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Telomerase Is a Highly Sensitive and Specific Molecular Marker in Fine-Needle Aspirates of Breast Lesions

From the Gerhard-Domagk-Institute of Pathology, Westfälische Wilhelms-University, Muenster, Germany; Department of Pathology, University of Colorado Health Sciences Center, and Department of Cardiac Research, Veterans Affairs Medical Center, Denver, CO; and Department of Pathology and Laboratory Medicine, Presbyterian Medical Center, University of Pennsylvania, Philadelphia, PA.

Address reprint requests to Barbara Dockhorn-Dworniczak, MD, PhD, Gerhard-Domagk-Institute of Pathology, University of Münster, Domagkstr 17, D-48149 Münster, Germany; email dwornib{at}uni-muenster.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Telomerase has been detected in a majority of human malignant tumors, making telomerase activity (TA) one key difference between mortal and immortal cells. In this study, we evaluated in blind-trial fashion the association of TA with cytologic and final clinical/pathologic diagnosis in fine-needle aspirates (FNAs) of breast lesions.

MATERIALS AND METHODS: In 172 FNAs, including 80 samples that were cytologically malignant, 18 that were atypical but not diagnostic for malignancy, and 74 that were cytologically benign, TA was determined by a modified nonradioactive telomeric repeat amplification protocol (TRAP) assay. Final diagnosis was made by pathologic examination of follow-up surgical material available for all the cytologically malignant samples, a majority of the cytologically atypical samples, and a portion of the cytologically benign samples.

RESULTS: TA was detected in 85 of 172 samples. Comparison of the cytologic and histologic diagnoseswith TA showed that 80 of 87 samples from patients with breast cancer were telomerase-positive, resulting in a sensitivity of 92%. TA was found in four of five FNAs from carcinomas that were considered cytologically atypical but not diagnostic for malignancy. Eighty of 85 samples from patients with benign breast lesions were telomerase-negative, revealing a specificity of 94%. The five positive cases in this group were all fibroadenomas with low TA. Among the 18 cases with a cytologic diagnosis of atypia, there was a strong positive relationship between TRAP findings and histologic diagnosis.

CONCLUSION: The detection of TA in FNAs of breast lesions is a highly sensitive and specific marker of malignancy and may be used as an adjunct in cases with an equivocal cytologic diagnosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
TELOMERASE IS a ribonucleoprotein enzyme complex that uses an internal RNA template to form the specially structured ends of chromosomal DNA (ie, telomeres) by directing the synthesis of new hexameric nucleotide repeats (TTAGGG)_n onto the 3' end of chromosomes.¹ Cells without telomerase activity (TA) display progressive shortening of telomeric repeats with each cell division due to the "end-replication problem," leading to the stage when the cell is likely to exit from the cell cycle and become senescent. A current hypothesis proposes that (re)activation or upregulation of telomerase allows tumor cells to escape from cellular senescence by compensating for the loss of telomeric DNA and thus maintaining telomere length, which allows cells to proliferate indefinitely.^2-6 Indeed, TA is present in a high proportion of specimens from a wide variety of malignant tumors, including breast cancer.^7-12 The absence of TA in most nonneoplastic tissues and somatic cells and its presence in almost all malignant tumors have raised much interest in its potential diagnostic, prognostic, and therapeutic implications in the management of human cancer.¹³ Analysis of telomerase could complement established histology or cytology-oriented methods for early detection of cancer. Moreover, the combination of TA assessment and cytology in minimally invasive procedures such as fine-needle aspiration (FNA) may result in more accurate diagnoses.^14-16

Previous studies on TA in breast cancer yielded positive results in 73% to 95% of cases of invasive carcinomas and as many as 75% of in situ carcinomas, whereas normal breast tissue and almost all benign lesions, with the exception of a small portion of fibroadenomas, were telomerase-negative.^11,17-20 FNA, in combination with telomeric repeat amplification protocol (TRAP) assay, might prove to be a sensitive test for detection of TA in breast lesions that is helpful for differentiating between benign and malignant lesions, particularly in cases with inconclusive cytologic observations.

