Quantitative telomerase expression in glioblastomas shows regional variation and down-regulation with therapy but no correlation with patient outcome

Quantitative Telomerase Expression in Glioblastomas Shows Regional Variation and Down-Regulation With Therapy but No Correlation With Patient Outcome B. K. KLEINSCHMIDT-DEMASTERS, MD, LYNNETTE C. EVANS, MS, JOANNA B. BOBAK, PHD, DANIEL LOPEZ-URIBE, BA, DEBORAH HOPPER, RN, A. LAURIE SHROYER, PHD, AND KENNETH R. SHROYER, MD, PHD Despite the nearly ubiquitous expression of telomerase in almost all types of malignant human tumors, studies have shown widely varying positivity in the highest-grade glioma, the glioblastomas (GBMs), ranging from 26% to 100% of tumors analyzed. We have previously shown significant variability in positive versus negative telomerase expression from region to region within the same GBM. In this study, we hypothesized that application of new quantitative methodology would extend our previous observations and identify whether there is heterogeneity in levels of protein expression even within areas positive for telomerase in high-grade gliomas. Finally, we sought to correlate quantitative telomerase expression with patient outcome and therapeutic response. Quantitative analysis was achieved by polymerase chain– based TRAP assay with phosphorimager analysis and compared with clinical information obtained from 19 patients, most with primary, untreated GBMs. Results showed up to 3-fold variability in telomerase levels across multiple regional samples from the same patient, as well as between patients. In 5 of 6

patients with recurrent tumors who had received intervening radiation therapy or chemotherapy, telomerase was downregulated in the second, post-therapy sample. These data provide in vivo corroboration of recent in vitro experiments showing telomerase downregulation after radiation therapy or chemotherapy treatment of cell lines. Our finding of variability in levels of telomerase expression in GBMs parallels the known heterogeneity of these tumors for histologic features and cell growth–related factors. Statistical analysis showed no relationship between TRAP score and either time to clinical progression or time to death. HUM PATHOL 31:905-913. Copyright © 2000 by W.B. Saunders Company Key words: glioblastoma, prognosis, radiation therapy, therapy, telomerase. Abbreviations: GBM, glomerular basement membrane; AA, anaplastic astrocytoma; TRAP, telomeric repeat amplification protocol; PCR, polymerase chain reaction; PG, product generated.

Intense research has recently been directed toward studying telomerase, an enzyme ribonucleoprotein complex that plays a critical role in cellular immortalization and cancer.1 Its nearly ubiquitous expression in most types of cancer has led to investigations of its utility as a diagnostic marker of malignancy in difficult clinical situations, including in cytology preparations and as a marker for residual or recurrent disease after surgical resection.1-4 The expression of telomerase in most human tumors has led to numerous studies ex-

ploring its role in multistep tumorigenesis. Although telomerase expression is most characteristic of late stages of tumor development, the timing of its expression varies considerably for different tumor types in various organs.1 Most recently, large numbers of studies have focused on the correlation between telomerase expression and prognosis in various tumor types derived from virtually all organs of the body. A burgeoning number of studies have emerged on the latter subject, and a review of the literature is included in this article in table format. In adult gliomas, we have previously shown that telomerase expression correlates with tumor grade and is most commonly demonstrated in malignant gliomas (grades 3 and 4 of the 4-tiered World Health Organization grading scheme for astrocytomas).5 Studies in the literature, however, have generated conflicting information regarding the extent of telomerase expression in the most frequent adult high-grade glioma, the glioblastoma multiforme (grade 4 of 4 glioma, glomerular basement membrane [GBM]). Despite using similar qualitative methodology for telomerase analysis on single tissue samples from each tumor, investigators have shown positive telomerase expression in anywhere from 26.3%,6 to 55%,5 60%,7 73%,8 83.3%,9 89%,10 or 100%11,12 of GBMs. The necessity of assessing viability of tumor in tissue samples to be used for telomerase analysis by histologic examination was emphasized in both our original5 and subsequent studies11 and may

From the Departments of Pathology and Neurology and Surgery (Neurosurgery), University of Colorado Health Sciences Center, Denver, CO; and the Department of Cardiac Research, Denver, CO. Accepted for publication March 10, 2000. Presented in abstract format at the United States and Canadian Academy of Pathology meeting, San Francisco, CA, March 1999. This study received approval from the Colorado Multiple Institutional Review Board. Funding for this study was provided in part by a grant from the Colorado Cancer Center. A. L. Shroyer’s participation was funded in part by the Department of Veterans Affairs Health Services Research and Development Service. K. R. Shroyer and B. K. Kleinschmidt-DeMasters are members of the Colorado Cancer Center. Address correspondence and reprint requests to B. K. Kleinschmidt-DeMasters, MD, Department of Pathology, Box B216, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3108-0005$10.00/0 doi: 10.1053/hupa.2000.9086

