Accepted Manuscript
Androgen production in pediatric adrenocortical tumors may occur via both the classic and/or the alternative backdoor pathway
Nesa Marti, Jana Malikova, José A. Galván, Maude Aebischer, Marco Janner, Zdenek Sumnik, Barbora Obermannova, Genevieve Escher, Aurel Perren, Christa E. Flück
195H 0000-720P
ELSEVIER
Molecular and Cellular Endocrinology
| PII: | S0303-7207(17)30272-1 |
| DOI: | 10.1016/j.mce.2017.05.014 |
| Reference: | MCE 9945 |
| To appear in: | Molecular and Cellular Endocrinology |
| Received Date: | 4 February 2017 |
| Revised Date: | 24 April 2017 |
| Accepted Date: | 9 May 2017 |
Please cite this article as: Marti, N., Malikova, J., Galván, José.A., Aebischer, M., Janner, M., Sumnik, Z., Obermannova, B., Escher, G., Perren, A., Flück, C.E., Androgen production in pediatric adrenocortical tumors may occur via both the classic and/or the alternative backdoor pathway, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.05.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
HSD382
RoOH
CYP17A1
SRD5A1
AKR1CZ
AKRIC3
AKR1C4
Normal Adrenal
ACC Case 2
ACC Case 3
SCRIPT
Chameleons with Respect to Androgen Production
ACCEPTED MA
1
Androgen production in pediatric adrenocortical tumors may occur via both the classic and/or the
2 alternative backdoor pathway
3 4 5 6 *co-first authors for equal contributions
Nesa Marti1,2,3*, Jana Malikova1,2,5*, José A. Galván4, Maude Aebischer1, Marco Janner1, Zdenek Sumnik5,
Barbora Obermannova5, Genevieve Escher2,6, Aurel Perren4, Christa E. Flück1,2
7
8 1Pediatric Endocrinology and Diabetology, Department of Pediatrics; 2Department of Clinical Research, Inselspital, Bern University Hospital, University of Bern, Switzerland; 3Graduate School Bern, University
of Bern, Switzerland; 4Institute of Pathology, University of Bern, Switzerland; 5Department of Pediatrics,
2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech
9 10 11 12 Republic; ‘Department of Nephrology and Hypertension, Bern University Hospital, University of Bern, Switzerland
Abbreviated title: Androgen production by pediatric ACT
Key words: pediatric adrenocortical tumors, dihydrotestosterone, androgen biosynthesis, classic pathway, alternative (backdoor) pathway
Word count: (3591)
Corresponding author and address for reprint requests:
Christa E. Flück
Pediatric Endocrinology and Diabetology 23 University Children’s Hospital Bern
Inselspital Bern
24 25 26 3010 Bern
Freiburgstrasse 15 / C845
13 14 15 16 17 18 19 20 21 22
27 Switzerland
28 Email: christa.flueck@dkf.unibe.ch
29 30 Disclosure summary: The listed authors have nothing to disclose.
ACCEPTED MANUS CRIPT
Abstract (146)
Children with adrenocortical tumors (ACTs) often present with virilization due to high tumoral androgen production, with dihydrotestosterone (DHT) as most potent androgen. Recent work revealed two pathways for DHT biosynthesis, the classic and the backdoor pathway. Usage of alternate routes for DHT production has been reported in castration-resistant prostate cancer, CAH and PCOS. To assess whether the backdoor pathway may contribute to the virilization of pediatric ACTs, we investigated seven children suffering from androgen producing tumors using steroid profiling and immunohistochemical expression studies. All cases produced large amounts of androgens of the classic and/or backdoor pathway. Variable expression of steroid enzymes was observed in carcinomas and adenomas. We found no discriminative pattern. This suggests that enhanced androgen production in pediatric ACTs is the result of deregulated steroidogenesis through multiple steroid pathways. Thus future treatments of ACTs targeting androgen overproduction should consider these novel steroid production pathways.
ACCEPTED Mesle melhoresdelniemandenla .
31 32 33 34 35 36 37 38 39 40 41 42 43
1. Introduction
Adrenocortical tumors (ACTs) in children are rare and may be classified in benign bilateral adrenocortical hyperplasias (BAH) and adrenocortical adenomas (ACA) or malignant adrenocortical carcinomas (ACC) (Stratakis et al, 2007). ACC often imply a life-threatening diagnosis, even in children. Surgery is the best curative treatment option for localized tumors, while drug treatments and radiotherapy for extended 49 disease remain uncertain. ACTs comprise a heterogeneous group of hormone active or hormone inactive disorders. They exhibit different characteristics and variable prognosis. Incidence of ACC in adults is 0,7 50 51
- 1,0 per million and 0,3 -0,4 per million in children (Fassnacht et al, 2013; Kerkhofs et al, 2014). Only in 52 Brazil a 10-15-fold higher incidence is observed (Sandrini et al, 1997). For ACC a bimodal age 53 distribution is typically observed with a first peak of presentation in childhood at around 4 years of age 54 and a second peak in the fourth decade of life (Kerkhofs et al, 2014). Female gender is consistently predominant. ACCs in pediatric patients are often associated with germline mutations in the tumor suppressor gene TP53 and with the Li-Fraumeni syndrome (OMIM ID #151623) or the Li-Fraumeni-like syndrome (OMIM ID #151623) (Giacomazzi et al, 2013; Wasserman et al, 2015; Wasserman et al, 2012);
55 56 57 58 59 60 61 62
although other hereditary tumor syndromes may also be associated, e.g. the Beckwith-Wiedemann syndrome (OMIM ID #130650), which in its familial form is linked to loss of imprinting at the insulin- like growth factor II locus on chromosome 15 (Giacomazzi et al, 2013; Wasserman et al, 2015), and the syndrome of multiple endocrine neoplasia type 1 due to mutations in the MENI gene (OMIM ID #131100) (Gatta-Cherifi et al, 2012; Simonds et al, 2012). The remarkably higher incidence of pediatric
63 ACCs in Southern Brazil is caused by a high prevalence of the germline pR337H mutation in TP53
(Custodio et al, 2012; Giacomazzi et al, 2013).
Pediatric ACCs are mostly hormonally active. Androgen excess with or without hypercortisolism are typical in children. Clinical symptoms include signs of virilisation (e.g. hirsutism, acne) and precocious
64 65 66 67 68 69
pseudopuberty combined with Cushing syndrome. Although the prognosis of ACCs is generally poor, many authors report a better outcome for children than adult patients, even when similar histopathological features of malignancy are present (Dehner & Hill, 2009; Wieneke et al, 2003). In adults, ACCs are
44 45 46 47 48
70 71 72 73 74 75 76 77 classified according to the Weiss score, which considers the histological architecture of the tumor, cell nuclei characteristics, necrosis and extension of invasion of the tumor to vascular structures and the capsule (Weiss, 1984). In children, this score is less useful as studies have shown that ACTs presenting with a histology, which is associated with malignancy in adults, might behave benign in children (Lau & Weiss, 2009). Predictors of long term survival in children with ACCs are age, tumor size, weight and extension, metastatic disease and tumor spillage during surgery (Gulack et al, 2016). Thus, small tumor size, androgen production only, localized lesion and young age (0-3 years) seem associated with a better outcome. Actually the 3-year progression-free survival (PFS) and overall survival (OS) were 39% and 78 55% for the whole population, respectively, and 51% and 73% for localized diseases, respectively (Cecchetto et al, 2016).
