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Original Studies |
Sainte-Justine Hospital Research Center, Departments of Pediatrics and Pathology (L.L.O.), Université de Montréal, Montréal, Québec, Canada H3T 1C5
Address all correspondence and requests for reprints to: Cheri Deal, Ph.D., M.D., Endocrine Service, Room 1706, Ste-Justine Hospital, 3175 Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5. E-mail: dealc{at}ere.umontreal.ca
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In adult ACT, genetic alterations of chromosome 11p have been found in 23–50% of sporadic ACT, whereas those involving 17p have been found in 14–64% (3, 4, 5). Other alterations involving candidate genes, such as gsp, gip2, ras, ACTH-R, and p16, have not been found or are seen only rarely (2). However, using comparative genomic hybridization of a larger series of sporadic adult tumors, chromosomal aberrations were often seen, and the frequency of multiple chromosomal losses and gains increased with tumor size (5). Indeed, the incidence of genetic alterations specifically involving 11p and 17p is much higher in carcinomas than in adenomas, with 9.5% of adenomas vs. 78.5% of carcinomas demonstrating 11p uniparental disomy (UPD), 8.7% of adenomas vs. 80% of carcinomas showing insulin-like growth factor II gene (IGF2) overexpression, and 12.5% of adenomas vs. 100% of carcinomas showing loss of heterozygosity (LOH) for 17p (3). It has therefore been suggested that the presence of these genetic alterations may have a bearing on prognosis (3, 5, 6, 7), which is traditionally difficult to predict because, unlike other carcinomas, histological diagnosis is unreliable in ACT in both adults and children (8).
Unlike the dismal survival statistics in adult ACT series (only a 20% survival rate after 5 yr), pediatric series, including one at our own center (8), report 43–89% survival. Clearly, the extent of surgical resection is the most important factor in patient outcome, but as children seem to have a better prognosis, it is important to determine whether the same molecular alterations described in adults are also present in children, and whether this is related to prognosis. Molecular studies of sporadic ACT in the pediatric age group are limited to a very few patients, most of them in the adolescent age range or coming from southern Brazil, where the incidence is strikingly high. This may be related to agrotoxic compounds and may not reflect the same pathogenic mechanisms (9).
In this study we report extensive molecular characterization of ACT from four pediatric patients (three under age 5 yr) with respect to p53 mutations (germline and somatic) and chromosomal rearrangements involving 11p15. In addition, tumor IGF2 steady state messenger ribonucleic acid (mRNA) levels were examined and compared to immunohistochemical results. IGF-II and p53 immunohistochemistry were performed on six other tumors from patients discussed in our previously published series (8). Finally, we provide evidence that the mechanisms involved in IGF2 deregulation in pediatric tumors, including IGF2 dosage effects, loss of tumor suppressor genes on the maternal chromosome 11, germline and/or somatic p53 mutations, and IGF2 methylation changes, are identical to those seen in adult adrenocortical carcinomas regardless of treatment outcome in this small series. They all lead to IGF2 overexpression and a phenotype reminiscent of the fetal adrenal cortex.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Four girls presenting at our tertiary care hospital were
included in this study (Table 1
). Patient
1 presented at 2 yr, 5 months of age with signs of virilization (pubic
hair, clitoromegaly, growth acceleration, and aggressiveness) as well
as signs of Cushing’s syndrome (obesity and acne) that had developed
over a 6-month period. She had a positive family history that was
suggestive of the Li-Fraumeni syndrome, with lung cancer, breast
cancer, intestinal cancer, and thyroid cancer present in some family
members. On physical examination she had a palpable abdominal mass.
Patient 2 presented at 10 months of age with signs of virilization
(pubic hair, clitoromegaly, and growth acceleration) of 2-month
duration. Family history was positive for an ACT in a maternal aunt,
who had been diagnosed at age 5 yr and treated by excision alone and is
now alive and well at 23 yr of age. The physical exam in patient 2
revealed virilization as described, but no abdominal mass was palpable.
