The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 91, Issue 4

Scientific Articles | April 01, 2009

Telomere-Maintenance Mechanisms in Soft-Tissue Malignant Fibrous Histiocytomas

Toshihiro Matsuo, MD, PhD¹; Jerry W. Shay, PhD²; Woodring E. Wright, MD, PhD²; Eiso Hiyama, MD, PhD¹; Shoji Shimose, MD, PhD¹; Tadahiko Kubo, MD, PhD¹; Takashi Sugita, MD, PhD¹; Yuji Yasunaga, MD, PhD¹; Mitsuo Ochi, MD, PhD¹

¹ Department of Artificial Joints and Biomaterials (T.M. and Y.Y.), Natural Science Center for Basic Research and Development (E.H.), and Department of Orthopaedic Surgery (S.S., T.K., T.S., and M.O.), Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail address for T. Matsuo: tomatsuo@hiroshima-u.ac.jp
² Department of Cell Biology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039

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Disclosure: The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.

Investigation performed at Hiroshima University, Hiroshima, Japan, and University of Texas, Southwestern Medical Center, Dallas, Texas

The Journal of Bone and Joint Surgery, Inc.

J Bone Joint Surg Am, 2009 Apr 01;91(4):928-937. doi: 10.2106/JBJS.G.01390

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Abstract

Background: Sarcomas are distinct from carcinomas in that a substantial portion of them use the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. The present study clarifies the prevalence of the ALT mechanism and examines the prognostic importance of telomere factors in soft-tissue malignant fibrous histiocytomas.

Methods: We investigated a series of forty-three soft-tissue malignant fibrous histiocytoma samples from forty-three patients with regard to telomere length, telomerase activity, and human telomerase reverse transcriptase (hTERT) mRNA expression. Tumor samples were obtained from surgical specimens and were stored at -80°C until use. Univariate analysis of the tumor samples from patients for whom data were available on age, sex, histological grade, tumor size, surgical margin, recurrence, and telomere factors was performed with use of the log-rank test. Multivariate analysis with only significant variables was then performed.

Results: Telomerase activity was detectable in 79.1% of the tumor samples, hTERT expression was demonstrated in 90.7% of the tumor samples, and evidence of engagement of the ALT mechanism of telomere length maintenance was observed in 32.6% of the tumor samples. Among the variables tested, ALT-positive status emerged as the only independent prognostic factor for death of the patient (hazard ratio, 0.275; 95% confidence interval, 0.104 to 0.724; p = 0.0089). There were no significant differences in survival rates between patients with ALT-positive, telomerase-positive tumors and those with ALT-positive, telomerase-negative tumors (p = 0.301) or between patients with ALT-positive tumors that showed above-average telomerase activity and those with ALT-positive tumors that showed below-average telomerase activity (p = 0.900). Therefore, telomerase activity does not affect the prognosis in patients with ALT-positive malignant fibrous histiocytoma. High telomerase expression is associated with a poor prognosis in patients with ALT-negative malignant fibrous histiocytoma (p = 0.0027).

Conclusions: More detailed analysis will be needed to identify the most valuable prognostic factor in patients with malignant fibrous histiocytoma, and a more thorough understanding of telomere biology may give an indication of telomere-targeting therapy in the future.

Level of Evidence: Prognostic Level II. See Instructions to Authors for a complete description of levels of evidence.

Figures in this Article

Introduction

Telomeres are specialized structures containing unique (TTAGGG)_n repeats at the ends of chromosomes, which are thought to be important for the protection and replication of chromosomes^1,2. Lagging strand DNA synthesis at the very end of linear chromosomes cannot be completed (a phenomenon known as the end-replication problem), and this situation results in the progressive shortening of telomeric repeats with each division³. When telomeres become critically short, further cell division is blocked, a process often referred to as replicative senescence⁴. Telomerase contains an RNA-dependent DNA polymerase, which compensates for the end-replication problem, and is expressed in germline cells but not in most somatic cells⁵. Recent studies have revealed that telomere and/or telomerase mechanisms are correlated with osteoarthritis⁶, rheumatoid arthritis⁷, sarcoma^8-19, and some benign tumors^20,21, so that telomere biology also plays an important role in musculoskeletal disease. Martin and Buckwalter reported that decreasing telomere length with increased age was closely correlated with increasing expression of beta-galactosidase and decreasing mitotic activity in articular cartilage chondrocytes⁶. Yamanishi et al. revealed that the presence of telomerase activity in rheumatoid synovial tissues was an indicator of a more aggressive phenotype⁷.

There are two telomere-maintenance mechanisms in human tumors: telomerase activity and alternative lengthening of telomeres (ALT)^22,23. Almost all carcinoma cells have engaged a mechanism to maintain stable telomere length by reactivating or upregulating telomerase activity^24,25. Many studies have revealed that telomerase activity is expressed in many different types of carcinomas and is detected in approximately 85% to 90% of the human carcinoma samples tested, and, in some instances, it has been found to be useful as a prognostic indicator^26-28.