In pediatric ACTs, virilisation is the most common presentation (Michalkiewicz et al, 2004). This is due to 81 tumoral androgen production, of which dihydrotestosterone (DHT) is the most potent natural androgen. Recently, it has been shown that there are at least two pathways for DHT synthesis in the human adrenals, namely the classic and an alternative, so called backdoor pathway (Auchus, 2004). In contrast to the classic pathway, the backdoor pathway does not produce DHT through the intermediate testosterone, but uses 17-hydroxyprogesterone and proceeds through the steroid metabolites 17OH-dihydroprogesterone, 17OH-allopregnanolone, androsterone and androstanediol (Figure 1). Conversion involves the following
enzymes for catalysis: 5a-reductase type1 (SRD5A1), aldo-keto reductase family 1 members C2/C4 (AKR1C2/C4), cytochrome P450c17 (CYP17A1), 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) / aldo-keto reductase family 1 C3 (AKR1C3) and aldo-keto reductase family 1 C4 (AKRIC4) / 17- hydroxysteroid dehydrogenase type 6 (RoDH) (Auchus, 2004). An additional connection between the classic and the backdoor pathway leading DHEA via androstenedione to androstanedione and DHT has also been described (Fassnacht et al, 2003).
However, the respective use of the classic and the backdoor pathway in health and disease states remains largely unknown. Urine steroid profiling of backdoor pathway metabolites suggested that hyperandrogenism in patients with 21-hydroxylase deficiency is related to DHT production via the
79 80 82 83 84 85 86 87 88 89 90 91 92 93 94 95
backdoor pathway (Homma et al, 2006). We showed recently that the alternative pathway is established in the human ovary and that it might contribute to the androgen excess observed in some patients with the polycystic ovary syndrome (PCOS) (Marti et al, 2016). Moreover, the backdoor pathway seems hyper- active in castration-resistant prostate cancers and seems counteracting androgen deprivation therapy (Chang et al, 2011; Fiandalo et al, 2014; Mohler et al, 2011). Studies suggest that prostate tumors are able to activate alternate routes of androgen synthesis, which are not targeted by current drugs, and that increased DHT production may occur via the backdoor pathway (Chang et al, 2011). Therefore, drugs targeting the backdoor pathway might provide new therapeutic opportunities against prostate cancer and other hyperandrogenic disease states including ACTs.
Whether the observed virilization in pediatric ACT patients is (at least in part) due to excessive androgen production via the backdoor pathway remains to be established. In this study, we investigated the steroid pathways used in 7 patients presenting with androgen producing adrenal tumors. We provide androgen profiles and immunohistochemical studies comparing classic and backdoor pathway androgen production in adrenal carcinomas and adenomas. Pediatric ACTs seem to produce androgens in excess using both the classic and/or the backdoor pathway, thereby following no obvious pattern. Targeting the backdoor pathway in addition to the classic pathway in drug regimens against hyperandrogenism in ACTs seems advisable.
2. Patients, Materials and Methods
2.1 Ethical approval and case reports
This study was approved by the Ethics commission of the University of Bern and the Canton of Bern, Switzerland. Patients and their parents gave informed consent for using their data in anonymous form for a case series study.
Detailed case descriptions of all seven cases can be found in Suppl Material A, Case Reports. Clinical data are summarized in Table 1 and biochemical findings in Table 2.
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
2.2 Steroid profiling
Serum steroids were measured by conventional, commercially available immunoassays. The QuantiChrom Creatinine Assay (DICT-500; BioAssay Systems, Hayward, CA, USA) was used to measure urinary creatinine by quantitative colorimetry. Urine steroid analysis was performed in 1.5 ml urine by an established in-house gas chromatography and mass spectrometry (GC-MS) method on either 24h urine collections (Ackermann et al, 2015; Henschkowski et al, 2008) or spot urine samples (Dhayat et al, 2017; Dhayat et al, 2015). In brief, medroxyprogesterone was added as a recovery standard, before the urine sample was extracted on a Sep-Pak C18 column, then hydrolyzed with sulfatase and ß- glucuronidase/arylsulfatase. Free steroids were then extracted on a Sep-Pak C18 cartridge. The two standards Stigmasterol and 365ß-TH-aldosterone were added to the extract, then methoxyamine HCI 2% in pyridine was added and the sample was incubated at 60℃ for one hour. After evaporation of the solvent, trimethylsilylimidazole (TMSI) was added to the extracts and derivatised for 16 h at 100℃. Samples were purified by gel filtration on Lipidex 5000 columns to remove excess derivatization reagent and analyzed by mass spectrometry on a gas chromatograph 7890A from Agilent Technologies (La Jolla, California, USA) coupled to a mass selective detector by Hewlett-Packard 5975C providing selected ion monitoring (SIM). Absolute excretion in 24h urine collection was calculated in ug/24h. In spot urine, measured steroids were expressed in u g/mmol creatinine.
2.3 Immunohistochemical studies (IHC)
Immunohistochemical analysis was performed on fresh frozen, and formalin fixed and paraffin embedded (FFPE) tissue samples with specific antibodies (Suppl Table A) as described (Marti et al, 2016). In brief, serial sections were cut at 3um thickness and dried at 60° for 30 minutes. Specificity of antibodies was tested using cell lines with known positive (H295R, HepG2) and negative (HEK293) immunoreactivity (Suppl Figure A). Additional characteristics of new custom-made antibodies anti-AKR1C2 and anti- AKR1C4 are given in Suppl Table B. The automated system BOND RX (Leica Biosystems, Newcastle, UK) was used to carry out the immunohistochemical detection of proteins. Therefore sections were
122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
deparaffinized and rehydrated in dewax solution (Leica Biosystems). Antigen retrieval was done with citrate buffer solution (pH 6.5) at 95° for 20 min. Endogenous peroxidase was blocked with H2O2 solution for 4 minutes. Primary antibodies and dilutions were used as given in Suppl Table A with an incubation time of 30 minutes at room temperature. The following secondary antibodies were used: rabbit anti- chicken antibody (ab97136, AbCam, Cambridge, UK.) at 1:2000 dilution and goat anti-rabbit (E0466, Dako, Glostrup, Denmark) at 1:400 dilution. Slides were visualized with the Bond Polymer Refine kit (with 3-3’-Diaminobenzidine-DAB as chromogen) (Leica Biosystems). Counterstaining was done with hematoxylin. Finally, all slides were mounted in Aquatex (Merck, Darmstadt, Germany) and scanned and photographed in a Pannoramic P250 scanner (3DHistech, Hungary).
2.4 Semi-quantitative assessment of protein expression revealed by IHC and statistics
Tumor protein/enzyme expression was semi-quantified using the Q-score scoring system (Galván et al, 2013), which assesses two parameters: one, the intensity of the staining (I; 0-1-2-3) and two, the percentage of positive cells (P; 0%-100%). Multiplication of the two parameters results in the Q-score (Q = I x P); minimum = 0, maximum = 300). The assessment was done by four independent examiners (JM, JAG, NM, CEF). The mean of the four assessments was calculated and the scatter blots and the median of all seven different tumor cases were produced. For comparison, expression of same proteins was also assessed in the very same manner for the three zones of normal adrenal cortex tissue.
