ANALYSIS OF NUMERICAL CHROMOSOMAL ABERRATIONS IN ADRENAL CORTICAL NEOPLASMS BY FLUORESCENCE IN SITU HYBRIDIZATION
TAKEFUMI SHONO, HIDEKI SAKAI,* KOUSUKE TAKEHARA, SUMIHISA HONDA AND HIROSHI KANETAKE
From the Departments of Urology and Radiation Epidemiology, Atomic-Bomb Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan
ABSTRACT
Purpose: We identified numerical chromosomal aberrations in adrenal cortical neoplasms using interphase fluorescence in situ hybridization (FISH) and correlated these aberrations with DNA ploidy and endocrine dysfunction.
Materials and Methods: Our study included 25 adenomas and 2 carcinomas associated with primary aldosteronism or Cushing’s syndrome. Eight normal adrenal tissue samples served as controls. Isolated nuclei from frozen samples were used for FISH and formalin fixed, paraffin embedded tissues from the same materials were analyzed by flow cytometry for DNA ploidy. For FISH we used centromere specific probes for chromosomes 3, 7, 8, 11 and 12.
Results: None of the normal adrenal tissues had any numerical chromosomal aberrations in any chromosome analyzed or any abnormal findings on DNA ploidy analysis. Tetrasomy of chromosomes 3, 7, 8, 11 and 12 was detected in 8, 13, 14, 11 and 12 of the 17 adenomas associated with primary aldosteronism, and in 2, 0, 0, 0 and 0 of the 8 associated with Cushing’s syndrome, respectively. DNA flow cytometry revealed tetraploidy in 11 of the 17 cases of primary aldoste- ronism and in 1 of the 8 of Cushing’s syndrome. Five diploid adenomas associated with primary aldosteronism also showed tetrasomy in 2 or more chromosomes. One of the 2 carcinomas showed aneuploidy and aneusomy of chromosomes 8, 11 and 12 but the other showed no abnormal peaks on DNA histography and no numerical chromosomal aberrations.
Conclusions: All chromosomes analyzed in adenomas associated with primary aldosteronism frequently showed tetrasomy, whereas few chromosomal abnormalities were detected in adenomas associated with Cushing’s syndrome. Our results indicate that DNA tetraploidy is common in adrenal cortical adenomas associated with primary aldosteronism. Interphase FISH strongly supported flow cytometry findings and could provide further information on individual chromosomes.
KEY WORDS: adrenal glands, adrenal neoplasms, Cushing’s syndrome, hyperaldosteronism, chromosome aberrations
Adrenal cortical adenoma, which arises from adrenal cor- tical cells and is frequently associated with endocrine syn- dromes, has various histological features, especially ade- noma associated with primary aldosteronism.1,2 Previously investigators have applied various methods to adrenal corti- cal tumors to distinguish malignant from benign lesions.3-7 Techniques such as image analysis and flow cytometry have estimated but not actually enumerated chromosomal abnor- malities in several neoplasms. Karyotypic change in adrenal cortical neoplasms, particularly nonrandom alterations in chromosomes 6, 7, 11, 13, 17, 19, 22 and Y, have recently been described.8-10 However, these results of these methods are uncertain because of the low frequency of such tumors and the limited information obtained from the few dividing cells available for such analyses.
We have previously identified a significant relationship of flow cytometry DNA ploidy with nuclear grade as well as with the clinical syndrome associated with adrenal cortical adenomas.11 Our results suggested that adenomas associ- ated with primary aldosteronism had significantly higher nuclear grade than tumors associated with Cushing’s syn- drome. We also noted that a larger population of adenomas
Accepted for publication May 10, 2002.
Supported by Grant-in-Aid for Scientific Research 08671825 from the Ministry of Education, Science, Sports and Culture of Japan. * Requests for reprints: Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.
associated with primary aldosteronism than with Cushing’s syndrome showed tetraploid DNA histography results. Therefore, our findings suggest that nuclear pleomorphism may be due to a tetraploid stem line in adrenal cortical adenomas associated with primary aldosteronism.