In the present study, we analyzed TA in 172 FNA samples of breast lesions. In contrast to the few previous studies on this subject, an almost identical number of samples from patients with malignant versus benign breast lesions were evaluated, and results were scored for TA in blind-trial fashion, without any knowledge of the corresponding cytologic or histologic results. Furthermore, we applied a nonradioactive, modified TRAP assay using fluorescence-labeled primers and an automated laser-fluorescence sequencer for semiquantitative and fast real-time determination of TA.^11,21

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cytologic Samples
Initially, a total of 181 FNA samples were collected from material previously obtained for routine cytologic examination. All cases were consecutively collected and were derived from palpable breast lesions. The study was approved by the local ethics committees. On cytologic examination, 82 samples were considered to be indicative of malignancy, 79 samples were benign, and 20 were atypical but not diagnostic for malignancy. The techniques of aspiration, preparation of smears, and staining were performed according to standard methods. Briefly, 1-inch 23-gauge disposable needles attached to standard disposable syringe holders were used. One half of the aspirated material was centrifuged and expressed onto glass slides for routine cytologic examination. The other half was rinsed into a saline buffer, collected by centrifugation, and stored at -80°C until use. Follow-up surgical material was available for all (82 of 82) of the cytologically malignant samples, 80% (16 of 20) of the cytologically atypical samples, and 34% (27 of 79) of the cytologically benign samples.

Extract Preparation and Telomerase Assay
Protein extracts of FNAs and corresponding tissue specimens were prepared as previously described,^11,21 with slight modifications. Briefly, cell pellets from FNAs were resuspended in 30 µL of ice-cold 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) lysis buffer containing 1 U/µL of RNase inhibitor (Boehringer Mannheim, Mannheim, Germany). After 30 minutes of incubation on ice, the lysates were centrifuged at 12,500 x g for 30 minutes at 4°C. The supernatant was frozen rapidly in liquid nitrogen and stored at -80°C until use. The residual pellet was relysed in 100 µL of lysis buffer, incubated, and centrifuged as described above. The protein concentrations were measured using the Coomassie Protein Assay Reagent (BioRad, Hercules, CA) on an enzyme-linked immunoadsorbent assay reader and adjusted to 1.5 µg/µL. Eight samples with no or low (< 0.3 µg/µL) protein after repeated extractions were excluded from the study, resulting in 173 samples for TRAP analysis.

For in vitro detection of TA, a modified version of the TRAP assay was used. In brief, 1 µL of each protein extract (containing 1.5 µg of protein) was suspended in 24 µL of reaction mix containing 200 mmol/L Tris-HCl pH 8.3; 15 mmol/L MgCl₂; 630 mmol/L KCl; 0.5% Tween-20; 10 mmol/L ethylene glycol-bis (beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid; 0.1% bovine serum albumin; 2.5 mmol/L each dATP, dTTP, dGTP, dCTP; 0.2 µL Taq-Polymerase (5 U/µL, Perkin-Elmer, Branchburg, NJ); 0.15 µmol/L fluorescence-labeled TS forward primer (5'-(Cy-5)-AATCCGTCGAGCAGAGTT-3'); and 19.8 µL polymerase chain reaction (PCR)–grade ddH₂O. Additionally, each reaction mixture contained 0.25 µL of CX reverse primer (5'-CCCTTACCCTTACCCTTACCCTTA-3') and an internal PCR-amplification control from a commercially available kit (TRAPeze telomerase detection kit, Intergen, Purchase, NY). The internal PCR-amplification control produces a 36–base pair (bp) product, which is coamplified with telomerase-elongated products in each reaction. Each analysis included a telomerase-positive control (Ewing's tumor cell line VH-64), heat-inactivated controls (telomerase-positive control incubated at 94°C for 5 minutes before reaction), and a negative control (CHAPS lysis buffer instead of sample). After 30 minutes of incubation at 30°C for telomerase-mediated extension from annealed TS-oligonucleotides, the reaction mixture was immediately heated to 94°C for 5 minutes and then subjected to 30 PCR cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds.

For analysis of amplification products, 1 µL of PCR product and 6.7 µL of loading buffer (90% formamide, 10% Blue dextran) were mixed and denatured at 94°C for 5 minutes. Six microliters were loaded on denaturing 8% polyacrylamide gels on an automated laser-fluorescence sequencer (ALFexpress, Pharmacia, Freiburg, Germany) and subjected to electrophoresis. Fluorescence data were collected automatically and analyzed by the Fragment Manager Program (Version 1.02, Pharmacia). Each fluorescent peak was quantitated in terms of size (bp), peak height, and peak area. For semiquantitative analysis, the mean value of peak area of the first five fluorescent peaks was compared with the internal PCR-amplification control. The accuracy of this semiquantitative analysis was previously established by several dilution series for samples with different cellularity and TA levels.¹¹ A single case failed to show amplification of the internal PCR-amplification control. This case carried a clinical/cytologic diagnosis of metastatic melanoma. A strong reduction in signal intensity was observed when this lysate was mixed 1:1 with unrelated TRAP-positive lysates from the Ewing's tumor cell line VH-64, which is consistent with the effect of an inhibitor of PCR. Therefore, this sample was excluded from further study.

