Evaluation of Telomere Length Maintenance Mechanisms in Adrenocortical Carcinoma

Tobias Else, Thomas J. Giordano, and Gary D. Hammer

Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine (T.E., T.J.G., G.D.H.), and Department of Pathology (T.J.G.), University of Michigan Health System, Ann Arbor, Michigan 48109-2200

Context: Adrenocortical cancer (ACC) is a rare disease with an often fatal outcome. The clinical and pathological diagnosis of a malignant vs. benign adrenocortical tumor is sometimes challenging. Telomere maintenance mechanisms (TMMs) are critical for the persistence of the malignant phe- notype, but little is known about these mechanisms or their diagnostic value in adrenocortical lesions.

Objective: Tissue samples of diagnostically known adrenocortical neoplasms were evaluated for parameters of known TMMs, telomerase activity (TA), and alternative telomere lengthening (ALT).

Design: The study analyzed retrospectively collected frozen adrenocortical tissue samples from the University of Michigan Health System.

Patient Samples: Samples included 24 ACCs, 11 adrenocortical adenomas (ACAs), and three normal adrenal tissues.

Main Outcome Measures: Telomerase activity (telomerase activity protocol assay), alternative telomere lengthening (telomere restriction fragment analysis, telomere associated promyelocyte leukemia bodies) were measured.

Results: A total of 22 of 24 ACCs (92%) could be definitively assigned to a TMM. The TMM classi- fication was: 19 of 24 TA (79%), two of which displayed very long telomeres, one of 24 ALT (4%) and two of 24 (8%) TA and ALT. Results of two of 24 (8%) were inconclusive (one negative for TA and positive in one ALT assay, one negative in all assays). None of the normal adrenal tissues (none of three) or ACA (none of 11) samples had signs of an active TMM.

Conclusions: TA is the main TMM in the majority of ACCs, but subsets of ACCs additionally or exclusively exhibit signs of ALT. Determination of telomere maintenance mechanisms in diagnos- tically challenging adrenocortical tumors might be of additional diagnostic value in the patho- logical diagnosis of malignant vs. benign lesions. (J Clin Endocrinol Metab 93: 1442-1449, 2008)

A drenocortical cancer (ACC) is a rare but often fatal dis- ease with an incidence of 1-2 per one million people. Despite the effort to improve therapy and implement new therapeutic regimens such as IGF-I receptor antagonists, the clinical course of the disease is often rapidly progressing (1,2). The 5-yr survival rate ranges from 16 to 38%, with a signif- icantly better prognosis for early stages (60% survival for stage I vs. 0% for stage IV) (1, 2). Even with established ther-

apeutic regimens such as mitotane, the overall benefit is rel- atively small (3). These dismal statistics provide motivation to improve early diagnosis and treatment. A special importance lies in the differentiation between malignant and benign le- sions, with the latter being a very common, often incidental finding as common as 3% over the age of 50 yr (2, 4). Whereas imaging studies and clinical evaluation often fail to make this critical distinction, fine-needle biopsy has been advocated as

Abbreviations: ACA, Adrenocortical adenoma; ACC, adrenocortical cancer; ALT, alterna- tive telomere lengthening; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; NAT, normal adrenocortical tissue; PBST, PBS and Tween 20; PML, promyelocyte leukemia; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TA, telomerase activity; TMM, telomere maintenance mechanism; TRAP, telomere repeat amplification protocol; TRF, telomere-length restriction fragment.