3. Results
3.1 Clinical and biochemical characteristics
We describe 7 children with androgen producing tumors aged 8 months to 17 years; six females, one male. Four suffered from an ACC, two from an ACA and one a steroid cell tumor in the ovary. Clinical and biochemical details are summarized in Case Reports (Suppl Material A) and Tables 1 and 2. Among these patients the common clinical features were typical signs of androgen excess, which manifested age- specific with signs of premature pubarche or pseudopubertas praecox in young children, while secondary
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173
amenorrhea was observed in adolescent girls. Other signs of androgen excess included hypertrichosis, hirsutism and acne. Cushingoid habitus was described in 6/7 with excessive weight gain and hypertension seen in 4/7. Accordingly, serum cortisol levels were elevated in all and ACTH was found suppressed in 6/7 (Table 2). Elevated serum androgens of any kind were measured in all cases except Case 5, in which only free testosterone was assessed and found below the detection limit. In cases 1, 2 and 3 a urinary steroid profile was performed. In all 3 cases, we found overall grossly elevated androgens with most measured androgen metabolites (from the classic and alternative pathway) increased above the upper normal limit for age and sex (Figure 2). In Case 1 suffering from a steroid cell tumor of the ovary, DHT was more than 10-fold elevated. In cases 2 and 3 presenting with ACCs, DHT was even more elevated and some other metabolites were increased by more than 100-fold, e.g. DHEA in Case 2, and androstenediol, androsterone and androstenetriol in cases 2 and 3. By contrast, excretion of mineralocorticoid and glucocorticoid metabolites were normal (data not shown). Geneting testing for Li-Fraumeni syndrome was performed according to Chompret criteria (Tinat et al., 2009). Thus 4/7 of our patients qualified for testing, but one ACC patient and her family refused genetic testing. One patient with a steroid producing 188 tumor of the ovary and two patients with ACAs did not fullfil the criteria. Of the three genetically tested ACC patients, two were found to have known TP53 mutations.
3.2 Immunohistochemical studies of tumor tissues
To assess the expression of steroid enzymes involved in androgen biosynthesis of the classic and the backdoor pathway in the tumor tissues, specific IHC staining for HSD3B2, RoDH, CYP17A1, SRD5A1 and AKR1C2/3/4 was performed and pictures were semi-quantitatively analyzed using the Q-score as previously described (Marti et al, 2016). Representative IHC pictures for a normal adrenal control tissue and two ACC tumor tissues are given in Figure 3 (all cases are summarized in Suppl Figure B). Comparing the seven tumor tissues, we found a very broad range of expression of the 7 studied steroid enzymes. Every tumor presented a unique profile. No pattern could be identified; even clinically similar ACC cases 2 and 3 were presenting with grossly different expression of steroid enzymes (Figure 3). IHC
174 175 176 177 178 179 180
181 182 183 184 185 186 187 189 190 191 192 193 194 195 196 197 198 199
200
enzyme expression profiles of tumor tissues were also not resembling the expression found in the three 201 layers of the normal adrenal cortex, which shows a typical zone specific expression of androgen producing enzymes.
202 203 204
In the normal adrenal, HSD3B2 is highly expressed in the zona glomerulosa and in the zona fasciculata, where its activity is needed for mineralocorticoid and glucocorticoid synthesis, while low expression is 205 characteristic for the androgen producing zona reticularis (Figure 3). HSD3B2 showed no uniform expression in the seven tumors. High expression was found in one adrenal adenoma (Case 5), but almost no expression in the other (Case 4). One of four ACC (Case 3) as well as the ovarian steroid-cell tumor showed moderate HSD3B2 expression. Three of four ACC did not express HSD3B2. RoDH showed similar variability in the tumors. RoDH was highly expressed in the ovarian steroid-cell tumor (Case 1) and in one ACC (Case 2), moderately expressed in two ACC (cases 3 and 7) and one ACA (Case 4), but low expressed in one ACC (Case 6) and one ACA (Case 5). CYP17A1, which is expressed at high levels 212 in the normal adrenal zona fasciculata and zona reticularis, showed high expression in all tumors, except one ACC (Case 7). Compared to the normal adrenal cortex, SRD5A1 was highly expressed in one ACC (Case 3) and one ACA (Case 5). Similar to the normal adrenal cortex, AKR1C2/4 showed low expression levels in all assessed tumor cases, except in one ACC (Case 7). AKR1C3 was strongly expressed in one ACC (Case 2) and in the ovarian steroid-cell tumor (Case 1). In the other androgen producing tumors, AKR1C3 expression was low compared to the normal adrenal.
Even when comparing steroid enzymes in the context of their position in the steroid pathway (e.g. classic versus backdoor), we found no signature in the expression pattern for a specific tumor. High or low expression of specific enzymes seemed to occur at random in both androgen producing pathways, especially also without correlation to other enzymes along the same path.
To quantify observations, pictures of IHC stained tumor samples were assessed for the expression of the androgen producing enzymes by the Q-Score. Figure 4 shows the Q-score for all 7 enzymes in the seven cases. AKR1C2/4 showed a low score in all tumors, while all other assessed enzymes presented with a
206 207 208 209 210 211 213 214 215 216 217 218 219 220 221 222 223 224
very variable score confirming the described observation that there is no pattern. For instance, ACC showed Q-scores for RoDH from 0 to 217, and also Q-scores for CYP17A1, HSD3B2 and AKR1C3 were found all over the place. Thus the variability between the tumor tissues was too high to allow a statistical comparison between tumors and normal adrenals.
4. Discussion
ACTs are rare tumors with very variable characteristics in both children and adults. They may be benign adenomas (ACA) or bilateral hyperplasias (BAH), or malignant carcinomas. They may be hormonally active or not. Our study on 7 androgen producing tumors identified in children and adolescents confirms this variability. Characteristic in all was androgen excess, while clinical signs of Cushings found in 6/7 were not unambiguously confirmed by biochemical investigations.
Av.pleaseletlenl.
To find better treatment options against ACTs for both anti-tumor growth and anti-hormone production, and to discriminate between benign ACAs and malignant ACCs, detailed characterization of such tumors is needed. This includes clinical, biochemical, histological and molecular phenotyping as well as follow- up. Moreover, pediatric ACTs have specific characteristics distinguishing them from adult ACTs, therefore pediatric and adult ACTs should be studied in separate groups (Faria & Almeida, 2012; Lalli & Figueiredo, 2015). In our study, we characterized androgen biosynthesis stimulated by the fact that recently novel avenues of androgen production had been found in castration-resistant prostate cancers (Chang et al, 2011), in hyperandrogenic disease states such CAH (Kamrath et al, 2012) and in PCOS (Marti et al, 2016). IHC investigations revealed that the ACT tumor tissues expressed essential proteins/enzymes for DHT synthesis through the classic and the backdoor pathway (Figure 3). However, expression levels of these enzymes varied enormously between tumors without any obvious pattern between ACCs and ACAs. Likewise, detailed urinary steroid profiling including metabolites of both pathways in 3 tumors showed similar androgen excess for one ACA and 2 ACCs (Figure 2). Thus ACTs produce excess androgens not only through the classic pathway, but also through alternate routes perhaps offering opportunities for novel treatments.
225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250
251 252 253 254 In the past two decades several studies have been published providing gene expression profiles of ACCs, ACAs and normal adrenal tissues in the search for diagnostic markers and drug targets (de Fraipont et al, 2005; Fernandez-Ranvier et al, 2008; Fujisawa et al, 2016; Giordano et al, 2009; Giordano et al, 2003; Bassett et al, 2005, Wilmot Roussel et al, 2013, Slater et al, 2006; Velazquez-Fernandez et al, 2005). 255 Most studies were performed in adults, and only few dealt with pediatric ACTs (Fujisawa et al, 2016; Pinto et al, 2015; West et al, 2007), or both (de Reynies et al, 2009; Zheng et al, 2016).