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partly explain these differing results. GBMs often contain large areas of coagulative necrosis, and frozen tissue samples from completely necrotic areas may have been inadvertently chosen for analysis in some studies and might be expected to be negative for telomerase. Considerable differences in tumor cell density are characteristic of most gliomas, and it is possible that some negative results in some previous studies may be attributable to assaying tumor areas with scanty numbers of tumor cells. It is unclear how many tumor cells need to be present in glioma tissue samples to yield positive telomerase expression results. Recently, eloquent microdissection studies on 8 gliomas of various types and grades by Weil and colleagues have also emphasized the need to assess optimal tumor-containing tissue samples for telomerase expression.12 These investigators have shown that telomerase expression could be identified in 8 of 8 gliomas in their study when areas of “pure” tumor were preselected.12 They did not study, however, whether variability in levels of telomerase expression existed within different areas from the same tumor or whether negative or lower levels of expression could be found when lesser numbers of tumor cells were present in a sample. An additional explanation for variability in results may be that true biologic differences exist for telomerase expression in differing tumor cell populations in GBMs, even after controlling for necrosis and optimizing tumor samples by histologic correlation. GBMS are highly heterogeneous tumors both histologically and biologically. These tumors express considerable variation in histologic features from area to area,13 as well as variability for labeling of tumor cells by immunocytochemical markers of cell proliferation11 or oncoproteins such as p53 protein14 or MMAC/PTEN.15 Using a qualitative assay for telomerase, we have previously shown regional variation in positive versus negative telomerase expression in high-grade gliomas.11 When we assessed 5 to 7 different regional samples from each tumor, we showed that 100% of GBMs we analyzed had positive expression of telomerase in at least 1 region of the tumor. We also showed that positive telomerase expression correlated with Mib-1 labeling index.11 Such heterogeneity of positive versus negative telomerase expression parallels heterogeneity of cell cycling rates in GBMs and also may help explain low percentages of telomerase positivity in these tumors reported in some previous studies if samples were taken from tumor areas with low Mib-1 labeling indices. In the current study, we sought to extend our observation of heterogeneity further, using a quantitative method for telomerase analysis, employing phosphorimaging for studying up to 3 regional samples from each of 19 high-grade gliomas, including 17 GBMs and 2 anaplastic astrocytomas (AAs). We postulated that we could not only show variation in positive versus negative telomerase expression in high-grade gliomas, but variability in levels of enzyme even within positive areas. We also hypothesized that the use of quantitative methods might resolve conflicting results regarding

whether telomerase expression in gliomas predicts patient outcome. Nakatani et al,7 in a qualitative study of telomerase expression in brain tumors, have reported that activity tended to correlate with patient prognosis, particularly for GBMs. In their series, they noted that the median survival time from the time of diagnosis for patients with telomerase-positive GBMs was 8.0 months. In contrast, a second group of GBM patients whose tumors showed undetectable telomerase activity had a median survival of 13.8 months. These authors concluded from their study that “the expression of telomerase activity may be an entirely new marker for malignancy in GBMs . . . and may be a useful prognostic factor for patients with malignant astrocytic tumors.”7 Falchetti et al,9 in a more recent 1999 study, seemed to support this view and similarly concluded that “assessment of telomerase activity may influence tumor prognosis, and possibly treatment, in malignant gliomas . . .” In contrast, Weil et al,12 also in a 1999 study, could find no correlation between quantitative telomerase levels and prognosis for GBMs and AAs, although the number of cases in their study was relatively small (3 AAs, 3 GBMs).12 In the current study, we performed quantitative evaluation of telomerase expression in a larger number of high-grade gliomas, predominantly GBMs, to test the hypothesis that telomerase expression levels are prognostic of time to tumor recurrence and death. We identified 6 patients on whom we had both primary and posttherapy, recurrent tumor samples, and specially addressed the issue as to whether intervening therapy had any effect on quantitative, regional telomerase levels.

MATERIALS AND METHODS Nineteen patients with malignant gliomas had specimens available for study. The patients included 6 women and 13 men, ranging in age from 41 to 77 years. Specimens were accumulated from August 1996 to September 1998 but were not eligible after that date, to allow for adequate follow-up time. Only patients with 2 to 3 banked tissue specimens taken from different regions of tumor, removed at a single surgical operation, were eligible. A total of 75 regional specimens from 19 patients were accrued. Most patients had undergone stereotactic volumetric resections rather than biopsies, insuring greater accuracy of diagnosis and grading. All tumors were histologically assessed and graded using the World Health Organization 4-tiered grading scale, with anaplastic astrocytomas corresponding to grade 3 of 4 glioma and GBMs to grade 4 of 4 glioma.16 Regional specimens from a single surgical procedure were available from 13 patients, and regional specimens from 2 or more surgical procedures performed at our institution, including posttherapy tumor samples, were available on 6 patients. In 13 patients, the first tumor sample available for analysis came from their untreated, primary glioblastoma. In 3 patients, the first frozen (banked) tissue sample available for analysis came from the patient’s recurrent GBM. Two patients whose initial surgical sample was diagnosed as anaplastic astrocytoma had frozen (banked) tissue samples available from secondary GBMs. One patient with anaplastic as-