TABLE 1. Patient and tumor characteristics: sample number, type of tissue, age, sex, hormone status, weight, size, grade, mitotic rate (mitosis/HPF), survival (months after primary surgery) and stage of disease (clinical data)
Patient no.	Diagnosis	Age (yr)	Sex	Hormone secretion	Weight (g)	Size (cm)	Grade (high/low)	Mitotic rate	Survival (months)	Stage
1	NAT	28	M	N/A/Pheo MEN2A	N/A	N/A	N/A	N/A	N/A	N/A
2	NAT	71	F	N/A/Met. Lung Ca	N/A	N/A	N/A	N/A	N/A	N/A
3	NAT	55	M	N/A/Conn syndrome	N/A	N/A	N/A	N/A	N/A	N/A
4	ACA	62	F	Cortisol	65	3.3	N/A	N/A	N/A	N/A
5	ACA	51	F	Aldosterone	4.95	2.5	N/A	N/A	N/A	N/A
6	ACA	48	M	NF	174.5	9	N/A	N/A	N/A	N/A
7	ACA	45	M	NF	45	5.5	N/A	N/A	N/A	N/A
8	ACA	64	F	NF	116	7.2	N/A	N/A	N/A	N/A
9	ACA	44	M	Aldosterone	10.5	UNK	N/A	N/A	N/A	N/A
10	ACA	33	F	Aldosterone	17.2	1.1	N/A	N/A	N/A	N/A
11	ACA	55	M	Cortisol	31	4.5	N/A	N/A	N/A	N/A
12	ACA	32	F	Aldosterone	14.9	5	N/A	N/A	N/A	N/A
13	ACA	25	F	Cortisol	11.1	2.7	N/A	N/A	N/A	N/A
14	ACA	57	F	Cortisol/bilateral MNH	50	5.5	N/A	N/A	N/A	N/A
15	ACC	48	M	NF	110	6.2	Low	4	21, alive	3
16	ACC	19	M	Cortisol	UNK	10.5	High	27	8	3
17	ACC	47	F	NF	UNK	16	High	41	21, alive	2
18	ACC	34	M	NF	266	9.2	High	74	9	3
19	ACC	49	F	NF	UNK	UNK	Low	6	58, alive	4
20	ACC	77	M	Cortisol	UNK	UNK	High	45	19	4
21	ACC	51	F	Cortisol	UNK	UNK	High	37	97, alive	1
22	ACC	60	F	Cortisol	1805	18.5	High	40	4	4
23	ACC	61	F	Cortisol	253	9.8	High	27	11	2
24	ACC	55	M	Cortisol	35.1	5.5	Low	4	Lost to follow-up	3
25	ACC	52	F	NF	2890	19	Low	2	34	2
26	ACC	39	F	Cortisol	900	18	High	109	1	3
27	ACC	45	F	NF	UNK	9	High	137	1	4
28	ACC	48	F	NF	UNK	UNK	High	51	37	4
29	ACC	71	M	NF	2440	22	High	22	8	4
30	ACC	2	F	Testosterone	12	3.2	High	22	152, alive	1
31	ACC	41	F	DHEA	2540	17	Low	12	58	4
32	ACC	51	F	Testosterone	1305	15.5	High	23	27	3
33	ACC	35	F	Cortisol	460	16	Low	8	15	4
34	ACC	25	F	Cortisol + Testosterone	2300	26	High	34	11	4
35	ACC	31	F	Cortisol	520	12	High	65	108, alive	4
36	ACC	45	M	NF	60	5	Low	2	61, alive	1
37	ACC	45	F	Testosterone	180	9	High	24	18	2
38	ACC	8	M	Testosterone	450	12	High	64	6	4

M, Male; F, female; Y, present; N, not present; NF, nonfunctioning; IVC, inferior vena cava; DHEA, dehydroepiandrosterone; N/A, not applicable; UNK, unknown or not determined; Pheo, pheochromocytoma; MEN2A, multiple endocrine neoplasia type 2A; Met, metastasized; MNH, macronodular hyperplasia.

a useful modality that may improve the accuracy of diagnosis (5). However, even after removal of a mass, the final patho- logical diagnosis can be challenging (5, 6). Whereas a variety of morphological criteria (e.g. Weiss score), immunohisto- chemical markers (e.g. Ki67, which mirrors proliferation rate), and molecular diagnostics (e.g. LOH 17q13 or DNA content) has been suggested, such approaches do not achieve the necessary diagnostic specificity (2, 6).

