Clonal analysis of human adrenocortical carcinomas and secreting adenomas
Christine Gicquel*, Marie Leblond-Francillardt, Xavier Bertagnat, Albert Louvelt, Yves Chapuis§, Jean-Pierre Lutont, François Girard* and Yves Le Bouc*
*Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, 75012 Paris; tClinique des Maladies Endocriniennes et Métaboliques, Service d’Anatomo-Pathologie and §Clinique chirurgicale, Hôpital Cochin, 75014 Paris, France
(Received 8 July 1993; returned for revision 29 July 1993; finally revised 27 September 1993; accepted 27 September 1993)
Summary
OBJECTIVES Adrenocortical tumours in man are charac- terized mainly on biochemical, anatomical and histologi- cal grounds which establish their secretory pattern and, with some uncertainty, their benign or malignant nature. To study further these tumours and eventually to shed some light on their pathogenesis, we determined their clonal composition.
METHODS Clonal composition was determined by X-chromosome inactivation analysis on tumour and leucocyte DNA using three markers: M278, phospho- glycero-kinase (PGK) and hypoxanthine-phosphoribosyl transferase (HPRT) with 88, 33 and 27% heterozygosity rates respectively.
PATIENTS Cional analysis was performed on 25 tumours from 19 heterozygous female patients: four had a carci- noma, 14 had a single secreting adenoma, and one had autonomous bilateral macronodular hyperplasia with Cushing’s syndrome (seven adenomas examined).
RESULTS The malignant tumours had patterns indicative of monoclonality. The single adenomas displayed con- trasting results with patterns indicative of monoclonality in eight cases, and patterns indicative of polyclonality in six cases; monoclonal adenomas were larger and had a higher prevalence of nuclear pleomorphism than the apparently polyclonal adenomas. In the patient with bilateral macronodular hyperplasia, different clonal pat- terns were present in different adenomas: whereas a clear monoclonal pattern was observed in the three adenomas
Correspondence: Dr Christine Gicquel, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, 26 Avenue Arnold Netter, 75012 Paris, France.
of the right gland, in which the active X-allele was not always the same, in two interpretable adenomas of the left gland, a moderately skewed pattern suggested a partial monoclonal component.
CONCLUSIONS These data show that adrenocortical car- cinomas are monoclonal and suggest that adenomas may arise from a single cell or from more than one cell under the putative action of local growth factors. In adenomas, which until now had appeared homogeneous, this genetic heterogeneity may reflect different pathophysiological mechanisms or it may represent different stages of a common multistep process exceptionally occurring in a single patient with bilateral macronodular hyperplasia.
Steroid hormone producing adrenocortical tumours in man have a low incidence and distribute roughly evenly between the benign adenomas and the malignant carcinomas (Hutter & Kayhoe, 1966; King & Lack, 1979; Bertagna & Orth, 1981; Nader et al., 1983; Freeman, 1986; Luton et al., 1990; Ross & Aron, 1990). Their most frequent clinical presentation is that of Cushing’s syndrome where they account for approxi- mately 30% of the cases (Baxter & Tyrrell, 1981). Adenomas typically have pure glucocorticoid oversecretion whereas carcinomas produce an array of steroids, especially andro- gens which may occasionally be the only secreted hormones. About half of the carcinomas present as non-secreting tumours although more subtle investigations often reveal their ability to produce non-bioactive precursors, hence their apparent endocrine silence (Bertagna & Orth, 1981; Luton et al., 1990). Aldosterone and oestrogen secreting adrenocorti- cal carcinomas are exceptional (Farge et al., 1987; McKenna et al., 1990).
To distinguish between benign and malignant tumours remains a challenge in many cases. The secretory pattern, the tumour weight and anatomical aspect, and the histological features all contribute to assess the likelihood of malignancy (King & Lack, 1979; Bertagna & Orth, 1981; Baxter & Tyrrell, 1981; Freeman, 1986); yet a certain diagnosis can be attained only when regional invasion, local recurrence or metastatic spread is demonstrated. Because malignant adre- nocortical tumours have a poor prognosis (King & Lack, 1979; Bertagna & Orth, 1981; Baxter & Tyrrell, 1981; Nader et al., 1983; Freeman, 1986; Luton et al., 1990) systematic adjuvant antimitotic therapy has been advocated after surgery even in purely localized disease (Luton et al., 1990), a
proposal which should, theoretically, imply an unequivocal diagnosis.
