic copy number changes at chr11p15.5 thus presenting a normal sample. (B) A tetraploid ACC sample (51), for which loss of a single allele resulted in triploidy of chr11p15. (C) An UPD-like

genotype (57) being tetraploid with loss of two alleles of the same parental origin. (D) A diploid sample (39) with a copy number neutral variation presenting an UPD. (E) The PCC 33 represents samples being diploid and with loss of a single allele of chr11p15.
<figure><img src="image_6.png" /><figcaption>**Figure 4**</figcaption></figure>

Doughnut diagrams integrating *IGF2* gene expression data with somatic copy number changes and DNA methylation alterations of the *IGF2/H19* locus. CAs and ACBTs were mainly characterized by normal *IGF2* expression, absence of copy number changes, and normal DNA methylation levels of the *H19* ICR. In contrast all PCCs showed overexpression of *IGF2*, where all samples with somatic copy number changes showed hypermethylation of the *H19* ICR. Seventeen out of 20 ACCs presented with overexpression of

*IGF2* and this mainly correlated with somatic copy number changes of chr11p15.5 and hypermethylation of the *H19* ICR. Somatic copy number changes of chr11p15.5 were not exclusively associated with high *IGF2* expression levels as a ACC sample (57) with an UPD-like genotype had *IGF2* expression levels comparable to the samples from patients with Conn's adenoma.

11 samples had normal DNA methylation levels of the *H19* ICR (three CAs (2, 6, and 12), three ACBTs (14, 20, and 32), four PCCs (35, 38, 40, and 41), and a single ACC sample (48)). The two remaining samples with no copy number alterations and *IGF2* overexpression were diploid ACCs (50 and 54) and they both showed hypermethylation of the *H19* ICR. The two diploid samples (39 and 45) presenting with UPD both had high *IGF2* expression levels and high *H19* ICR methylation levels.

For the PCCs and ACCs, loss of a single allele in diploid samples was observed for 12/29 (41%) samples and therefore a recurrent event associated with *IGF2* overexpression and high *H19* ICR methylation levels (Fig. 4C and D).

Tetraploidy was a unique feature for ACCs in our study and with a frequency of 37% (7/19) it was a frequent event often accompanied by loss of either a single or two allele(s) of the same parental origin at chr11p15.5. Both tetraploid
<figure><img src="image_6.png" /><figcaption>**Figure 4**</figcaption></figure>

Doughnut diagrams integrating *IGF2* gene expression data with somatic copy number changes and DNA methylation alterations of the *IGF2/H19* locus. CAs and ACBTs were mainly characterized by normal *IGF2* expression, absence of copy number changes, and normal DNA methylation levels of the *H19* ICR. In contrast all PCCs showed overexpression of *IGF2*, where all samples with somatic copy number changes showed hypermethylation of the *H19* ICR. Seventeen out of 20 ACCs presented with overexpression of

*IGF2* and this mainly correlated with somatic copy number changes of chr11p15.5 and hypermethylation of the *H19* ICR. Somatic copy number changes of chr11p15.5 were not exclusively associated with high *IGF2* expression levels as a ACC sample (57) with an UPD-like genotype had *IGF2* expression levels comparable to the samples from patients with Conn's adenoma.

11 samples had normal DNA methylation levels of the *H19* ICR (three CAs (2, 6, and 12), three ACBTs (14, 20, and 32), four PCCs (35, 38, 40, and 41), and a single ACC sample (48)). The two remaining samples with no copy number alterations and *IGF2* overexpression were diploid ACCs (50 and 54) and they both showed hypermethylation of the *H19* ICR. The two diploid samples (39 and 45) presenting with UPD both had high *IGF2* expression levels and high *H19* ICR methylation levels.

For the PCCs and ACCs, loss of a single allele in diploid samples was observed for 12/29 (41%) samples and therefore a recurrent event associated with *IGF2* overexpression and high *H19* ICR methylation levels (Fig. 4C and D).