Sarcomas are distinct from carcinomas in that many of them use ALT mechanisms. Some telomerase-negative cell lines that have been immortalized in vitro possess very long and heterogeneous telomeres as a result of ALT, and a substantial proportion of types of sarcomas have been reported to have elongated telomeres consistent with ALT in the absence of telomerase activity, indicating the existence of non-telomerase-based ALT mechanisms for telomere maintenance^{12,14,16,18,24,29}.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase and is a rate-limiting determination of the enzyme activity of human telomerase. The close relationship between hTERT mRNA expression and telomerase activity suggests that quantification of the mRNA expression of hTERT could be used as an alternative measure of telomerase activity^18,30-32. However, little is known concerning telomerase activity and hTERT expression in human sarcomas, especially malignant fibrous histiocytoma^10,11,31.

Panels of sarcomas have been investigated for telomerase and ALT telomere-maintenance mechanisms as well as characteristics such as tumor aggressiveness and patient survival, and the telomere-maintenance mechanism has emerged as a prognostic factor for sarcomas^8-19. We hypothesized that patients with malignant fibrous histiocytoma might present an opportunity to examine the impact of different telomere-maintenance mechanisms on clinical behavior. In the present study, we measured telomere factors in malignant fibrous histiocytoma samples to determine if these factors can be used as a prognostic indicator of malignant fibrous histiocytoma.

Materials and Methods

Patients and Tissue Procurement

Among the patients who underwent surgery between 1988 and 2005, a total of forty-three soft-tissue malignant fibrous histiocytoma samples from forty-three patients were obtained at the time of surgery, and, between 2005 and 2006, telomere factors were analyzed for one sample from each tumor at Hiroshima University Hospital and Southwestern Medical Center. The present study was approved by our institutional review boards. All clinical data at the time of the latest observation are shown in a table in the Appendix. Tumor samples were obtained from the resected surgical specimens, and all tumor samples were immediately frozen and stored at -80°C until use. We collected all primary tumor samples by means of biopsy or resection, and none of the patients had undergone chemotherapy before the surgical specimens were collected.

The tumor size was evaluated by measurement of the largest diameter on computed tomographic or magnetic resonance images at the time of diagnosis. Histological grades were assigned according to the system of the French Federation of Cancer Centers Sarcoma Group on the basis of tumor differentiation, mitotic count, and necrosis³³. Slides of each specimen that had been stained with hematoxylin and eosin were reviewed to confirm the diagnosis and histological grade of each tumor in a blinded fashion by one author (T.K.) and a pathologist. The surgical margins that were achieved were classified with use of the method described by Kawaguchi et al.³⁴. With this method, surgical margins are evaluated according to the distance of the margin from the tumor's reactive zone and are divided into four categories: curative, wide, marginal, and intralesional. The reactive zone is composed of hemorrhagic tissue, scar tissue, degenerated muscle, edema, or the tumor capsule. Barriers include muscle fascia, joint capsule, tendon, tendon sheath, epineurium, vascular sheath, and cartilage that have any resistance against tumor invasion. If the surgical margin is >5 cm outside the reactive zone, we grade the margin as curative. The surgical margin is referred to as wide if the margin is <5 cm but is outside the reactive zone. A margin in the reactive zone is graded as marginal, and a margin passing through a tumor as intralesional. A surgical margin that is outside a barrier, with normal tissue between the barrier and the reactive zone, is considered to be curative (Fig. 1). We performed brachytherapy or external radiation therapy following surgery for all patients who received a marginal resection.

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Fig. 1: Illustration depicting the evaluation of surgical margins according to the method of Kawaguchi et al.³⁴. A curative surgical margin passes through the region outside the reactive zone at a distance of >5 cm. A wide surgical margin is not enough to be curative but is outside the reactive zone. A marginal surgical margin passes through the reactive zone. An intralesional surgical margin passes inside the tumor.

Telomerase Assay

The evaluation of telomerase activity was performed by means of the telomeric repeat amplification protocol (TRAP) assay. In short, 50 to 100 mg of frozen tumor sample was homogenized in 200 µL of 3-([3-cholamidopropyl]-dimethylammonio)-1-propane sulfonic acid (CHAPS) lysis buffer. After twenty-five minutes of incubation on ice, the lysate was centrifuged at 16,000 times the force of gravity for twenty minutes at 4°C, and the supernatant was frozen at -80°C. The concentration of tumor protein was measured with use of the BCA Protein Assay Kit (Pierce Chemical, Rockford, Illinois). One microgram of tumor protein was used for each TRAP assay. The level of telomerase activity was measured with use of the TRAPeze XL Telomerase Detection Kit (Intergen, Purchase, New York), which is a quantitative fluorescein-labeled polymerase chain reaction system incorporating the use of a polymerase chain reaction internal control. For the direct detection of fluorescein and sulforhodamine, the polymerase chain reaction product was measured in a fluorescent plate reader with use of the appropriate excitation and emission filters. The telomerase activity was quantified as the ratio of the fluorescein intensity of the entire TRAP ladder to the sulforhodamine intensity of the internal control after the correction of each fluorescent intensity for negative control and background and was expressed as total product generated (TPG) units (units/µg protein). One amole of TSR8, which is a control template and an oligonucleotide with a sequence identical to the TS primer extended with eight telomeric repeats (AGGGTTAG)₇, equals 1000 TPG units.

Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction

Total cellular RNA was extracted with use of the RNeasy Mini Kit (QIAGEN, Valencia, California), and cDNA was synthesized with 1 µg of total RNA with use of the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany). Quantitative detection of hTERT mRNA was performed with the LightCycler TaqMan Master (Roche Applied Science) with use of the LightCycler Instrument (Roche Molecular Diagnostics, Pleasanton, California). Polymerase chain reaction lasted for ten seconds at 95°C, thirty seconds at 60°C, and 1 second at 72°C, with forty-five cycles. Expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was also analyzed in each tumor sample as an indicator of RNA quality. A 3 × 10⁶ of telomerase-positive HeLa cells was used as a positive control.

Telomere Length Analysis

Genomic DNA was isolated from the frozen tumor samples by means of proteinase K digestion followed by phenol-chloroform extraction³⁵. For the analysis of telomere length, 1 µg of DNA was digested to completion with 1.67 units of Alu I, Cfo I, Hae III, Hinf I, Msp I, and Rsa I, and was electrophoresed on 0.7% agarose gels. The gels were vacuum-dried, and then in-gel hybridization was performed with use of the labeled oligonucleotide containing TTAGGG probe. We scanned the dried gels with a PhosphorImager and visualized them with use of ImageQuant (Molecular Dynamics, Sunnyvale, California). We estimated the telomere lengths as the mean and semi-interquartile range of the hybridization signal with use of Telometric software³⁶. ALT status was determined on the basis of whether the mean and semi-interquartile range of telomere length distribution was >16 kb and >4 kb, respectively. Tumors were determined to be ALT-positive when all of these factors were met according to the report by Henson et al.¹⁸.

Statistical Analysis

The mean values (and standard deviations) for telomerase activity, hTERT mRNA expression, and telomere length were calculated for subgroups that were defined according to various factors. Between-subgroup differences and 95% confidence intervals were then calculated. Five-year survival rates were calculated with use of the Kaplan-Meier method for subgroups defined according to clinical factors, and univariate comparisons were performed with use of the log-rank test. Multivariate analysis with only significant variables that had been detected with the log-rank test was performed with use of the Cox proportional hazards model. Then, backward variable selection with an alpha of 0.05 was applied to determine the final model. Each prognostic factor was divided into two groups on the basis of an average value. Data are presented as the mean and the standard deviation. For all analyses, the level of significance was set at p < 0.05.

Source of Funding

There was no external funding source for the present study.

Results

Telomerase Activity

Telomerase activity was detectable in 79.1% (thirty-four) of the forty-three tumor samples (see Appendix). The mean level of telomerase activity was 7.57 ± 7.27 TPG (range, 0 to 25.6 TPG). The mean level of detectable telomerase activity (and standard deviation) was 9.57 ± 6.89 TPG (95% confidence interval, 7.17 to 12.0 TPG). Table I lists the association of telomerase activity with various clinical factors.

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TABLE I Association of Telomerase Activity and hTERT mRNA with Clinical Factors*

		Telomerase Activity (TPG)			hTERT mRNA
Clinical Factor	N	Mean and Standard Deviation	Difference (95% Confidence Interval)	Mean and Standard Deviation	Difference (95% Confidence Interval)
Age
=65 yr	23	6.76 ± 6.90	Reference	320 ± 633	Reference
<65 yr	20	8.50 ± 7.73	-1.74 (-6.25 to 2.77)	394 ± 693	-74 (-482 to 335)
Sex
Male	22	5.55 ± 5.83	Reference	276 ± 546	Reference
Female	21	9.69 ± 8.12	-4.14 (-8.47 to 0.20)	437 ± 756	-161 (-566 to 243)
Histological grade
1 or 2	24	6.50 ± 6.14	Reference	392 ± 678	Reference
3	19	8.93 ± 8.45	-2.43 (-6.93 to 2.07)	307 ± 638	85 (-324 to 495)
Tumor size
=10 cm	17	8.90 ± 8.01	Reference	429 ± 810	Reference
<10 cm	26	6.70 ± 6.76	2.20 (-2.38 to 6.78)	306 ± 542	123 (-292 to 539)
Surgical margin
Wide	27	7.90 ± 7.01	Reference	378 ± 618	Reference
Marginal	16	7.01 ± 7.88	0.89 (-3.79 to 5.56)	315 ± 731	63 (-359 to 484)
Recurrence
Presence	7	8.09 ± 5.66	Reference	969 ± 1079	Reference
Absence	36	7.47 ± 7.60	0.62 (-5.52 to 6.75)	235 ± 474	734 (232 to 1235)
Metastasis
Presence	18	9.02 ± 8.16	Reference	516 ± 819	Reference
Absence	25	6.53 ± 6.52	2.49 (-2.03 to 7.01)	238 ± 491	278 (-126 to 682)
ALT, telomerase
+, +	8	14.91 ± 5.18	Reference	407 ± 604	Reference
+, -	6	0.00 ± 0.00	14.91 (10.25 to 19.57)	571 ± 969	-164 (-1079 to 750)
Telomerase
=7.6 TPG	18	14.84 ± 4.85	Reference	338 ± 664	Reference
<7.6 TPG	25	2.34 ± 2.75	12.50 (10.15 to 14.85)	366 ± 661	-28 (-441 to 386)
hTERT
=355	9	5.77 ± 6.73	Reference	1424 ± 773	Reference
<355	34	8.05 ± 7.42	-2.28 (-7.80 to 3.24)	71 ± 74	1353 (1090 to 1617)