Telomerase is a ribonucleoprotein that elongates the 3’ end of chromosomes to circumvent the end replication problem that leads to a loss of telomeric sequences over consecutive cell cycles and eventually to a removal of cells with critically short telomeres from the proliferating pool by the induction of apoptosis or senescence. Normal human somatic cells lack telomerase activity (TA), and only some constantly prolifer- ating or self-renewing tissues such as germ cells, skin, and the lymphoid system display significant TA (7-9). Proliferating

tumor cell clones often reactivate telomerase expression and activity. Roughly 90% of malignant human tumors make use of this telomere maintenance mechanism (TMM) (10, 11). Unfortunately, the few studies that have surveyed TA as a potential marker for malignancy in the adrenal gland reported conflicting results, presumably due to the limitations of the following: 1) small sample sizes (fewer than eight ACCs), 2) lack of conclusive final pathological diagnosis, 3) question- able tissue quality, or 4) differences in assay systems used (12-16).

Recently telomerase-independent alternative telomere main- tenance mechanisms defined as alternative telomere lengthening (ALT) have been described in several human tumors, mainly sarcoma-derived tumor cell lines (17-20). In a subset of ALT cell lines, homologous recombination between sister chromatids is used as a TMM, leading to an increased overall telomere length as well as length variation, compared with TA cell populations

TABLE 2. TMM analysis and TMM status
Patient no.	Diagnosis	TA (TRAP)	ALT (PML/telomere-FISH)	ALT (TRF/median kb length)		TMM
1	NAT	-			11	UNK
2	NAT	-			13	UNK
3	NAT				9	UNK
4	ACA				14	UNK
5	ACA				7	UNK
6	ACA				9	UNK
7	ACA				11	UNK
8	ACA				7	UNK
9	ACA				8	UNK
10	ACA				9	UNK
11	ACA				10	UNK
12	ACA				14	UNK
13	ACA				12	UNK
14	ACA				10	UNK
15	ACC	+			5	TA
16	ACC	+			8	TA
17	ACC	+			7	TA
18	ACC	+			9	TA
19	ACC	+			6	TA
20	ACC	+			6	TA
21	ACC	+			6	TA
22	ACC	+			10	TA
23	ACC	+		+	22	TA/ALT
24	ACC	+			10	TA
25	ACC	+			5	TA
26	ACC	+			3	TA
27	ACC	+			6	TA
28	ACC	+			5	TA
29	ACC	+		+	78	TA/ALT
30	ACC	+			7	TA
31	ACC	+			8	TA
32	ACC	+			8	TA
33	ACC	+			15	TA
34	ACC			ND	ND	UNK
35	ACC		+	+	20	ALT
36	ACC		+		9	UNK/ALT
37	ACC	+	+	+	20	TA/ALT
38	ACC	+	+	+	20	TA/ALT
NCIh295A		+	+	+	23	TA/ALT
SW13		+			10	TA
RL251		+			4	TA
HeLa		+			6	TA

Results for the individual assays are shown as positive (+) or negative (-) for TA (TRAP assay, positive: visible ladder) and ALT [immunohistochemical analysis for telomere-associated PML bodies (telomere-FISH/anti-PML-immunohistochemistry, positive: bright telomere signal and anti-PML colocalization of >8/high-power field), telomere length (TRF, positive: length > 19 kb)]. For each sample an overall classification is given. Upper-case superscript ALT is positive in one assay in addition to TA or UNK. N/A, Not Applicable; UNK, Unknown, not classifiable for any TMM due to absence of TA and lack of positivity in both or one ALT assays; ND, not detectable.

(21, 22). Although the exact mechanisms of ALT need to be elucidated, the presence of telomere-associated promyelocyte leukemia (PML) bodies correlates well with the existence of ALT (23). PML nuclear bodies are multifunctional subnuclear components, which have been shown to play a role in tumor- suppressive functions such as p53-mediated apoptosis and senescence or Rb-mediated transcriptional repression (24, 25). In ALT cell lines a bright telomere fluorescence in situ hybridization (FISH) signal colocalizes with PML immunostaining.