Tetraploidy was a unique feature for ACCs in our study and with a frequency of 37% (7/19) it was a frequent event often accompanied by loss of either a single or two allele(s) of the same parental origin at chr11p15.5. Both tetraploid
samples, which had lost a single allele and were therefore
triploid for chr11p15.5 (51 and 53) had *IGF2* over-
expressed and high *H19* ICR DNA methylation levels
(Fig. 4D). Three tetraploid samples (44, 46, and 57) had
lost two alleles of same parental origin resulting in an
UPD-like genotype. Whereas the samples 44 and 46
showing an UPD-like genotype had *IGF2* overexpressed
and high *H19* ICR methylation levels, sample 57 had *IGF2*
expression levels equivalent to the CA samples, but still
had the *H19* ICR hypermethylated (Fig. 4D) (Table 2).
The tetraploid ACC sample 61 had lost one of each allele
thus having normal allelic dosage accompanied by normal
*H19* ICR DNA methylation levels and *IGF2* overexpres-
sion. The remaining tetraploid sample (62) presented with
four allelic copies and was associated with normal *IGF2*
expression levels and normal *H19* ICR methylation levels.
The ACC samples 61 and 62 were the only tetraploid
samples with equal number of parental allelic copies,
which could explain the absence of high *H19* ICR DNA
methylation levels.

Tumor samples with a higher number of paternal alleles compared to maternal ones had an increased probability of being metastatic (*P*<0.00001, χ²-test), but none of the other clinical parameters was associated to copy number changes or ploidy.

High DNA methylation levels of the imprinting control region were thus mainly observed for samples showing copy number changes of chr11p15.5 as 13/15 ACCs and 6/6 PCCs with copy number changes had increased DNA methylation levels (Fig. 4C and D) and the majority of adrenal tumors (90%) with somatic copy number changes also showed overexpression of *IGF2* (Fig. 4D).

Discussion

ACCs and PCCs develop in the adrenal cortex and the
adrenal medulla respectively, which are two distinct
organs of separate embryonic origin. However, ACCs and
PCCs share the characteristic *IGF2* overexpression. Unra-
veling the molecular mechanisms underlying *IGF2* over-
expression is important as IGF2 contributes to cell
proliferation and tumor progression (Livingstone 2013,
Guillaud-Bataille *et al.* 2014). Studies investigating the
underlying mechanisms of *IGF2* overexpression in adrenal
tumors at more than a single molecular level are limited
(Gicquel *et al.* 1994, 1997, Gao *et al.* 2002) and no study
has so far integrated epigenetic and copy number variation
obtained with quantitative high-resolution technologies
with gene expression. In the present study we therefore

integrated *IGF2* and *H19* expression levels with the DNA
methylation levels of three regulatory DMRs located
throughout the *IGF2/H19* locus and compared this to the
copy number status and ploidy of chr11p15.5 in a cohort
of 62 adrenal tumor samples.

In concordance with previous findings, we found a
correlation between *IGF2* overexpression and the presence
of malignant adrenocortical tumors (Ilvesmaki *et al.*
1993a, Gicquel *et al.* 1997, Slater *et al.* 2006, Soon *et al.*
2009, Ragazzon *et al.* 2011) as 85% of the ACCs over-
expressed *IGF2*. However, similar to recently published
data, the degree of overexpression did not correlate with
the Weiss score (Guillaud-Bataille *et al.* 2014) or other
clinical parameters. Moreover, *IGF2* overexpression was
found in all PCCs, but here the degree of malignancy,
as given by the PASS score, correlated with the level of
overexpression, similar to previously published results
(Margetts *et al.* 2005, Meyer-Rochow *et al.* 2010, Sandgren
*et al.* 2010). While due to lack of normal tissue from the
adrenal medulla, the gene expression level of the PCCs
was compared to the expression level of adenoma in the
context of Conn's Syndrome as reference and thus to a
different tissue, the degree of the overexpression was
probably not overestimated as the expression of *IGF2*
in adults is restricted to the adrenal capsule and the
periphery of the cortex (Baquedano *et al.* 2005), and the
expression of *IGF2* in normal adrenal medullary cells is
below the expression level in benign tumors (Meyer-
Rochow *et al.* 2010). Furthermore, healthy adrenal
medulla did not show IGF2 expression by immuno-
histochemical staining in human samples (Soon *et al.*
2009) or mouse models (Drelon *et al.* 2012), providing
conclusive evidence that the detected high levels
of *IGF2* in the PCCs are a tumor-specific alteration.
Our data is further supported by the fact that the
magnitude of overexpression detected in our study is
very similar to a previously published study demonstrat-
ing the overexpression of *IGF2* in PCCs when compared to
normal adrenal medulla (Waldmann *et al.* 2010).