TPG = total product generated (units/µg protein).

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TABLE I Association of Telomerase Activity and hTERT mRNA with Clinical Factors*

		Telomerase Activity (TPG)			hTERT mRNA
Clinical Factor	N	Mean and Standard Deviation	Difference (95% Confidence Interval)	Mean and Standard Deviation	Difference (95% Confidence Interval)
Age
=65 yr	23	6.76 ± 6.90	Reference	320 ± 633	Reference
<65 yr	20	8.50 ± 7.73	-1.74 (-6.25 to 2.77)	394 ± 693	-74 (-482 to 335)
Sex
Male	22	5.55 ± 5.83	Reference	276 ± 546	Reference
Female	21	9.69 ± 8.12	-4.14 (-8.47 to 0.20)	437 ± 756	-161 (-566 to 243)
Histological grade
1 or 2	24	6.50 ± 6.14	Reference	392 ± 678	Reference
3	19	8.93 ± 8.45	-2.43 (-6.93 to 2.07)	307 ± 638	85 (-324 to 495)
Tumor size
=10 cm	17	8.90 ± 8.01	Reference	429 ± 810	Reference
<10 cm	26	6.70 ± 6.76	2.20 (-2.38 to 6.78)	306 ± 542	123 (-292 to 539)
Surgical margin
Wide	27	7.90 ± 7.01	Reference	378 ± 618	Reference
Marginal	16	7.01 ± 7.88	0.89 (-3.79 to 5.56)	315 ± 731	63 (-359 to 484)
Recurrence
Presence	7	8.09 ± 5.66	Reference	969 ± 1079	Reference
Absence	36	7.47 ± 7.60	0.62 (-5.52 to 6.75)	235 ± 474	734 (232 to 1235)
Metastasis
Presence	18	9.02 ± 8.16	Reference	516 ± 819	Reference
Absence	25	6.53 ± 6.52	2.49 (-2.03 to 7.01)	238 ± 491	278 (-126 to 682)
ALT, telomerase
+, +	8	14.91 ± 5.18	Reference	407 ± 604	Reference
+, -	6	0.00 ± 0.00	14.91 (10.25 to 19.57)	571 ± 969	-164 (-1079 to 750)
Telomerase
=7.6 TPG	18	14.84 ± 4.85	Reference	338 ± 664	Reference
<7.6 TPG	25	2.34 ± 2.75	12.50 (10.15 to 14.85)	366 ± 661	-28 (-441 to 386)
hTERT
=355	9	5.77 ± 6.73	Reference	1424 ± 773	Reference
<355	34	8.05 ± 7.42	-2.28 (-7.80 to 3.24)	71 ± 74	1353 (1090 to 1617)

TPG = total product generated (units/µg protein).

hTERT mRNA Expression

hTERT mRNA expression was demonstrated in 90.7% (thirty-nine) of the forty-three tumor samples. The mean level of hTERT in the tumor samples was 354 ± 654 (range, 0 to 2656) (see Appendix). The level of detectable hTERT mRNA was 391 ± 677 (95% confidence interval, 171 to 610). Table I lists the association of hTERT mRNA expression with various clinical factors.

Telomere Length

The mean telomere length was 17.7 ± 1.94 kb (range, 13.5 to 20.7 kb; 95% confidence interval, 17.1 to 18.3 kb) (see Appendix). The mean semi-interquartile range was 3.8 ± 0.6 kb (range, 2.6 to 5.0 kb; 95% confidence interval, 3.66 to 4.01 kb). According to the characteristic high mean and range of the telomere length distribution, 32.6% (fourteen) of the forty-three tumor samples were ALT-positive. In addition, 18.6% (eight) of forty-three tumor samples were positive for both telomerase activity and ALT. There were no significant variations in the mean telomere length between samples that were positive for both ALT and telomerase activity and samples that were positive for ALT but negative for telomerase activity (difference, -0.36 kb; 95% confidence interval, -1.42 to 0.70 kb).

