Adrenocortical Carcinoma Is Characterized by a High Frequency of Chromosomal Gains and High-Level Amplifications
Martha Dohna,’ Martin Reincke,2 Antoaneta Mincheva,’ Bruno Allolio,3 Sabina Solinas-Toldo,’ and Peter Lichter1*
‘Organisation komplexer Genome, Deutsches Krebsforschungszentrum, Heidelberg, Germany
2Abteilung Innere Medizin II, Medizinische Klinik und Poliklinik, Universität Freiburg, Freiburg, Germany
3Endokrinologie, Medizinische Universitätsklinik Würzburg, Würzburg, Germany
Distinction of adrenocortical carcinoma from benign adrenocortical lesions by standard criteria is often difficult. In order to search for additional diagnostic parameters, a series of 25 adrenocortical tumors, 8 adenomas, 14 primary carcinomas, 1 metastasis, and the 2 adrenocortical carcinoma cell lines SW13 and NCI-H295 were analyzed by the approach of comparative genomic hybridization (CGH). Except for the two smallest adenomas, all tumors showed chromosomal imbalances with a high incidence of chromosomal gains, most frequently involving chromosomes or chromosome arms 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q. The only significant loss of material concerned the distal part of 9p. Furthermore, 21 high-level amplifications were identified in 15 different regions of the genome. The consensus regions of recurrent gains and the focal high-level amplifications allowed identification of a series of chromosomal subregions containing candidate proto-oncogenes of potential pathogenic function in adrenocortical tumors: Ip34.3-pter, Iq22-q25, 3p24-pter, 3q29, 7p11.2-p14, 9q34, Ilq12-11q13, 12q13, 12q24.3, 13q34, 14q1 1.2-q12, 14q32, 16p, 17q24-q25, 19p13.3, 19q13.4, and 22ql 1.2-q12. A subset of the CGH data was independently confirmed by interphase cytogenetics. Interestingly, the adenomas larger than 4 cm contained gained material of regions also overrepresented in carcinomas. In addition, several chromosomal gains, in particular the high-level amplifications, were exclusive for the malignant status of the tumors. These data indicate that the larger adrenal lesions need to be carefully considered in the diagnosis of adrenocortical tumors, and that genetic aberrations might provide useful markers for a better diagnostic differentiation. Genes Chromosomes Cancer 28:145-152, 2000. @ 2000 Wiley-Liss, Inc.
INTRODUCTION
Adrenocortical carcinoma is a rare malignancy with an estimated incidence of one to two cases per million population per year in the United States (Thompson et al., 1983). More than two-thirds of the patients already suffer from an advanced tumor stage with local invasion or distant metastases, mainly in the liver, lungs, bones, and brain, at the time of diagnosis (Soreide et al., 1992). Once diag- nosed, the prognosis of adrenocortical carcinoma is poor, with the median survival reported to be less than 2 years (Bodie et al., 1989). In contrast, benign adrenocortical lesions are frequent, with a preva- lence of 1% in the population (Copeland, 1983). Most of these benign lesions are clinically silent and are detected incidentally by imaging tech- niques such as ultrasound or computed tomography (Ross and Aron, 1990). The pathogenesis of these lesions is still unknown. Tumor size and histology are presently the two main criteria for distinguish- ing between benign and malignant adrenocortical tumors. Because these criteria are often insuffi-
cient, genetic studies have been performed aimed at the identification of better diagnostic markers.
Progress in the understanding of adrenocortical tumorigenesis has been slow, mainly because tu- mor tissue is difficult to obtain. Studies of genetic factors using the candidate gene approach showed a low prevalence of mutations in tumor suppressor genes and oncogenes in adenomas (Gicquel et al., 1995; Reincke, 1998). Mutations in G-protein-cou- pled receptors and G-protein identified in growth hormone-secreting pituitary tumors and thyroid tu- mors have not been found in adrenal-cortex neo- plasms (Latronico et al., 1995). This supports the concept that the signal transduction cascade con- trolling tumor growth in adrenocortical neoplasms is different from that of other endocrine tumors.
Supported by: Deutsche Krebshilfe (to P.L.); Wilhelm-Sander- Stiftung, München (to M.R.).
