2564050

Genetic Changes in Human Adrenocortical Carcinomas

Takahiko Yano, Marston Linehan, Patrick Anglard, Michael I. Lerman, Lambert N. Daniel, Cy A. Stein, Cary N. Robertson, Renata LaRocca, Berton Zbar*

Recent studies have suggested that loss of heterozygosity at loci on the short arm of human chromosome 11 (11p) may be important in the pathogenesis of benign and malignant adrenal cortical tumors. To test this concept, adrenocortical carcinomas from nine patients and benign adrenal corti- cal lesions from eight patients were tested for loss of alleles at loci on human chromosomes 11, 13, and 17. All patients with adrenocortical carcinoma whose normal somatic tissues were heterozygous for a locus on chromosome 17p had lost alleles in the tumor. Four of six patients with adrenocortical carcinoma who were heterozygous for one or more alleles on chromosome 11p in normal tissues had lost 11p alleles in the tumor. Three of six patients with adrenocortical carcinoma showed loss of 13q alleles in the tumor. Loss of alleles on chromosomes 11p, 13q, and 17p was observed in primary tumors and metastases but not in adrenocortical adenomas or hyperplastic lesions of the adrenal cortex. One patient with adrenocortical carcinoma had a somatic mutation in the HRAS1 gene in the normal adrenal gland. The consis- tency of the genetic changes on chromosomes 11p, 13q, and 17p suggests that they are important in the pathogenesis of adrenocortical carcinoma. [J Natl Cancer Inst 81:518-523, 1989]

Adrenocortical carcinoma is an exceedingly rare hu- man malignancy; it accounts for 0.2% of all cancers in the United States (1). There is increased interest in this rare malignancy because susceptibility to this neoplasm ap- pears to be inherited in some individuals. Children with Beckwith-Wiedemann syndrome, a growth disorder charac- terized by macroglossia, gigantism, and omphalocele (2,3), have an increased incidence of a number of tumors, includ- ing adrenal adenomas and adrenocortical carcinomas (4,5). Adrenocortical carcinoma is part of a constellation of tu- mors inherited in the sarcoma, breast, lung, adrenocortical carcinoma syndrome described by Li and Fraumeni (6) and Lynch et al. (7).

Koufos et al. (8) suggested that a recessive oncogene, with pleiotropic action, located on chromosome 11p confers pre- disposition to adrenal cortical tumors, hepatoblastoma, and rhabdomyosarcoma. Support for this suggestion was pro- vided by the demonstration that loss of heterozygosity at loci on chromosome 11p was consistently observed in pa-

tients with rhabdomyosarcoma and hepatoblastoma (9-11) and by analysis of a family with two children affected with adrenal carcinoma (12). Loss of heterozygosity at 11p was found in the tumor from one of the children. The chromo- some 11p region retained in the tumor was present in both affected children and was inherited from the same parent.

We examined 17 adrenal cortical tumors, both benign and malignant, for genetic changes on chromosomes 11, 13q, and 17. Because patients with adrenocortical carcinoma are usually treated by surgical excision of the primary tumor before referral to the National Cancer Institute, in most cases we analyzed local tumor recurrences and metastases.

All patients with adrenocortical carcinoma whose normal somatic tissues were heterozygous for a locus on chromo- some 17p (D17S30) had lost one allele in the tumor. Four of six patients whose normal tissues were heterozygous for one or more loci on chromosome 11p had a loss of 11p alleles in the tumor. Three of six patients whose normal tissues were heterozygous for one or more loci on chromosome 13q had lost 13q alleles in the tumor. We found no evidence for loss of heterozygosity at loci on chromosomes 11p, 13q, or 17p among eight patients with benign adrenocortical lesions.

Patients and Methods

Tumor and nonneoplastic tissues were obtained from pa- tients treated at the National Cancer Institute, Bethesda, MD, and the Memorial Sloan-Kettering Hospital, New York, NY.

Received November 15, 1988; revised January 17, 1989; accepted January 19, 1989.

T. Yano, M. I. Lerman, L. N. Daniel, and B. Zbar, Laboratory of Im- munobiology, National Cancer Institute-Frederick Cancer Research Fa- cility, Frederick, MD.

Surgery Branch (M. Linehan, ‘P. Anglard, and C. N. Robertson) and Medicine Branch (C. A. Stein and R. LaRocca), National Cancer Institute, National Institutes of Health, Bethesda, MD.