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trocytoma was included in the study because he represented one of the few cases in our tumor bank of high-grade gliomas with 2 or more sequential samples accrued over time, including posttherapy samples, as noted above (Table 1). Despite the extensive tumor tissue bank available to us at our institution, only these 6 patients were identified that had 2 or more sequential tumor samples frozen and banked. This reflects the infrequency of reoperation for many GBM patients and the referral nature of our hospital (University of Colorado Health Sciences Center) such that only a portion of any given patient’s care may occur at any 1 institution. Patients with sequential samples had all received intervening gene therapy, chemotherapy, or radiation therapy and details were obtained from patient records in the neurosurgery department. Time of demise was obtained from records in the neurosurgery department as well as the hospital Medical Records files. For calculating the time to death, the study was terminated on September 16, 1999; 2 patients were still alive at that time. Clinical charts were reviewed in detail to determine time to clinical progression. Time to progression was identified on the basis of both clinical and radiographic deterioration, not radiographic changes alone, to better insure that the patient’s worsening symptoms and signs were attributable to tumor growth, not simply therapy-induced changes. For patients with multiple surgical resections, time to progression was calculated only from the first surgical procedure to the first identifiable clinical deterioration. From one patient who had received several types of experimental therapies, multiple surgical operations had yielded 3 specimens suitable for banking, culminating in a final, fourth tissue sampling at the time of his death and autopsy. Details of therapy regimens and patient demographics are outlined in Table 1.

Tissue Specimens All surgical tumor tissue samples were obtained from resection specimens within 15 minutes of surgical tissue removal. Tissue samples were taken from viable areas of tumor, avoiding areas of gross necrosis, especially in patients treated with radiation therapy or chemotherapy. Up to 3 anatomically separate areas of tumor were sampled from each resection specimen, with the number of regional specimens taken from each case determined by the volume of resected tissue available. Tissue samples for telomerase analysis were immediately snap frozen in a dry ice/ethanol mixture and stored at ⫺70°C. Adjacent, matching tissue samples for histologic analysis were fixed in 10% formalin. Formalin-fixed, paraffin-embedded sections were cut at 4 ␮m and stained with Harris hematoxylin and eosin. Both the adjacent tissue samples matched to the frozen tissue, as well as additional tissues submitted in toto from the resection specimens, were used for diagnosis and grading according to World Health Organization criteria.

EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L beta-mercaptoethanol, 0.5% CHAPS, 10% glycerol), and homogenized for 5 minutes using a disposable hand-held homogenizer. The cellular suspension was incubated on ice for 30 minutes and then centrifuged 30 minutes at 14,000g at 4°C. The supernatant was removed and quickly frozen in a dry ice bath.

Telomeric Repeat Amplification Protocol TRAP Assay The TRAP assay was performed on 5-␮g protein aliquots of the tissue samples as described by the manufacturer, using the Oncor TRAPeze Telomerase Detection Kit (Gaithersburg, MD). Protein concentration of the lysate was standardized to 2.5 ␮g/uL according to Bradford assay.17 Extension of the TS template was performed by incubation at 30°C for 30 minutes, followed by 30 cycles of polymerase chain reaction (PCR) with a thermal profile of 94°C/30 seconds, 60°C/30 seconds. The telomerase-positive control was a serial dilution of HeLa cells (103 to 10 cell equivalents) (American Type Culture Collection, Rockville, MD). The negative control consisted of an RNase treatment of the cellular lysate for 30 minutes, before TRAP. Electrophoresis of the TRAP assay products was performed on a nondenaturing 12% polyacrylamide gel at 50 v/cm. Quantitation of telomerase expression was determined by measurement of the internal telomerase assay standard band and the sum of the 56, 62, and 68 base pair bands, using a phosphorimager (Storm 860, Molecular Dynamics Inc., Sunnyvale, CA). The units of product generated (PG units) were calculated as follows: [(sample band ⫺ RNase treated control band)/RNase treated internal standard band]/ [(TSR8 quantitation control band ⫺ negative control band)/ TSR8 quantitation internal standard band] ⫻ 100. A quantitation number was assigned to the PG units as follows: 0: Negative telomerase signal, 1: ⬍101 PG units, 2: ⬎ or ⫽ 101 PG units, 3: ⬎ or ⫽ 102 PG units, and 4: ⬎ or ⫽ 103 PG units. A positive result was defined by the detection of multiple (more than 2) 6-nucleotide repeat bands by phosphorimager analysis. Two or less bands consistent with a size of 50 base pairs or less were considered primer-dimer artifacts and were not considered positive.