Patient 3 presented at 12 yr of age with a history of pubarche at age 9
yr, but more recent temporal balding, deepening of the voice, and
increased muscle mass. She also presented clitoromegaly and a palpable
abdominal mass on physical examination. Patient 4 presented at 2 yr, 2
months of age with signs of virilization (pubic hair, clitoromegaly,
growth acceleration, and excessive muscular mass) as well as mild
obesity and an abdominal mass on palpation.
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Patients 2, 3, and 4 have shown no signs of tumor recurrence and are alive and well 3.8, 3.7, and 0.5 yr after their interventions. Patient 1 had her first local relapse 24 months after her first surgery, and despite a second surgical intervention and chemotherapy with cis-diaminedichloroplatinum II (Cisplatin; Faulding Canada, Inc., Kirkland, Quebec, Canada) and VP16–213 (Etoposide, Novopharm, Ltd., Toronto, Ontario, Canada), she had a second local relapse 1 month after that. She then had a third debulking surgery, received adjuvant radiotherapy (4500 cGy), and was given cyclophosphamide (Procytox; Carter-Horner Inc., Mississauga, Ontario, Canada) and topotecan (Hycamtin; SmithKline Beecham Pharma, Oakville, Ontario, Canada), with no response of the residual tumor. She received, in succession, irinotecan, liposomal doxorubicin [Doxyl, Doxorubicin (ribosomal); Sequus Pharmaceuticals, Inc., Menlo Park, Ca], and paclitaxel (Taxol; Bristol-Myers Squibb). Despite this her tumor volume increased, and after tumor resection, which included a portion of the liver, she received local radiotherapy (3600 cGy) and gemcitabin, but died 3.3 yr after her initial diagnosis. The secretory potential of the tumor was still noted at the last endocrine evaluation, 2 months before death, with a marked elevation of androgens.
The tumor histology of the first 3 patients was previously reported in detail (patients 1, 4, and 9, in Ref. 8). In summary, all 4 tumors were, at least focally, very pleomorphic. Mitotic activity was brisk in the first surgical specimen from patient 1 (up to 120 mitoses/50 high power fields), and atypical mitoses were numerous. Mitoses were scarce in the other 3 patients; some of these were atypical. Patient 1’s tumor was also unique in that it transgressed the capsule with a positive resection margin.
Tissue preparation and nucleic acid purification
ACT fragments were immediately frozen in liquid nitrogen and kept at -70 C until genomic DNA isolation was performed. Additional tumor fragments were preserved in 10% buffered formalin for microscopic examination and immunohistochemistry. Peripheral blood was collected in ethylenediamine tetraacetate-containing Vacutainer collection tubes (Becton-Dickinson, Franklin Lakes, NJ) after obtaining informed consent from parents and/or patients. Nuclear pellets were centrifuged at 1100 x g for 20 min and kept frozen at -20 C. Leukocyte genomic DNA was purified with phenol extractions at neutral pH as previously described (10). Genomic DNA from ACT was recovered from phenol/chloroform/isoamyl alcohol extractions after proteinase K and ribonuclease A digestion of the tissue. Total RNA from ACT was isolated by acid guanidinium isothiocyanate followed by phenol-chloroform extraction, and contaminating genomic DNA was digested with ribonuclease-free RQ1 deoxyribonuclease (Promega Corp., Madison, WI) as previously described (11).
Chromosome 11 haplotype analysis and tumor loss of heterozygosity study
Microsatellite markers D11S1246 on 11p14 (GDB ID: G00–198-082),
D11S1252 on 11p12-p11.2 (GDB ID: G00–198-088), and D11S29 (GDB ID:
187701) and D11S490 (GDB ID: 178512) on 11q23 were amplified using
Human MapPairs and PCR conditions from the protocol of Genome Services
(Research Genetics, Inc., Huntsville, AL). We routinely
used 0.2–1 µCi [
-32P]deoxy-ATP
(Amersham Pharmacia Biotech, Arlington Heights, IL) and
Taq DNA polymerase from Life Technologies, Inc.