In a study investigating TMMs (TA and ALT) in liposar- comas, TMMs could be identified in approximately half of the neoplasms. A total of 25.9% of tumors had TA, 26.6% were

classified as ALT, and a small number was positive for both TMMs. This study shows that different TMMs are not mu- tually exclusive and that the underlying molecular mechanism needs to be further elucidated (19). The primary purpose of our study was to evaluate the different TMMs in ACCs, using the high-quality tissues collected at the University of Michigan Health System. A pathologically defined large sample set of ACCs (24 samples) and control tissues were scored for TMMs, using the telomere repeat amplification protocol (TRAP) as- say as a direct measurement of TA and immunostaining for telomere-associated PML bodies and telomere-length restric- tion fragment (TRF) analysis as surrogate parameters for ALT.

TABLE 3. Summary of results of TMM analysis in ACC
	TA (TRAP assay)	ALT-telomere length (TRF)	ALT-IHC (telomere-FISH/PML)	TMM classification
	TA (TRAP assay)	ALT-telomere length (TRF)	ALT-IHC (telomere-FISH/PML)	TA	ALT	TA/ALT	Positive
NAT	0/3	0/3	0/3	0/3	0/3	0/3	0/3
ACA	0/11	0/11	0/11	0/11	0/11	0/11	0/11
ACC	21/24	5/24	4/24	19/24	1/24	2/24	22/24

Number of samples positive in each assay and final classification with regard to the different TMMs. All samples that were tested in all assays are included. Analysis for the remaining two samples were the following: one sample only positive in the ALT immunostaining, negative in the TRF analysis (<15 kb) and negative for TA, the other sample negative in all assays. IHC, Immunohistochemistry.

Patients and Methods

Tissue samples

Patient samples (Table 1) were collected at the University of Michigan via the Tissue Procurement Service with institutional review board ap- proval. All samples were obtained from primary surgery except samples 20, 21, and 28, which were from surgery of recurrent ACC. Tissue sec- tions were reviewed by an endocrine pathologist, and representative parts were processed to paraffin tissue array blocks containing normal adrenocortical tissues (NATs), adrenocortical adenomas (ACAs), ACCs, and other reference organ tissues. Tissue samples were frozen in opti- mum cutting temperature compound and stored at -80 C. To select representative tissue samples, a section of tissue (NAT, ACA, or ACC) was marked on a standard hematoxylin and eosin-stained frozen section. The corresponding tissue was then microdissected from the tissue block and further kept at -80 C or on dry ice. This method of tissue prepa- ration avoids multiple freeze and thaw cycles and ensures tissue integrity as well as tissue pathology of the obtained tissue sample. Three NATs, 11 ACAs, and 24 ACCs were analyzed for telomere length, TA, and telomere-FISH/PML staining. Initial samples (two NATs, two ACAs, and 24 ACCs) were blind coded for the scoring researcher. The number of ACAs and NATs was increased for revision of this publication, and additional samples were therefore not blind coded.

Immunohistochemistry/telomere-FISH

An adrenocortical-specific tissue array was constructed using 1-mm cores from paraffin-embedded blocks of the University of Michigan ar- chives. Combined telomere-FISH and immunohistochemistry was car-

NAT	ACA		ACC				NCIh295A	HeLa
		ALT/TA	ALT	TA		ALT	ALT/TA	TA
2HI 2	14 14HI	38HI 38	35HI 35	19	15HI 15 19HI	35/+ 35	5x103 HI 5x102	〒 5x102

FIG. 1. Telomerase activity in adrenocortical tissue samples and the NCIh295A cell line. Representative samples and the according telomere maintenance mechanisms as well as controls are shown. Samples have been run on different gels, and therefore, signal intensity varies [all sets were run with negative heat inactivated (HI) samples and positive controls (HeLa cell extract)].