Statistical Analysis Univariate associations were explored by using Fisher’s exact, chi-square, Wilcoxon rank sum, and Kruskall-Wallis tests, as appropriate. Survival time was modeled by using Kaplan-Maier survival curves stratified for the TRAP score, censoring for survivors.

Tissue Processing for Telomerase Analysis

RESULTS

The frozen tissue specimens were stored in the Immunology/Neuro-oncology Brain Tumor Bank at the University of Colorado and retrieved just before analysis. Approximately a 2- to 3-mm3 tissue sample was removed from the larger frozen sample on dry ice to minimize possible enzymatic deterioration. The frozen tissue blocks (10 to 50 mg) were washed in 200 ␮L ice-cold wash buffer (10 mmol/L HEPES-KOH, pH 7.5, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L dithiothreitol), resuspended in 100 ␮L ice-cold lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L MgCl2, 1 mmol/L

Seventeen patients had at least 1 sample from at least 1 resection specimen that showed telomerase expression (Table 1). Regional tumor samples taken for quantitative telomerase assessment from 2 to 3 regions of tumor obtained from a single surgical resection procedure showed up to 3-fold variation in telomerase levels (Fig 1). Most patients showed more limited variation in levels, ranging from 1- to 2-fold increments across the 2 to 3 regions analyzed per tumor. In all cases, negative or low telomerase levels could not be

907

Age/ Gender Tumor Type/Location

Treatment Received Between Specimen Collections

67/F

54/M

57/M

3

4

5

908

Intervening therapy3 Recurrent GBM

Primary GBM/left frontal Intervening therapy3 Recurrent GBM Intervening therapy3 Recurrent GBM Intervening therapy3 Recurrent GBM Primary GBM/left temporal Intervening therapy3 Recurrent GBM Intervening therapy3 Recurrent GBM Primary GBM/left temporal Intervening therapy3 Recurrent GBM Intervening therapy3 Recurrent GBM AA/Bifrontal Intervening therapy3 Recurrent AA Primary GBM/left temporal Intervening therapy3 Recurrent GBM Primary GBM/right frontal

Surgery with specimen #1 collection Gene therapy; radiation therapy, 6,000 cGy Surgery with specimen #2 collection BCNU; reoperation; vincristine Rx Surgery with specimen #3 collection Stereotactic radiosurgery Autopsy with specimen #4 collection Surgery without specimen collection Radiation therapy, 5,940 cGy; BCNU Surgery and specimen #1 collection PCV Rx Surgery and specimen #2 collection Surgery without specimen collection Radiation therapy, 5,940 cGy; BCNU Surgery with specimen #1 collection VP16 and tamoxifen Rx Surgery with specimen #2 collection Surgery with specimen #1 collection Radiation therapy, 5,940 cGy; PCV Rx Surgery with specimen #2 collection Surgery with specimen #1 collection Gene therapy; radiation therapy, 6,000 cGy Surgery with specimen #2 collection Surgery with specimen #1 collection Gene therapy; Radiation therapy, 6,000 cGy Surgery with specimen #2 collection

69/M 41/F 31/M

77/F 67/M 49/M 55/F

13 14 15

16 17 18 19

Primary GBM/right frontal Primary GBM/right temporal Primary GBM/left frontal Primary GBM/left occipital Primary GBM/left temporal Recurrent AA/right frontal Intervening therapy3 Secondary GBM Primary GBM/right temporal Primary GBM/right frontal Primary GBM/right frontal Intervening therapy3 Recurrent GBM Primary GBM/left parietal Primary GBM/left temporal Primary GBM/right temporal Recurrent AA/right parietal Intervening therapy3 Secondary GBM

Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery without specimen collection Radiation therapy 6,000 cGy; BCNU Rx Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery without specimen collection Radiation therapy; PCV Rx Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery with specimen collection Surgery without specimen collection Radiation therapy 5,940 cGy; PCV Rx Surgery with specimen collection