Expression levels of steroid hormones are mentioned in few ACT studies (de Fraipont et al, 2005; Bassett et al, 2005, Wilmot Roussel et al, 2013, de Reynies et al, 2009; Slater et al, 2006). De Fraipont et al (de 259 Fraipont et al, 2005) identified a steroidogenic cluster (including CYP11A1, CYP11B1, CYP17A1, 260 CYP21A2, HSD3B1, StAR), which correlated with ACC in the low expression group and with ACA in the high expression group. Similarly, Bassett et al (Bassett et al, 2005) observed higher levels of HSD3B2, CYP21A2 and CYP11B2 mRNA in aldosterone producing adenomas and higher levels of CYP11A1, CYP17A1, HSD3B2, and CYP11B1 in cortisol producing adenomas compared to normal tissue. Roussel et al (Wilmot Roussel et al, 2013) defined through gene expression profiling clusters of non-secreting, subclinical cortisol producing and cortisol producing ACAs. In this study the transcriptome identified two types of cortisol-producing adenomas, one prone to overt cortisol production the other heterogeneous. In another study downregulation of steroid enzymes HSD3B2 and CYP11B1 was observed in ACC compared to ACA, but other genes were suggested for discriminating ACC from ACA, and for predicting outcome (de Reynies et al, 2009). In contrast to these studies, the newest study of Zheng et al. (Zheng et al. 2007) assessed the expression of 25 genes (including CYP21A2, CYP11B1, CYP11B2, HSD3B2, STAR, CYP17A1, SULT2A1, CYP11A1, cholesterol transporters, SF1, MC2R) in ACT samples originating from teenagers and adults and found a negative correlation between a calculated gene expression score and adrenocortical differentiation for ACC and survival; e.g. well-differentiated tumors were associated with poor prognosis. This discrepancy between reported studies together with the matter of fact that expression of steroid hormones may vary in ACC and ACA samples indicate that prediction of tumor dignity or prognosis on the basis of steroid expression profiling is not reliable.
256 257 258 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
Until now only few studies looked at steroid genes in microarray data obtained from pediatric ACAs, ACCs and normal tissues (Fujisawa et al, 2016; West et al, 2007;). West at al found no difference in the expression of steroid genes from ACAs and ACCs (West et al, 2007). But expression of HSD3B2 and CYP11B2 were markedly lower in pediatric ACAs and ACCs compared to normal adrenal tissues. In the newest case study from the Fujisawa group (Fujisawa et al, 2016), expression of HSD3B2, AKR1C3 and CYP21A2 were reduced in an ACC compared to a healthy control, while CYP17A1 was increased. In line with these data, we found low expression of HSD3B2 in 3 out of 4 ACC cases and 1 out of 2 ACAs. However, in the ACA of Case 5, HSD3B2 was higher expressed than in the normal adrenal zona reticularis. Furthermore, similar to Fujisawa’s case, almost all ACTs of our study (except one ACC, Case 7) showed elevated CYP17 expression. Other genes of the backdoor pathway (e.g. AKR1C3, SRD5A1, RoDH) were found unaltered (Fujisawa et al, 2016), while we found variable results. Overall, currently available data suggest that studying expression of steroid genes may not help in discriminating ACAs from ACCs, but it may help in estimating outcome in ACCs.
Most recently ACCs have been characterized in a great detail using a pan-genomic approach (Assie et al, 2014; Pinto et al, 2015; Zheng et al, 2016). These comprehensive studies try to cluster characteristics of ACCs based on the analysis of chromosomal instability (especially loss of heterozygosity of chromosome 11p and 17 with overexpression of IGF2, and loss of wild-type allele of TP53, respectively), whole genome sequencing (mutations in driver genes such as TP53, CTNNB1, ZNFR, ATRX, TERT, PRKARIA, RPL22), mRNA expression, miRNA expression and methylation patterns. These studies show that changes in methylation are prognostic in ACCs (Assie et al, 2014; Jouinot et al, 2016; Zheng et al, 2016). Assie et al (Assie et al, 2014) used miRNA expression and methylation characteristics to divide previously established aggressive malignant ACC (C1A) from malignant ACC with good prognosis (C1B) (de Reynies et al, 2009). Zheng et al (Zheng et al, 2016) identified three clusters of ACCs according to DNA copy numbers, DNA-methylation, mRNA and miRNA expression performing a Cluster of Cluster
277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302
analysis (CoC). A comparison between CoC and previously identified C1A/C1B clusters showed that the majority of CoCI subtype were classified as C1B, while most CoCII and CoCIII were predicted as C1A (de Reynies et al, 2009). Defining ACCs by clusters using pan-genomic methods may improve patient care and outcome and therefore has clinical implications.
Steroid metabolomic studies of ACTs are also able to discriminate ACAs from ACCs in adult patients (Arlt et al, 2011; Imperiale et al, 2013). Metabolic profiling of 32 adrenal steroid metabolites in urine of adults with ACCs and ACAs using GC/MS analysis and computer assisted prediction models was able to 311 discriminate malignancy with a diagnostic sensitivity of 88-90% (Arlt et al, 2011). Among the 32 measured metabolites, 9 (predominantly precursor) steroids were essential to discriminate between ACA and ACC, with tetrahydrocortisol (THS) scoring highest, before 5a-pregnanetriol and 5a-pregnanediol. However, whether this metabolic signature also applies to pediatric ACTs remains to be demonstrated. Looking at 8 of the 9 prediction markers in the steroid profiles of our patients, we don’t see a discriminating picture between the benign tumor of Case 1 and the ACCs of cases 2 and 3. Remarkably, THS was normal in all. But of course, studies of larger pediatric cohorts are needed to solve this question. Finally, studies of adult ACT tissue samples using HRMAS NMR spectroscopy also showed significant metabolic differences between ACAs, ACCs and normal adrenals (Imperiale et al, 2013). ACC exhibited typical characteristics of neoplastic tissues such as choline-containing compounds, markers of anaerobic processes and glycolysis. Unfortunately for children, also these studies have not yet been performed in pediatric ACTs.
Major limitation of this pilot study is clearly the small sample size. Because of the low incidence of pediatric ACA and ACC, we were only able to collect a heterogeneous group of seven children presenting with mixed tumor types. IHC studies and steroid profiling of these restricted samples suggest that pediatric ACTs are a heterogeneous group with respect to expression of steroidogenic genes and androgen metabolites. However the question remains, whether there would be group discriminating (e.g. ACA
303 304 305 306 307 308 309 310
312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328
versus ACC) characteristics serving as diagnostic pattern, when analyzing larger cohorts. Collaborative studies employing larger cohorts of pediatric ACTs similar to efforts undertaken in adults are needed to better characterize the tumors in order to improve treatment options and prognosis of children diagnosed with ACC. In addition, for final proof of function of the alternative pathway in the pediatric ACTs, backdoor pathway metabolites need to be measured in tumoral tissues directly.
In conclusion, we show that pediatric ACTs seem to produce excess androgens through the classic biosynthesis pathway and/or through alternate routes such as the backdoor pathway. This may provide novel opportunities to target androgen excess in ACTs, which often leads to severe adverse effects (e.g. on growth or on sexual maturation). Our pilot study however suggests that there is no discriminative pattern for androgen pathway usage and androgen metabolites for pediatric ACAs versus ACCs; rather every tumor possesses unique properties concerning androgen biosynthesis and metabolism. But this needs to be verified in larger studies.
Acknowledgments
We thank all patients, families and care givers for providing data. This work has been supported by a grant of the Swiss National Science Foundation (320030-146127) to CEF and a Swiss Government Excellence Scholarship to J.M.
References
Ackermann, D., Pruijm, M., Ponte, B., Guessous, I., Ehret, G., Escher, G., Dick, B., Al-Alwan, H., Vuistiner, P., Paccaud, F., Burnier, M., Pechere-Bertschi, A., Martin, P. Y., Vogt, B., Mohaupt, M. & Bochud, M. (2015) CYP17Al Enzyme Activity Is Linked to Ambulatory Blood Pressure in a Family- Based Population Study. Am J Hypertens, 29(4), 484-93.
Arlt, W., Biehl, M., Taylor, A. E., Hahner, S., Libe, R., Hughes, B. A., Schneider, P., Smith, D. J., Stiekema, H., Krone, N., Porfiri, E., Opocher, G., Bertherat, J., Mantero, F., Allolio, B., Terzolo, M., Nightingale, P., Shackleton, C. H., Bertagna, X., Fassnacht, M. & Stewart, P. M. (2011) Urine steroid
329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357
metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab, 96(12), 3775-84.