ried using the protocol of Meeker et al. (26) with slight modifications. Briefly, deparaffinized slides were hydrated through a graded ethanol series, transferred to ddH2O, and then boiled in citrate buffer [10 mM sodium citrate, 0.05% Tween 20 (pH 6.0)]. Slides were then washed in PBST (PBS, 0.05% Tween 20), rinsed in ddH2O, transferred to 95% ethanol, and air dried. The hybridization mixture [0.3 µg/ml peptide nucleic acid probe Cy3(CCCTAA)3, 70% formamide, 10 mM Tris, (pH 7.5), 0.5% blocking reagent] was applied, and slides were heated to 83 Con a glass plate in a prewarmed hybridization oven. Hybridization was carried out for at least 2 h at room temperature in the dark. Slides were washed twice in 70% formamide, 10 mM Tris (pH 7.5), and 0.1% BSA and once in PBST and then incubated overnight o/n at 4 C with anti-PML (no. 05-718; Upstate, Lake Placid, NY) 1:100 in PBST. Primary antibody was detected with a goat antimouse Alexa-Fluor 486-coupled antimouse IgG (no. A11029; Invitrogen, Carlsbad, CA; 1:200 in PBST, 1 h, room temperature), counterstained with 4’,6’-diamino-2-phenylindole (Sigma, St. Louis, MO; 1:2000, PBS), and washed twice in PBS. Cells grown on slides were subjected to the same protocol but not air dried before hybridization. Images were analyzed using an Optiphot-2 micro- scope (Nikon, Melville, NY) with a DP-70 camera and software system (Olympus, Hauppauge, NY). Only samples with a bright nuclear te- lomere-FISH signal (detectable at much lower exposure time than the usual telomere signal) colocalizing with anti-PML staining were counted as ALT positive in this procedure (>8 colocalization/high-power field HPF).

Cell culture

Cell culture media used in this study were as follows: NCIh295A: RPMI 1640, 2.5% bovine serum, and 1% insulin/transferrin/selenium; U2OS: Mc- Coy’s 5a medium, 10% fetal bovine serum (FBS); HeLa: DMEM, 10% FBS; SW13: DMEM, 10% FBS; RL251: RPMI 1640 10% FBS (27, 28). For immunohistochemi- cal analysis, cells were grown on fibronectin (Sigma; 1:40 in serum-free media)-coated slides overnight, fixed in 3.7% formalde- hyde in PBS for 10 min, and permeabalized 2 min at room temperature in 0.5% Nonidet P-40 in PBS. Slides were stored in 100% methanol at -20 C.

TRAP

Telomerase activity was determined by the TRAP using a commercially available kit (no. S7700; Chemicon, Temecula, CA) ac- cording to the manufacturer’s recommenda- tion. Tissue samples were gently crushed on liquid nitrogen and resuspended in [(3-chol- amidopropyl) dimethyl-ammonio]-1-pro- pane-sulfonate buffer. One microgram of protein was used for the TRAP assay. For cell lines and controls, the equivalent of 5 × 103 and 5 × 102 cells was used. A 32P-end labeled

FIG. 2. Telomere length analysis of adrenocortical tissue samples and the NCIh295A cell line. Representative TRF gels are shown for NAT and ACA (A), ACCs (B), and HeLa and NCIh295A cells (C). ACCs are annotated with their final categorization of TMM.

NAT

ACA

TA/ALT

ALT

HeLa

NCIh295A

145.5

97.0

48.5

23.1

9.42

6.55

4.36

primer was used in the TRAP reaction and dried gels were exposed to film for an average of 4 h. Sample lanes with a clearly visible ladder-type pattern were assessed as telomerase positive. All samples were run with a heat-inactivated negative control, and every run was controlled by a sample of 5 × 102 Hela cells. Negative samples were spiked with the equivalent of 5 × 102 Hela cells and reevaluated to exclude telomerase or polymerase inhibitors interfering with the assay.