The pathophysiological mechanisms responsible for the growth and/or the steroid overproduction of adrenocortical tumours remain largely mysterious. In all cases the secretory activity is, apparently, autonomous, that is, non-ACTH dependent. Yet about half of the adenomas respond in vivo to exogenous ACTH; the other half and most carcinomas do not (Bertagna & Orth, 1981). The molecular defect respon- sible for this lack of responsiveness is unknown; functional studies in vitro merely indicate that it is different in different tumours, and may be at the receptor or post-receptor level (Riou et al., 1977; Sharma et al., 1977; Lamberts et al., 1990).
Recent studies performed in some rare pathological conditions, the Beckwith-Wiedemann syndrome (Hayward et al., 1988; Henry et al., 1989a), the McCune-Albright syndrome (Weinstein et al., 1991) and the Li-Fraumeni syndrome (Malkin et al., 1990) have added further evidence that variable mechanisms may result in adrenocortical tumours. It has still to be proven whether similar, or related, defects are implied as well in sporadic tumours.
The study of clonality will provide important clues about the origins of adrenocortical tumours. Monoclonality sug- gests that a somatic mutation contributes to tumorigenesis whereas polyclonality suggests that adrenocortical cells respond to local (paracrine) or systemic stimuli. Technical means have been developed which determine the X-inactiva- tion pattern in heterozygous females and permit recognition of whether a given tissue is monoclonal or polyclonal (Vogelstein et al., 1985, 1987; Fearon et al., 1987; Abraham- son et al., 1990; Fey et al., 1992). These techniques have been successfully used in the endocrine field and have already shown that most endocrine tumours are monoclonal (Arnold et al., 1988; Alexander et al., 1990; Herman et al., 1990; Namba et al., 1990; Schulte et al., 1991; Gicquel et al., 1992). To examine adrenocortical tumours, we used three X- chromosome probes: hypoxanthine-phosphoribosyl trans- ferase (HPRT) and phospho-glycero-kinase (PGK) (Vogelstein et al., 1987) and the more recently described anonymous M27฿ probe (Abrahamson et al., 1990; Fey et al., 1992). They were used to analyse the clonal composition of 25 tumours from 19 female patients: we found that whereas carcinomas were monoclonal, adenomas could be either monoclonal or polyclonal.
Patients and methods
Patients
Patients evaluation. Twenty-two female patients, 16-64 years old, admitted to the Clinique des Maladies Endocriniennes et Métaboliques, Hôpital Cochin, between 1989 and 1991 for
the investigation of adrenocortical tumours, were included in this study.
The hormonal evaluation was performed as previously described (Luton et al., 1990). The diagnosis of Cushing’s syndrome was made on both clinical features and biological evidence of glucocorticoid over-secretion (increased urinary free cortisol (UFC) and abnormal response to the high dose dexamethasone test).
Staging of the tumour as localized, regional, or metastatic disease was based on clinical data, radiological studies and CT scanning, and was confirmed by the findings at surgery and on pathological examination (Luton et al., 1990).
All patients were operated by the same surgical team in our institution. Tissue fragments were cautiously selected by the same pathologist in the homogeneous and non-necrotic areas of the tumour, immediately frozen in liquid nitrogen, then stored at - 80℃ until extraction. Control DNA studies were performed on peripheral blood leucocytes in each patient but one (patient 12), and on normal (peritumoral) adrenocortical tissue in two patients with adenomas (patients 10 and 11, Table 1).
All anatomical and histological analyses were performed by the same examiner. Tissues were fixed in formaldehyde, embedded in paraffin and stained with haematoxylin and eosin. A tumour weight above 80 g and/or the presence of haemorrhages, necrosis, broad fibrous bands, capsular or blood vessel invasion, increased mitotic activity and nuclear pleomorphism were diagnostic of carcinomas. Tumours with benign appearance were classified as adenomas even when they exhibited mild nuclear pleomorphism. The proportion of clear or vacuolated cells resembling the normal zona fasciculata cells and of compact cells containing dense granules and resembling the zona reticularis cells was evaluated in each tumour (Table 1). In some tumours, several tissue specimens were selectively picked up in macroscopic- ally different areas made of clear or compact cells.