(Gaithersburg, MD). Several restriction fragment length polymorphisms
markers (RFLP) and other polymorphic markers on 11p15.5 up to 11q23
were used to study LOH in ACT as previously described: Harvey ras
(Hras; 11p15.5) (12), H19 (11p15.5) (13),
IGF2 (11p15.5) (11), insulin (Ins; 11p15.5) (14),
tyrosine hydroxylase (TH; 11p15.5) (15),
p57KIP2 (16), apolipoprotein A-I (APO
A-I; 11q23) (17), and apolipoprotein A-IV (APO A-IV;
11q23) (18, 19). For primers and PCR conditions of the 5'-insulin
variable number of tandem repeats (5'-INS VNTR; 11p15.5),
see the report by Bennett et al. (20), except that we used
strand labeling with [-32P]deoxy-ATP and the
antisense primer VNTR (5'-TCG TCA GCA CCT CTT CCT CAG GAC CAG
CG-3').
LOH vs. paternal isodisomy
For patients 1 and 4, Southern blotting of an ApaI digest of genomic leukocytes and tumor DNA was hybridized with the IGF2 exon 9 PCR product (11). For patient 3, RsaI digests were blotted and hybridized with an exon 4–5 H19 PCR fragment probe (13). The same membranes were reprobed with a cloned PCR fragment from IGFBP3 exon 3 to normalize band intensity ratios. This probe was chosen because this locus (7p) has not been observed to be involved in the chromosomal anomalies previously reported in ACT (9). Specific bands were revealed by autoradiography and quantitated by densitometry or phosphorimaging.
Quantitative RT-PCR
RNA isolation and complementary DNA (cDNA) synthesis were
performed as described by Paquette et al. (11). The internal
competitor standard was generated using PCR-based in vitro
mutagenesis of IGF2, H19, or 
-actin
as indicated (21, 22). For IGF2 PCR, the standard is 190 bp
and consists of the same sequence as that of the exon 9-derived
IGF2 cDNA PCR product, with an internal deletion of 46 bp.
For H19 PCR, the standard is 488 bp and consists of the same
sequence as that of the exon 4- and 5-derived H19 cDNA PCR
product, with an internal deletion of 87 bp (13). For
-actin PCR, the modified standard has lost a
restriction site (BstEII) and gives a fragment of 612 bp
instead of two smaller fragments of 360 and 252 bp. IGF2 and
H19 PCR conditions have been described previously (11, 13).
For the -actin PCR, the cDNA and competitor were mixed
with sense (5'-GAC ACC AGG GCG TCA TGG TG-3') and antisense (5'-GCA GCT
CGT AGC TCT TCT CC-3') -actin primers (50 pmol each) in
the buffer supplied by Life Technologies, Inc.,
supplemented by 2 mM MgCl2,
0.2 mM of each deoxy-NTP including 2.5 µCi
[-32P]deoxy-ATP, and 2 U Taq DNA
polymerase. The cycling parameters consisted of 30 cycles of 60 s
at 94 C and 60 s at 72 C, followed by a final extension time of 10
min at 72 C. PCR-amplified products were resolved electrophoretically
and quantified by phosphorimager analysis (Molecular Dynamics, Inc., Sunnyvale, CA). All competitive PCRs were performed on
triplicate RT reactions with three concentrations of internal
standard.
Methylation analysis
The methylation status of IGF2 was analyzed by Southern blotting. Ten micrograms of genomic DNA from leukocytes and tumor were digested with the methylation-sensitive restriction enzyme AvaII, blotted, and hybridized with an IGF2 cDNA probe (exons 3, 7, 8, and 9 up to the XhoI site provided by Dr. P. E. Holthuizen). The completeness of digestion was confirmed using a plasmid as an internal control.
p53 mutation analysis
Because of a positive family history, peripheral blood from patients 1 and 2 was sent to OncorMed (Gaithersburg, MD) for p53 mutation analysis by direct sequencing.