*IGF2* overexpression has previously been associated
with LOH at the *IGF2/H19* locus in adrenocortical tumor
samples. 28/38 adrenal tumors with LOH of the *IGF2/H19*
locus had lost the maternal allele and gained an extra copy
of the paternal allele (Gicquel *et al.* 1994, 1997). In our
cohort 15 out of 19 ACC samples analyzed had somatic
copy number alterations at the *IGF2/H19* locus, with 6/15
samples having an extra copy of a single allele (Fig. 4D).
As all six samples with UPD or an UPD-like genotype
displayed hypermethylation of the *H19* ICR, we can
assume that the unmethylated maternal allele is lost and
samples, which had lost a single allele and were therefore
triploid for chr11p15.5 (51 and 53) had *IGF2* over-
expressed and high *H19* ICR DNA methylation levels
(Fig. 4D). Three tetraploid samples (44, 46, and 57) had
lost two alleles of same parental origin resulting in an
UPD-like genotype. Whereas the samples 44 and 46
showing an UPD-like genotype had *IGF2* overexpressed
and high *H19* ICR methylation levels, sample 57 had *IGF2*
expression levels equivalent to the CA samples, but still
had the *H19* ICR hypermethylated (Fig. 4D) (Table 2).
The tetraploid ACC sample 61 had lost one of each allele
thus having normal allelic dosage accompanied by normal
*H19* ICR DNA methylation levels and *IGF2* overexpres-
sion. The remaining tetraploid sample (62) presented with
four allelic copies and was associated with normal *IGF2*
expression levels and normal *H19* ICR methylation levels.
The ACC samples 61 and 62 were the only tetraploid
samples with equal number of parental allelic copies,
which could explain the absence of high *H19* ICR DNA
methylation levels.

Tumor samples with a higher number of paternal alleles compared to maternal ones had an increased probability of being metastatic (*P*<0.00001, χ²-test), but none of the other clinical parameters was associated to copy number changes or ploidy.

High DNA methylation levels of the imprinting control region were thus mainly observed for samples showing copy number changes of chr11p15.5 as 13/15 ACCs and 6/6 PCCs with copy number changes had increased DNA methylation levels (Fig. 4C and D) and the majority of adrenal tumors (90%) with somatic copy number changes also showed overexpression of *IGF2* (Fig. 4D).

Discussion

ACCs and PCCs develop in the adrenal cortex and the
adrenal medulla respectively, which are two distinct
organs of separate embryonic origin. However, ACCs and
PCCs share the characteristic *IGF2* overexpression. Unra-
veling the molecular mechanisms underlying *IGF2* over-
expression is important as IGF2 contributes to cell
proliferation and tumor progression (Livingstone 2013,
Guillaud-Bataille *et al.* 2014). Studies investigating the
underlying mechanisms of *IGF2* overexpression in adrenal
tumors at more than a single molecular level are limited
(Gicquel *et al.* 1994, 1997, Gao *et al.* 2002) and no study
has so far integrated epigenetic and copy number variation
obtained with quantitative high-resolution technologies
with gene expression. In the present study we therefore

integrated *IGF2* and *H19* expression levels with the DNA
methylation levels of three regulatory DMRs located
throughout the *IGF2/H19* locus and compared this to the
copy number status and ploidy of chr11p15.5 in a cohort
of 62 adrenal tumor samples.