*Correspondence to: Dr. Peter Lichter, Deutsches Krebsfor- schungszentrum, Abt. Organisation komplexer Genome, Im Neuen- heimer Feld 280, 69120 Heidelberg, Germany.
E-mail: p.lichter@dkfz-heidelberg.de
@ 2000 Wiley-Liss, Inc.
| Tumor | Age (years) | Sex | Tumor size (cm) | Hormone excess | Histology |
|---|---|---|---|---|---|
| 1 | 43 | F | 2.0 | Cushing's syndrome | Adenoma |
| 2 | 31 | F | 3.5 | Cushing's syndrome | Adenoma |
| 3 | 48 | F | 3.6 | Cushing's syndrome | Adenoma |
| 4 | 54 | F | 4.0 | Nonfunctional | Adenoma |
| 5 | 67 | F | 4.3 | Nonfunctional | Adenoma |
| 6 | 68 | F | 4.5 | Nonfunctional | Oncocytic adenoma |
| 7 | 44 | F | 7.0 | Cushing's syndrome | Adenoma |
| 8 | 35 | F | 7.0 | Nonfunctional Cushing's syndrome, | Adenoma |
| 9 | 17 | F | 8.0 | virilization | First recurrence, carcinoma |
| 10 | 79 | F | 8.0 | Conn's syndrome | Carcinosarcoma |
| 11 | 57 | F | 8.0 | Cushing's syndrome | Carcinoma |
| 12 | 38 | M | 10.0 | Cushing's syndrome | Carcinoma |
| 13 | 64 | F | 12.0 | Unknown | Carcinoma |
| 14 | 22 | F | 12.0 | Virilization | Carcinoma |
| 15 | 75 | F | 12.5 | Virilization | Carcinoma |
| 16 | 26 | F | 14.0 | Nonfunctional | Carcinoma |
| 17 | 38 | M | 15.0 | Unknown | Carcinoma |
| 18 | 14 | F | 16.0 | Virilization | Carcinoma |
| 19 | 13 | F | >16.0 | Cushing's syndrome | Second recurrence, carcinoma |
| 20 | 46 | F | 17.0 | Nonfunctional | Carcinoma |
| 21 | 46 | F | 18.0 | Virilization | Carcinoma |
| 22 | 15 | F | 25.0 | Cushing's syndrome | First recurrence, carcinoma |
| 15A | Metastatic lymph node of tumor 15 Immortalized adrenocortical tumor cell line | ||||
| SW13 | F | Nonfunctional | (ATCC) Immortalized adrenocortical tumor cell line | ||
| NCI-H295 | F | Steroid-producing | (ATCC) |
There are only few data on the genetic aberra- tions in this tumor. The most frequently recurring alterations described are loss of heterozygosity (LOH) in 11p, 11q, 13q, and 17p affecting the TP53 locus (Henry et al., 1989; Yano et al., 1989; Sameshima et al., 1992; Ohgaki et al., 1993; Herr- mann et al., 1994; Reincke et al., 1994; Wagner et al., 1994; Iida et al., 1995; Reincke, 1998). A mo- lecular cytogenetic analysis of eight adrenocortical carcinomas by comparative genomic hybridization (CGH) revealed gains or losses affecting all chro- mosomes, while only in a minor fraction of adeno- mas changes were detected (Kjellman et al., 1996). In contrast, a very recent study revealed character- istic gains and losses in adrenocortical carcinomas (six cases) as well as adenomas (three cases) (Figueiredo et al. 1999). We here report on a CGH study of 25 samples of adrenocortical tumors, in- cluding 14 primary carcinomas, revealing a set of characteristic recurrent imbalances. A subset of the CGH data was independently confirmed by inter- phase cytogenetics. The implication of the genetic data concerning a presumed progression from ade- noma to carcinoma is discussed.
MATERIALS AND METHODS
Tumor Samples
The tumor samples obtained from a total of 22 adult patients (20 female, 2 male) with adrenocortical tumors and from 2 established cell lines of this tumor type were collected at the Department of Medicine of the University of Würzburg (Germany) and are listed in Table 1. From the 22 patients, 8 adenomas, 14 carcinomas, and 1 metastatic lymph node of 1 of the carcinomas were examined. Neither the histo- logic analysis of the adenomas nor the clinical fol- low-up of the patients with adenoma suggested the presence of a malignant tumor. For all the tumor samples, genomic DNA was prepared from tissue blocks, which were either frozen in liquid nitrogen or formalin-fixed and paraffin-embedded (tumors 11, 15, 21, 15A) immediately after surgical removal. Stan- dard protocols based on proteinase K digestion and phenol-chloroform extraction were applied (Lichter et al., 1996).