We thank Drs. Y. Nakamura, P. O’Connell, and R. White for the gift of probes pYNZ22, pTHH59, pRMU3, pPTH-LF, D3S2, and D15S1; Dr. B. Carritt for DNF15S2; Dr. R. A. Weinberg for HRAS1 (pUCEJ6.6); Dr. H. H. Kazazian, Jr., for HBBC (JW151); Dr. C. Dickson for INT2 (SS6); and Dr. M. Brennan, Dr. Harry Keiser, Dr. John Gill, and Dr. Jeff Norton for tissue samples. We thank Ms. E. Miller and Ms. M. L. Orcutt for technical support and Mr. C. Riggs for statistical support.

*Correspondence to: Dr. Berton Zbar, Cellular Immunity Section, Labora- tory of Immunobiology, Bldg. 560, Rm. 12-71, National Cancer Institute- Frederick Cancer Research Facility, Frederick, MD 21701.

Tumor samples were also obtained from the Cooperative Human Tissue Network, The University of Alabama, Bir- mingham. DNA polymorphisms were compared in the neo- plastic and nonneoplastic tissues of individual patients with adrenal tumors. Therefore, it was essential to obtain paired samples of tumor and normal tissue from each individual patient. Patients were considered evaluable if their normal tissue was heterozygous for the restriction fragment length polymorphism. The source of normal DNA varied; it was extracted from peripheral blood leukocytes, adrenal tissue, kidney, liver, muscle, heart, and lung. The source of tumor DNA also varied; it was extracted from primary tumors, lo- cal tumor recurrences, or metastases. Samples were collected at the time of surgical resection of adrenal tumors or at au- topsy. Paired normal and neoplastic tissues were obtained from six patients with adrenocortical carcinomas; adreno- cortical carcinomas without accompanying normal tissues (unpaired samples) were obtained from two patients. Paired tissue samples were obtained from six patients with adrenal adenomas and two patients with adrenal cortical hyperpla- sia. Information about the characteristics of the samples and of the patients who donated the samples is available from us on request.

DNA extraction, Southern blotting, and hybridization were performed as described previously (13). Tumor samples used for DNA extraction were monitored histologically with the cryostat sectioning procedure (14). Laser densitometry, was performed as described (15); the reference probes were D3S2 (chromosome 3) and HP (chromosome 16). To interpret the results for primary tumor samples without corresponding normal tissue, we determined the ratio of Al and A2 al- leles for a panel of normal tissues and compared this ratio with that obtained for two unpaired tumor samples (16). The

unpaired tumor samples were analyzed by comparing the A1/A2 ratio for the tumor with the A1/A2 ratio for normal tissues from a population sample. When the ratio A1/A2 for the tumor was significantly different from that for the pop- ulation sample, we concluded that there was probable allele loss in that tumor sample. We also used quantitative densi- tometry to determine chromosome copy number. Blots were hybridized with the chromosome 11p probe (D11S12) and 3p probe (D3S2), and band densities were determined. The ratio [(D3S2 tumor/D3S2 normal)/(D11S12 tumor/D11S12 normal)] was calculated for each tumor sample.

The following probes were used: D3S2 (p12-32) (17), HRASI (pUCEJ) (18), INS (pHins310) (19), D11S12 (pADJ762) (20), HBBC (JW151) (21), PTH (pPTH-LF) (22), CAT(pCAT41)(23),INT2(SS6)(24), D13S1(p7F12) (25), D13S2 (p9D11) (25), HP (hp-2 a) (26), D17S30 (pYNZ22) (27), D17S4 (pTHH59) (27), and D17S24 (pRMU3) (27).

Results

Adrenocortical Carcinoma

Chromosome 11p. DNA from normal tissues of six pa- tients with adrenocortical carcinoma was heterozygous for one or more restriction fragment length polymorphisms de- tected with recombinant DNA probes for chromosome 11p (table 1, fig. 1). In four of six patients, one of two codomi- nant alleles was lost in the adrenocortical carcinoma. In two patients for whom tumor tissue was available without accom- panying normal tissue, loss of alleles at loci on chromosome 1 lp was suspected in the primary tumors (see below, fig. 2).