Statistical Analysis
To preclude bias, all samples were coded and TA levels were scored without knowledge of the cytologic or histologic diagnoses. If biopsy was not performed, a final clinical/pathologic diagnosis was used in place of the histologic diagnosis as the "gold standard" assessment for presence or absence of disease. The sensitivity of the TRAP assay in FNAs was determined as the frequency of samples correctly identified as malignant by the TRAP assay among the total number of cases with a final histologic diagnosis of malignancy. The specificity was calculated as the frequency of samples correctly identified as benign by the TRAP assay among the total number of cases with a clinical and/or histologic diagnosis of benign lesions. Positive and negative predictive values of the test were determined according to the method illustrated in Fig 1. A Fisher's exact test or ² test were used where appropriate to examine for a potential association between dichotomous study variables of interest.^22,23 Pending a statistically significant association being documented, a Cramer V was used as a measure of the strength of association.²⁴ To determine the relative improvement in cytologic diagnosis by the addition of TRAP assay findings, a McNemar's test as a matched pair comparison was performed.²⁵ For the matched pair analysis, the cases were stratified by histologic diagnosis (85 cases with negative histologic diagnosis and 87 cases with positive histologic diagnosis). As part of the McNemar's test, a continuity correction was performed, to be most conservative, in assessment of the potential association between paired test results.

View larger version (30K): [in this window] [in a new window]   Fig 1. Positive and negative predictive values of TRAP assay. "Diagnosis" refers to the final clinical/pathologic diagnosis. Two of seven cases represent false-negative diagnoses on both cytologic examination and TRAP assay as a result of sampling error (Table 1; see also Discussion).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Positive telomerase expression was defined as amplification of six or more bands including the 50-bp, 56-bp, 62-bp, 68-bp, 74-bp, and 80-bp bands (Fig 2). TRAP analysis revealed TA in 85 (49%) of 172 samples. Among those, 16 (19%) of 85 samples that had originally been telomerase-negative became positive after the first protein extraction when the residual pellet was relysed and the TRAP assay repeated. In 88 (51%) of 172 samples, no TA was detectable. TA was detected in 76 (95%) of 80 FNAs from patients with FNA cytology that was positive for malignant cells. Comparison with the final histologic diagnosis revealed TA in 80 (92%) of 87 cases of carcinoma of the breast, including five cases with a cytologic diagnosis of atypia, and two cases with a false negative cytologic diagnosis. Conversely, TA was detected in four (5%) of 74 FNAs with a benign cytologic diagnosis, and comparison with the final clinical and/or histologic diagnosis revealed TA in five (6%) of 85 cases of benign breast lesions. Thus the specificity of the TRAP assay compared with the FNA cytologic diagnosis in our study was 95%. The five telomerase-positive FNAs of benign breast lesions were all fibroadenomas, of which four samples had been assigned to the group of benign breast cytology and one sample as fibroadenoma with atypical features to atypical breast cytology. TA levels for all these samples were equally low.

View larger version (86K): [in this window] [in a new window]   Fig 2. Histology, cytology, and TA of breast FNAs. Lane A, infiltrating ductal carcinoma (GII) with high TA level (x400). Lane B, infiltrating ductal carcinoma (GI) with intermediate TA level (x400). Lane C, fibroadenoma with no detectable TA (x400).