TRF length assay

TRF analysis used published protocols (29) with modifications. Tissues (~100 µg) gently ground in liquid nitrogen as well as the nonsoluable fraction of the TRAP protocol [(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate lysis were resuspended in 100 ul PBS and mixed with 100 ul 2% LMP-agarose (SeaPlaque; Lonza, Rockland, ME). Plugs of this mixture were digested for 36 h in 2% sarcosyl, 500 mM EDTA, and 1 mg/ml proteinase K. After three washes in Tris/EDTA and preincubation in 500 ul Dpn II buffer (NEB, Ipswich, MA), genomic DNA was digested with 60 U Dpn II in fresh buffer for 36 h. Plugs and a low range PFG marker (NEB) were run in 1% agarose (Seakem, Lonza) using a CHEF mapper (Bio-Rad, Her- cules, CA). The automatic algorithm was set to a range of 1-80 kb. The DNA gels were depurinated (0.25 N HCl), denatured (0.5 N NaOH, 1.5 M NaCl), neutralized [0.5 M Tris, 1.5 M NaCl (pH 6)], and transferred to ¿- probe GT membrane (Bio-Rad). The membrane was baked 2 h at 80 C, prehybridized [4× Denhardts, 6X saline sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS)[, and hybridized with purified 32P-end labeled (TTAGGG)3 [5 ul 32P-ATP, 2 ul T4 polynucleotide kinase buffer, 1 µl PNK (NEB), 12 ul ddH2O, 30 min at 37 C, and cleaned through G25 spin columns (Amersham, Pisca- taway, NJ)] in 6× SSC and 0.1% SDS. Membranes were washed three times for 1 h in 6× SSC and 0.1% SDS and exposed to film for up to 4 dat -80 C. Scanned films were analyzed using the telemetric software (http:// bioinformatics.fccc.edu/software/opensource/telometric/telometric.shtml).

Results and Discussion

Telomerase activity

Twenty-four ACCs, 11 ACAs, and three NATs were analyzed for TA. Of the ACCs, 21 of 24 (88%) displayed clearly detectable TA, and three of 24 (12%) were TA negative (Tables 2 and 3 and Fig. 1). None of the ACAs and NATs had detectable TA. Samples

FIG. 3. Immunohistochemical analysis for ALT of tissue array samples. A, Classical colocalization of telomere signal (red) and anti-PML signal (green) in an ALT-positive ACC. B, Lack of colocalization in a ALT-negative ACC. C, Lack of colocalization in an ACA sample. Note the extremely bright telomere signal in ALT-positive samples in A vs. the normal telomere signal in B and C, which were taken with a longer exposure time.

negative in the primary TRAP assay were spiked with an extract of the equivalent of 5 X 102 Hela cells and reassayed in the TRAP to exclude inhibitors of TA in the sample preparation. All but one of the samples negative in the primary assay then dis- played significant TA (not significantly different from the HeLa cell positive control). The remaining sample showed a slightly diminished but clearly detectable TA. This excludes the presence of a telomerase inhibitor, and these samples were therefore clas- sified as TA negative. Extracts obtained from all adrenocortical cell lines, NCIh295A, SW13, and RL251, also displayed signif- icant telomerase activity.

Telomere length

To evaluate telomere length, we ran Dpn II-digested genomic DNA of ACCs, ACAs, and NATs in a pulsed-field gel (Table 2 and Fig. 2). In this analysis, three of three NATs and 11 of 11 ACAs displayed a homogeneous length distribution with a me- dian telomere length less than 15 kb (7-14 kb) in accordance with the reported length for human somatic cells. The ACC sam- ples displayed a much broader variation between samples (3-78 kb) with five of 24 samples showing a TRF pattern reminiscent of ALT with a very long telomere length (median > 19 kb) (18). Of the adrenocortical cell lines tested, only NCIh295A had long telomeres (median 23 kb), whereas telomere length of HeLa, SW13, and RL251 were in a range less than 10 kb.

FIG. 4. Immunohistochemical analysis for ALT in the NCIh295A cell line. A, Immunohistochemical analysis for ALT-associated telomere-associated PML bodies in NCIh295A cells showing a bright telomere signal colocalizing with anti- PML staining. B, U2OS cells as a positive control for ALT. C, Hela cells as a negative control (note the visible normal telomere signal due to a longer exposure time).