Immunohistochemistry
IGF-II immunohistochemical staining was performed using standard methodology. Sections were pretreated with hydrogen peroxide for 20 min. Mouse anti-IGF-II (Upstate Biotechnonology, Inc., Lake Placid, NY), at a dilution of 1:100 was applied to tissue for an overnight incubation at 4 C. Phosphate-buffered saline was used to wash tissue before incubation with a nondiluted biotinylated secondary antirabbit, goat and mouse antibody (Lipshaw, Pittsburgh, PA), for 20 min. Visualization was performed with streptavidin peroxidase (Lipshaw), followed by diaminobenzidine coloring and hematoxylin nuclear counterstaining.
For p53 immunohistochemistry, deparaffinized slides were put in boiling water for 10 min, rinsed with phosphate-buffered saline, and pretreated with hydrogen peroxide for 20 min. Universal Blocking Solution was then applied undiluted for 10 min (DAKO Corp. Diagnostics Canada, Mississauga, Canada) followed by an overnight incubation with anti-p53 monoclonal antibody (NCL-p53-DO7, Novocastra Laboratories, Newcastle upon Tyne, UK). All subsequent incubations were the same as described for IGF-II.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Leukocyte and tumor DNA were analyzed in all four patients and
their parents at 11p15.5, 11p14, 11p12, and 11q23 as detailed in
Materials and Methods. Results are summarized in Table 2. LOH was demonstrated in all four
patients for part (patient 1) or all (patients 2–4) of chromosome 11.
In all cases, the lost allele was of maternal origin.
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shows
the Southern blot probed with H19 for patient 3. The
densitometric analysis of H19-specific bands showed a
decrease in the intensity of the maternal allele with a concomitant
increase in the intensity of the paternal allele in the tumor compared
to the leukocytes. When normalized to a nonchromosome 11 probe
(IGFBP3 exon 3) to control for DNA loading (see also Fig. 1), the ratio (tumor/leukocytes) between the intensity of the remaining
paternal allele is 0.9 for patient 1, suggesting only LOH, and 1.6 and
1.8 for patients 3 and 4, respectively, suggesting paternal isodisomy
in a subpopulation of cells in these two patients. For patient 2, we
were not able to determine whether there was duplication of the
paternal allele as well as LOH.
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Steady state mRNA levels for IGF2 and H19
were measured in triplicate in the tumors of all four patients and
compared to normal adrenal by competitive RT-PCR using an internal
competitor. mRNA content was corrected for -actin.
Patient 1 had three surgical interventions, but only the third tumor
was analyzed by RT-PCR, as dissection of the first tumor proved
difficult because of numerous zones of necrosis, and the second tumor
contained large amounts of intermixed normal tissue. Tumors from
patients 1, 3, and 4 had significantly increased levels of
IGF2 mRNA relative to normal adrenal tissue and tumors from
patients 1, 2, and 3 had undetectable levels of H19 mRNA
relative to normal adrenal (Fig. 2). In
contrast, patient 4 showed relatively high levels of both mRNA. By
comparison to the IGF2 overexpression data reported by
Gicquel et al. (7) in adrenal carcinomas (2- to 100-fold),
our four patients had similar fold increases in IGF2 mRNA
(patient 1, 27-fold; patient 2, 2-fold; patient 3, 19-fold; patient 4,
36-fold).
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As the 5' INS VNTR has been shown to affect IGF2 transcription in at least some tissues (11), it was also genotyped in peripheral blood from parents and patients as well as in tumor tissue by PCR using primers that flank the polymorphic region as well as for an INS PstI RFLP that is in linkage disequilibrium with the VNTR (11, 20). Patients 1–3 were class I/I homozygotes, and in all three tumor specimens, only class I alleles were retained as expected from the LOH studies described above. Class I allele size analysis in tumor tissue revealed retention of the following subclasses: allele 814 in patients 1 and 2, and allele 683 in patient 3. For patient 4 and her mother, we were unable to confirm VNTR status by direct PCR. However, they could theoretically have inherited two class III alleles, because 5% of PstI (+) are class III alleles (23) (data not shown).