Assie, G., Letouze, E., Fassnacht, M., Jouinot, A., Luscap, W., Barreau, O., Omeiri, H., Rodriguez, S., Perlemoine, K., Rene-Corail, F., Elarouci, N., Sbiera, S., Kroiss, M., Allolio, B., Waldmann, J., Quinkler, M., Mannelli, M., Mantero, F., Papathomas, T., De Krijger, R., Tabarin, A., Kerlan, V., Baudin, E., Tissier, F., Dousset, B., Groussin, L., Amar, L., Clauser, E., Bertagna, X., Ragazzon, B., Beuschlein, F., Libe, R., de Reynies, A. & Bertherat, J. (2014) Integrated genomic characterization of adrenocortical carcinoma. Nat Genet, 46(6), 607-12.
Auchus, R. J. (2004) The backdoor pathway to dihydrotestosterone. Trends Endocrinol Metab, 15(9), 432- 8.
Bassett, M. H., Mayhew, B., Rehman, K., White, P. C., Mantero, F., Arnaldi, G., Stewart, P. M., Bujalska, I. & Rainey, W. E. (2005) Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab, 90(9), 5446-55.
Cecchetto, G., Ganarin, A., Bien, E., Vorwerk, P., Bisogno, G., Godzinski, J., Dall’Igna, P., Reguerre, Y., Schneider, D., Brugieres, L., Leblond, P., Ferrari, A., Brecht, I., De Paoli, A. & Orbach, D. (2016) Outcome and prognostic factors in high-risk childhood adrenocortical carcinomas: A report from the European Cooperative Study Group on Pediatric Rare Tumors (EXPERT). Pediatr Blood Cancer, doi:10.1002/pbc.26368.
Chang, K. H., Li, R., Papari-Zareei, M., Watumull, L., Zhao, Y. D., Auchus, R. J. & Sharifi, N. (2011) Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer. Proc Natl Acad Sci U S A, 16, 13728-33.
Custodio, G., Komechen, H., Figueiredo, F. R., Fachin, N. D., Pianovski, M. A. & Figueiredo, B. C. (2012) Molecular epidemiology of adrenocortical tumors in southern Brazil. Mol Cell Endocrinol, 351(1), 44-51.
de Fraipont, F., El Atifi, M., Cherradi, N., Le Moigne, G., Defaye, G., Houlgatte, R., Bertherat, J., Bertagna, X., Plouin, P. F., Baudin, E., Berger, F., Gicquel, C., Chabre, O. & Feige, J. J. (2005) Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab, 90(3), 1819-29.
de Reynies, A., Assie, G., Rickman, D. S., Tissier, F., Groussin, L., Rene-Corail, F., Dousset, B., Bertagna, X., Clauser, E. & Bertherat, J. (2009) Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol, 27(7), 1108-15.
Dehner, L. P. & Hill, D. A. (2009) Adrenal cortical neoplasms in children: why so many carcinomas and yet so many survivors? Pediatr Dev Pathol., 12, 284-91.
Dhayat, N. A., Dick, B., Frey, B. M., d’Uscio, C. H., Vogt, B. & Fluck, C. E. (2017) Androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway: Insights into enzyme activities and steroid fluxes in healthy infants during the first year of life from the urinary steroid metabolome. J Steroid Biochem Mol Biol, 165(Pt B), 312-322.
Dhayat, N. A., Frey, A. C., Frey, B. M., d’Uscio, C. H., Vogt, B., Rousson, V., Dick, B. & Fluck, C. E. (2015) Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy
358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408
children: Tracing the complexity of human postnatal steroidogenesis. J Steroid Biochem Mol Biol, 154, 226-36.
Faria, A. M. & Almeida, M. Q. (2012) Differences in the molecular mechanisms of adrenocortical tumorigenesis between children and adults. Mol Cell Endocrinol, 351(1), 52-7.
Fassnacht, M., Kroiss, M. & Allolio, B. (2013) Update in adrenocortical carcinoma. J Clin Endocrinol Metab, 98(12), 4551-64.
Fassnacht, M., Schlenz, N., Schneider, S. B., Wudy, S. A., Allolio, B. & Arlt, W. (2003) Beyond adrenal and ovarian androgen generation: Increased peripheral 5 alpha-reductase activity in women with polycystic ovary syndrome. J Clin Endocrinol Metab, 88(6), 2760-6.
Fernandez-Ranvier, G. G., Weng, J., Yeh, R. F., Khanafshar, E., Suh, I., Barker, C., Duh, Q. Y., Clark, O. H. & Kebebew, E. (2008) Identification of biomarkers of adrenocortical carcinoma using genomewide gene expression profiling. Arch Surg., 143, 841-6.
Fiandalo, M. V., Wilton, J. & Mohler, J. L. (2014) Roles for the backdoor pathway of androgen metabolism in prostate cancer response to castration and drug treatment. Int J Biol Sci, 10(6), 596-601.
Fujisawa, Y., Sakaguchi, K., Ono, H., Yamaguchi, R., Kato, F., Kagami, M., Fukami, M. & Ogata, T. (2016) Combined steroidogenic characters of fetal adrenal and Leydig cells in childhood adrenocortical carcinoma. J Steroid Biochem Mol Biol, 159, 86-93.
Galván, J. A., Astudillon, A., Vallina, A., Fonseca, P. J., Gómez-Izquierdo, L., García-Carbonero, R. & González, M. V. (2013) Epithelial-mesenchymal transition markers in the differential diagnosis of gastroenteropancreatic neuroendocrine tumors. Am J Clin Pathol., 140, 61-72.
Gatta-Cherifi, B., Chabre, O., Murat, A., Niccoli, P., Cardot-Bauters, C., Rohmer, V., Young, J., Delemer, B., Du Boullay, H., Verger, M. F., Kuhn, J. M., Sadoul, J. L., Ruszniewski, P., Beckers, A., Monsaingeon, 438 M., Baudin, E., Goudet, P. & Tabarin, A. (2012) Adrenal involvement in MEN1. Analysis of 715 cases from the Groupe d’etude des Tumeurs Endocrines database. Eur J Endocrinol, 166(2), 269-79.
Giacomazzi, J., Selistre, S. G., Rossi, C., Alemar, B., Santos-Silva, P., Pereira, F. S., Netto, C. B., Cossio, S. L., Roth, D. E., Brunetto, A. L., Zagonel-Oliveira, M., Martel-Planche, G., Goldim, J. R., Hainaut, P., Camey, S. A. & Ashton-Prolla, P. (2013) Li-Fraumeni and Li-Fraumeni-like syndrome among children diagnosed with pediatric cancer in Southern Brazil. Cancer, 119(24), 4341-9.
Giordano, T. J., Kuick, R., Else, T., Gauger, P. G., Vinco, M., Bauersfeld, J., Sanders, D., Thomas, D. G., Doherty, G. & Hammer, G. (2009) Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling. Clin Cancer Res, 15(2), 668-76.
Giordano, T. J., Thomas, D. G., Kuick, R., Lizyness, M., Misek, D. E., Smith, A. L., Sanders, D., Aljundi, R. T., Gauger, P. G., Thompson, N. W., Taylor, J. M. G. & Hanash, S. M. (2003) Distinct Transcriptional Profiles of Adrenocortical Tumors Uncovered by DNA Microarray Analysis. Am J Pathol, 162(2), 521- 531.