Patients’ classification (Table 1). Five patients were diag- nosed as having adrenocortical carcinomas. Four of them presented with clinical and biochemical features of Cushing’s syndrome; in one patient (patient 4), the presenting com- plaint was flank pain. All had large tumours with weights ranging from 135 to 2010 g. Two patients had lung metastases at the time of diagnosis (patients 1 and 3). One presented with a local recurrence after surgery (patient 5). Kidney invasion was present in patient 2. Only one patient (patient 4) had localized disease; in this patient, the likeli- hood of malignancy was assumed on suggestive anatomical and histological data. Two tumours were exclusively com- posed of compact cells (patients 2 and 3), one contained exclusively clear cells (patient 1), one had mixed populations
| Clinical data | Hormonal data | Histological data | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient | Age (years) | Clinical presentation | Ist symptom to diagnosis (months) | Tumour stage at diagnosis | UFC* (nmol/day) N=55-250 | ACTH (pmol/l) N=8±3.9 | Increased androgenst | Tumour weight (g) | Comp§ % | Clear§ % | Haemorrhage/ necrosis/ invasion | Nuclear pleomorphism |
| Carcinomas | (n=5) | |||||||||||
| 1 | 20 | Cushing's | 12 | metastases | 1 | <1 | yes | 135 | 1 | 100 | yes | no |
| 2 | 49 | Cushing's | 6 | regional | 257 | <1 | yes | 1550 | 100 | 1 | yes | yes |
| 3 | 42 | Cushing's | 24 | metastases | 480 | 1 | no | 1230 | 100 | 1 | yes | yes |
| 4 | 22 | flank pain | 2 | localized | 138 | 1.8 | no | 160 | Il | yes | yes | |
| 5 | 16 | Cushing's | 36 | recurrence | 7395 | <1 | yes | 2010 | 70 | 30 | yes | yes |
| Adenomas (n= 16) | ||||||||||||
| 6 | 29 | Cushing's | 30 | localized | 1245 | < 1 | no | 17 | 20 | 80 | no | yes |
| 7 | 62 | Cushing's | 42 | localized | 295 | <1 | no | 9 | 40 | 60 | no | no |
| 8 | 57 | Cushing's | 48 | localized | 2420 | <1 | no | 17 | >80 | <20 | no | no |
| 9 | 26 | Cushing's | 24 | localized | 745 | <1 | no | 10 | 50 | 50 | no | no |
| 10 | 39 | Cushing's | 48 | localized | 1357 | <1 | no | 15 | ? | ? | no | no |
| 11 | 36 | Cushing's | 12 | localized | 361 | <1 | no | 18 | 20 | 80 | no | no |
| 12 | 39 | Cushing's | 36 | localized | 841 | 1 | no | 30 | 50 | 50 | no | no |
| 13 | 27 | Cushing's | 36 | localized | 268 | <1 | no | 12 | 60 | 40 | no | no |
| 14 | 53 | Cushing's | 48 | localized | 436 | <1 | no | 30 | 50 | 50 | no | yes |
| 15 | 24 | virilization | 84 | localized | 108 | 8.8 | yes | 34 | >95 | <5 | no | yes |
| 16 | 35 | Cushing's | 9 | localized | 803 | 1 | no | 12 | 75 | 25 | no | yes |
| 17 | 40 | Cushing's | 66 | localized | 411 | <1 | no | 15 | 80 | 20 | no | no |
| 18 | 39 | Cushing's | 60 | localized | 1 | <1 | 1 | 12 | 80 | 20 | no | yes |
| 19 | 48 | Cushing's | 30 | localized | 709 | <1 | no | 10 | 100 | 1 | no | no |
| 20 | 64 | HBP | 36 | localized | 127 | <1 | yes li | 20 | 20 | 80 | no | no |
| 21 | 55 | Cushing's | 24 | localized | 728 | <1 | no | 20 | 30 | 70 | no | yes |
| Macronodular hyperplasia (n=1) | ||||||||||||
| 22 | 38 | Cushing's | 180 | localized | 530 | <1 | no | ** R: 37 | 1 | 100++ | no | no |
| L: 47 | ||||||||||||
* UFC, Urinary free cortisol; t increased secretion of androgens was defined as testosterone level greater than 2 nmol/l and/or androstenedione level greater than 12.9 nmol/l and/or dehydroepiandrosterone sulphate greater than 8-4 pmol/l; § repartition of compact versus clear cells at histological examination; || 100% undifferentiated cells; 1 increased androgens from gonadal origin; ** weight of right (R) and left (L) adrenals; tt all adenomas were made exclusively of clear cells except adenoma G which was made equally of clear and compact cells.
(patient 5), and one contained undifferentiated cells (patient 4).
Sixteen patients were diagnosed as having adrenocortical adenomas. Fourteen presented with typical clinical and biochemical features of Cushing’s syndrome. In patient 20, who had only high blood pressure, the diagnosis of Cushing’s syndrome was assumed on the basis of abnormal response to the low dose dexamethasone test with undetectable ACTH and corticotrophic insufficiency after unilateral adrenalec- tomy. A single patient presented with clinical and biochemi- cal features of androgen oversecretion (patient 15). Tumour weight ranged from 9 to 34 g (mean+SD=18±8). The likelihood of benignity was assumed on the basis of pure glucocorticoid secretion (in 15 cases) and/or lack of tumour spread (in all 16 cases). Histological data, as usual, were not completely unequivocal: six tumours (patients 6, 14, 15, 16, 18, 21) showed nuclear pleomorphism. This feature was particularly important in patient 15 who also had androgen oversecretion and had the largest tumour (34 g) of the series; thus some doubt might be raised in this patient. Except for two tumours which had an exclusive or > 95% component of compact cells, all others had variable amounts of compact and clear cells.