CGH
Labeling of the probe, hybridization, and image acquisition were performed as reported previously
(Lichter et al., 1995). Metaphase chromosome spreads were prepared from phytohemagglutinin- stimulated peripheral blood leukocytes of a healthy female individual according to standard proce- dures. Control DNA was isolated from peripheral blood lymphocytes of a healthy male individual. One microgram of biotin-labeled tumor DNA and 1 µg of digoxigenin-labeled control DNA were com- bined with 80 µg of human Cot-1 DNA in a 12-ul hybridization cocktail. After denaturation and hy- bridization for 2 to 3 days, slides were washed to a stringency of 0.1 × SSC at 60℃. Hybridized tumor DNA was detected via avidin-FITC and control DNA via mouse-antidigoxigenin antibody conju- gated to TRITC. The chromosomes were counter- stained with DAPI (4,6-diamidino-2-phenylindole) to allow their identification.
For each tumor sample, images from 20 meta- phase cells were acquired separately for each fluo- rochrome using a fluorescence microscope equipped with a cooled CCD camera (Photometrics, Tucson, AZ). The ratio of FITC:TRITC fluorescence in- tensities along each individual chromosome was calculated using dedicated software (Du Manoir et al., 1995). For each chromosome, ratio values were averaged from a minimum of seven metaphase cells and converted into a profile plotted next to each chromosomal ideogram. Chromosomal imbal- ances were defined as a deviation of the ratio pro- file from the balanced value of 1. The diagnostic values 0.75 and 1.25 were used as cutoff levels indicating under- and overrepresentations, respec- tively. These values had previously been tested in many CGH studies using this analysis program (Bentz et al., 1995; Du Manoir et al., 1995). Criteria for high-level amplifications were either overrepre- sentations exceeding the value of 2.0 or strong focal hybridization signals clearly detectable by visual inspection and with a ratio profile diagnostic for overrepresentation. Of the chromosome regions recognized to be problematic in CGH analyses (Kallioniemi et al., 1994; Du Manoir et al., 1995), region 1p32-pter and chromosome 19 were not scored in the results for reasons specified elsewhere (Solinas-Toldo et al., 1996). However, in accor- dance with previous studies, alterations found on these two regions were scored in the case of high- level amplifications or if they were confirmed by interphase cytogenetics.
Interphase Cytogenetics
Five chromosomal regions, which were found to be gained by CGH, were selected for additional analysis by interphase cytogenetics: 1p36.3, 9q34.1,
| Tumor | Probe | Region | Increased number of signalsª (in %) |
|---|---|---|---|
| 5 | YAC35B2 | 22q12.2 | 36.4 |
| 7 | pSE16-2 | 16cen | 15 |
| YAC35B2 | 22q12.2 | 18 | |
| 9 | YAC147e10 | 9q34.1 | 83.3 |
| 10 | pSE16-2 | 16cen | 31 |
| 16399 | 19p13.3 | 43.3 | |
| 13 | YAC147e10 | 9q34.1 | 11 |
| pSE16-2 | 16cen | 14 |
aPercentage of cells with the number of signals greater than the cutoff levels defined on control cells.