IGF-II methylation studies
Using the methylation-sensitive restriction enzyme
AvaII, we looked for demethylation in the genomic DNA from
tumor tissue of each patient. AvaII is a restriction enzyme
that cuts the site G/G(A or T)CC but not G/G(A or T)Cm5C (m5C =
5-methyl cytosine), i.e. only when it is demethylated. This
generates a 1.1-kb and an additional 0.5-kb fragment, which brings
about a concomitant decrease in the 1.6-kb fragment. This
AvaII site was previously reported to reflect the general
methylation status of IGF2 and showed a specific profile
depending on the tissue of origin (24). Figure 3 shows the Southern blot used to
calculate the degree of demethylation in the first three tumors
compared with that in normal adrenal. The degree of demethylation was
expressed as the ratio of the intensity of 0.5-kb band divided by the
sum of the intensity of the 0.5- and 1.6-kb band (x100) as described
by Schneid et al. (25). Demethylation was 76% for patient
1, 66% for patient 2, and 68% for patient 3 compared to 19% for the
normal adrenal. Our figure of 19% demethylation in normal adrenal is
similar to that reported by Gicquel et al. (10 ±
4.5%; n = 4) (26). Compared with leukocytes, all four tumors
showed demethylation of the AvaII site located at the 3'-end
of IGF2 exon 7 (94%, 81%, 90%, and 84%; mean, 87%).
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p53 germline mutations were looked for in patients 1 and 2 for exons 5–9 of the p53 gene, where over 95% of the mutations have been found to date. A p53 germline mutation was found in patient 2, who had a positive family history for an adrenal tumor in a maternal aunt. This mutation involves a C to T transversion that causes a change from proline (CCC) to serine (TCC) at codon 219 in exon 6 and was previously reported in the maternal aunt (27).
Immunohistochemistry
Tumor tissue from patients 1–4 in addition to tumor tissue
from six other patients previously described (8) was examined for the
presence of p53 somatic mutations by immunohistochemistry (peroxidase
antiperoxidase) and compared to each patient’s normal adrenal
tissue. Representative p53 immunohistochemistry results are shown in
Fig. 4: for patient 2 (Fig. 4A), for
patient 1 at the first, second, and third surgeries (Fig. 4, C, E, and
G, respectively), and for patient 4 (Fig. 4I). Positive staining by
this technique occurs only when p53 is mutated and retained in the
nucleus, as evidenced by an orange-brown staining. Normal p53 does not
stain because it has a very short half-life (28). The first three
patients show somatic mutations of p53. Of note is the intensity of
staining, which increased (light brown to black) with each tumor
recurrence in patient 1. Also note that the patient with the p53
germline mutation (patient 2) had a more diffuse and intense staining
than the other two patients, although her maternal aunt, who harbors
this same mutation, did not show intense staining (patient 7 from Table 1
). Immunohistochemistry of p53 for patient 4 show only rare positive
cells. Table 3 summarizes the
immunohistochemistry results for the four patients reported in this
article as well as six others previously reported (8).
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. Representative photos of IGF-II
immunohistochemistry are shown in Fig. 4: for patient 2 (Fig. 4B), for
patient 1 at the first, second, and third surgeries (Fig. 4, D, F, and
H, respectively), and for patient 4 (Fig. 4J). All tumors demonstrated
increased IGF-II content compared to the corresponding normal adrenal.
All tumors except the one from patient 4 showed a positive correlation
between p53 immunostaining and IGF-II peptide content. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGF2 is imprinted, and in adrenal tissue, mRNA is transcribed only from the paternal allele. During the fetal period, IGF-II is especially plentiful in the adrenal cortex, but postnatally it is found mainly in stromal tissue (32, 33). Recent studies in phosphoenolpyruvate carboxykinase-IGF-II transgenic mice have shown that postnatal overexpression of IGF2 is associated with an increase in adrenal weight and enhanced steroidogenesis, presumably by a direct mitogenic effect of IGF-II on adrenocortical fasciculata cells. Mice did not develop tumors, suggesting that IGF2 overexpression alone is not sufficient for tumorigenesis (34).