The positive predictive value of the test, defined as the probability that a positive TRAP assay is true-positive compared with the final clinical/pathologic diagnosis, was 94% in this study. Congruously, the negative predictive value, calculated as the probability that a negative TRAP assay is true-negative compared with the final clinical/pathologic diagnosis, was 92% (Fig 1). Based on the analysis of the full study population (172 cases), there was a strong positive relationship between TRAP findings and final clinical/pathologic diagnosis (P < .01; Cramer's V = 0.86). Match-pair comparisons within strata of clinical/pathologic diagnosis revealed no difference in the cytologic diagnosis in comparison to the TRAP findings (P > .07 for both histologic positive and negative cases, independent of the classification methodology used for cytologic diagnosis). For the subset of 18 cases with cytologic diagnosis of atypia, there was a strong positive relationship between TRAP findings and histologic diagnosis (P < .01; Cramer's V = 0.72).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recent publications have revealed the potential of assessing TA as an adjunct to the cytologic diagnosis of malignancy for different body cavity washings, exfoliative cells, or body fluids. These studies have yielded encouraging results with good sensitivity and specificity of telomerase expression for detecting malignant cells. Studies on voided urine or bladder washings for the detection of transitional-cell carcinoma revealed sensitivities of 62% to 97% and specificities of 96% to 100%.^26-29 The analysis of TA in 12 brush samples from the pancreatic duct for detection of ductal adenocarcinoma showed both a sensitivity and specificity of 100%, representing the most promising telomerase/cytology correlation to date.³⁰ Yang et al³¹ found that the detection of TA may be a useful adjunct to cytopathologic methods in the diagnosis of pleural effusions, yielding a sensitivity of 91.4% and a specificity of 94.2% based on 144 samples. The largest study to date on telomerase expression in body fluids of any kind was performed by Kleinschmidt-DeMasters et al¹⁵ using the TRAP protocol for 281 CSFs from a wide variety of patients with neurologic and nonneurologic conditions. In this study, the adjusted specificity was 90% and the sensitivity was 64% for detection of malignant cells in CSF by TA.

For cytologic breast FNA diagnosis of malignancy, sensitivity ranges from 66% to 100% in the literature and specificity from 82% to 99%.^32-37 Therefore, the idea of using telomerase as a marker of malignancy is tempting to improve the accuracy of FNA diagnosis in breast lesions. To date, only a few studies have been published on this subject, all of them plagued by small sample numbers, a predominance of benign cases, FNA samples obtained from excised breast tumors rather than in situ lesions, or lack of follow-up surgical data. In the first study published on TA in breast FNAs, 44 samples (15 cytologically malignant, 29 cytologically benign) were included, and the study yielded a sensitivity of 67% and a specificity of 90%.¹⁴ Among the 33 FNAs analyzed by Hiyama et al,¹⁹ 15 (45%) showed TA. Of these 15 samples, 14 (93%) were diagnosed as malignant by cytology and follow-up surgery. Pearson et al³⁸ analyzed 46 FNAs obtained in the operating room from excised breast tumor material. TA was found in one (12%) of eight benign samples, nine (56%) of 16 fibroadenomas, two (100%) of two carcinomas-in-situ, and 17 (90%) of 19 invasive cancers, with FNA-TRAP results and gross tissue specimen TRAP results coinciding in 95% of the cases. In the most recent and largest study published, Villa et al³⁹ analyzed 116 FNAs, 80 of which were diagnosed as benign and 36 as malignant. Unfortunately, the sensitivity and specificity only ranged from 39% to 75% and 51% to 85%, respectively, taking into account the results from TRAP assays performed at two protein concentrations. The authors, therefore, stated that their poor predictive values for both malignant and benign lesions were far from acceptable for routine diagnostic application and stressed the need for assay improvements.

In our study, we analyzed a total of 172 FNA samples from an approximately equal number of patients with malignant (n = 87) versus benign (n = 85) lesions of the breast. Results were scored in a blind trial fashion without any knowledge of corresponding cytologic or histologic results. Compared with the aforementioned studies, we found both a sensitivity and a specificity for the TRAP assay of 95% compared with the cytologic diagnosis. Our study was limited, however, by the lack of histologic follow-up on the majority of patents with a benign cytologic diagnosis. Despite this theoretical limitation, our data suggest that a clear separation of TA between benign and malignant samples might prove useful in routine practice as a marker for FNA biopsies to help distinguish benign from malignant disease in indeterminate FNA samples. In our study, for example, cytologic diagnoses were signed out as atypical but not diagnostic for malignancy in 18 cases. Five of these cases were shown to be derived from invasive ductal carcinomas by subsequent surgical excision and 13 were found to be derived from benign breast lesions. TA was detected in four of five cases of ductal carcinoma with a cytologic diagnosis of atypia but was detected in only one of 13 benign breast lesions with a cytologic diagnosis of atypia. Review of the FNA specimens from breast cancer with a cytologic diagnosis of atypia showed scant cellularity in three of five cases, including the single case in this category that was telomerase negative. The other two cases were highly cellular, including one with both numerous benign-appearing ductal cells and discohesive cells that were suspicious for carcinoma. The other case in this series was limited by lack of clinical data from the clinician that performed the aspirate but was considered on final review to be consistent with ductal carcinoma. Thus telomerase analysis was positive in four of five cases of ductal carcinoma with an equivocal FNA diagnosis but was also positive in one of 13 cases from benign breast tissue with an equivocal FNA diagnosis.