16cen, 19p13.3, and 22q12.2. The following DNA probes were used: p1-79, a repetitive probe map- ping to chromosome arm 1p (Buroker et al., 1987); YAC147e10 coding for the ABL1 gene in chromo- some region 9q34.1 (Chumakov et al., 1995); pSE16-2, an alpha-satellite derived from the cen- tromere of chromosome 16 (Greig et al., 1989); cosmid 16399 coding for the gene TCF3 on 19p13.3 (kindly provided by Ann Olsen, Livermore, CA); and CEPH YAC 35B2 coding for the immunoglob- ulin locus on chromosome region 22q12.2 (Frippiat et al., 1995). The alpha satellite p&10RP8 (Devilee et al., 1988), specific for the centromere of chromo- some 10, served as a positive control for hybridiza- tion efficiency, because it was known from the CGH results that chromosome 10 was disomic in all of the tumors analyzed by interphase cytogenetics. The combinations of probes and tumors are given in Table 2. For hybridization, nuclei were isolated from frozen tissue samples as described elsewhere (Hopman et al., 1992). After two-color FISH using differently labeled test and control DNA probes, 200 cells were evaluated for the presence of hy- bridization signals. Diagnostic cutoff values were defined for each probe using five different popula- tions of normal lymphocytes as described (mean of signal number + three times the standard devia- tion) (Stilgenbauer et al., 1993), scoring all nuclei with more signals derived from probe DNA than from control DNA. Because the control cells orig- inated from a tissue different from the tumors, probes were also hybridized to one available sam- ple of interphase nuclei extracted from the cortex of an adrenal gland of a healthy individual, yielding the same distribution of hybridization signals as did the control lymphocytes. It should be noted that there were discrepancies between interphase FISH and CGH results in the two regions 1p32-pter and chromosome 19, which can be attributed to the
occurrence of false-positive CGH data in these two regions. In one case, the interphase analysis of chromosome 19 confirmed the CGH ratio values; it is therefore the only one listed in Table 2.
RESULTS
CGH
Of a total of 25 samples of adrenocortical tumors (22 primary tumors, 1 metastasis, and 2 cell lines) examined by CGH, all except the 2 smallest ade- nomas showed chromosomal imbalances (Fig. 1). Gains of chromosomal material clearly outweighed losses and were observed on each chromosome. The most frequent gains of chromosomal material were on chromosomes 5 (12 tumors), 7p (11 tu- mors), 8 (9 tumors), 9q (12 tumors), 11q (10 tu- mors), 12q (18 tumors), 14q (10 tumors), 16 (18 tumors), 17q (12 tumors), 19 (8 tumors), 20 (15 tumors), and 22q (7 tumors). For several recur- rently overrepresented regions, comparison of the extension of the gained material allowed us to narrow the critical region down to a considerably smaller subregion, namely, distal 9q34, 11q12-q13, 12q24.3, and 14q32. Significant loss of chromo- somal material was found in the distal part of chro- mosome arm 9p (six tumors). The cell lines exhib- ited the same pattern of imbalances as did the primary tumors.
In small tumors, the number of chromosomal imbalances was small; however, larger tumors showed considerably more gains and losses (Fig. 2). Genetic aberrations were detected only when the tumor size exceeded 3.5 cm, and the adenomas larger than 4 cm showed 5-7 gains of chromosomal regions. Interestingly, the changes discovered in adenomas affected the same regions as those in carcinomas.
Numerous high-level DNA amplifications were identified, which are located at 1p34.3-pter (one tumor), 1q22-q25 (two tumors), 3p24-pter and 3q29 (one tumor each), 7p11.2-p14 (one tumor), 12q13 and 12q24.1-qter (one tumor), 13q34 (one tumor), 14q11.2-q12 and 14q31-q32 (one tumor each), 16p (one tumor), 17q24-q25 (one tumor), 19p and 19q (five and three tumors, with the com- monly involved regions 19p13.3 and 19q13.4, re- spectively), and 22q11.2-q12 (one tumor). For each tumor with a high-level amplification, the respective CGH profile is shown in Figure 3.
Interphase Cytogenetics
In order to confirm independently chromosomal imbalances identified by CGH ratios in our series
of tumors, certain chromosomal regions were sub- jected to a copy number analysis by interphase cytogenetics as detailed above. The respective re- gions, the DNA probes, as well as the results of the interphase study are listed in Table 2. Interphase cytogenetics confirmed all gains affecting 9q34.1, 16cen, and 22q12.2.