Chromosome 13q. DNA from normal tissues of six pa- tients was heterozygous for one or more restriction fragment

Table 1. Chromosome 11 alleles in adrenocortical carcinomas*

Patient No.	Tissue		llp						11q
		HRASI (B)	INS (P)	D11S12 (Bcl)	HBBCt (H)	PTH (Pst)	CAT		INT2
							A	K	(B)
1	Recurrent tumor			22	22/44
	Liver metastasis			22	22/44
2	Recurrent tumor	12	12	12					12
	Diaphragm metastasis	22	22	22					22
	Lung metastasis	22	22	22					22
	Liver metastasis	22	22	22					22
3	Recurrent tumor	-¥	22				12
4	Kidney metastasis				12	12			12
	Lymph node metastasis				12	12			12
	Liver metastasis				12	12			12
5	Recurrent tumor	12	12		12
6	Recurrent tumor					ND	ND	ND	ND
9	Primary tumor		22	22

*The tumor phenotype is shown in every case where the normal tissue showed heterozygosity. Symbols: 12 = heterozygosity in tumor sample; 1 = retention of larger allele and loss of smaller allele; 2 = retention of smaller allele and loss of larger allele; 11 = loss of smaller allele and reduplication of larger allele; 22 = loss of larger allele and reduplication of smaller allele. A blank space indicates that the constitutional DNA was homozygous at that locus. Abbreviations for restriction endonucleases: B = BamHI, P = PvuII, Bcl = Bell, H = HindIII, A = AvalI, K = KpnI, Pst = Psrl. ND = not done. tThe HBBC probe detects two polymorphic systems: the alleles of the first system are designated 1 and 2; the alleles of the second system are designated 3 and 4. An individual who is doubly heterozygous would be designated 1234. A listing of 22/44 indicates that the normal tissue of that patient was heterozygous at both loci; the tumor showed loss of the 1 and 3 alleles and duplication of the 2 and 4 alleles. # Mutation in the adrenal gland of patient 3. See table 3.

Figure 1. Loss of alleles at loci on chro- mosome 11p in adrenocortical carcino- mas. Hybridization pattern of 32P-labeled probes for D11S12, HBBC, HRAS1, and INS to DNA from patients 1-3. Seven micrograms each of normal tissue and adrenocortical carcinoma DNA were di- gested with BamHI or PvuII, subjected to electrophoresis on a 0.75% agarose gel, and transferred to nylon membranes. Hy- bridization with 32P-labeled DNA (specific activity, 1 X 109-2 × 109 cpm/ug) was carried out at 65 ℃ as described in Pa- tients and Methods. The filter was washed and dried, and bands were visualized by autoradiography. We used TaqI instead of PvuII for analysis of the INS probe in pa- tient 2. Patient 3 was homozygous when tested with the Taql-digested INS probe. Abbreviations: N. = normal, Rec. = re- current, Met. = metastasis, Diaph. = di- aphragm.

N. Liver

Rec. Tumor

Liver Met.

N. Liver

Rec. Tumor

Diaph. Met.

Lung Met.

Liver Met.

N. Liver

N. Heart

N. Lung

N. Adrenal

Rec. Tumor

kb

11.6-

7.2-

6.6- 6.2-

7.0-

6.6~

HRASI BamHI

HRAS1 BamHI

4.3-

3.3-

2.1-

D11S12 BclI

2.5-

8.0- 7.2-

INS TaqI

0.74-

INS PvuII

3.5-

2.7-

HBBC HindIII

1

2

3

length polymorphisms detected with recombinant chromo- some 13q probes. In three of six patients, one of two codom- inant alleles was lost in the tumor (see table 2).

Chromosome 17p. All six patients whose normal tissues were heterozygous for a locus (D17S30) on 17p showed a loss of one allele in the tumor (see fig. 3). In one of two patients for whom primary tumor tissue was available with- out accompanying normal tissue, a loss of alleles at a locus (D17S4) on chromosome 17 was suspected (see below).

Analysis of Unpaired Samples

Because of the rarity of adrenocortical carcinoma, we an- alyzed two primary adrenocortical carcinomas even though corresponding normal tissue was not available. Allele loss on chromosomes 11p and 17 was suggested by the presence in both primary adrenocortical carcinomas of DNA fragments of markedly different intensity (fig. 2). When DNA from pa- tient 7 was digested with BamHI and hybridized with the HRAS1 probe, there was an intense signal at 8.0 kilobases (kb) and a faint signal at 6.6 kb. When DNA was digested with Pvull and hybridized with the THH59 probe, there was an intense signal at 1.2 kb and a faint signal at 0.7 kb. The ratio of signal intensity (A1/A2) for HRAS1 and THH59 in tumor tissue was outside the 95% tolerance limits of signal intensity ratio for a panel of normal individuals.