In concordance with previous findings, we found a
correlation between *IGF2* overexpression and the presence
of malignant adrenocortical tumors (Ilvesmaki *et al.*
1993a, Gicquel *et al.* 1997, Slater *et al.* 2006, Soon *et al.*
2009, Ragazzon *et al.* 2011) as 85% of the ACCs over-
expressed *IGF2*. However, similar to recently published
data, the degree of overexpression did not correlate with
the Weiss score (Guillaud-Bataille *et al.* 2014) or other
clinical parameters. Moreover, *IGF2* overexpression was
found in all PCCs, but here the degree of malignancy,
as given by the PASS score, correlated with the level of
overexpression, similar to previously published results
(Margetts *et al.* 2005, Meyer-Rochow *et al.* 2010, Sandgren
*et al.* 2010). While due to lack of normal tissue from the
adrenal medulla, the gene expression level of the PCCs
was compared to the expression level of adenoma in the
context of Conn's Syndrome as reference and thus to a
different tissue, the degree of the overexpression was
probably not overestimated as the expression of *IGF2*
in adults is restricted to the adrenal capsule and the
periphery of the cortex (Baquedano *et al.* 2005), and the
expression of *IGF2* in normal adrenal medullary cells is
below the expression level in benign tumors (Meyer-
Rochow *et al.* 2010). Furthermore, healthy adrenal
medulla did not show IGF2 expression by immuno-
histochemical staining in human samples (Soon *et al.*
2009) or mouse models (Drelon *et al.* 2012), providing
conclusive evidence that the detected high levels
of *IGF2* in the PCCs are a tumor-specific alteration.
Our data is further supported by the fact that the
magnitude of overexpression detected in our study is
very similar to a previously published study demonstrat-
ing the overexpression of *IGF2* in PCCs when compared to
normal adrenal medulla (Waldmann *et al.* 2010).

*IGF2* overexpression has previously been associated
with LOH at the *IGF2/H19* locus in adrenocortical tumor
samples. 28/38 adrenal tumors with LOH of the *IGF2/H19*
locus had lost the maternal allele and gained an extra copy
of the paternal allele (Gicquel *et al.* 1994, 1997). In our
cohort 15 out of 19 ACC samples analyzed had somatic
copy number alterations at the *IGF2/H19* locus, with 6/15
samples having an extra copy of a single allele (Fig. 4D).
As all six samples with UPD or an UPD-like genotype
displayed hypermethylation of the *H19* ICR, we can
assume that the unmethylated maternal allele is lost and
accompanied by a duplication of the normally methylated
paternal allele. However, it should be noted that no
parental DNA was available to confirm this hypothesis.
Due to the limitations of the method used in previous
studies, the frequency of UPD cannot be easily compared
as the ploidy of the tumors was not determined in the
study of Gicquel *et al*. Even though they observed that loss
of the maternal allele is accompanied by duplication of the
paternal allele, it remains unknown whether this was due
to an UPD in a diploid cell or loss of two alleles of the same
parental origin in a tetraploid cell leading to an UPD-like
genotype. As only a single ACC and a single PCC sample
displayed UPD in our cohort, our data suggest that loss
of the unmethylated maternal allele rather than UPD is
the critical mechanism underlying *IGF2* overexpression.
The presence of more paternal alleles than maternal alleles
was significantly associated with the presence of metas-
tases. However, the low number of metastatic samples
in our study (n=4) makes it difficult to draw general
conclusions from these results and will require validation
in larger cohorts.

Tetraploidy was found to be a unique feature for the ACCs, which is supported by previous findings (Pignatelli *et al.* 1998, Blanes & Diaz-Cano 2006). In contrast to our study, tetraploidy has also been observed for PCCs using flow cytometry (Shono *et al.* 2002) and likewise has tetrasomy of single chromosomes been observed in samples of patients with CA (Shono *et al.* 2002), which also is in contrast to our and others (Lu *et al.* 1996). However, it cannot be ruled out that some PCCs could be tetraploid as only a limited number of tumors were analyzed in our study. It should be pointed out, that although the polyploidy of ACCs has previously been reported, none of the studies investigating the mechanism of *IGF2* overexpression took this information into account.

Gicquel *et al.* (1997) did not find *IGF2* overexpression restricted to samples with allelic imbalance of the *IGF2/H19* locus as 7/82 samples had *IGF2* overexpressed without any somatic copy number changes of chr11p15.5. This is in concordance with our findings as we found 13/36 diploid tumors overexpressed *IGF2*, of which 11 had normal *H19* ICR methylation levels. This indicates the involvement of an additional so far unknown mechanism leading to the characteristic overexpression of *IGF2* for PCCs and ACCs as well as for a subset of the ACBTs, which were found to be in general chromosomally stable in our study. The amplitude of the expression changes was not directly correlated to the different somatic copy number changes. A tetraploid sample with UPD-like genotype could present with a similar high expression as a diploid

sample with no genetic alterations supporting again the
hypothesis of other changes probably in trans influencing
*IGF2* overexpression. In contrast to recently reported
differences in DNA methylation patterns in the *H19*
DMR between high and low IGF2 expressing ACCs
(Guillaud-Bataille *et al.* 2014), we did not find any
correlation between methylation patterns and expression
levels of *IGF2* in the ACCs with their complex genetic
background. However, a positive correlation was detected
in the PCCs, which remained diploid.