DISCUSSION
The analysis of 25 samples of adrenocortical tu- mors by comparative genomic hybridization re- vealed numerous, previously unknown chromo- somal changes. Of the characteristic imbalances, gains of chromosomal regions were by far more frequent than losses. Whereas 12 regions were found to be overrepresented in at least 7 tumors, the only frequently occurring underrepresentation was loss of distal 9p in 6 samples. These data add substantially to the genetic aberrations found asso- ciated with this tumor type, because very little is known about specific overrepresented regions. In contrast to our results, another study did not iden- tify a predominance of chromosomal gains (Kjell- man et al., 1996). However, a recently published CGH analysis of six adrenocortical carcinomas and three adenomas revealed a set of recurrent chro- mosomal imbalances, which are in part concordant to the data presented here (Figueiredo et al., 1999). Interestingly, of those chromosome regions for which frequent LOH (Yano et al., 1989; Iida et al., 1995) has been described in this tumor type, CGH revealed loss of material only for 11p and 13q. Chromosome arms 11q and 17p were found to be balanced or gained, indicating that either the re- spective regions are too small to be detected by CGH-only regions ≥ 10 Mb can be reliably de- tected as loss by chromosomal CGH (Bentz et al., 1998)-or that there is a high frequency of unipa- rental disomy in the analyzed tumor type.
In the present CGH study, numerous high-level amplifications were discovered (Figs. 1 and 3). This is in contrast to the above-mentioned report ana- lyzing eight adrenocortical carcinomas, in which no high-level amplifications were found (Kjellman et al., 1996), but in agreement with the study by Figueiredo et al. (1999), who identified six high- level amplifications, which matched with amplifi- cations (three cases) or low copy number gains (three cases) detected in our study. The recur- rently gained or lost regions identified in our study provide the basis for the identification of proto- oncogenes or tumor suppressor genes, respectively, residing within the respective regions and possibly playing an important role in the etiology or devel-
tions are indicated by thicker lines. Different colors represent carci-
correspond to gain of genetic material. High-copy-number amplifica-
Figure 1. Summary of chromosomal imbalances identified in 25 adrenocortical tumor samples by CGH. Vertical lines on the left side of chromosome ideograms indicate loss, and vertical lines on the right
3
9
13
19
a
19
DEI
13
7
SW13
1
13
9
18
9
22
16
6
22
20
13
22
SW13
9
13
19
12
22
NCI-H295
17
20
15A
10
11
NCI-H295
NCI-H295
16
9
1
14
SW13
11
10
14
12
12
18
20
22
22
10
17
15
15A
9
18
SW13
11
0
20
NCI-H295
N
22
18
13
20
NCI-H295
15A
17
-
SW13
22
9
16
13
16
15A
15
18
20
SW13
80
5
6
8
7
16
10
NCI-H295
SW13
15
11
14
12
21
20
9
10
20
18
NCI-H295
8
SW13
SW13
7
NCI-H295
19
SW13
18
NCI-H295
w
E
SW13
17
14
10
13
12
16
22
20
0
SW13
14
10
13
16
22
16
22
9
17
Methods.
Numbers above or below each line refer to the tumor sample analyzed. Imbalances found on the chromosomal region Ip32-pter and on chro- mosome 19 were neglected (*) for reasons outlined in Materials and
noma (red), metastasis (gray), adenoma (blue), and cell line (green).
ADRENOCORTICAL CARCINOMA
21
16
20
15A
13
4
5
22
SW13
11
10
NCI-H295
15
12
20
18
13
21
10
17
16
15A
19
6
22
11
9
16
7
8
SW13
11
5
NCI-H295
22
SW13
NCI-H295
SW13
16
12
NCI-H295
9
+
19
15
17
20
11
5
23
22
20
7
NCI-H295
17
17
13
NCI-H295
00
20
18
19
20
15A
17
13
19
NO
8
22
9
SW13
16
5
7
SW13
12
20
16
8
NCI-H295
SW13
13
SW13
9
11
X
18
15
9
21
i
10
22
11
14
12
- 17
12
13
20
12
18
15
17
6
21
19
22
18
11
9
6
15A
15A
19
6
18
8
5
8
NCI-H295
NCI-H295
SW13
22
Adenoma
18
Total number of imbalances
16
14
12
10
8
6.