One particular patient had bilateral macronodular hyper- plasia (patient 22). She presented with typical clinical features of Cushing’s syndrome and ACTH-independent glucorticoid oversecretion. An abdominal CT scan revealed huge nodular enlargement of both glands which harboured numerous adenomatous lesions varying from 0.5 to 5 cm. Three were collected in the right adrenal (A-C) and four in the left (D-G). Histologically, all adenomas, except one (G), were made exclusively of clear cells; this latter adenoma had mixed, compact and clear populations.
Clonal analysis
DN A extraction. Leucocyte DNA was prepared according to Miller et al. (1988) with minor modifications (Schneid et al., 1990). Tumour DNA was extracted simultaneously with RNA by the guanidinium thiocyanate caesium method (Schneid et al., 1992). DNA was dialysed overnight against 0.001 M EDTA, 0-01 M Tris-HC1, pH 7.5, and further treated as leucocyte DNA.
DNA probes and clonal analysis. Clonal analysis was per- formed using three different X-chromosome probes: PGK (pSPT 19 -1), HPRT (pPB 1-7) and M27B as previously described (Fearon et al., 1987; Vogelstein et al., 1987; Abrahamson et al., 1990; Fey et al., 1992; Gicquel et al., 1992). DNA was digested in two steps. A first digestion revealed the restriction fragment length polymorphism
(RFLP) and distinguished the maternal and paternal copies of the gene in a heterozygous female. The second digestion with a methylation sensitive enzyme (Hpall or Hhal) estab- lished the methylation state of each allele and distinguished active from inactive alleles.
Clonal analysis at the PGK locus (Fig. 1). DNA was first digested with BstXI +Pstl or Eco RI+Bgl I+Bgl II, giving rise to two polymorphic fragments of respectively 1.05 and 0.9 kb or 1.7 and 1.3 kb. One aliquot was further digested with Hpall.
Clonal analysis at the HPRT locus (Fig. 1). DNA was first digested with BamHI + Pvull giving rise to two polymorphic fragments of 18 and 12 kb. One aliquot was further digested with Hhal.
Clonal analysis at the M27B locus (Fig. 1). M27 detects a mutiallelic polymorphism due to variable numbers of tan- dem repeats. DNA was first digested with Pstl giving rise to two polymorphic fragments in the 5-10 kb range. One-third of Pstl digested product was further digested with Hpall and one-third with Mspl, the methylation non-sensitive iso- schizomer of Hpall.
The products of each reaction were precipitated with ethanol before electrophoresis on a 0.7% agarose gel for
(a)
BstX | EcoRI
BstX| Pst l Bgll
士
+
Bgl II
PGK
500 bp
(b)
BamHI
+
BamH I
Pvu li
.
HPRT
1 kb
(c)
Pstl
VNTR
Pstl
M27/3
500 bo
M27B and HPRT and 1% agarose gel for PGK. Transfer and hybridization conditions were performed as previously described (Chomczynski & Qasba, 1984; Feinberg & Vogelstein, 1983).
Quantification of X-inactivation. Autoradiographs were scanned with a Densitometer Model GS 300 Hoefer Scien- tific Instruments (San Francisco, USA). Each Hpall (or Hhal) band was compared to its homologous ‘first digestion’ band and the allelic cleavage ratio (ACR) was calculated according to the formula (Fey et al., 1992):
upper first digestion band/upper Hpall (or Hhal) band lower first digestion band/lower Hpall (or Hhal) band
Reciprocal values were reported when the ratio was <1.0.
Results
Clonal analysis of tumours examines X inactivation patterns in females heterozygous for X linked polymorphisms. According to Lyon’s hypothesis (Lyon, 1961) a single X chromosome is active in each somatic cell of a female. Inactivation of either the maternal or paternal X chromo- some is a random, balanced process and the pattern of X chromosome inactivation of a cell is transmitted in a highly stable fashion to its progeny cells. Changes in the activity of genes are accompanied by changes in the methylation of cytosine residues.
In a polyclonal tissue with random and balanced X- inactivation, both the maternal and the paternal alleles will be partly and equally digested by a methylation-sensitive enzyme whereas in a monoclonal tissue the same allele will be completely digested by the methylation-sensitive enzyme.