Fluorescence in situ hybridization (FISH) facilitates the study of chromosomal alterations in a large number of inter- phase and metaphase cells on glass slides. However, few FISH studies of adrenal cortical neoplasms have been reported.12,13 Therefore, to our knowledge the importance of this approach for characterizing chromosomal changes in these tumors has never been underscored. The combination of FISH and DNA flow cytometry may provide additional information on chromosomal abnormalities and their relationship with clonal DNA ploidy in adrenal cortical adenomas. In the current study we applied FISH to fresh tumor samples and normal adrenal tissue to investigate numerical chromosomal alterations in adrenal cor- tical neoplasms, correlate interphase numerical aberrations with corresponding DNA ploidy and correlate these findings with the clinicopathological features of these tumors.
MATERIALS AND METHODS
Patients. Our study included 25 adrenal cortical adenomas and 2 adrenal cortical carcinomas associated with primary aldosteronism or Cushing’s syndrome. All patients under- went surgery at our institution between 1994 and 2000.
Patient age and sex as well as the associated endocrine syndrome were obtained from clinical and pathological re- ports.
Histopathological examination. All specimens were rou- tinely processed with 10% neutral buffered formalin fixation and paraffin embedding. Sections (4 um.) from representa- tive blocks were prepared and stained with hematoxylin and eosin.
FISH. A total of 27 frozen sections obtained at open or laparoscopic adrenalectomy in 27 patients formed the mate- rial for in situ hybridization. Eight normal adrenal tissues, each excised at least 1.5 cm. from the edge of an adrenal tumor, served as controls. Small fragments (8 to 10 mm.3) of frozen tissue samples were thawed in 1.5 ml. polypropylene tubes and gently touched onto the bottom of the tubes to attach cells. After removing the tissue samples from the tube the attached cells were immediately treated with 0.05 M. KCI and fixed in 3:1 methanol acetic acid (volume per volume). Nuclei suspensions were dropped onto silane coated slides and air dried. Slides were dipped in phosphate buffered sa- line for 15 minutes for immediate use or stored at -20C. FISH was performed according to standard procedures with some modifications.14
Pretreated slides were directly added to 72 × denaturation solution (70% formamide/2 × saline sodium citrate for 5 minutes), then dehydrated through a series of ethanol solu- tions and air dried. Biotinylated probes for chromosomes 3 (D3Z1), 7 (D7Z2), 8 (D8Z1), 11 (D11Z1) and 12(D12Z1) were used (Oncor, Gaithersburg, Maryland). Immunodetection was accomplished by incubation with fluorescein isothiocya- nate conjugated avidin. Slides were counterstained with pro- pidium iodide in anti-fade solution and examined under a confocal laser scanning microscope.
Evaluation of FISH results. Evaluation of results was per- formed by counting at least 100 interphase nuclei per slide, as described by Hopman et al15 with slight modifications.14 Counting was done while blinded to clinical data associated with the sample. In our study the percent of control nuclei containing 1 to 8 spots per nucleus was counted per probe. Polysomy, including trisomy and tetrasomy, was frequently
observed even in normal adrenal cortex samples. The mean incidence plus or minus standard deviation of nuclei from normal tissue showing 3 signals was 3.3% + 2.3%, 5.5% + 3.9%, 4.1% + 2.4%, 4.4% + 3.2% and 4.9% + 2.8% for chro- mosomes 3, 7, 8, 11 and 12, respectively. On the other hand, the incidence of nuclei with 4 signals was 4.3% + 2.8%, 5.2% + 2.3%, 3.7% + 2.7%, 5.5% + 3.0% and 3.6% + 1.8%, respec- tively. If the percent of nuclei with 3 or 4 signals for a chromosome was more than the mean +3 standard deviation of control samples, the chromosome was considered to show trisomy or tetrasomy.16 When trisomy and tetrasomy were identified in the same sample, the chromosome was consid- ered to show aneusomy. The nuclear DNA content of 27 adrenal cortical neoplasms and 8 normal adrenal glands was analyzed by flow cytometry, as described previously.11 The Mantel extension test was used for comparing the distribu- tion of number of signals per nucleus among endocrine syn- drome groups with p < 0.05 considered statistically signifi- cant.