The factors contributing to IGF2 overexpression are numerous. Gene dosage effects are common; in our four most recent patients, LOH was found in all (and always involved the maternal allele), and two patients had UPD of chromosome 11. Demethylation of IGF2 using the enzyme AvaII was also demonstrated in all four tumors, supporting the idea that increased IGF2 expression is correlated with demethylation at this locus. This has been shown in other neoplasms as well (24). Schneid et al. (25) showed that the maternal allele of IGF2 was specifically hypomethylated in control leukocyte DNA on the 5'-portion of exon 9 using the AvaII/HpaII double digest.
The expression of human IGF2 has been found to be under the allelic effect of the minisatellite DNA polymorphism, consisting of a variable number of tandem repeats (VNTR) located in the human insulin gene (INS) 5'-flanking region. Shorter alleles (class I) have been associated with higher steady state IGF2 mRNA levels in vivo and in vitro than the longer, class III alleles (11). In three of our patients, only VNTR class I alleles were found. Although not significant with regard to allele frequency data in the general population given the small numbers of samples, it is interesting to speculate that the VNTR may be acting as a locus control region, and this may be yet another mechanism by which IGF2 is overexpressed in these tumors.
Loss of the p53 tumor suppressor gene will also lead to increased IGF2 expression (35). p53 germline mutations have been previously found in three of eight pediatric patients (27, 31). In Li-Fraumeni families with germline mutations of p53, ACT are the earliest tumors to appear, generally occurring before 5 yr of age (36). Only patient 2 was found to have a p53 germline mutation that was passed on through the maternal lineage, as her maternal aunt carried this same mutation. It should be noted that we only sought germline mutations if a family history was suggestive of p53-related tumors in first and second degree relatives (patients 1 and 2). Somatic mutations as demonstrated by immunohistochemistry were found in all but one of the remaining nine patients.
IGF2 lies only 110–200 kb downstream of H19 (37), a gene whose transcript is not translated (38, 39), but whose molecular evolution suggests a functional role for its mRNA (40). Recent data from our laboratory suggest that H19 mRNA decreases steady state IGF2 mRNA levels (Wilkin, F., J. Paquette, E. Ledru, M. Pollak, and C. Deal, unpublished data), supporting a role for this gene in tumor suppression (41, 42), and like IGF2, it is an imprinted gene, but expressed from the maternal allele (43, 44). Three studies (7, 45, 46) reported very low levels of H19 mRNA in active adrenocortical carcinomas from adults, in contrast with high levels observed in adult human adrenals and benign adrenal neoplasms. Our patient 4 was unique with regard to the high H19 mRNA levels observed in her tumor. We were unable to determine whether H19 transcription was occurring only in the few cells that had not lost the maternal allele or whether it represented abnormal H19 imprinting (loss of imprinting). However, the concomitant presence of high levels of IGF2 mRNA and peptide, the size of her tumor (9 cm in diameter), and the early age of presentation raise the interesting question of whether this patient may have an inactivating mutation in H19; this is currently under investigation.
p57KIP2 is another imprinted gene on chromosome 11p15.5 that is maternally expressed and probably involved in tumor suppression (47, 48). Expression of p57KIP2 mRNA correlated positively with H19 and negatively with IGF2 mRNA in active adult adrenocortical carcinomas (46). We have not explored expression levels of this gene, but we would expect to see decreased levels given the loss of the maternal alleles in our patients.
Previous studies have attempted to correlate molecular changes at 11p with prognosis (adenoma vs. carcinoma). However, our data show that genetic alterations involving IGF2, H19, and p53 were found in our four most recent patients for whom frozen tissue was available and in the other six patients based on immunohistochemistry for IGF-II and p53. It is of interest to note the more benign clinical course of patients 2–10 compared with patient 1, who suffered multiple tumor relapses and died 3.3 yr after her initial presentation. Patient 1 differed from the other three recent cases in several respects, including clinically (virilization and Cushing’s syndrome), per-operatively (capsule rupture), histologically (atypical mitosis, necrosis, and capsular invasion), and molecularly (partial LOH). It is impossible to attribute principle causality in her death to any of these features, although from past experience in large series, incomplete surgical resection places all patients at high risk (2).