We used three systems to detect polymorphic loci on X- chromosomes: the M27฿ probe reveals a RFLP in a variable number tandem repeat region of the X-chromosome with 88% heterozygosity rate (Fraser et al., 1987, 1989; Fey et al., 1992). Two other X-linked loci, the PGK and HPRT genes, have RFLP with heterozygosity rates of 33 and 27% respectively (Vogelstein et al., 1987).
With the M270 system, in a polyclonal tissue, the methyla- tion-sensitive Hpall enzyme shortens partially the two polymorphic fragments giving rise to a four-band pattern; in a monoclonal tissue, a single allele is entirely digested whereas the other is unchanged.
With PGK or HPRT markers, in a polyclonal tissue, the two polymorphic alleles will show a roughly equal reduction in intensity after Hpall or Hhal digestion. In a monoclonal tissue a single allele (maternal or paternal) will be entirely digested and the other totally undigested by the methylation- sensitive enzyme and a single PGK or HPRT allele will disappear.
Adrenocortical carcinomas
Four of the five patients were heterozygous for at least one marker (Table 2). The results of the X-inactivation analysis are shown in Table 2 and Fig. 2. The three tumours, which were interpretable, displayed a monoclonal pattern.
For the three patients heterozygous at the M278 locus (patients 2, 3 and 4), X-inactivation analysis was performed as followed and as shown for tumours 2 and 4, on Fig. 2 (upper part). DNA was first digested by Pstl; for a given patient, an identical pattern was obtained with leucocyte DNA (L, lane a) and tumour DNA (T, lane a). In all cases, the methylation non-sensitive Mspl enzyme further digested each allele entirely, generating a downward shift of each of these two fragments, again in leucocyte DNA (L, lane b) and tumour DNA (T, lane b). The results obtained with the methylation-sensitive Hpall enzyme were different: with leucocyte DNA (L, lane c), the two alleles were partly digested giving rise to four restriction fragments, a pattern of polyclonality as expected, even if the pattern was sometimes slightly skewed; the ACR values were close to 1 (1-6-2-4) (Table 2). With tumour DNA, a single allele was entirely digested and the other totally undigested giving rise to a pattern typical of monoclonality (T, lane c). The ACR values in tumours 2, 3 and 4 were very different from 1 (21, > 100 and > 100, respectively, Table 2). Two of these patients were also heterozygous at the PGK (patients 3 and 4) and/or the HPRT (patient 4) locus. Their X-inactivation patterns in these systems agreed with that found in the M27B system as shown for patient 4 (Fig. 2, lower part). The fourth patient (patient 5) was heterozygous for HPRT only: analysis of leucocyte DNA showed the preferential reduction of the upper band with Hhal giving an ACR of 1.7 and tumour DNA also had a skewed pattern with an intermediary ACR value of 3.3, not allowing to state strictly tumoral mono- clonality (Fig. 2, lower part).
Adrenocortical adenomas
Fifteen of the 16 patients were heterozygous for at least one of the X-chromosome markers (Table 2). One patient (patient 18) was heterozygous at the M278 locus but had an additional Mspl/Hpall restriction site which precluded its study. Clonal analysis was performed in 14 tumours: using the M27B probe in 11 cases, the PGK probe in six cases and the HPRT probe in two cases. As opposed to the results obtained in malignant tumours, those in adenomas were contrasted: eight tumours showed a monoclonal pattern whereas six had apparently a polyclonal pattern. Figure 3 shows the results of two monoclonal (patients 8 and 20) and one polyclonal (patient 12) adenomas examined in the M27฿ system. Figure 4 shows the results of one monoclonal adenoma (patient 11) examined with the PGK and the
| Patient | M278 | PGK | HPRT | Tumour weight (g) | Nuclear pleomorphism | Leucocyte ACR* | Tumoral ACR* | Tumoral pattern |
|---|---|---|---|---|---|---|---|---|
| Carcinomas (n == 5) | ||||||||
| 1 | - | - | - | 135 | no | / | / | non informative |
| 2 | + | - | - | 1550 | yes | 2.4 | 21 | Monoclonal |
| 3 | + | + | - | 1230 | yes | + | >100 | Monoclonal |
| 4 | + | + | + | 160 | yes | 1.6 | >100 | Monoclonal |
| 5 | - | - | + | 2010 | yes | 1.7 | 3.3 | Monoclonal? |
| Adenomas (n=16) | ||||||||
| 6 | - | + | - | 17 | yes | 2.8 | 13.6 | Monoclonal |