89 days

7/8/97 2/20/98

9/17/98

118 37 182 250

1/29/98 1/2/98 4/16/98 6/12/98 1/27/97

147

days days days days

141 days 49 days 454 days

12/2/96 12/19/96 5/8/97 4/21/97

days days days days days

104 105 75 219 202

1/31/96 8/2/96 8/9/96 10/14/96 10/24/96 8/4/88

6/8/98

233 days

72 days

10/23/97 2/13/97

5/29/98 9/22/97

139 days

12/13/96

5/1/97

days days days days days

365—Alive as of 9/16/99

181 days 37 days 306 days 462—Alive as of 9/16/99

180 days 65 days 844 days

294 258 100 423 403

91 days

276 days 350 days

55 days 374 days

10 days 200 days

324 days

137 days

1⫹, 1⫹, 0

0†, 0, 0† 1⫹, 0 0, 0, 0 1⫹, 1⫹, 0 No sample available

3⫹, 1⫹, 3⫹ 3⫹, 3⫹, 0 3⫹, 3⫹ No sample available

4⫹, 3⫹, 3⫹ 0, 0, 3⫹ 3⫹, 3⫹, 3⫹ 2⫹, 3⫹, 2⫹ 3⫹, 2⫹, 3⫹ No sample available

0, 2⫹, 2⫹

0, 1⫹, 1⫹ 3⫹, 3⫹

1⫹, 1⫹, 1⫹ 2⫹, 0, 0

2⫹, 0 4⫹, 3⫹, 3⫹

3⫹, 2⫹, 3⫹

4⫹, 3⫹, 3⫹ No sample available

2⫹, 3⫹, 3⫹

357 days

9/23/96

198 days

0*, 0‡ No sample available

0*, 0

3⫹, 2⫹, 2⫹

0, 0, 0

Quantitative Telomerase Values on 2-3 Regional Samples

7/12/98

174 days

1/12/98

541 days

Time to Demise

322 days

201 days

Time to Progression

8/18/97

1/10/97

Specimen Collection Date

Abbreviations: M, male; F, female; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine); PCV, procarbazine; 1-(2-chloroethyl)-3-cyclohexyl-1nitrosourea (CCNU, lomustine), vincristine; Rx, therapy. *Tissue taken from infiltrating edge of tumor, as assessed on matching, adjacent tissue taken for histologic analysis. ‡Tissue with radiation damage and questionable rare tumor cells. †Very scant numbers of viable tumor cells present.

68/M 68/M 34/M 63/M 59/M 41/F

7 8 9 10 11 12

Patients with specimens from a single surgical resection for telomerase analysis

50/M

50/F

2

6

49/M

1

Patients with multiple surgical resections and sequential specimens for telomerase analysis

Patient No.

TABLE 1. Quantitative Telomerase Analysis in High-Grade Gliomas on Two to Three Regional Samples per Specimen

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FIGURE 1. TRAP assay showing the characteristic 6 – base pair ladders diagnostic of telomerase expression. Arrow indicates the 36 – base pair internal standard (ITAS). The first and second samples from patient 6, third sample from patient 19, the first and second samples from patient 5, and third sample from patient 18 tested positive for telomerase activity (See Table 1 for quantitative values). The second sample from patient 19 and second sample from patient 18 were faintly positive for telomerase activity (quantitative values ⫽ 1). The remaining samples tested negative for telomerase. Each regional sample was also pretreated with RNase before TRAP assay to exclude PCR contamination. The RNase-pretreated samples (⫹) are shown in the lanes immediately to the left of the regional samples. TSR8 indicates additional internal controls of 2 and 1 ␮L of telomerase-positive extract; LB indicates lysis buffer control.

accounted for by completely necrotic tumor, based on histologic assessment of formalin-fixed, paraffin-embedded mirror-image tissue sections. In several instances, however, low or negative telomerase levels appeared to correlate with small numbers of tumor cells being present (Table 1). Matching histologic sections suggested that samples in the cases that showed low or negative telomerase levels and contained lower numbers of tumor cells were taken either from the infiltrating edge of the neoplasm or from areas with radiation damage. These areas contained a higher proportion of non-neoplastic tissue or necrotic tissue, respectively, than tumor. Two of our 19 tumors were negative for telomerase in the 3 regions of tumor that were assessed (patients 15 and 17). Patient 17 had an extremely necrotic primary GBM, and although the specimens banked for telomerase were severely necrotic as well, at least a few viable tumor cells were present in each adjacent matched tissue sample. Patient 15 had received radiation therapy and chemotherapy before the time of acquisition of the tumor sample we had available for telomerase analysis, but assessment of the matched histologic sections showed abundant viable tumor with little or no radionecrosis in these areas. Unfortunately, in this patient, we did not have the original primary GBM tumor specimen for comparison of telomerase levels pretherapy and posttherapy, to prove whether this patient also was showing downregulation of telomerase levels posttherapy.