Gulack, B. C., Rialon, K. L., Englum, B. R., Kim, J., Talbot, L. J., Adibe, O. O., Rice, H. E. & Tracy, E. T. (2016) Factors associated with survival in pediatric adrenocortical carcinoma: An analysis of the National Cancer Data Base (NCDB). J Pediatr Surg, 51(1), 172-7.
409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458
Henschkowski, J., Stuck, A. E., Frey, B. M., Gillmann, G., Dick, B., Frey, F. J. & Mohaupt, M. G. (2008) Age-dependent decrease in 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in hypertensive patients. Am J Hypertens,21(6), 644-9.
Homma, K., Hasegawa, T., Nagai, T., Adachi, M., Horikawa, R., Fujiwara, I., Tajima, T., Takeda, R., Fukami, M. & Ogata, T. (2006) Urine steroid hormone profile analysis in cytochrome P450 oxidoreductase deficiency: implication for the backdoor pathway to dihydrotestosterone. J Clin Endocrinol Metab, 91(7), 2643-9.
Ide, H., Terado, Y., Tokiwa, S., Nishio, K., Saito, K., Isotani, S., Kamiyama, Y., Muto, S., Imamura, T. & Horie, S. (2010) Novel germ line mutation p53-P177R in adult adrenocortical carcinoma producing neuron-specific enolase as a possible marker. Jpn J Clin Oncol, 40(8), 815-8.
Imperiale, A., Elbayed, K., Moussallieh, F. M., Reix, N., Piotto, M., Bellocq, J. P., Goichot, B., Bachellier, P. & Namer, I. J. (2013) Metabolomic profile of the adrenal gland: from physiology to pathological conditions. Endocr Relat Cancer, 20(5), 705-16.
Jouinot, A., Assie, G., Libe, R., Fassnacht, M., Papathomas, T., Barreau, O., B, D. L. V., Faillot, S., Hamzaoui, N., Neou, M., Perlemoine, K., Rene-Corail, F., Rodriguez, S., Sibony, M., Tissier, F., Dousset, B., Sbiera, S., Ronchi, C., Kroiss, M., Korpershoek, E., R, D. E. K., Waldmann, J., Bartsch, D. K., Quinkler, M., Haissaguerre, M., Tabarin, A., Chabre, O., Sturm, N., Luconi, M., Mantero, F., Mannelli, M., Cohen, R., Kerlan, V., Touraine, P., Barrande, G., Groussin, L., Bertagna, X., Baudin, E., Amar, L., Beuschlein, F., Clauser, E., Coste, J. & Bertherat, J. (2016) DNA methylation is an independent prognostic marker of survival in adrenocortical cancer. J Clin Endocrinol Metab, jc20163205.
Kamrath, C., Hochberg, Z., Hartmann, M. F., Remer, T. & Wudy, S. A. (2012) Increased activation of the alternative “backdoor” pathway in patients with 21-hydroxylase deficiency: evidence from urinary steroid hormone analysis. J Clin Endocrinol Metab, 97(3), E367-75.
JE
Kerkhofs, T. M., Ettaieb, M. H., Verhoeven, R. H., Kaspers, G. J., Tissing, W. J., Loeffen, J., Van den Heuvel-Eibrink, M. M., De Krijger, R. R. & Haak, H. R. (2014) Adrenocortical carcinoma in children: first population-based clinicopathological study with long-term follow-up. Oncol Rep, 32(6), 2836-44.
Lalli, E. & Figueiredo, B. C. (2015) Pediatric adrenocortical tumors: what they can tell us on adrenal development and comparison with adult adrenal tumors. Front Endocrinol (Lausanne), 6, 23. Lau, S. K. & Weiss, L. M. (2009) The Weiss system for evaluating adrenocortical neoplasms: 25 years later. Hum Pathol, 40(6), 757-68.
Marti, N., Galvan, J. A., Pandey, A. V., Trippel, M., Tapia, C., Muller, M., Perren, A. & Fluck, C. E. (2016) Genes and proteins of the alternative steroid backdoor pathway for dihydrotestosterone synthesis are expressed in the human ovary and seem enhanced in the polycystic ovary syndrome. Mol Cell Endocrinol, 441, 116-123.
Michalkiewicz, E., Sandrini, R., Figueiredo, B., Miranda, E. C., Caran, E., Oliveira-Filho, A. G., Marques, R., Pianovski, M. A., Lacerda, L., Cristofani, L. M., Jenkins, J., Rodriguez-Galindo, C. & Ribeiro, R. C. (2004) Clinical and outcome characteristics of children with adrenocortical tumors: a report from the International Pediatric Adrenocortical Tumor Registry. J Clin Oncol, 22(5), 838-45.
Missouris, C. G., Markandu, N. D., He, F. J., Papavasileiou, M. V., Sever, P. & MacGregor, G. A. (2016) Urinary catecholamines and the relationship with blood pressure and pharmacological therapy. J Hypertens, 34, 704-9.
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510
Mohler, J. L., Titus, M. A., Bai, S., Kennerley, B. J., Lih, F. B., Tomer, K. B. & Wilson, E. M. (2011) Activation of the androgen receptor by intratumoral bioconversion of androstanediol to dihydrotestosterone in prostate cancer. Cancer Res, 71(4), 1486-96.
Pinto, E. M., Chen, X., Easton, J., Finkelstein, D., Liu, Z., Pounds, S., Rodriguez-Galindo, C., Lund, T. C., Mardis, E. R., Wilson, R. K., Boggs, K., Yergeau, D., Cheng, J., Mulder, H. L., Manne, J., Jenkins, J., Mastellaro, M. J., Figueiredo, B. C., Dyer, M. A., Pappo, A., Zhang, J., Downing, J. R., Ribeiro, R. C. & Zambetti, G. P. (2015) Genomic landscape of paediatric adrenocortical tumours. Nat Commun, 6, 6302.
Sandrini, R., Ribeiro, R. C. & De Lacerda, L. (1997) Childhood Adrenocortical Tumors. J Clin Endocrinol Metab, 82, 2027-31.
Simonds, W. F., Varghese, S., Marx, S. J. & Nieman, L. K. (2012) Cushing’s syndrome in multiple endocrine neoplasia type 1. Clin Endocrinol (Oxf), 76(3), 379-86.
Slater, E. P., Diehl, S. M., Langer, P., Samans, B., Ramaswamy, A., Zielke, A. & Bartsch, D. K. (2006) Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors. Eur J Endocrinol, 154(4), 587-98.
Stratakis, C. A. and Boikos, S. A. (2007) Genetics of adrenal tumors associated with Cushing’s syndrom: a new classification for bilateral adrenocortical hyperplasias. Clin Pract Endocrinol Metab, 3(11), 748-57.
version rets, B., Berthet, P., Dugast C. S Vasso was. Nat
Tinat, J., Bougeard, G., Beart-Desurmont, Vasseur, S., Martin, C., Bouvignies, E., Caron, O., Bressac- de Paillerets, C., Bonaïti-Pellié, C., Stoppa-Lyonnet, D., Frébourg, T. (2009) 2009 of the Chompret criteria for Li Fraumeni syndrome. J Clin Oncol, 27(26):e108-9, doi: 10.1200/JCO.2009.22.7967.
Velazquez-Fernandez, D., Laurell, C., Geli, J., Hoog, A., Odeberg, J., Kjellman, M., Lundeberg, J., Hamberger, B., Nilsson, P. & Backdahl, M. (2005) Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery, 138(6), 1087-94.
Wasserman, J. D., Novokmet, A., Eichler-Jonsson, C., Ribeiro, R. C., Rodriguez-Galindo, C., Zambetti, G. P. & Malkin, D. (2015) Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical carcinoma: a children’s oncology group study. J Clin Oncol, 33(6), 602-9.
Wasserman, J. D., Zambetti, G. P. & Malkin, D. (2012) Towards an understanding of the role of p53 in adrenocortical carcinogenesis. Mol Cell Endocrinol, 351(1), 101-10.