In patient 8, loss of alleles at a locus on chromosome 1 lp (D11S12) was suspected in the primary tumor (fig. 2). When DNA was digested with Bc/I and hybridized with the D11S12 probe, there was an intense signal at 11.6 kb and a faint signal at 4.3 kb. The signal intensity ratio for D11S12 in the tumor was outside the 95% tolerance limits of the signal intensity ratio for a panel of normal individuals. To test for incomplete Bc/I digestion, the filter shown in figure 2 was melted and rehybridized with a probe for chromosome 3 (D3S2); a single band was observed, consistent with com- plete Bc/I digestion.

These hybridization patterns are consistent with the con- cept that patient 7 was constitutionally heterozygous at the HRAS1 and THH59 loci and that patient 8 was constitution- ally heterozygous at the D11S12 locus. The weak signal in the tumors reflects loss of heterozygosity at that locus, with the residual signal originating from contaminating normal tissue.

Patterns of Allele Loss

In tumors from patients 4, 5, and 6, loss of alleles was detected at loci on chromosome 17p but not at loci on chromosome 11p or 13q. In tumors from patient 1, loss of heterozygosity was demonstrated at loci on chromosomes

Figure 2. Analysis of DNA from primary adre- nocortical carcinomas of patients 7 and 8. When DNA from primary tu- mor of patient 7 was hybridized with HRASI, an 8.0-kb band and a faint 6.6-kb band were observed; with the THH59 probe, a 1.2-kb band and a faint 0.9- kb band were observed. When DNA from the primary tumor of patient 8 was hybridized with the D11S12 probe, an 11.6-kb band and a faint 4.3-kb band were ob- served.

N. Liver

Rec. Tumor

Liver Met.

N. Liver

Rec. Tumor

Diaph. Met.

Lung Met.

Liver Met.

N. Liver

N. Heart

N. Lung

N. Adrenal

Rec. Tumor

kb

1.4-

1.5-

1.2-

1.1-

1.1-

1.2-

YNZ22 BamHI

YNZ22 BamHI

YNZ22 BamHI

1.4-

1.4-

1.5-

1.3-

THH59 PvuII

0.9-

THH59 PvuII

0.9-

THH59 PvuII

1

2

3

11p and 17q but not at a locus on 13q. In tumors from patients 2, 3, and 9, loss of heterozygosity was demonstrated at loci on chromosomes 11p, 13q, and 17p (table 2).

Multiple tumor samples were obtained from some patients at the time of surgical resection or autopsy. In patient 2, a recurrent adrenocortical tumor did not show loss at loci on chromosomes 11p, 13q, and 17p, but three distinct metas- tases did show such allele loss.

Evidence for Genetic Change in Adrenal Gland

DNA extracted from the liver, heart, and lung of pa- tient 3 showed a single 6.6-kb fragment when digested with

the restriction endonuclease BamHI and hybridized with a full-length probe for the HRASI gene; under the same con- ditions, DNA extracted from the normal adrenal gland and adrenal carcinoma of patient 3 showed two fragments, 6.6 and 6.2 kb (fig. 1, table 3). This result suggested that a so- matic mutation in the HRAS1 gene had occurred during the development of the normal adrenal gland. Other possible ex- planations for these findings were considered: (a) incorrect sample identification; the normal adrenal might have orig- inated from another patient or the “normal” adrenal might actually have been tumor tissue; (b) differences in suscepti- bility to restriction endonuclease digestion of DNAs from

Table 2. Chromosomes 13 and 17 alleles in adrenocortical carcinomas*

Patient No.	Tissue	Chromosome 13		Chromosome 17
		D13SI (M)	D13S2 (M)	YNZ22 (B)	THH59 (P)	RMU3 (T)
1	Recurrent tumor	12		22	22	11
	Liver metastasis	12		22	22	11
2	Recurrent tumor		12	12	12	12
	Diaphragm metastasis		22	11	11	11
	Lung metastasis		22	11	11	11
	Liver metastasis		22	11	11	11
3	Recurrent tumor		22	22	22	22
4	Kidney metastasis		12			22
	Lymph node metastasis		12			22
	· Liver metastasis		12			12
5	Recurrent tumor	12	12	1
6	Recurrent tumor	ND	ND	1
9	Primary tumor		2	2

*See table, footnote *. Additional abbreviations: M = MspI, T = TaqI.