It is interesting to note that the phenotype of childhood ACT is reminiscent of the cells populating the fetal adrenal cortex. The human fetal adrenal cortex is characterized by rapid growth, high steroidogenic activity, and a morphology composed primarily of two zones: the outer definitive zone and the inner fetal zone, which normally undergoes involution during the postnatal period (for review, see Ref. 49). Recent studies by Spencer et al. (50) indicate that fetal zone remodeling in the human neonatal adrenal is an apoptotic process. The dynamics of fetal adrenal cortical growth involve 1) cellular hyperplasia, mainly in the definitive zone; 2) cell migration from the periphery to the center of the gland; and 3) hypertrophy, limited proliferation, and apoptosis in the fetal zone. Apoptosis is stimulated by p53 and inhibited by IGF-II (51).
As in our pediatric ACT, in situ hybridization analysis in
intact human fetal adrenal glands revealed that IGF2 is
expressed in relatively high abundance by all fetal cortical cells. In
cultured human fetal adrenal cortical cells, IGF2 mRNA is
also abundant and is markedly up-regulated by ACTH (33). These data
strongly suggest the implication of IGF-II in regulation of the
proliferation and development of fetal adrenals and in a local role in
the trophic action of ACTH. It should be noted that IGF-II is also
involved in the differentiation of human fetal adrenal cells by
increasing the ACTH-stimulated expression of the steroidogenic enzymes
(cholesterol side-chain cleavage cytochrome P450, cytochrome
P450 17-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid
dehydrogenase) and in cortisol and dehydroepiandrosterone sulfate
production (52).
In conclusion, genetic alterations in 11p and 17p in ACT are frequent in children as well as adults. Although these abnormalities seem to correlate with prognosis in adults, we were not able to affirm this in children, because in our center, the only two deaths (of 12) (this paper and Ref. 8) occurred in one patient with metastatic disease at presentation and in our patient with per-operative tumor spillage. The consistent phenotype of our patients’ tumors with respect to high IGF2 expression, cell multiplication, and steroidogenesis is highly reminiscent of the fetal adrenal cortex and supports the hypothesis that childhood ACT arise because of the persistence of the fetal adrenal cell, triggered by defective apoptosis, particularly in patients whose tumors present within the first 3 yr of life.
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Acknowledgments |
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Footnotes |
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Received September 27, 1999.
Revised January 18, 1999.
Accepted January 18, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A transition) in the -78 position of the
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J. Bertherat, L. Groussin, F. Sandrini, L. Matyakhina, T. Bei, S. Stergiopoulos, T. Papageorgiou, I. Bourdeau, L. S. Kirschner, C. Vincent-Dejean, et al. Molecular and Functional Analysis of PRKAR1A and its Locus (17q22-24) in Sporadic Adrenocortical Tumors: 17q Losses, Somatic Mutations, and Protein Kinase A Expression and Activity Cancer Res., September 1, 2003; 63(17): 5308 - 5319. [Abstract] [Full Text] [PDF]
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Z.-H. Gao, S. Suppola, J. Liu, P. Heikkila, J. Janne, and R. Voutilainen Association of H19 Promoter Methylation with the Expression of H19 and IGF-II Genes in Adrenocortical Tumors J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1170 - 1176. [Abstract] [Full Text] [PDF]
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M. A. Godil, M. P. Atlas, R. I. Parker, C. J. Priebe, M. M. Zerah, P. Kane, J. Tsung, and T. A. Wilson Metastatic Congenital Adrenocortical Carcinoma: A Case Report with Tumor Remission at 31/2 Years J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 3964 - 3967. [Abstract] [Full Text]
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, negative cells. D, F, and H,
Immunohistochemistry for IGF-II of patient 1 at each surgical
intervention at x20 magnification. I and J, Immunohistochemistry for
p53 and IGF-II, respectively, of patient 4 at x40 magnification.
Arrow, A rare positive cell for p53 and rare negative
cells for IGF-II.