Weiss, L. M. (1984) Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol., 8, 163-9.
West, A. N., Neale, G. A., Pounds, S., Figueredo, B. C., Rodriguez Galindo, C., Pianovski, M. A., Oliveira Filho, A. G., Malkin, D., Lalli, E., Ribeiro, R. & Zambetti, G. P. (2007) Gene expression profiling of childhood adrenocortical tumors. Cancer Res, 67(2), 600-8.
Wieneke, J. A., Thompson, L. D. & Heffess, C. S. (2003) Adrenal cortical neoplasms in the pediatric population: a clinicopathologic and immunophenotypic analysis of 83 patients. Am J Surg Pathol., 27, 867-81.
511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560
Wilmot Roussel, H., Vezzosi, D., Rizk-Rabin, M., Barreau, O., Ragazzon, B., Rene-Corail, F., de Reynies, A., Bertherat, J. & Assie, G. (2013) Identification of gene expression profiles associated with cortisol secretion in adrenocortical adenomas. J Clin Endocrinol Metab, 98(6), E1109-21.
Zheng, S., Cherniack, A. D., Dewal, N., Moffitt, R. A., Danilova, L., Murray, B. A., Lerario, A. M., Else, T., Knijnenburg, T. A., Ciriello, G., Kim, S., Assie, G., Morozova, O., Akbani, R., Shih, J., Hoadley, K. A., Choueiri, T. K., Waldmann, J., Mete, O., Robertson, A. G., Wu, H. T., Raphael, B. J., Shao, L., Meyerson, M., Demeure, M. J., Beuschlein, F., Gill, A. J., Sidhu, S. B., Almeida, M. Q., Fragoso, M. C., Cope, L. M., Kebebew, E., Habra, M. A., Whitsett, T. G., Bussey, K. J., Rainey, W. E., Asa, S. L., Bertherat, J., Fassnacht, M., Wheeler, D. A., Cancer Genome Atlas Research, N., Hammer, G. D., Giordano, T. J. & Verhaak, R. G. (2016) Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma. Cancer Cell, 29(5), 723-36.
Figure Legends
Figure 1. Scheme of androgen producing pathways depicting steroid metabolites and genes involved in the conversion of pregnenolone (Preg) to dihydrotestosterone (DHT). The classic pathway of DHT biosynthesis via testosterone (T) is shown in light grey, while the more recently described alternative, backdoor pathway for DHT production is shown in darker grey. Enzymes are given in italic. Prog - progesterone, DHProg/DHP - dihydroprogesterone, 17OHPreg - 17-hydroxy-pregnenolone, 17OHP - 17- hydroxy-progesterone, 17OHDHP - 17-hydroxy-dihydroprogesterone, Allo - allopregnanolone, 17OHAllo - 17-hydroxy-allopregnanolone, DHEA - dehydroepiandrosterone, 16OH-DHEA - 16- hydroxy-dehydroepiandrosterone, 44A - androstenedione, A’dione - androstanedione, A’diol - androstanediol, AST - androsterone, 110HAST - 11-hydroxy-androsterone.
Figure 2. Urine androgen profiles. In 3 patients (cases 1-3), we were able to perform urinary steroid profiling either from a 24h urine collection (case 1) or on spot urines (cases 2 and 3) using established GC/MS methods (Ackermann et al, 2015; Dhayat et al, 2015; Henschkowski et al, 2008). In all cases, 2 ACC and one ACA, all measured androgen metabolites synthesized via both pathways, the classic and alternative, were found far above the upper limit for age and sex.
Figure 3. Immunohistochemistry for androgen producing enzymes in normal adrenal tissue in comparison to tumor tissues of adrenocortical carcinomas (ACT). Androgen producing tumors (n=7)
561 562 563 564 565 566 567 568 569 570 571 572 573 574
575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591
592 593 and normal adrenal tissue were stained using specific antibodies against androgen producing proteins (Suppl Table 1). With the depicted adrenocortical carcinomas the big variability in enzyme expression 594 between tissues is illustrated. Case 3 differed markedly from normal tissue in the expression of HSD3B2, RoDH, CYP17A1, SRD5A1 and AKR1C3. By contrast, case 2 showed a similar enzyme expression pattern as normal adrenal tissue. Representative pictures are given in a magnification of 10x (scale bar = 200um). HSD3B2, 3ß-hydroxysteroid dehydrogenase, type 2; RoDH, retinol dehydrogenase/17ß- hydroxysteroid dehydrogenase type 6; CYP17A1, 17a-hydroxylase/17,20-lyase; SRD5A1, 5a-reductase, type 1; AKR1C2/3/4, aldo-keto reductase family 1, member C2/C3/C4.
Figure 4. Semi-quantitative analysis of the expression of backdoor pathway enzymes in seven androgen producing tumors and in the zones of the normal adrenal cortex. Protein/enzyme expression in tumor tissues were assessed by two parameters, a) the intensity of the IHC staining (0-1-2-3) and b) the percentage of positive cells (0-100%); both parameters revealed the Q-score for each androgen producing tumor (n=7). A. The diagram shows the final Q-scores representing the mean for each tumor assessed by 4 independent investigators. For each enzyme the median Q-score of the seven cases is depicted as black bar. The non-uniform pattern of the scatter blot illustrates the high variability of enzyme expression between the seven tumors. B. Q-scores of enzyme expression in the three layers of the adrenal cortex. ACC, adrenocortical carcinoma; ACA, adrenocortical adenoma; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; HSD3B2, 3ß-hydroxysteroid dehydrogenase, type 2; RoDH, retinol dehydrogenase/17ß-hydroxysteroid dehydrogenase type 6; CYP17A1, 17a-hydroxylase/17, 20-lyase; SRD5A1, 5a-reductase, type 1; AKR1C2/3/4, aldo-keto reductase family 1, member C2/C3/C4
ACCESO ME
595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611
Table 1: Summary of clinical data and histological findings of reported patients with adrenal and ovarian tumors
F - female, M - male, ND - not done
| Case 1 Bern | Case 2 Bern | Case 3 Bern | Case 4 Bern | Case 5 Prague | Case 6 Prague | Case 7 Prague | |
|---|---|---|---|---|---|---|---|
| Gender | F | F | M | F | F | F | F |
| Age at manifestation | 13.5 years | 8 months | 8 months | 10.5 years | 13 years | 6 months | 17 years |
| Age at diagnosis | 2 years | 8 months | 11.5 years | 14 years | 7 months | 17 years | |