Comparisons of Telomerase Activity, hTERT, and Telomere Length According to Histological Grade

We compared two groups (patients with grade-1 and 2 tumors and those with grade-3 tumors) in terms of levels of telomerase activity, hTERT, and telomere length (expressed as the mean and the semi-interquartile range). The patients who had grade-3 tumors had significantly longer telomere lengths than those who had grade-1 or 2 tumors in terms of both the mean length (difference, -1.24 kb; 95% confidence interval, -2.39 to -0.09 kb) and the semi-interquartile range (difference, -0.44 kb; 95% confidence interval, -0.77 to -0.11 kb). There were no significant differences between the groups in terms of telomerase activity (difference, -2.43TPG; 95% confidence interval, -6.93 to 2.07TPG) or hTERT (difference, 85; 95% confidence interval, -324 to 495) (Table II).

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TABLE II Analysis of Prognostic Factors

					Cox Regression
					Initial Model					Final Model with Backward Selection
Clinical Factor	N	No. of Deaths	Five-Year Survival	P Value (Log-Rank Test)	Hazard Ratio	95% Confidence Interval	P Value	Hazard Ratio	95% Confidence Interval	P Value
Age				0.862
=65 yr	23	9	54.8%
<65 yr	20	8	57.8%
Sex				0.245
Male	22	7	66.1%
Female	21	10	44.4%
Histological grade				0.273
1 or 2	24	8	56.7%
3	19	9	52.6%
Tumor size				0.0404*
=10 cm	17	10	36.8%		Reference
<10 cm	26	7	70.4%		0.599	0.219 to 1.634	0.317
Surgical margin				0.555
Wide	27	10	59.5%
Marginal	16	7	49.1%
Recurrence				0.953
Presence	7	3	42.9%
Absence	36	14	58.5%
ALT				0.0049†
Positive	14	10	25.0%		Reference			Reference
Negative	29	7	73.6%		0.367	0.135 to 0.998	0.0495*	0.275	0.104 to 0.724	0.0089†
Telomerase				0.152
Positive	34	12	62.2%
Negative	9	5	0.0%
Telomerase‡				0.0183*
=7.6 TPG	18	11	36.7%		Reference
<7.6 TPG	25	6	73.3%		0.403	0.147 to 1.107	0.0779
hTERT				0.464
Positive	39	16	55.1%
Negative	4	1	66.7%
hTERT				0.379
=355	9	5	33.3%
<355	34	12	61.7%

P < 0.05. †P < 0.01. ‡TPG = total product generated (units/µg protein).

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TABLE II Analysis of Prognostic Factors

					Cox Regression
					Initial Model					Final Model with Backward Selection
Clinical Factor	N	No. of Deaths	Five-Year Survival	P Value (Log-Rank Test)	Hazard Ratio	95% Confidence Interval	P Value	Hazard Ratio	95% Confidence Interval	P Value
Age				0.862
=65 yr	23	9	54.8%
<65 yr	20	8	57.8%
Sex				0.245
Male	22	7	66.1%
Female	21	10	44.4%
Histological grade				0.273
1 or 2	24	8	56.7%
3	19	9	52.6%
Tumor size				0.0404*
=10 cm	17	10	36.8%		Reference
<10 cm	26	7	70.4%		0.599	0.219 to 1.634	0.317
Surgical margin				0.555
Wide	27	10	59.5%
Marginal	16	7	49.1%
Recurrence				0.953
Presence	7	3	42.9%
Absence	36	14	58.5%
ALT				0.0049†
Positive	14	10	25.0%		Reference			Reference
Negative	29	7	73.6%		0.367	0.135 to 0.998	0.0495*	0.275	0.104 to 0.724	0.0089†
Telomerase				0.152
Positive	34	12	62.2%
Negative	9	5	0.0%
Telomerase‡				0.0183*
=7.6 TPG	18	11	36.7%		Reference
<7.6 TPG	25	6	73.3%		0.403	0.147 to 1.107	0.0779
hTERT				0.464
Positive	39	16	55.1%
Negative	4	1	66.7%
hTERT				0.379
=355	9	5	33.3%
<355	34	12	61.7%

P < 0.05. †P < 0.01. ‡TPG = total product generated (units/µg protein).

Prognostic Factors

Data on the analyses of prognostic factors are shown in Tables II and III. The variables are all derived from telomerase and ALT, and they are associated with each other. If they had been included in the same model with telomerase and ALT, parameter estimation would have been unstable and the interpretation might have been difficult. Therefore, the significant predictors in Table III were not advanced to the proportional-hazards model.