| 7 | + | - | 9 | no | 3 | 3 | Polyclonal? | |
| 8 | + | 17 | no | 2.1 | > 100 | Monoclonal | ||
| 9 | + | + | 10 | no | 1 | + | Polyclonal? | |
| 10 | + | + | 15 | no | 1.2 | 2.4 | Polyclonal | |
| 11 | + | + | 18 | no | 1.4 | 5.6 | Monoclonal? | |
| 12 | + | 30 | no | 1 | 2.5 | Polyclonal | ||
| 13 | + | - | + | 12 | no | 1 | 1.2 | Polyclonal |
| [4 | + | 30 | yes | 1.2 | 7.7 | Monoclonal | ||
| 15 | + | 34 | yes | 1.3 | >100 | Monoclonal | ||
| 16 | 12 | yes | 1 | 1 | non informative | |||
| 17 | + | 15 | no | 2.2 | >100 | Monoclonal | ||
| 18 | + | 12 | yes | / | 1 | § | ||
| 19 | + | + | 10 | no | 1.1 | 1.2 | Polyclonal | |
| 20 | + | 20 | no | + | >100 | Monoclonal | ||
| 21 | + | 20 | yes | 1-4 | 4 | Monoclonal? | ||
| Macronodular hyperplasia (n=1) | ||||||||
| 22 | + | - | fRight: 37 | A | 1 | >100 | Monoclonal | |
| B | 1 | >100 | Monoclonal | |||||
| C | 1 | >100 | Monoclonal | |||||
| TLeft: 47 | D | 1 | 8 | Monoclonal? | ||||
| E | / | 1 | Demethylated | |||||
| F | 1 | 1 | Hypermethylated | |||||
| G | 1 | 5 | Monoclonal? | |||||
* ACR; allelic cleavage ratio.
t Because of too close restriction fragment lengths, evaluation of ACR was not possible.
§ Additional Mspl/Hpall restriction site precluding clonal analysis.
{ Weight of each adrenal.
HPRT systems and two polyclonal adenomas examined with the PGK (patient 10) or the HPRT systems (patient 13). For patient 10, two different areas of the tumour selected for the predominance of compact cells (T comp) or of clear cells (T clear) showed a similar polyclonal pattern. The five adeno- mas which were examined with two different systems had the same pattern: for example, patient 11 was monoclonal in the PGK and in the HPRT systems (Fig. 4).
The leucocyte ACR values ranged from 1.1 to 3. The polyclonal and monoclonal tumour ACR values ranged respectively from 1.2 to 3 and 4 to > 100 (Table 2). Among the 14 studied adenomas, six (patients 6, 8, 14, 15, 17 and 20)
are unequivocally monoclonal, with tumoral ACR respect- ively at 13.6, > 100, 7.7, >100, >100 and >100; four (patients 10, 12, 13 and 19) are unequivocally polyclonal, with tumoral ACR respectively at 2-4, 2.5, 1.2 and 1.2; the other adenomas (patients 7, 9, 11 and 21) showed more skewed patterns which could be ambiguous. However, tumoral ACR values and DNA patterns were in favour of monoclonality (patients 11 and 21) or polyclonality (patients 7 and 9) (Table 2).
Monoclonal adenomas were larger than apparently poly- clonal adenomas (mean, 21.4 vs 14.3 g, P<0.05, Mann- Whitney test).
M273
M273
L
T
L
T
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b
1
C
a
b
C
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pat 2
pat 4
HPRT
HPRT
L
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a
b
a
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b
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pat 4
pat 5
Among the six tumours with nuclear pleomorphism, the four which could be analysed were monoclonal; none of the six polyclonal adenomas had nuclear pleomorphism. No difference could be found in the age of patients, in the length of evolution or in the biochemical evaluation of the two different types of tumours.
Bilateral macronodular hyperplasia
Seven different macroadenomas were carefully selected by the pathologist: three from the right adrenal (adenomas A to C) and four from the left adrenal (adenomas D to G) (Fig. 5). Clonal analysis with the M270 system showed different restriction profiles. The three adenomas of the right adrenal all had unequivocally a monoclonal composition with ACR > 100. Moreover, they did not have the same X-inactivation
pattern (Fig. 6): in adenomas A and C, the inferior allele alone was entirely digested by Hpall; in adenoma B, the superior allele alone was entirely digested by Hpall. In the left adrenal, restriction profiles observed (Fig. 6) in adenomas D and G were different: the two alleles were partially digested by Hpall giving rise to a four-fragment pseudopolyclonal pattern: yet, ACR of 8 and 5, respectively were compatible with monoclonality. These patterns, however, were clearly different from those observed in the adenomas of the right adrenal. Moreover, DNA from adenoma E was hypomethy- lated and completely digested by Hpall and DNA from adenoma F was completely insensitive to Hpall. These latter data were reproducible.