Immunohistochemical signs of ALT

Tissue arrays including all samples used for TRF and TRAP analysis as well as additional ACA, NAT, and control tissue samples (24 ACCs, 22 ACAs, four NATs, and 16 normal control tissues) were subjected to a FISH procedure with a Cy3-labeled peptide nucleic acid telomere probe followed by immunohisto- chemistry for PML bodies. The association of a telomere signal with PML bodies has been shown to define tumors using ALT (23). Using this procedure, four of 24 ACC samples displayed a striking colocalization of PML staining and a bright telomere signal, which was detectable at exposure times significantly lower (~ one of six) than that necessary for the visualization of the usual telomeric telomere signal (Table 2 and Fig. 3). All but one of these samples were also determined to be ALT positive in the TRF analysis. Finally, two of the three ALT-positive ACCs were assayed positive for TA. None of the other 20 ACCs, 22 ACAs, four NATs, or control tissue samples was positive in this assay. Some samples showed a positive PML signal but lacked bright telomere signals (Fig. 3). None of the array samples dis- played a bright telomere signal in the absence of positive PML staining. We also tested the adrenocortical cell line NCIh295A for telomere-associated PML bodies as well as U2OS cells, a defined ALT cell line, and HeLa cells, a well-described TA cell line, as a positive and negative control, respectively (Fig. 4). De- spite their telomerase activity, NCIh295A cells displayed a clas-

sically colocalized ALT staining, indicative of dual TMMs in these cells. We did not observe a typical ALT staining in either SW13 or RL251 cells.

This study represents the largest analysis of TMMs in ACCs. In summary, we were able to assign 22 of 24 ACCs to at least one definitive TMM. In contrast, none of the NAT or ACA controls displayed signs of a TMM (Tables 2 and 3). The majority of ACC samples were positive for TA (21 of 24, 88%), with 19 of 24 (79%) using TA as their only TMM. In the ALT analysis, three ACCs showed definitive signs of ALT (positive in the immuno- histochemical assay and long telomeres >19 kb), with two of three having TA as well and one of three being definitively cat- egorized as ALT. Two samples were inconclusive in data inter- pretation. One sample was ALT positive in the telomere-asso- ciated PML body staining but ALT negative in the TRF analysis (length <15 kb) and negative for TA. The other sample was negative for TA and ALT-associated immunostaining, whereas telomere length was uninterpretable due to the lack of DNA in this lane. In this current analysis, TMMs could be determined in the vast majority of samples. This is in contrast to analyses of other tumor entities, in which nearly half of the samples could not be definitively assigned to a class of TMM and underscores the importance of active TMMs in ACCs (17, 19).

The fact that a relatively high percentage of our ACC samples were TA positive is in contrast to previous studies and might be due to differences in assays, sample quality, and tissue integrity. In this study tissues were handled as safely as possible from sam- ple site through storage to final analysis. For the latter two stages, thawing did not occur. Although we tried to ensure to capture the right sample area from tissue blocks guided by hematoxylin and eosin-stained mirror slides, the sample lacking TA activity as well as interpretable TRF analysis may represent an autolytic, par- tially degraded sample. Also, tissue underlying the target area on the frozen section slide may have been of extremely low cellu- larity or necrosis. The variability of percentage of TA-positive samples between studies may also reflect epidemiological heter- ogeneity between different studies.

These data identify TA as the major TMM in ACCs and pro- vide evidence for ALT as a TMM in a small subset of ACCs as well as for the coexistence of telomerase-dependent and -inde- pendent mechanisms in the same tumor. Indeed, the existence of ALT is in accordance with an earlier study, which showed long telomere length in two of six ACCs (18). Interestingly, our find- ings in the adrenocortical cell line NCIh295A support the usage of TA as well as ALT mechanisms. It has recently been shown that NCIh295R cells, another subclone of the original parental NCIh295 cell line, uses ALT but is TA negative, which highlights the variability of TMM in the same cell line over passages and subcloning procedures (30). These findings are well in accor- dance with data from liposarcomas showing a coexistence of both mechanisms in the same tumor presumably due to the ex- istence of different subpopulations using different, or even both, mechanisms (17, 19, 31).