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TABLE III Analysis of Prognostic Factors with Multiple Factors

Clinical Factor	N	No. of Deaths	Five-Year Survival	P Value (Log-Rank Test)
Telomerase (-), ALT (+)	6	5	0.0%	0.0128*
Other	37	12	63.8%
Telomerase (low), ALT (-)	18	1	94.4%	0.0004†
Other	25	16	32.9%
Telomerase (-), ALT (+)	6	5	0.0%	0.0091‡
Telomerase (+), ALT (-)	26	7	71.6%
Telomerase (high), ALT (-)	11	6	42.4%	0.0027‡
Telomerase (low), ALT (-)	18	1	94.4%
Telomerase (+), ALT (+)	8	5	37.5%	0.188
Other	35	12	62.2%
Telomerase (+), ALT (+)	8	5	37.5%	0.301
Telomerase (-), ALT (+)	6	5	0.0%
Telomerase (high), ALT (+)	7	5	28.6%	0.067
Other	36	12	63.4%
Telomerase (high), ALT (+)	7	5	28.6%	0.900
Telomerase (low), ALT (+)	7	5	21.4%

P < 0.05. †P < 0.001. ‡P < 0.01.

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TABLE III Analysis of Prognostic Factors with Multiple Factors

Clinical Factor	N	No. of Deaths	Five-Year Survival	P Value (Log-Rank Test)
Telomerase (-), ALT (+)	6	5	0.0%	0.0128*
Other	37	12	63.8%
Telomerase (low), ALT (-)	18	1	94.4%	0.0004†
Other	25	16	32.9%
Telomerase (-), ALT (+)	6	5	0.0%	0.0091‡
Telomerase (+), ALT (-)	26	7	71.6%
Telomerase (high), ALT (-)	11	6	42.4%	0.0027‡
Telomerase (low), ALT (-)	18	1	94.4%
Telomerase (+), ALT (+)	8	5	37.5%	0.188
Other	35	12	62.2%
Telomerase (+), ALT (+)	8	5	37.5%	0.301
Telomerase (-), ALT (+)	6	5	0.0%
Telomerase (high), ALT (+)	7	5	28.6%	0.067
Other	36	12	63.4%
Telomerase (high), ALT (+)	7	5	28.6%	0.900
Telomerase (low), ALT (+)	7	5	21.4%

P < 0.05. †P < 0.001. ‡P < 0.01.

Univariate analysis revealed that tumor size, ALT, and telomerase activity were significant prognostic risk factors for death. With regard to tumor size, patients with lesions that were =10 cm in diameter had a worse prognosis than those with lesions that were <10 cm (p = 0.0404). Patients with ALT-positive tumors had a worse prognosis than those who did not (p = 0.0049) (Fig. 2). Patients with tumors that had above-average telomerase activity (=7.6 TPG) had a significantly worse prognosis than those with tumors that had below-average telomerase activity (p = 0.0183). Multivariate analysis of tumor size, ALT status, and telomerase activity revealed that ALT-positive status (hazard ratio, 0.275; 95% confidence interval, 0.104 to 0.724; p = 0.0089) was the only independent prognostic factor for death.

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Fig. 2: Kaplan-Meier curve illustrating the association between survival rate and ALT status in patients with malignant fibrous histiocytoma. Patients with ALT-positive tumors have a worse prognosis, with five-year survival of 25.0% (95% confidence interval, 11.0% to 48.9%) as compared with 73.6% (95% confidence interval, 56.3% to 90.8%) for patients with ALT-negative tumors (p = 0.0049). DOD = died of disease.

Patients with ALT-positive, telomerase-negative tumors had a significantly worse prognosis than other patients did (p = 0.0128). Patients with ALT-negative tumors that had below-average telomerase activity had a significantly better prognosis than other patients did (p = 0.0004). Patients with ALT-positive, telomerase-negative tumors had a significantly worse prognosis than patients with ALT-negative, telomerase-positive tumors did (p = 0.0091) (Fig. 3). Patients with ALT-negative tumors that had above-average telomerase activity had a significantly worse prognosis than patients with ALT-negative tumors that had below-average telomerase activity (p = 0.0027) (Fig. 3).

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Fig. 3: Kaplan-Meier curves showing the survival rates for patients with malignant fibrous histiocytoma samples that were ALT-negative and telomerase-positive, ALT-positive and telomerase-negative, and ALT-negative with different levels of telomerase activity. Patients with ALT-positive, telomerase-negative tumors had a worse prognosis than those with ALT-negative, telomerase-positive tumors (five-year survival rate, 0% compared with 71.6% [95% confidence interval, 53.6% to 89.7%]; p = 0.0091). Patients with ALT-negative tumors that had above-average telomerase activity had a worse prognosis than those with ALT-negative tumors with below-average telomerase activity (five-year survival rate, 42.4% [95% confidence interval, 11.8% to 73.0%] compared with 94.4% [95% confidence interval, 83.9% to 100%] [p = 0.0027]). DOD = died of disease.