Five of 6 patients with sequential specimens available for telomerase analysis who had intervening radiation therapy or chemotherapy showed decrease of telomerase quantitative levels across the multiple regions sampled from the second, posttherapy specimen (patients 1, 3, 4, 5, 6). These patients included 2 individuals who had received experimental gene therapy and radiation therapy, but not standard chemotherapy (patients 5 and 6). In 1 of the 6 patients, experimental gene therapy and radiation therapy was followed by increased telomerase levels (patient 1). Nevertheless, later treatment with standard chemotherapy resulted in a decrease in telomerase levels. This patient was very aggressively treated with multiple regimens and survived 541 days from time of diagnosis with a GBM. This patient was, however, in a favorable age-group (less than 50 years). We could identify no correlation between patient age and quantitative telomerase levels. Specifically, patients younger than age 50 years did not routinely show low telomerase levels, and conversely elderly did not uniformly show high levels (see patients 15 and 17). We did note, however, that several of our patients with longer survivals (patients 17, 18, and 19) displayed low telomerase levels (either 0 or 1⫹) across all regions of their tumor. Nevertheless, 3 patients with intervals to death exceeding 400 days (patients 10, 11, and 14) showed quantitative regional telomerase values that were uniformly high (2⫹ and 3⫹) across all regions sampled in their tumors. Statistical analysis

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showed no relationship between TRAP score and either time to clinical progression (P ⫽ .68) or time to death (P ⫽ .87) in our patient cohort. DISCUSSION Glioblastomas have an overall poor survival rate, with most patients dying within 2 years of diagnosis.13 Nevertheless, some variability in prognosis exists, with relatively better clinical outcomes associated with younger patient age, better preoperative clinical performance status, and the addition of radiation therapy.18 Beyond these factors, the exact biologic mechanisms influencing the relatively favorable outcome seen for some patients with GBMs are poorly understood and have been the subject of numerous studies.15,19-34 Long-term survival in GBMs occurs in approximately 1% of patients, usually those of younger age.19,28 An attempt to study whether these long-term survivors showed unique molecular genetic characteristics indicated that there was no distinct difference in this subset of long-term GBM survivors compared with GBMs in general.19 Specifically, accumulation of wild-type p53 protein and overexpression of epidermal growth factor receptor, 2 factors know to be frequent in GBMs in general, were found with nearly equal frequency in long-term survivors as in all GBMs.19 Hence, no distinctive molecular profile emerged from this study that could identify features unique to these long-term survivors or possibly predict favorable outcome in GBMs. A number of studies have addressed whether other genetic and cell-growth–related factors might correlate with prognosis in GBMs. Newcomb et al25 showed that survival of patients with GBM was not influenced by altered expression of p16, p53, epidermal growth factor receptor, MDM2, or bcl-2, as assessed by immunohistochemistry and additional analysis of gene sequencing for p53. Other studies, in contrast, have reported positive prognostic value for various markers in GBMs, including immunohistochemical staining for cathepsin B,20 MMAC/PTEN,15 c-Met protein,22 and p53 protein.24 Also studied, and believed to have prognostic significance in GBMs and high-grade gliomas, have been the c-myc oncogene family of proto-oncogenes,21 glutathione S-transferase pi,23 epidermal growth factor receptor,25 and genetic sub-typing.27 More groups have investigated markers of cell proliferation such as Mib-1, either alone or in conjunction with markers of cell apoptosis, to evaluate prognosis in gliomas, but with mixed success.29-32 In some studies, markers of cell proliferation did not add significant prognostic information to traditional histologic-based grading systems, after adjustment for tumor grade and patient age.32 Hence, attempts to identify new prognostic markers within tumor grade continue to be active and promising areas of investigation in GBMs. That telomerase expression might be a useful new prognostic marker in high-grade gliomas seemed reasonable, based on numerous studies in the literature on its prognostic implications in other tumor types, as

summarized in Table 2. These studies come to diverse conclusions as to whether telomerase expression has prognostic utility and emphasize that the question needs to be addressed for each individual tumor type. Most studies have employed qualitative rather than quantitative methodology. In regard to central nervous system tumors, Langford et al33 observed a highly significant correlation in ordinary meningiomas between the presence of telomerase activity and a poor prognosis for the patient, a finding for meningiomas that was corroborated by Falchetti et al.9 Most relevant to our current study, studies by Nakatani et al7 in 1997, Falchetti et al9 in 1999, and Weil et al12 in 1999 have generated conflicting results regarding the prognostic utility of telomerase expression in gliomas, predominantly GBMs. Considering the regional variability that has been reported for other tumor markers such as Mib-1 in GBMs, we chose to address the question of relationship between telomerase expression and prognosis by a detailed, regional, quantitative study. In the current study, we analyzed 2 to 3 regions from each tumor and applied quantitative methodology not available to us when we undertook our original regional study of high-grade gliomas. In this technique, the widely used telomeric repeat amplification protocol results in the formation of a characteristic progressive 6 – base pair PCR amplification product that is evaluated by polyacrylamide gel electrophoresis. Phosphorimager analysis of the PCR product correlates with the amount of telomerase activity that was present in the solubilized tissue sample. We noted a trend between lower telomerase levels and longer duration of survival in a few of our patients, but higher telomerase levels across multiple regions of the patient’s tumor did not automatically result in adverse clinical outcome. Univariate analysis of quantitatively analyzed regional tissue samples showed no statistical association between telomerase levels and time to progression, an end point favored by most clinicians when assessing efficacy of therapeutic regimens (P ⫽ .68). Similarly, there was no statistical association between the level of telomerase expression and time to patient death (P ⫽ .87). Thus, when regional telomerase expression was addressed by quantitative methodology, we were unable to support the earlier observation of Nakatani et al7 that telomerase expression is a useful prognostic factor for patients with malignant astrocytic tumors, particularly within the highest grade tumor of the spectrum, the GBMs. Our results are also discordant with the conclusion of Falchetti et al9 that “the assessment of telomerase activity may influence tumor prognosis . . . in malignant gliomas . . .” Our findings do agree, however, with Weil et al and extend the observations made by that group to a larger number of systematically studied high-grade glioma cases.12 Our study also furthered our understanding regarding heterogeneity of telomerase expression in high-grade gliomas. In our preceding qualitative study of large numbers of regional specimens from GBMs, at least 1 sample was positive for telomerase in each of the tumors studied. In our current quantitative study, an up