One candidate transcription factor that could be
interesting to investigate for its potential role in modulating
*IGF2* expression levels is PLAG1 as its oncogenic function is
mediated through the IGF2 pathway by binding to promoter
3 located between exon 4 and 5, resulting in *IGF2* over-
expression (Hensen *et al.* 2002, Voz *et al.* 2004, Akhtar *et al.*
2012). Upstream enhancers have also been suggested to be
involved in *IGF2* overexpression (Ulaner *et al.* 2003).
However, whether the broad range from 4 to ~600 fold of
*IGF2* overexpression observed in the ACC group could be
caused by transcription factors, upstream enhancers or other
trans acting elements needs to be elucidated.

In conclusion, our data suggest that *IGF2* overexpres-
sion in adrenal tumors correlates mainly with allelic loss
leading to an imbalance of the ratio between paternal and
maternal alleles and that the aberrant DNA methylation
levels observed for the *H19* ICR are a consequence thereof.
Whether demethylation of the *IGF2* DMR2 contributes to
overexpression of a subset of *IGF2* transcripts needs further
investigation. Our study further underlines the import-
ance to take ploidy into account to accurately discern the
mechanism of gene-specific overexpression in tumors.

**Supplementary data**

This is linked to the online version of the paper at http://dx.doi.org/10.1530/ERC-15-0086.

**Declaration of interest**

The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.

**Funding**

The project was funded by the institutional budget of CEPH and CNG.

**Author contribution statement**

H M Nielsen, A How-Kit, A Daunay and C Guerin performed laboratory experiments. H M Nielsen, A How-Kit, H K M Vollan, V N Kristensen, P V Loo, L L Hansen, J Tost were involved in the data analysis. C Guerin, F Castinetti,
accompanied by a duplication of the normally methylated
paternal allele. However, it should be noted that no
parental DNA was available to confirm this hypothesis.
Due to the limitations of the method used in previous
studies, the frequency of UPD cannot be easily compared
as the ploidy of the tumors was not determined in the
study of Gicquel *et al*. Even though they observed that loss
of the maternal allele is accompanied by duplication of the
paternal allele, it remains unknown whether this was due
to an UPD in a diploid cell or loss of two alleles of the same
parental origin in a tetraploid cell leading to an UPD-like
genotype. As only a single ACC and a single PCC sample
displayed UPD in our cohort, our data suggest that loss
of the unmethylated maternal allele rather than UPD is
the critical mechanism underlying *IGF2* overexpression.
The presence of more paternal alleles than maternal alleles
was significantly associated with the presence of metas-
tases. However, the low number of metastatic samples
in our study (n=4) makes it difficult to draw general
conclusions from these results and will require validation
in larger cohorts.

Tetraploidy was found to be a unique feature for the ACCs, which is supported by previous findings (Pignatelli *et al.* 1998, Blanes & Diaz-Cano 2006). In contrast to our study, tetraploidy has also been observed for PCCs using flow cytometry (Shono *et al.* 2002) and likewise has tetrasomy of single chromosomes been observed in samples of patients with CA (Shono *et al.* 2002), which also is in contrast to our and others (Lu *et al.* 1996). However, it cannot be ruled out that some PCCs could be tetraploid as only a limited number of tumors were analyzed in our study. It should be pointed out, that although the polyploidy of ACCs has previously been reported, none of the studies investigating the mechanism of *IGF2* overexpression took this information into account.