Thus clonal analysis was possible for five of the seven adenomas, and three different clonal profiles were found. Histologically, all the adenomas, except adenoma G, had the
M27B
L
T
L
T
T
abc abc abcabc abc
pat 8
pat 20
pat 12
same composition with a great majority (>95%) of clear cells; in adenoma G distinct regions of clear (G clear) and compact (G comp) cells were present which were separately analysed: both showed the same four-fragment, pseudo- polyclonal, pattern.
Discussion
Endocrine tumours have been the source of recent break- throughs unravelling several mechanisms of tumorigenesis (Hayward et al., 1988; Landis et al., 1989; Larsson et al., 1988; Lyons et al., 1990). At least four different molecular defects are now known which may be the cause of adrenocor- tical tumours and possibly steroid oversecretion: allelic loss, probably responsible for the loss of an antioncogen, was found in 11p15 for one adrenocortical adenoma (Hayward et al., 1988) and several carcinomas of Beckwith-Wiedmann syndrome (Henry et al., 1989a) or familial carcinomas (Henry et al., 1989b); somatic mutations of Gsx and Gi2x were found respectively in the McCune-Albright syndrome (Weinstein et al., 1991) and in some sporadic adrenocortical tumours (Lyons et al., 1990); p53 mutations were found in the Li-Fraumeni syndrome (Malkin et al., 1990). Yet these discoveries concern rare congenital diseases (Hayward et al., 1988; Henry et al., 1989a, b; Malkin et al., 1990; Weinstein et al., 1991) or only a minor proportion of sporadic and acquired tumours (Lyons et al., 1990). Alternatively, long standing stimulation of adrenocortical glands by ACTH
produces diffuse hyperplasia often accompanied by the occurrence of local nodules as observed for example in Cushing’s disease (Doppman et al., 1988) and in congenital adrenal hyperplasia (Jaresch et al., 1992). In these situations the local action of auto or para-crine growth factors is suggested and the formation of polyclonal lesions is antici- pated.
Determining the clonal composition of tumoral tissues has established the cellular origins of many human tumours (Woodruff, 1988). To elucidate the pathophysiological mechanism(s) which generate acquired steroid producing adrenocortical tumours in man, and eventually distinguish between different processes, it was necessary first to study their clonal composition: a polyclonal tumour would favour the idea that it developed from a group of cells under the common stimulus of a growth factor from extra or intra- adrenocortical origin; conversely, a monoclonal tumour would suggest that it developed from a single genetically aberrant cell; this latter type of tissue only would therefore provide a useful specimen for DNA studies in search of a molecular defect.
Determining the clonal pattern of a tissue is now feasible by X-inactivation analysis in heterozygous females. Because steroid producing adrenocortical tumours are rare, and because the classical PGK - and HPRT- loci only have a 50% heterozygosity rate when they are used in combination, we have primarily developed our study with the M278 probe directed toward an anonymous X-region and having 90%
L
PGK T comp T clear
PGK L PTT T
a
b
a
b
a
b
pat 10
a
b
a
b
a
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pat 11
HPRT
HPRT
L
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pat 13
pat 11
22
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M273 pat 22
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heterozygosity rate. This probe has been used already to determine the clonal composition of endocrine tumours (Fey et al., 1992; Gicquel et al., 1992); it allowed us to study one carcinoma (out of four) and seven adenomas (out of 16) which were homozygous with either PGK or HPRT.
It is somehow arbitrary to choose a definite cut-off point of ACR to separate monoclonal from polyclonal tumours. There are two difficulties which are inherent to the technique and emphasize its limitations: the tissue specimen may not be homogeneous with regard to cell population, and control tissue-particularly blood leucocytes-may have a skewed pattern with elevated ACR up to 3 in this study and 11 in another (Fey et al., 1992). In some cases, as in the adenomas of patients 13 and 19, both alleles were equally digested by the methylation sensitive enzyme giving a genuine polyclonal pattern tumour with ACR~1, made of randomly inacti- vated X chromosomes; these tumours were stated as poly- clonal; in other tumours like those of patients 2, 3, 4, 6, 8, 15, 17, 20, 22 (adenomas A, B, C) ACRs were definitely elevated (between 13.6 and >100): these tumours were stated as monoclonal. Finally, some tumours showed a pattern where
both alleles were partly digested by the methylsensitive enzyme (patients 5, 11, 14, 21, 22: adenomas D and F); although these tissues are apparently polyclonal the skewed ACR (>3) strongly suggests that at least part of them is made of a monoclonal component: they were stated as possibly monoclonal (monoclonal ?: Table 2).