| Manifestation | Secondary amenorrhea, hypertrichosis, weight gain | Premature pubarche, recurrent facial acne - like exanthema, sweating, clitoris hypertrophy | Pseudopubertas praecox, Cushing | Weight loss, lack of growth, hypertrichosis, behavioral problems | Back pain, secondary amenorrhea, hirsutism/hypertrichosis | Excessive weigh gain, premature pubarche, Cushing, hirsutism, acne | High blood pressure, edema, hirsutism/hypertrichosis. |
| Pubertal development (Tanner stage) | B5, P6 | B1, P3 | G2-3, P3 | B1, P3 | B5, P6 | B1, P2 | B5, P5 |
| Acne | No | Yes | Yes | Yes | No | Yes | Yes |
| Hypertrichosis/hirsutism | Yes | No | No | Yes | Yes | Yes | Yes |
| Cushing | Yes | No | Yes | Yes | Yes | Yes | Yes |
| Hypertension | No | Yes | Yes | No | No | Yes | Yes |
| Other complications | Relapse | Brain tumor diagnosed at 12 months of age | Severe osteomalacia | Hypertrophic myocarditis | Pregnancy | ||
| Imaging | Tumor of the left ovary | Tumor of the left adrenal gland - 10x12cm | Tumor of the right adrenal gland - 5.0×4.0×3.5cm | Tumor of the left adrenal gland - 2.5x2.4x3.2cm | Tumor of the right adrenal gland - 2.4×2.9cm | Tumor of the left adrenal gland - 4.8x4.8×4.6cm | Tumor of the right adrenal gland - 10.6x9.0×9.7cm |
| Histology | Steroid-cell tumor NOS ("not otherwise specified") | Adrenocortical carcinoma | Adrenocortical carcinoma | Adrenocortical adenoma | Adrenocortical adenoma | Adrenocortical carcinoma | Adrenocortical carcinoma |
| Genetics | ND | Li-Fraumeni syndrome (TP53 mutation) | Li-Fraumeni syndrome (TP53 mutation) | ND | ND | Negative for Li- Fraumeni syndrome (no TP53 mutation) | ND |
| Treatment | Salpingo - oophorectomy | Adrenalectomy with nephrectomy | Adrenalectomy, 4 months later inoperable brain tumor, palliative care | Adrenalectomy | Adrenalectomy, treatment of osteomalacia | Adrenalectomy, antihypertensive therapy | Adrenalectomy, chemotherapy |
| Outcome (age) | Alive | Alive | Died (13 months) | Alive | Alive | Alive | Alive |
| Case 1 Bern | Case 2 Bern | Case 3 Bern | Case 4 Bern | Case 5 Prague | Case 6 Prague | Case 7 Prague | |
|---|---|---|---|---|---|---|---|
| Age (months/years) | 13.5 years | 8 months | 8 months | 10.5 years | 13 years | 6 months | 17 years |
| ACTH (ng/l) | 86.1* (N 7.2 - 63.3) | 1.8 (N 7.2 - 63.3) | <5 (N 5.0 - 50.0) | <2 (N 9.0 - 50.0) | <4 (N 7.2 - 63.3) | <4 (N 7.2 - 63.3) | <4 (N 7.2 - 63.3) |
| LH (IU/l) | 5.4 (N 0.5 - 41.7) | <0.07 (N 0.2 - 6.0) | <0.07 (N 0.2 - 6.0) | ||||
| FSH (IU/l ) | 5.2 (N 1.6-17.0) | 0.42 (N 1.2 - 7.8) | 0.92 (N 1.2 - 7.8) | ||||
| Progesterone (nmol/l) | 6.05 (N 0.01 - 3.20) | ||||||
| 17-OHP (nmol/l) | 66 (N < 9.0) | 14.2 (N < 6.0) | 20 (N < 6.0) | 4.25 (N 0.64 - 7.44) | 15.59 (N 0.64 - 7.44) | ||
| Cortisol (nmol/l) | 835* (N 171 - 536) | 410 (N 171 - 536) | 475 (N 140 - 470) | 774 (N 137 - 600) | 901 (N 263 - 724) | 1810 (N 263 - 724) | 5462 (N 263 - 724) |
| DHEA (nmol/l) | 36.0 (N 7.0 - 30.0) | 71.0 (N 0.7 - 4.6) | 107 (N 0.7 - 4.6) | ||||
| DHEAS (umol/l) | 10.77 (N 1.77 - 9.99) | > 27 (N 0.01 - 0.53) | > 27 (N < 3.0) | 0.97 (N < 3.0) | 12.50 (N 0.06 - 1.00) | 21.07 (N 0.06 - 1.00) | |
| Androstendione (nmol/l) | > 35 (N < 8.4) | 31.8 (N < 1.8) | > 35 (N 0.3 - 1.7) | 3.5 (N 1.5 - 6.6) | |||
| Estradiol (pmol/l) | 165 (N 110 - 367) | 23.0 (N 22.0 - 99.1) | <18.35 (N 22.0 - 99.1) | ||||
| Free Testosterone (pmol/l) | 61.8 (N 1.13 - 9.80) | 108 (N 0.1 - 5.4) | 47.9 (N < 3.6) | < 0.1 (N 0.1 - 6.0) | 48.55 (N 0.1 - 6.0) | 11.56 (N 0.1 - 6.0) | |
| Testosterone (nmol/l) | 47.8 (N 0.4 - 1.7) | 2.07 (N 0.4 - 1.7) | |||||
| 11-Desoxycortisol (nmol/l) | 22.0 (N < 12.0) | 11.9 (N < 12.0) | |||||
| Prolactine (μg/Ι) | 69.3* (N 3.0 - 14.4) | 11.8 (N 3.0 - 14.4) | 27.3 (N 3.0 - 14.4) | ||||
| Urinary free cortisol nmol/24h | 11.0 (N < 60.0) | 1547 (N < 40.0) |
ACCEPTED MANUSCRIPT
16OHDHEA
Androstenetriol
Classic Pathway
Preg
CYP17A1
17OHPreg
CYP17A1
DHEA
HSD17B
Androstenediol
HSD3B
HSD3B
HSD3B
HSD3B
CYP17A1
HSD17B
Prog
170HP
44A
T
SRD5A
SRD5A
SRD5A
1
SRD5A
Etiocholanolone
DHProg
CYP17A1
CYP17A1
HSD17B
17OHDHP
A’dione
DHT
AKR1C
AKR1C
RoDH
AKR1C
RoDH
AKR1C
Allo
CYP17A1
CYP17A1
HSD17B
17OHAllo
AST
A’diol
Backdoor Pathway
110HAST
ACCEPTED MANUS
ACCEPTED MANUSCRIPT
8000
4000
Case 1
-
-
1000
Upper limit
[ug/24h]
1000
-
500
-
100
-
100
50
-
0
Dehydroepiandrosterone
16a-OH-Dehydroepiandrosterone
Androstenediol -
Testosterone -
5a-DH-Testosterone -
Androstanediol/Dihydroandrosterone -
Androsterone -
5-Androstenetriol -
116-OH-Androsterone -
Etiocholanolone
ug/mmol Creatinine
100000
50000
5000
☒
Case 2
· Case 3
1000
☒
☒
Upper limit sex independant
100
- Upper limit boy
50
- Upper limit girl
0
Dehydroepiandrosterone
16a-OH-Dehydroepiandrosterone
Androstenediol -
Testosterone-
5a-DH-Testosterone-
Androstanediol/Dihydroandrosterone -
Androsterone
5-Androstenetriol -
11B-OH-Androsterone-
Etiocholanolone
ACCEPTED MANUSCRIPT
| HSD3B2 | RoDH | CYP17A1 | SRD5A1 | AKR1C2 | AKR1C3 | AKR1C4 |
|---|---|---|---|---|---|---|
| Normal Adrenal | ||||||
| ACC Case 2 | ||||||
| ACC Case 3 |
ACCEP THEY
ACCEPTED MANUSCRIPT
300
· ACC, n = 4
300
XX
+ ZG
ACA, n = 2
X ZF
250
Ovarian steroid
250
* ZR
cell tumor, n = 1
200
200
×
Q-score
150
Q-score
150
100
100
50
50
0
0
HSD3B2
RoDH
CYP17A1
SRD5A1
AKR1C2
AKR1C3
AKR1C4
HSD3B2
RoDH
CYP17A1
ACCEPTED MANUS CI
SRD5A1
AKR1C2
AKR1C3
AKR1C4
ACCEPTED MANUSCRIPT
Marti et al. Androgen production by pediatric ACT
Highlights
1) Pediatric adrenocortical tumors (pACT) typically produce large amounts of androgens
2) Androgen production in pACT may occur through the classic and/or backdoor pathway
3) Both adenomas and carcinomas may use the backdoor androgen production pathway
4) Alternate pathways may offer novel treatment targets for androgen excess disorders
ACCEPTED MANUS CRINE