Gicquel *et al.* (1997) did not find *IGF2* overexpression restricted to samples with allelic imbalance of the *IGF2/H19* locus as 7/82 samples had *IGF2* overexpressed without any somatic copy number changes of chr11p15.5. This is in concordance with our findings as we found 13/36 diploid tumors overexpressed *IGF2*, of which 11 had normal *H19* ICR methylation levels. This indicates the involvement of an additional so far unknown mechanism leading to the characteristic overexpression of *IGF2* for PCCs and ACCs as well as for a subset of the ACBTs, which were found to be in general chromosomally stable in our study. The amplitude of the expression changes was not directly correlated to the different somatic copy number changes. A tetraploid sample with UPD-like genotype could present with a similar high expression as a diploid

sample with no genetic alterations supporting again the
hypothesis of other changes probably in trans influencing
*IGF2* overexpression. In contrast to recently reported
differences in DNA methylation patterns in the *H19*
DMR between high and low IGF2 expressing ACCs
(Guillaud-Bataille *et al.* 2014), we did not find any
correlation between methylation patterns and expression
levels of *IGF2* in the ACCs with their complex genetic
background. However, a positive correlation was detected
in the PCCs, which remained diploid.

One candidate transcription factor that could be
interesting to investigate for its potential role in modulating
*IGF2* expression levels is PLAG1 as its oncogenic function is
mediated through the IGF2 pathway by binding to promoter
3 located between exon 4 and 5, resulting in *IGF2* over-
expression (Hensen *et al.* 2002, Voz *et al.* 2004, Akhtar *et al.*
2012). Upstream enhancers have also been suggested to be
involved in *IGF2* overexpression (Ulaner *et al.* 2003).
However, whether the broad range from 4 to ~600 fold of
*IGF2* overexpression observed in the ACC group could be
caused by transcription factors, upstream enhancers or other
trans acting elements needs to be elucidated.

In conclusion, our data suggest that *IGF2* overexpres-
sion in adrenal tumors correlates mainly with allelic loss
leading to an imbalance of the ratio between paternal and
maternal alleles and that the aberrant DNA methylation
levels observed for the *H19* ICR are a consequence thereof.
Whether demethylation of the *IGF2* DMR2 contributes to
overexpression of a subset of *IGF2* transcripts needs further
investigation. Our study further underlines the import-
ance to take ploidy into account to accurately discern the
mechanism of gene-specific overexpression in tumors.

**Supplementary data**

This is linked to the online version of the paper at http://dx.doi.org/10.1530/ERC-15-0086.

**Declaration of interest**

The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.

**Funding**

The project was funded by the institutional budget of CEPH and CNG.

**Author contribution statement**

H M Nielsen, A How-Kit, A Daunay and C Guerin performed laboratory experiments. H M Nielsen, A How-Kit, H K M Vollan, V N Kristensen, P V Loo, L L Hansen, J Tost were involved in the data analysis. C Guerin, F Castinetti,
C D Micco, D Taieb, F Sebag and A Barlier were responsible for the patient cohorts, clinical and pathological analyses and sample preparation. H M Nielsen and J Tost provided the first draft of the manuscript. All authors participated in writing of the manuscript and approved the final version of the manuscript. A Barlier and J Tost initiated and designed the study. F Castinetti and C Guerin, and A Barlier and F Sebag contributed equally, and should be considered joint third and second last authors, respectively.

## Acknowledgements

We thank Lasse Sommer Kristensen for critical reading and suggestions for improvement of the manuscript, Anne-Lise Børresen Dale for the intellectual input and financial support of H K M Vollan and Anne Laure Germanetti for excellent technical assistance. Tumor specimens were stored in the AP-HM tumor bank AC 2013–1786.

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Grumbach MM, Biller BM, Braunstein GD, Campbell KK, Carney JA, Godley PA, Harris EL, Lee JK, Oertel YC, Posner MC *et al*. 2003 Management of the clinically inapparent adrenal mass ("incidentaloma"). *Annals of Internal Medicine* **138** 424–429. (doi:10.7326/0003-4819-138-5-200303040-00013)

Guillaud-Bataille M, Ragazzon B, de Reynies A, Chevalier C, Francillard I, Barreau O, Steunou V, Guillemot J, Tissier F, Rizk-Rabin M *et al*. 2014 IGF2 Promotes growth of adrenocortical carcinoma cells, but its overexpression does not modify phenotypic and molecular features of adrenocortical carcinoma. *PLoS ONE* **9** e103744. (doi:10.1371/journal. pone.0103744)

Gunderson KL, Steemers FJ, Lee G, Mendoza LG & Chee MS 2005 A genome-wide scalable SNP genotyping assay using microarray technology. *Nature Genetics* **37** 549–554. (doi:10.1038/ng1547)

Hao Y, Crenshaw T, Moulton T, Newcomb E & Tycko B 1993 Tumour-suppressor activity of H19 RNA. *Nature* **365** 764–767. (doi:10.1038/ 365764a0)

Hensen K, Van Valckenborgh IC, Kas K, Van de Ven WJ & Voz ML 2002 The tumorigenic diversity of the three PLAG family members is associated with different DNA binding capacities. *Cancer Research* **62** 1510–1517.