RESULTS
We analyzed 25 samples of adrenal cortical adenoma, 2 of adrenal cortical carcinoma and 8 of normal adrenal cortex. All 8 normal adrenal glands showed diploidy on flow cytom- etry and disomy for the 5 chromosomes analyzed by FISH. The table lists clinical data, flow cytometry and FISH find- ings on the 5 chromosomes tested in the 25 adenomas. The patient population comprised 15 females and 10 males 26 to 66 years old (mean age 46). DNA ploidy analysis revealed 11 diploid (44%) and 12 tetraploid (48%) tumors. Only 2 aneu- ploid cases were detected in this study but subsequently recurrence or metastasis did not develop in them or in the other adenoma cases. Six of the 8 adenomas (75%) associated with Cushing’s syndrome showed DNA diploidy and no dif- ference was noted in tumor size or microscopic findings in diploid and nondiploid adenomas. On the other hand, 11 of the 17 adenomas (65%) associated with primary aldosteron- ism showed tetraploidy. FISH revealed tetrasomy of chromo- somes 3, 7, 8, 11 and 12 in 8, 13, 14, 11 and 12, respectively,
| Pt. - Age - Sex No. | Clinical Manifestation | DNA Ploidy | Chromosome Change | ||||
|---|---|---|---|---|---|---|---|
| 3 | 7 | 8 | 11 | 12 | |||
| Adenoma | |||||||
| 1- 31-M | Cushing's syndrome | Diploidy | Disomy | Disomy | Disomy | Disomy | Disomy |
| 2- 40 -F | Cushing's syndrome | Diploidy | Disomy | Disomy | Disomy | Disomy | Disomy |
| 3- 26 -F | Cushing's syndrome | Diploidy | Disomy | Disomy | Disomy | Disomy | Disomy |
| 4- 66 -M | Cushing's syndrome | Diploidy | Disomy | Disomy | Disomy | Disomy | Disomy |
| 5- 33-F | Cushing's syndrome | Diploidy | Disomy | Disomy | Disomy | Disomy | Tetrasomy |
| 6- 51 -F | Cushing's syndrome | Diploidy | Tetrasomy | Disomy | Disomy | Disomy | Disomy |
| 7- 43-M | Cushing's syndrome | Tetraploidy | Tetrasomy | Disomy | Disomy | Disomy | Tetrasomy |
| 8- 45-F | Cushing's syndrome | Aneuploidy | Disomy | Disomy | Disomy | Trisomy | Aneusomy |
| 9- 44-F | Aldosteronism | Diploidy | Disomy | Disomy | Tetrasomy | Tetrasomy | Disomy |
| 10- 62 -F | Aldosteronism | Diploidy | Disomy | Disomy | Tetrasomy | Tetrasomy | Disomy |
| 11- 31 -M | Aldosteronism | Diploidy | Tetrasomy | Tetrasomy | Tetrasomy | Disomy | Disomy |
| 12- 54-M | Aldosteronism | Diploidy | Aneusomy | Tetrasomy | Tetrasomy | Disomy | Tetrasomy |
| 13- 44 -F | Aldosteronism | Diploidy | Tetrasomy | Tetrasomy | Trisomy | Tetrasomy | Tetrasomy |
| 14- 49 -F | Aldosteronism | Tetraploidy | Disomy | Tetrasomy | Tetrasomy | Tetrasomy | Disomy |
| 15- 51 -F | Aldosteronism | Tetraploidy | Disomy | Tetrasomy | Tetrasomy | Disomy | Tetrasomy |
| 16- 40 -M | Aldosteronism | Tetraploidy | Tetrasomy | Tetrasomy | Trisomy | Tetrasomy | Disomy |
| 17- 54-M | Aldosteronism | Tetraploidy | Tetrasomy | Aneusomy | Tetrasomy | Disomy | Tetrasomy |
| 18- 43 -M | Aldosteronism | Tetraploidy | Aneusomy | Tetrasomy | Tetrasomy | Disomy | Tetrasomy |