4-
2
0
Tumor 2 3.5 3.6 4 4.3 4.5 7
7
8 8 8 10 12 12 12.5 14 15 16 >16 17 18 25
size (cm)
Gain
Loss
tumor 22 Chr.1
NCI-H295 Chr.1
tumor 22
NCI-H295 Chr. 12
Chr.3
SW13 Chr. 3
tumor 17 Chr. 7
NCI-H295 Chr. 13
NCI-H295 Chr. 14
SW13 Chr.14
NCI-H295 Chr. 16
tumor 22 Chr.17
NCI-H295 Chr.22
tumor 12 Chr. 19
tumor 16 Chr. 19
tumor 19 Chr. 19
tumor 20 Chr. 19
tumor 22 Chr. 19
tumor 23 Chr. 19
NCI-H295 Chr. 19
opment of adrenocortical carcinomas. Comparison of the extensions of the gains or losses of a given chromosomal region allows us to define the small- est commonly imbalanced region, and thus to nar-
row the genomic region of interest considerably (Fig. 1); for a review of chromosomal losses in human tumors detected by CGH, see Knuutila et al. (1999). Focal hybridization signals, which are
generated by high-level amplifications, are partic- ularly suitable for the definition of small genomic intervals harboring a putative oncogene of patho- genic relevance; for a review of high-level amplifi- cations in human tumors detected by CGH, see Knuutila (1998). There are numerous genes within the highly amplified regions of the analyzed adre- nocortical tumors for which an oncogenic function can be assumed, but two groups of genes deserve particular consideration.
Fibroblast growth factors 12 and 14 are localized within two of the amplified regions, namely, 3q28- qter and 13q34, respectively. It has previously been shown that the adrenocortical carcinoma cell line SW13 produces autocrine fibroblast growth factors mediating anchorage-independent growth (Corin et al., 1990). CGH of SW13 revealed amplification of the chromosomal segment harboring FGF12. Be- cause genomic amplification is a hallmark for over- expression of a gene, it is tempting to speculate that overexpressed FGF12 provides the autocrine factor responsible for the ability of SW13 to grow in soft agar. By analogy, such a role could also be postulated for FGF14 in the adrenocortical carci- noma cell line NCI-H295. It should be pointed out, however, that the chromosomal regions harboring FGF12 and FGF14 were found amplified only in cell lines, while in primary tumors the respective regions were not highly overrepresented. However, amplification of the chromosomal region containing FGF14 has recently been reported for one primary tumor (Figueiredo et al. 1999).
Interestingly, of the numerous candidate genes localized at the highly amplified chromosome re- gions, several code for proteins of the insulin, the somatostatin, or the growth hormone signaling pathway. Overexpression of IGF1 and IGF2 as well as their receptors and the insulin receptors has been reported, including their putative role in the autocrine/paracrine regulation of adrenocortical tu- mors (Kamio et al., 1991; Ilvesmaki et al., 1993).
To this date, it remains difficult to differentiate adrenocortical adenomas from carcinomas in the absence of metastasis. Tumor size and histology are presently the two main criteria to distinguish between benign and malignant tumor growth. Un- fortunately, these criteria are often not sufficient, and therefore there is a strong need for better diagnostic markers. Analyses of genetic aberrations in adrenocortical tumors aim at the identification of new prognostic markers to supplement the present diagnostic approach. In this context, it seems note- worthy that gains of chromosome 7, 14, or 19 and, more strikingly, high-level amplifications were ob-
served only in carcinomas but not in adenomas. Whereas the overall number of analyzed tumors is still too small to define a prognostic marker of statistical significance, the fact that high-level am- plifications are found exclusively in carcinomas is in line with the data obtained in other tumors, where such amplifications are late events during tumorigenesis (Alitalo and Schwab, 1986). The fact that CGH revealed a high-level amplification in a metastasis (tumor 15A), whereas no such amplifi- cation was present in its primary tumor (tumor 15, which otherwise exhibited almost the same imbal- ances as 15A) might also serve as an argument for the concept of high-level amplifications as late events in the development of adrenocortical tu- mors. In view of this concept, the detection of high-level amplifications in adrenocortical carcino- mas might become a useful diagnostic marker of possible prognostic relevance.
At present, it is still debatable whether there is a definite progression from adenoma to carcinoma. A comparison between the genomic imbalances ob- served in the present study in both groups clearly shows that, although there are considerably fewer aberrations in adenomas, the same chromosomal regions are affected as in carcinomas, strongly fa- voring the concept of a progression toward carci- noma. It is particularly striking that these imbal- ances are observed in adenomas of increased size. In the light of these data, it seems important to revise the weighting of the size of adenomas among the diagnostic criteria.
ACKNOWLEDGMENTS
We thank Andreas Rätsch, Konstanze Döhner (both Heidelberg), and Ann Olsen (Livermore) for providing DNA probes for the interphase analysis.
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