Among the 27 cases in our study with a benign cytologic diagnosis, one case was diagnosed as a poorly differentiated ductal carcinoma, 8 mm in maximal dimension, on subsequent core needle biopsy. Review of the FNA slides from this case showed only rare benign ductal cells. A second case was diagnosed as papillary carcinoma, 2.4 cm in maximal dimension, on histologic evaluation of the subsequent surgical excision specimen. Thus these cases represented false-negative diagnoses on both cytologic examination and TRAP assay as a result of sampling error.

The detection of low telomerase levels in FNAs from fibroadenomas (five TRAP-positive cases) demands careful interpretation of FNA-TRAP results, because low TA in particular may not necessarily be an indicator of malignancy in breast tissue but may just as well be associated with cell proliferation. In a previous study,¹¹ we found TA to be associated with an enhanced cell-proliferation rate demonstrated by MIB1 staining in tissue specimens from nine of 16 fibroadenomas. Other groups also found TA in fibroadenomas, although at a lower frequency.^10,14,18,19 A possible explanation might be that TA in fibroadenomas may originate from committed stem cells in breast tissue, because it is hypothesized that renewal of mammary gland epithelium originates from committed stem cells capable of differentiating into luminal epithelial or myoepithelial cells.^40,41 This explanation is in agreement with the findings of Hiyama et al⁴² that stem cells of renewal tissues may have TA for continuos proliferative ability throughout their lives.

For application of FNA-TRAP in daily routine use as a potential adjunct to the cytologic diagnosis of malignancy, it is of vital importance to obtain the results from analysis of TA as fast as possible. The use of a radioactive TRAP assay for daily routine diagnosis is severely hampered by the fact that autoradiographic exposure for visualizing telomerase products of FNA samples or body fluid specimens may take up to 7 days.⁴³ Therefore, we applied a novel, nonradioactive, modified TRAP assay, using fluorescence-labeled primers and an automated laser-fluorescence sequencer.^11,44 This method allows semiquantitative and fast real-time determination of TA within 2 to 3 hours after applying samples and was shown to be at least as sensitive and specific as the conventional radioactive TRAP assay.⁴⁵ Furthermore, to prevent false-negative results due to insufficient protein extraction, we found it useful to relyse the cell pellet after the first extraction in cases with no TA. Norton et al⁴⁶ found that CHAPS buffer, which is most often used for protein extraction before the TRAP assay, may not lead to complete protein extraction. Because relysis of TRAP-negative cases is time-consuming, especially when the test is to be used as an adjunct for routine diagnostics, new lysis buffers as a combination of NP-40 and sodium deoxycholate, as suggested by Norton et al,⁴⁶ may be applied to extract telomerase more efficiently.

In summary, this retrospective blind-trial study demonstrates that TA, provided that test conditions are optimized, is a highly sensitive and specific molecular marker in FNAs of breast lesions that may prove helpful as an adjunct to routine diagnostic cytology, especially when cytologic features of the specimen are inconclusive. To our knowledge, this is the first study on telomerase in breast FNAs that yields predictive values acceptable for routine diagnostic application for both malignant and benign lesions by using a modified TRAP assay for semiquantitative and fast real-time determination of TA. The performance of the TRAP assay as a cytologic adjunct, however, will depend on the prevalence of disease in the study population. Thus future studies are needed to evaluate the potential role of telomerase analysis for the detection of breast cancer in high-risk and low-risk populations.

	ACKNOWLEDGMENTS

 
We acknowledge Dagmar Henke for skillful technical assistance, Dr Friedrich Otterbach for help with collecting samples, and Susanne Kölsch for proofreading the manuscript.

	NOTES

 
This work was supported in part by the German Research Foundation "Deutsche Forschungsgemeinschaft" (DFG, Po 529/4-1).

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Submitted October 1, 1998; accepted March 16, 1999.

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