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QUANTITATIVE TELOMERASE IN GBMs (Kleinschmidt-DeMasters et al)

TABLE 2. Recent Studies Correlating Telomerase Activity With Prognosis in Various Tumor Types Author & Year

Cancer Type

Ebina et al; Int J Cancer 84:529-532, 1999 Premba et al; Ann Oncol 10:715-721, 1999 Harada et al; Cancer 86:1050-1055, 1999 Zhang et al, Eur J Cancer 35:154-160, 1999 Nagai et al, Oncol Rep 6:325-328, 1999

Endometrial cancer

Umbricht et al, Oncogene 18:34073414, 1999 Mokbel et al, Eur J Surg Oncol 25:269272, 1999 Kishimoto et al, J Surg Oncol 69:119124, 1998 Sakurai et al, Cancer 83:2060-2066, 1998

Breast cancer

Bechter et al, Cancer Res 58:49184922, 1998

B cell chronic lymphocytic leukemia

Okayasu et al, J Cancer Res & Clin Oncol, 124:444-449, 1998

Colorectal carcinoma

Roos et al, Int J Cancer, 79:343-348, 1998

Breast cancer

Kagata et al, J Oto-RhinoLaryngological Society of Japan, 101: 205-211, 1998 Oishi et al, Obstet Gynecol 91:568-571, 1998

Squamous cell carcinomas of head and neck Epithelial ovarian cancer

Kleinschmidt-DeMasters et al, J Neurol Sci 161(II):1160-1223, 1998 Hoos et al, Int J Cancer, 79:8-12, 1998

Metastatic brain tumors Breast cancer

Suda et al, Hepatology, 27:402-406, 1998

Hepatocellular carcinoma

Authors’ Conclusions From Study High telomerase activity correlated with advanced surgical stage and pelvic lymph node metastases Telomerase activity is independent prognostic marker

Neuroblastoma Central nervous system lymphoma Cervical cancer

Significant correlation between telomerase activity and duration of survival and interval to tumor progression High telomerase activity more frequently detected in advanced disease Semiquantitative telomerase activity significantly higher in the subjects with invasive cancer than in those with cervical intraepithelial neoplasia Telomerase activity useful adjunct in stratifying the risk of developing invasive breast cancer in patients with ductal carcinoma in situ Telomerase activity not associated with nodal status or disease outcome All carcinomas telomerase positive. Telomerase activity level correlated with the degree of differentiation and patient survival Telomerase activity–positive gastrointestinal stromal tumors significantly larger and showed a significantly higher rate of proliferation than telomerase activity–negative tumors High telomerase activity significantly associated with shorter median survival and telomerase activity, the most significant prognostic factor for overall survival in B-CLL Moderately or poorly differentiated subtypes more predominant in the telomerase-positive than in the telomerase-negative groups. Telomerase positivity correlated with high stage and lymph node metastases. Tumors with high telomerase levels significantly associated with a poor prognosis for node-negative patients, but not for node-positive patients Therapy-resistant tumors had higher telomerase activity. No relationship between telomerase activity and clinical stage. Telomerase activity level useful for predicting prognosis. Intensity of telomerase activity significantly higher in tumors with lymph node involvement, but intensity of telomerase activity was not independent factor for prognosis in patients with ovarian cancer Quantitative telomerase levels showed fourfold logarithmic variability but no correlation with tumor type or interval to patient death Telomerase activity strongly decreased in all chemotherapy-treated tumors compared with untreated tumors. Telomerase activity associated with aggressiveness of breast tumors Quantitative telomerase values in 9 patients suffering from early recurrences after surgery significantly higher than that in 11 patients without early intrahepatic recurrences