Discussion

Telomere maintenance is regarded as an important mechanism for evading senescence in tumor cells, and two types of telomere-maintenance mechanisms have been described in human tumors: telomerase activation and ALT^22,23. Although approximately 85% to 90% of human carcinomas rely on telomerase activation, many sarcomas rely on the ALT mechanism for the maintenance of telomeres. Henson et al. reported that ALT appears to be much more common in mesenchymal-originated sarcomas as compared with other tumor types, but the prevalence of ALT varied greatly among different soft-tissue sarcoma subtypes (range, 6% to 77%)¹⁸. In addition, the frequency of ALT in sarcomas has differed among previous reports^{8,12,14,16,18}. In the present study, 32.6% (fourteen) of the forty-three soft-tissue malignant fibrous histiocytoma samples were ALT-positive. The reason for the differing frequency of ALT among different tumor types is not clearly understood. Sarcoma subtypes can be classified into those with specific translocations displaying monotonous cytomorphology and those with complex karyotypes, which are characteristic of severe genetic and chromosomal instability³⁷. A previous report suggested an association between ALT and complex-karyotype soft-tissue tumors and between normal or reduced telomere lengths and specific translocations, which have few karyotypic abnormalities¹³, although it was suggested that ALT is not the only source of chromosomal instability, and typical complex-karyotype sarcomas showed a low frequency of ALT¹⁸, indicating heterogeneous characteristics of sarcomas.

Perrem et al. demonstrated the fusion of ALT cells to telomerase-positive immortal cells exhibiting regression of the ALT telomere, which suggests that the coexistence of ALT and telomerase activity is unlikely³⁸. However, several reports have shown evidence of the presence of both ALT and telomerase activity^8,12,14,18. In the present study, 18.6% (eight) of the forty-three tumor samples had both telomerase and ALT activity. It is unclear whether this phenomenon means that both the ALT mechanism and telomerase are activated in the same sarcoma cells or in different subpopulations within the same sarcomas. Further analysis of telomerase activity and telomere length in the single cell is necessary to clarify these mechanisms in ALT-positive, telomerase-positive sarcomas.

Scheel et al. reported that low telomerase activity was not sufficient to prevent telomere shortening in the osteosarcoma cell line Saos-2³⁹, indicating the presence of ALT. In the present study, there were no significant differences in mean telomere length between patients with ALT-positive, telomerase-positive tumors (19.3 ± 0.9 kb) and those with ALT-positive, telomerase-negative tumors (19.7 ± 0.9 kb) (difference, -0.36 kb; 95% confidence interval, -1.42 to 0.70 kb). Thus, our data suggest that telomerase activity may not act to prevent telomere shortening in patients who have ALT-positive tumors with telomerase activity.

The reported frequency of telomerase activity expression in malignant fibrous histiocytoma samples has ranged from 22% to 80%^{9-11,15,17,18}, which may be due to the small number of tumor samples analyzed. In the present study, telomerase activity was detectable in 79.1% (thirty-four) of the forty-three tumor samples. Aogi et al. reported that 81% (thirty) of thirty-seven primary bone and soft-tissue sarcomas, including ten cases of soft-tissue malignant fibrous histiocytomas, were telomerase-positive⁹, figures that are similar to our data. These findings suggest that telomerase activation may be prevalent in malignant fibrous histiocytomas and similar to findings in many carcinomas.

Telomerase is a ribonucleoprotein complex with a rate-limiting protein catalytic subunit component, termed the human telomerase reverse transcriptase (hTERT), that copies a template region of its functional RNA subunit (human telomerase RNA or human telomerase RNA component) to the end of the telomere. The levels of telomerase activity are significantly correlated with the levels of hTERT mRNA expression in human carcinomas^40,41. In the present study, hTERT mRNA expression was identified in 90.7% (thirty-nine) of the forty-three tumor samples, although there was a wide range of expression values. We observed discordant results when the presence of hTERT mRNA expression was not associated with telomerase activity in tumor samples. In addition, hTERT expression without telomerase activity was observed in seven tumor samples. While we do not have a clear understanding of these findings, the discordance between hTERT mRNA and enzyme activity may be due to the presence of unknown telomerase inhibitors⁴² and/or dephosphorylation of hTERT⁴³.

Despite numerous reports on telomere factors, almost all of the previous studies have evaluated one sample from each tumor^{7-10,12,14-21,26,27,35,39-42}. Yan et al. reported that telomerase activity and hTERT mRNA expression were heterogeneous in sarcomas and could vary according to the tumor areas that were examined, although those regions indicated similar histological findings¹¹. Therefore, we have to consider that analysis of only a single tumor sample might not yield a representative result.

The telomere-maintenance mechanism has recently emerged as a prognostic factor for sarcomas^8-19, although some of the data are controversial. In the present study, patients with ALT-positive tumors had a worse prognosis than those who did not (p = 0.0049), and, with multivariate analysis, ALT-positive status emerged as the only independent prognostic factor in patients with soft-tissue malignant fibrous histiocytoma. In addition, patients with ALT-positive, telomerase-negative tumors had a significantly worse prognosis than those who had ALT-negative, telomerase-positive tumors. Patients with ALT-negative tumors that had below-average telomerase activity had a significantly better prognosis than other patients did. Patients with ALT-positive, telomerase-negative tumors had a significantly worse prognosis than other patients did. These data suggest that the ALT mechanism for patients with soft-tissue malignant fibrous histiocytoma correlates more strongly with a worse prognosis than typical clinical factors such as the histological grade, tumor size, and local recurrence do.