Three of the four patients with adrenocortical carcinomas and which could be studied, exhibited a monoclonal pattern within their tumour DNA. This result was not unexpected and agrees with the classic mutational theory of carcinogene- sis (Fialkow, 1976). For the other heterozygous patient (patient 5), monoclonality could not be unequivocally stated. In contrast, the 14 patients diagnosed as having adrenocorti- cal adenomas which could be studied distributed roughly equally between apparently monoclonal (8/14) and appar- ently polyclonal (6/14) tumours. The finding that benign tumours could be monoclonal was not unexpected since similar results have been observed in benign tumours of the pituitary (Herman et al., 1990; Alexander et al., 1990; Schulte et al., 1991; Gicquel et al., 1992), the parathyroid (Arnold et al., 1988), the thyroid (Namba et al., 1990) and the colon
(Fearon et al., 1987). That adrenocortical adenomas could be polyclonal came as a surprise. These latter tumours, like the others, were well encapsulated and it can be ruled out that the tumour specimen was contaminated by adjacent normal adrenocortical tissue. Their content of non-tumoral cells providing the vascular supply and some stromal tissue, presumably of polyclonal origin, contributed only a neglig- ible cellular proportion of the tumour (less than 5%), again in similar proportion to that observed in monoclonal tumours. Lastly, in some cases, the polyclonal pattern was confirmed in the same tumour with different DNA probes, and with the same DNA probe in different regions of the same tumour. Tumours with skewed pattern are, probably, cell-mosaics with a monoclonal contingent.
How a polyclonal tumour supposedly responding to the stimulatory action of growth promoting factor(s), can develop and remain purely localized in an adrenal gland is mysterious. A local growth promoting event can be imagined since many factors (bFGF, IGFs) are normally locally produced which exert a mitotic effect on adrenocortical cells. Whatever the exact nature of this event, it might contribute to recruit normal (and polyclonal) cells around it. Because adrenal glands often have an intrinsic capacity to develop nodules within hyperplasia, even in response to systemic stimulation with ACTH (Jaresch et al., 1992), it is possibly this same and, so far, unexplained capacity which restricts the expression of a primary adrenal event to a single nodular or adenomatous region.
In any case we are left with two general hypotheses which are not mutually exclusive: the genetic heterogeneity which distinguishes monoclonal from polyclonal adrenocortical adenomas either indicates different pathological mechan- isms, or points at different stages of a common multistep process. Patient 22 concentrated the various possibilities in her own two adrenal glands which harboured adenomas with contrasting clonal patterns. Her genetic heterogeneity had a biochemical correlate: in-vitro studies showed that one of her adenomas (D) with an intermediary ACR secreted mainly cortisol like a normal gland, whereas one of her monoclonal adenomas (B) secreted cortisol precursors (Pham-Huu- Trung et al., 1992). This exceptional case may well be an example of the second hypothesis exhibiting at the same time, but in different locations, several stages of a common multistep process. Progression to monoclonal tumours could be imagined as follows: a first event would initiate the growth of a polyclonal or partially monoclonal tumour with the maintainance of a normal steroid secretory pattern; a second event provoked by a somatic change would confer a further growth advantage in a selected clone of cells with a concomitant loss of differentiated functions and a more or less aberrant steroid secretory pattern.
Monoclonal and polyclonal compositions have been reported also in benign thyroid tumours (Namba et al., 1990; Fey et al., 1992). Clonal analyses of parathyroid adenomas have also reported variable results. Losses of alleles from chromosome 11 (11q13) were found in some but not all parathyroid tumours of the multiple endocrine neoplasia type 1; this allelic loss correlated with a greater tumour mass (Friedman et al., 1989). Allelic losses support monoclonal composition. Sporadic adenomas were almost always mono- clonal (Arnold et al., 1988) whereas hyperplastic glands had a polyclonal pattern. So, it has been suggested that hyper- plasia preceded the occurrence of a monoclonal adenoma (Friedman et al., 1989).
In this study we show that the clonal composition of adrenocortical carcinomas and steroid producing adenomas can be determined in the majority of the cases. This technique showed that adenomas which until now appeared as a homogeneous group actually segregate into monoclonal and polyclonal tumours; this genetic heterogeneity may reflect different pathophysiological mechanisms or represent differ- ent stages of a common multistep process.
Acknowledgements
We are greatly indebted to Drs J. Singer-Sam, S. K. Kim and I. W. Craig for the generous gift of PGK, HPRT and M27 probes. This work was supported by Assistance Publique de Paris, Contrats de Recherche Clinique nº 913104 and nº 9104, by the University Paris-VI, Faculté Saint-Antoine and by INSERM, Contrat de Recherche Externe nº. 920709.
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