Cervical cancer

Breast cancer Hepatocellular carcinoma Gastrointestinal stromal tumors

to 3-fold regional variation in telomerase levels was noted within the same tumor, although most tumors showed a 1- to 2-fold variability in positive areas across multiple regions sampled. We conclude that even in GBMs that show positivity for telomerase expression, there is variability in the level of expression from region to region within the tumor. Interestingly, a very recent study of telomerase in prostate cancer, another notoriously histologically variable tumor, has also shown heterogeneity of telomerase expression.34 An additional observation we have made on a small number of samples in this current study, as well as our previous study,5 is that areas of tissue were negative for telomerase expression when they contained small numbers of tumor cells. Some negative telomerase-expressing tumor samples appeared to be taken from the infiltrating edge of tumor, where smaller numbers of tumor cells were present relative to reactive or normal tissue (Table 1). This observation needs to be investi-

gated further. Unlike some systemic malignancies such as colon cancer where assays for telomerase may serve to detect minimal residual disease,4 telomerase may not be a good marker of minimal residual disease in high-grade gliomas. Specifically, the possibility that telomerase analysis can be used as a method to distinguish reactive gliosis versus infiltrating glioma, or radiation damage versus small amounts of residual glioma, seems unlikely, at least in our experience with the soluble TRAP assay. We previously have shown the exquisite sensitivity of the TRAP assay and shown that as few as 10 cells from a HeLa cervical carcinoma cell line generate the characteristic 6 – base pair ladder diagnostic of positive telomerase expression.2 Whether our inability to detect minimal numbers of glioma tumor cells in our current samples taken from infiltrating tumor edges or areas of tumor with more necrosis and fewer numbers of tumor cells lies in the fact that the residual tumor

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Volume 31, No. 8 (August 2000)

cells have lower telomerase levels on a per-cell basis is completely unknown. Our in vivo results shown down-regulation of telomerase expression in tumor obtained within weeks or months after administration of chemotherapy or radiation therapy. We, and others, have shown that telomerase expression parallels Mib-1 labeling indices in gliomas11,35 and in ependymomas.36 Our finding of down-regulation of telomerase appears to correlate with the demonstration by other investigators of downregulation of Mib-1 after radiation therapy of GBMs.37 Relatively few mitotic figures are usually present in areas of paucicellular tumor after ionizing radiation therapy or chemotherapy of GBMs, and Bigio et al37 have shown that Mib-1 labeling indices are usually lower in areas of radiation damage than in areas of recurrent glioma, although notable exceptions were found. Hence, our finding of down-regulation of telomerase after radiation therapy and chemotherapy for gliomas is not surprising, given its known correlation with Mib-1 labeling index.11 However, it should be emphasized that measurement of telomerase activity is not a direct measurement of cell proliferation.1 Cell culture studies have shown that telomerase expression is not regulated directly by cell cycle status but is related to both the induction of apoptosis and the rate of cell proliferation.38 Thus, telomerase expression is an independent marker1 that correlates with previous studies that have reported a suppression of the Mib-1 labeling index after therapy for gliomas.37 Our work corroborates several recent in vitro studies on cell lines.38,39 Holt et al38 showed that when cells in culture were treated with either nocodazole or doxorubicin, the cells underwent growth arrest at the G2/M phase of the cell cycle, and decreased telomerase levels occurred. Sawant et al39 evaluated telomerase activity in response to ionizing radiation and found that telomerase activity was decreased in a dose-dependent manner that correlated with cell death in in vitro tests, as well as with tumor regression. They suggested that “detection of telomerase activity may be a useful monitor of radiotherapeutic efficacy and an early predictor of outcome.”39 Certainly, our results from this regional quantitative study suggest that down-regulation of telomerase in GBMs does occur in response to chemotherapy and radiation therapy in vivo, as well as in vitro. In the current study, we cannot exclude the possibility that the re-emergence of even a tiny area of high telomerase-expressing tumor cells (impossible to detect without analyzing the entire specimen, which currently cannot be done using the soluble assay) might be sufficient to cause excess tumor growth and patient death. A future area of research may, in fact, be to monitor the efficacy of therapeutic response in patients with GBMs with quantitative telomerase levels. Telomerase levels might be used to compare direct tumor effects of various chemotherapy, radiation therapy, or novel therapy paradigms, such as gene therapy or immunotherapy. The results of this study, however, suggest that multiple areas of tumor may have to be analyzed for telomerase

in these heterogeneous neoplasms to obtain meaningful data regarding therapeutic efficacy. Acknowledgment. The authors thank Vickie McHenry for histotechnology work, Ginger Woodward for manuscript preparation, and Bob McCullough for photographic assistance.

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