Although our current study is too small to correlate TMMs with patient and disease characteristics, it is remarkable that both patients from which the samples lacking TA were derived showed increased survival and have a long standing history of

ACC (108 and 61 months, both alive). This suggests a survival advantage for patients with tumors lacking TA, regardless of other TMM.

There is good evidence from other studies that the existence of TMMs generally correlates with a worse prognosis. In accor- dance with our results, in one study investigating TMM in as- trocytoma IV, length of patient survival correlated with the pres- ence of ALT, whereas in two studies of osteosarcomas and liposarcomas, any kind of TMM was correlated with a worse prognosis (19, 32, 33).

In transformed human and bovine adrenocortical cells, TA is not necessary for the development of a malignant phenotype but is required for immortalization and maintenance of an in vivo malignant tumor growth over extended passage numbers. In a study conducted by Sun et al. (34), cultures of transformed TA- negative bovine or human adrenocortical cells did not maintain the ability of indefinite clonal expansion but progressed to se- nescence after a limited number of serial transplantations in nude mice. Only after the additional expression of telomerase did transformed adrenocortical cells gain the characteristic of main- taining a malignant phenotype over an extended number of serial xenograft transplantation (34).

The analyses of TMMs presented in this study were con- ducted on extremely small tissue samples. As little as less than 5 mg can be sufficient for these procedures. This fact makes the determination of TMM interesting for samples from procedures, in which only very small tissue samples can be obtained, such as fine-needle biopsies. A biopsy approach is currently not part of the general diagnostic work-up of incidentally discovered adre- nocortical lesion of any size, and for large adrenal lesions, a surgical approach is warranted (35-37). Future studies need to address the safety of biopsy procedures and determine the inci- dence of associated periprocedural risks such as hemorrhage or needle-track metastasis. However, because an increasing num- ber of studies reports the feasibility of transgastral ultrasound and computed tomography-guided fine-needle aspirates or bi- opsies, these tissues may become more commonly available (38- 40). TMM analysis could be used as a molecular diagnostic tool to aid in differentiation of benign and malignant lesions and may complement simple morphological and immunocytochemical analysis of these samples. However, the validation of this diag- nostic procedure in a larger sample set or a prospective clinical trial is warranted.

The high percentage of samples positive for TMM in general and TA specifically identifies telomeres and the ribonucleopro- tein telomerase as a potential molecular target for ACC therapy. In preclinical studies, several telomerase inhibitors as well as immunological approaches using telomerase as an antigen show promising results in cancer therapy models (41). Further moti- vation to use TMM as a therapeutic target comes from the ob- servation that transformed adrenocortical cells need TA (or po- tentially any other kind of TMM) for maintenance of a malignant phenotype (34).

In summary, our study demonstrates the existence of different TMMs and telomerase-dependent (TA) as well as telomerase- independent (ALT) mechanisms in ACCs, but neither of these is evident in NAT or ACA. These findings suggest that the analysis

of TMM may not only be of potential diagnostic value but also represent a novel molecular therapeutic target in ACCs for which new therapeutic strategies are desperately needed.

Acknowledgments

The authors thank Michelle Vinco for tissue preparation and patient data collection; Jose Garcia-Perez, Tammy Morrish, and Marc Prindle for extremely valuable technical advice; and members of the Hammer Lab- oratory and Sonalee Shah for critical reading of the manuscript.

Address all correspondence and requests for reprints to: Gary D. Hammer, University of Michigan Medical School, 109 Zina Pitcher Place, BSRB 1502, Ann Arbor, Michigan 48109-2200. E-mail: ghammer@umich.edu; or Tobias Else, University of Michigan Medical School, 109 Zina Pitcher Place, BSRB 1860, Ann Arbor, Michigan 48109-2200. E-mail: telse@umich.edu.

This work was supported in part by Schembechler Fund for Adre- nocortical Cancer Research at the University of Michigan. T.E. is funded through the Garry Betty Foundation. G.D.H. receives support from American Cancer Society Grant RSG-04-236-01-DDC.

Disclosure Statement: T.E., T.J.G., and G.D.H. have nothing to declare.

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