Hodin R, Lubitz C, Phitayakorn R & Stephen A 2014 Diagnosis and management of pheochromocytoma. *Current Problems in Surgery* **51** 151–187. (doi:10.1067/j.cpsurg.2013.12.001)
C D Micco, D Taieb, F Sebag and A Barlier were responsible for the patient cohorts, clinical and pathological analyses and sample preparation. H M Nielsen and J Tost provided the first draft of the manuscript. All authors participated in writing of the manuscript and approved the final version of the manuscript. A Barlier and J Tost initiated and designed the study. F Castinetti and C Guerin, and A Barlier and F Sebag contributed equally, and should be considered joint third and second last authors, respectively.

### Acknowledgements

We thank Lasse Sommer Kristensen for critical reading and suggestions for improvement of the manuscript, Anne-Lise Børresen Dale for the intellectual input and financial support of H K M Vollan and Anne Laure Germanetti for excellent technical assistance. Tumor specimens were stored in the AP-HM tumor bank AC 2013–1786.

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Brunt LM & Moley JF 2001 Adrenal incidentaloma. *World Journal of Surgery* **25** 905–913. (doi:10.1007/s00268-001-0029-0)

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Staaf J, Vallon-Christersson J, Lindgren D, Juliusson G, Rosenquist R,
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Weiss LM 1984 Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. *American Journal of Surgical Pathology* **8** 163–169. (doi:10.1097/00000478-198403000-00001)

Received in final form 18 September 2015

Accepted 22 September 2015

Made available online as an Accepted Preprint

23 September 2015
Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagnere AM, Rene-
Corail F, Jullian E, Gicquel C, Bertagna X *et al.* 2005 Mutations of
β-catenin in adrenocortical tumors: activation of the Wnt signaling
pathway is a frequent event in both benign and malignant adreno-
cortical tumors. *Cancer Research* **65** 7622–7627. (doi:10.1158/0008-
5472.CAN-05-0593)

Tost J & Gut IG 2007 DNA methylation analysis by pyrosequencing. *Nature Protocols* **2** 2265–2275. (doi:10.1038/nprot.2007.314)

Ulaner GA, Yang Y, Hu JF, Li T, Vu TH & Hoffman AR 2003 CTCF binding at the insulin-like growth factor-II (IGF2)/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues. *Endocrinology* **144** 4420–4426. (doi:10.1210/en.2003-0681)

Van Loo P, Nordgard SH, Lingjaerde OC, Russnes HG, Rye IH, Sun W, Weigman VJ, Marynen P, Zetterberg A, Naume B *et al.* 2010 Allele-specific copy number analysis of tumors. *PNAS* **107** 16910–16915. (doi:10.1073/pnas.1009843107)

Volante M, Buttigliero C, Greco E, Berruti A & Papotti M 2008 Pathological and molecular features of adrenocortical carcinoma: an update. *Journal of Clinical Pathology* **61** 787–793. (doi:10.1136/jcp.2007.050625)

Voz ML, Mathys J, Hensen K, Pendeville H, Van Valckenborgh I, Van Huffel C, Chavez M, Van Damme B, De Moor B, Moreau Y *et al.* 2004 Microarray screening for target genes of the proto-oncogene PLAG1. *Oncogene* **23** 179–191. (doi:10.1038/sj.onc.1207013)

Waldmann J, Fendrich V, Holler J, Buchholz M, Heinmoller E, Langer P, Ramaswamy A, Samans B, Walz MK, Rothmund M *et al.* 2010 Microarray analysis reveals differential expression of benign and malignant pheochromocytoma. *Endocrine-Related Cancer* **17** 743–756. (doi:10.1677/ERC-09-0118)

Weiss LM 1984 Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. *American Journal of Surgical Pathology* **8** 163–169. (doi:10.1097/00000478-198403000-00001)

Received in final form 18 September 2015

Accepted 22 September 2015

Made available online as an Accepted Preprint

23 September 2015