| 19- 58 -F | Aldosteronism | Tetraploidy | Aneusomy | Disomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 20- 51 -M | Aldosteronism | Tetraploidy | Disomy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 21- 42 -F | Aldosteronism | Tetraploidy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 22- 45 -F | Aldosteronism | Tetraploidy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 23- 46 -M | Aldosteronism | Tetraploidy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 24- 44 -F | Aldosteronism | Tetraploidy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy | Tetrasomy |
| 25- 51 -M | Aldosteronism | Aneuploidy | Disomy | Tetrasomy | Disomy | Disomy | Tetrasomy |
| Ca | |||||||
| 1- 43 -F | Cushing's syndrome | Aneuploidy | Not analyzed | Tetrasomy | Aneusomy | Aneusomy | Aneusomy |
| 2- 79 -F | Aldosteronism | Diploidy | Not analyzed | Disomy | Disomy | Disomy | Disomy |
of the 17 adenomas associated with primary aldosteronism. Tetrasomy of those same chromosomes was demonstrated in only 2, 0, 0, 0 and 0, respectively, of the 8 adenomas associ- ated with Cushing’s syndrome. Furthermore, 5 diploid ade- nomas associated with primary aldosteronism showed tetra- somy in 1 or more chromosomes. In this study aneusomy was identified in 4 cases and trisomy was identified in 3.
The table also shows clinical data, flow cytometry and FISH findings on the 4 chromosomes tested in the 2 adrenal cortical carcinomas. Each patient had recurrence after sur- gery and died of disease progression. Patient 1 had an ane- uploid tumor with aneusomy of chromosomes 8, 11 and 12. In contrast, patient 2 showed diploidy on flow cytometry and disomy for all chromosomes analyzed by FISH.
Figure 1 shows representative examples of interphase cy- togenetics specific for chromosome 8 in normal adrenal tissue
a
b
C
and adenoma. In normal adrenal tissue and adrenal cortical adenomas associated with Cushing’s syndrome most nuclei showed 2 signals (fig. 1, a and b). In contrast, the nuclei of adenomas associated with primary aldosteronism frequently showed 4 signals and some nuclei showed 8 or more (fig. 1, c).
Figure 2 shows interphase cytogenetics results for normal adrenal tissues and adrenal cortical adenomas. The histo- gram represents the frequency distribution of the number of signals per nucleus detected by FISH with probes specific for chromosomes 3, 7, 8, 11 and 12. In normal adrenal tissues the proportion of nuclei with 3 or more signals was small in each chromosome. Adrenal cortical adenomas associated with pri- mary aldosteronism showed a higher frequency of cells with 4 signals in each chromosome compared with normal adrenal glands and adenomas associated with Cushing’s syndrome. In addition, the proportion of cells with 8 signals in adeno- mas associated with primary aldosteronism was also in- creased in the 5 chromosomes analyzed. By comparing the distribution of the number of signals per nucleus among endocrine syndrome groups for each chromosome tested more signals were detected for primary aldosteronism than for normal adrenal cortex or Cushing’s syndrome (p <0.001).
DISCUSSION
Adrenal cortical adenomas can show various histopatho- logical features, although they follow a benign clinical course.