In the present study, with the number of cases available for evaluation, there were no significant differences in the survival rate between patients with ALT-positive, telomerase-positive tumors and those with ALT-positive, telomerasenegative tumors or between patients with ALT-positive tumors that showed above-average telomerase activity and those with ALT-positive tumors that showed below-average telomerase activity. These data suggest that the telomerase activity does not affect the prognosis in patients with ALT-positive soft-tissue malignant fibrous histiocytoma.

Again, with the numbers available, there were no significant differences in the survival rate between patients with telomerase-positive and telomerase-negative soft-tissue malignant fibrous histiocytoma. However, patients with ALT-negative tumors that had above-average telomerase activity had a significantly worse prognosis than patients with ALT-negative tumors that had below-average telomerase activity. In addition, all patients with ALT-negative, telomerase-negative tumors were alive, but seven of twenty-six patients with ALT-negative, telomerase-positive tumors had died. These data suggest that telomerase expression (the presence of telomerase and higher levels of telomerase) is associated with a poor prognosis in patients with ALT-negative tumors, but not in those with ALT-positive tumors. Hence, telomerase activity is not associated with a good or bad prognosis in all cases of malignant fibrous histiocytoma as telomerase activity does not affect the prognosis in patients with ALT-positive malignant fibrous histiocytoma. Therefore, in terms of prognosis, patients with soft-tissue malignant fibrous histiocytoma must be divided into two groups. In patients with ALT-positive tumors, telomerase activity is not associated with prognosis, although ALT is a most significant prognostic risk factor. In patients with ALT-negative tumors, telomerase expression is a significant prognostic risk factor.

One of the factors of prognostic importance in patients with soft-tissue sarcomas, including malignant fibrous histiocytomas, is the histological grade⁴⁴. In the present study, patients who had grade-3 tumors had a significantly longer mean telomere length and semi-interquartile range than those who had grade-1 or 2 tumors. The mean levels of telomerase activity in grade-3 tumors were higher than those in grade-1 and 2 tumors (Table I). This finding suggests that elongation of telomeres may require upregulation of telomerase activity. Mean telomere length and mean telomerase activity in high-grade tumors were longer and higher than those in low-grade tumors, indicating that telomerase might stabilize telomeres in high-grade tumors.

After its acceptance as a separate diagnostic entity in the 1970s⁴⁵, malignant fibrous histiocytoma became the most commonly diagnosed type of soft-tissue sarcoma^46,47. However, based in part on current classification practices, many have questioned whether malignant fibrous histiocytoma should be considered a separate diagnostic entity^48,49. Malignant fibrous histiocytoma was a commonly used diagnosis during the time when our cases were collected; currently, however, malignant fibrous histiocytoma refers to pleomorphic sarcomas without defined differentiation, and some of these tumors today probably would be classified as "high-grade pleomorphic sarcoma," dedifferentiated liposarcoma, or myxofibrosarcoma.

Jackson et al.⁵⁰ recently reported that a telomerase inhibitor in early-stage clinical trials also induced an anti-adhesive effect against ALT cell lines (SUSM-1 from normal human fibroblasts AD387 by treatment with 4-nitroquinoline 1-oxide⁵¹, and VA 13 from fetal human diploid fibroblast cell line WI-38 after transformation with simian virus 40 [SV40]⁵²). As >79% of the malignant fibrous histiocytoma samples in the present study also had telomerase activity and the presence of ALT telomere-maintenance mechanisms indicates a poor prognosis for patients with soft-tissue malignant fibrous histiocytoma, telomerase activity and ALT may also be novel targets of adjuvant therapy for patients with malignant fibrous histiocytoma.

Appendix

A table showing details on all study specimens is available with the electronic versions of this article, on our web site at (go to the article citation and click on "Supplementary Material") and on our quarterly CD/DVD (call our subscription department, at 781-449-9780, to order the CD or DVD). Image Not Available

Acknowledgements

Note: The authors thank Gregory Thompson, Jennifer Costley, and Nuno Gomes for superb technical assistance.

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Topics

rna, messenger ; telomerase ; telomere ; neoplasms ; histiocytoma, malignant fibrous ; prognostic factors ; telomere maintenance

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Toshihiro Matsuo, Jerry W. Shay, Woodring E. Wright, Eiso Hiyama, Shoji Shimose, Tadahiko Kubo, Takashi Sugita, Yuji Yasunaga, Mitsuo Ochi; Telomere-Maintenance Mechanisms in Soft-Tissue Malignant Fibrous Histiocytomas. The Journal of Bone & Joint Surgery. 2009 Apr;91(4):928-937.

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