Table 3. Chromosomal changes in patient 3 on chromosomes 11p, 13, and 17*

Tissue	11p					13	17
	HRAS1			INS (P)	CAT (A)	D13S2 (M)	YNZ22 (B)	THH59 (P)	RMU3 (T)
	B	M	T
Normal liver	11	11	11	12	12	12	12	12	12
Normal heart	11	11	11	12	12	12	12	12	12
Normal lung	11	11	11 .	12	12	12	12	12	12
Normal adrenal	12	11	11	12	12	12	12	12	12
Adrenal carcinoma	12	11	11	11	12	22	22	22	22

*The genotype is given for normal tissues and adrenal carcinoma of patient 3. Symbols: 11 and 22 = two identical alleles at the indicated locus; 12 = two different alleles at the indicated locus. Abbreviations for restriction endonucleases: B = BamHI, M = MspI, T = TaqI, P = PvuII, A = Avall.

heart, lung, and liver compared with adrenal DNA; and (c) tissue-specific differences in methylation of the BamHI site in the HRAS1 gene.

Molecular genetic tests provided no evidence for incorrect sample identification or tissue-specific differences in suscep- tibility to digestion with the restriction endonuclease BamHI. Hybridization of DNA from normal lung, liver, heart, and adrenal gland with three variable tandem-repeat probes gave DNA fragments of identical sizes, indicating that these tis- sues came from the same individual (fig. 1 and 3). The identity of the normal adrenal sample was verified by pathology; the normal adrenal sample was distinct from the adrenal carci- noma as measured by allele loss. DNA fragments detected with the full-length HRASI probe in normal adrenal and other normal tissues were unchanged with increased amounts of restriction endonuclease. The difference in band size was also detected with an isoschizomer of BamHI, BstI. Restric- tion mapping suggested that cells in the normal adrenal gland contained a deletion or point mutation in the promoter region of one copy of the HRASI gene (data not shown).

Figure 3. Loss of alleles at loci on chromosome 17 in adrenocorti- cal carcinomas. Hybridization pattern of 32P-labeled probes YNZ22 and THH59 to DNA from patients 1-3. Hybridization conditions are described in the legend to figure I.

Tumor, 7

Tumor, 8

Tumor, 7

Tumor, 8

Tumor, 7

Tumor, 8

kb

8.0-

11.6-

1.2-

6.6-

HRAS1 BamHI

0.7-

7.0-

THH59 PvuII

4.3-

D11S 12 BclI

Adrenal Adenomas and Adrenal Hyperplasia

No loss at loci on chromosome 11p, 13q, or 17p was detected in samples from eight patients.

Discussion

We found consistent loss of heterozygosity at loci on chromosomes 11p, 13q, and 17p in adrenocortical carci- nomas but not adrenal adenomas or hyperplastic lesions of the adrenal gland. The genetic changes detected in adreno- cortical carcinoma may be interpreted in terms of the two-mutation theory of cancer, which postulates that tumors develop as a consequence of inactivation of both copies of specific genes (28). Consistent allele loss at specific loci sug- gests that the region identified harbors a tumor suppressor gene. When a tumor shows evidence of allele loss on multi- ple chromosomes (29,30), it is difficult to determine which locus or loci are important in tumor origin, which are impor- tant in tumor progression, and which are of little importance to tumor biology.

Our studies with adrenocortical carcinoma illustrate this difficulty. From our results, it is not possible to derive a se- quential order of genetic changes on chromosomes 11p, 13q, and 17p. The results of studies of tumors from individual pa- tients support different conclusions. A mutation in the HRAS1 gene (11p) in the normal adrenal gland may have been an early event in the development of malignancy in patient 3. The observation that tumors in three patients showed allele loss on 17p but not 11p or 13q suggests that changes on chromosome 17p occur earlier in the evolution of adreno- cortical carcinoma than changes on 11p or 13q. The detec- tion of changes on chromosomes 11p, 13q, and 17p in the metastases of patient 2 but not in the recurrent tumor sug- gests that these changes were important in tumor progression rather than tumor origin.

These apparently contradictory results might be inter- preted by postulating that the same type of malignancy may originate as a consequence of inactivation of genes at differ- ent loci. Changes at tumor suppressor loci (in adrenocortical carcinoma, on chromosome 11p, 13q, or 17p) could lead independently to the same histologic tumor type. Another possibility is that, for certain tumors, inactivation of several tumor suppressor genes may be essential for the development of malignancy.

Resolving these questions will require linkage studies of families with inherited adrenocortical carcinoma and attempts to suppress malignancy with microcell-mediated chromosome transfer (31).

References

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