100
Chromosome 3
100
Chromosome 7
80
80
60
60
%
%
40
40
20
20
0
2
Ø
V
9
0
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
Number of signals
Number of signals
100
Chromosome 8
100
Chromosome 11
80
80
60
60
%
%
40
40
20
20
0
2
5
2
4
2
8
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
Number of signals
Number of signals
100
Chromosome 12
80
normal adrenal gland
60
%
40
Cushing syndrome
20
primary aldosteronism
0
8
D
0
1
2
3
4
5
6
7
8
Number of signals
Others have previously distinguished malignant from benign adrenal neoplasms based on methods such as histological features, 4,6,7 immunohistochemical findings and flow cytom- etry DNA.5 However, cytogenetic studies of adrenal cortical neoplasms have been scant.8-10 FISH in interphase cells can overcome the limitations of cell culture and offer the possi- bility of analyzing high cell numbers.16 The combination analysis of numerical chromosomal abnormalities and DNA ploidy provides additional information that may be useful for the histological and biological evaluation of adrenal cortical adenoma.
In the current study we used 5 centromeric sequences to investigate numerical abnormalities involved in adrenal cor- tical tumors and we identified several novel genetic alter- ations. Numerical chromosomal abnormalities correlated with the DNA ploidy pattern determined by flow cytometry in 8 normal adrenal tissues and 25 adenomas. Results in adrenal cortical adenomas associated with Cushing’s syn- drome were similar to those in normal tissues except for 1. All were diploid and had few numerical chromosomal abnor- malities.
With respect to adrenal cortical adenomas associated with primary aldosteronism we identified some discrepancies in the results of FISH and DNA ploidy. Although only 5 of 17 tumors (29%) were diploid on flow cytometry, all showed tetrasomic changes in at least 2 chromosomes. The identity of chromosomes with tetrasomy differed among tumors. In ad- dition, FISH of tetrasomic tumors also revealed an increased number of nuclei containing 8 signals. These results sug- gested that adrenal cortical adenomas associated with pri- mary aldosteronism may have originated from a stem line that was aberrant (tetrasomic) for several chromosomes. We speculate that certain factors could arrest the cell cycle be- tween G2 and M, thus, generating an aberrant stem line with twice the normal DNA content. We also speculate that this alteration may occur to some degree in each adrenal cortical adenoma and flow cytometry could sometimes not detect changes in a small number of cells.
In our study 1 case of adrenal cortical carcinoma associated with Cushing’s syndrome showed aneuploidy by flow cytom- etry as well as aneusomy of chromosomes 8, 11 and 12, although the other case of carcinoma with primary aldoste- ronism did not show abnormal peaks on DNA histography and no numerical chromosomal aberrations on FISH. Al- though FISH failed to discriminate malignant from benign tumors in the latter case, multiple aneusomic chromosomes may suggest the malignant nature of adrenal cortical tumors but not vice versa.
We showed that interphase cytogenetics is a more valuable tool than flow cytometry for investigating chromosomal al- terations in adrenal cortical neoplasms. To our knowledge this report represents the first study of the relationship of interphase cytogenetics and DNA ploidy in adrenal cortical adenoma. Our results emphasize that one should not inter- pret tetrasomy or tetraploidy as malignant findings since they are most often evident in adrenal cortical adenomas associated with primary aldosteronism. Even DNA aneu- ploidy on flow cytometry does not always indicate malig- nancy. Our results also indicate that FISH is not necessarily useful for discriminating malignant and benign lesions but multiple aneusomic chromosomes in aneuploid adrenal cor- tical tumors are suggestive of malignancy. In situ hybridiza- tion can identify chromosomes involved in numerical aberra- tions and facilitate further detailed structural and molecular characterization. Clarification of the mechanism of tumori- genesis in adrenal cortical neoplasms requires further stud- ies.
CONCLUSIONS
In adrenal cortical adenomas associated with primary al- dosteronism all chromosomes analyzed frequently showed tetrasomy, whereas few chromosomal abnormalities were de- tected in adenomas associated with Cushing’s syndrome. Our results indicate that DNA tetraploidy is common in adrenal cortical adenomas associated with primary aldosteronism. Interphase FISH strongly confirmed flow cytometry findings and could provide further information on individual chromo- somes.
Dr. Morimasa Matsuta, Iwate Medical University assisted with FISH.
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