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Part II. Telomerase expression in cerebrospinal fluid specimens as an adjunct to cytologic diagnosis1
Journal of the Neurological Sciences 161 (1998) 124–134
Part II. Telomerase expression in cerebrospinal fluid specimens as an adjunct to cytologic diagnosis 1 B.K. Kleinschmidt-DeMasters
a ,b ,
*, Lynnette C. Evans a , Mitchell A. Bitter a ,b , A. Laurie Shroyer c , Kenneth R. Shroyer a
a
University of Colorado Health Sciences Center, Department of Pathology, 4200 East Ninth Avenue, Denver, CO 80262, USA University of Colorado Health Sciences Center, Department of Neurology, 4200 East Ninth Avenue, Denver, CO 80262, USA c Department of Cardiac Research, Veterans Affairs Medical Center, 1055 Clermont Street, Denver, CO 80220, USA
b
Received 18 March 1998; received in revised form 25 June 1998; accepted 26 June 1998
Abstract The diagnosis of meningeal carcinomatosis hinges on the cytologic examination of cerebrospinal fluid (CSF), which has a known low sensitivity for the identification of malignant cells. Often only ‘suspicious’ or ‘atypical’ diagnoses can be rendered, and specimens are commonly unsatisfactory for evaluation due to poor morphologic preservation. Telomerase is widely expressed in most brain metastases, medulloblastomas, lymphomas, oligodendrogliomas, and is expressed focally in glioblastomas. Little is known about the level of telomerase expression in these tumors, except for brain metastases, where a four-fold variation in telomerase levels exists. In our laboratory, as few as ten carcinoma cells can be detected by a sensitive polymerase chain reaction-based assay, the telomeric repeat amplification protocol (TRAP), for telomerase, but it was unclear whether varying levels of telomerase expressed by different types of metastases would influence detection. Using the TRAP protocol, we studied 281 CSF samples from a wide variety of patients with neurologic and non-neurologic conditions for telomerase expression. An adjusted specificity of 90% and a sensitivity of 64% were achieved for detection of malignant cells in CSF by telomerase expression. The TRAP assay for telomerase detection may serve as an adjunct to the traditional examination of CSF. Neither previously documented four-fold variation in the levels of telomerase expression in brain metastases, high CSF protein levels nor high white blood cell counts precluded detection of malignant cells in CSF. 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Cerebrospinal fluid; Cytology; Meningeal carcinomatosis; Metastases; Telomerase; Central nervous system; Brain; Prognosis
1. Introduction Telomerase is a ribonucleoprotein enzyme complex that synthesizes G-rich, six base pair sequences onto the ends of chromosomes, known as telomeres [1]. Telomeric reduction signals the cessation of cellular division and may serve as a biological clock to regulate the lifespan of the cell [15,22,30]. Telomerase is down-regulated in cells as they undergo senescence or differentiation, but continues *Corresponding author. Tel.: 1303-315-7298; fax: 1303-315-4792. 1 Presented in abstract form at the 74th annual meeting of the American Association of Neuropathologists, Minneapolis, MN, USA, on June 19 1998, where it was the recipient of the Rubinstein award for the best paper in Neuro-oncology for 1998.
to be expressed at low levels in proliferative tissues, including germ cells, endometrium, basal keratinocytes and lymphoid cells [22,30,31]. High telomerase expression, by contrast, is characteristic of immortalized cells and is found in the vast majority of all cancers [23]. While the grade and stage at which telomerase is expressed differs for each given tumor type and site, high telomerase levels are virtually always found in late-stage malignancies [4,5,7,17,18,21,23,33]. This nearly ubiquitous expression of telomerase in a wide variety of human neoplasms has prompted a plethora of reports addressing telomerase as a universal cancer marker or therapeutic target [4,15,16,28,29,31,32,35, 37,39]. Most studies have concentrated on analyzing tissue
0022-510X / 98 / $ – see front matter 1998 Published by Elsevier Science B.V. All rights reserved. PII: S0022-510X( 98 )00254-8
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
extracts of primary tumors [8,11,17,19,21,26,34], although several have specifically addressed telomerase expression in metastases [16,17,12]. Nakatani et al. [26] found positive telomerase expression in three of three tumors metastatic to the brain. In a study preliminary to the current investigation, our laboratory investigated telomerase expression in 35 brain metastases of different types, correlating telomerase expression levels with prognosis and interval to patient demise [12]. From this previous work, we found that at least 32 of 35 advanced-staged malignancies metastatic to the brain demonstrated positive telomerase expression, albeit at widely varying levels. It was unclear from that study whether the four-fold variation in telomerase levels would influence detection of metastatic cells in cerebrospinal fluid (CSF). Previous work by our group had also shown that primary cerebral malignancies that were likely to disseminate to the meninges, i.e. medulloblastomas [11], oligodendrogliomas and glioblastoma multiformes [10], were all telomerase-positive, at least focally. These findings prompted us to explore the utility of testing CSF for telomerase expression, as a potential adjunct to the diagnosis of tumors with metastases and dissemination to the meninges. Several papers had confirmed the potential for utilizing telomerase detection as an adjunct to the cytologic diagnosis of malignancy for a variety of body fluids, exfoliative cells, or body cavity washings [6,9,14,17,20,24,25,35,37–40]. Most of these studies had shown encouraging results, with good sensitivity and specificity of telomerase expression for detecting cytologically positive tumors. However, in most of these studies, sample numbers have been small, and only modest attempts have been made to explain discrepancies between telomerase expression and cytologic diagnosis. Also, few have included patients with possible confounding inflammatory or other non-neoplastic diseases for comparison. The current study is the first, to our knowledge, that addresses the utility of the telomeric repeat amplification protocol (TRAP) assay for telomerase expression as an adjunct to the cytologic diagnosis of CSF, and is the largest, to date, to study telomerase expression in fluids of any kind. In this study of 281 specimens, we paid particular attention to possible influential parameters, such as time interval between the clinical procedure and processing of the fluid, CSF protein levels and white blood cell counts. We also undertook thorough medical chart review to investigate all the differences between telomerase expression results and cytologic diagnoses. 2. Materials and methods
2.1. Patient information All available CSF specimens were collected from pa-
125
tients seen at the University of Colorado Health Sciences Center (UCHSC) between September 1996 to December 1997 for clinically indicated reasons. No patient had CSF fluid drawn specifically and solely for this study. Two hundred and eighty-one specimens from 165 patients were available for the TRAP analysis. CSF specimens that were unsatisfactory or inconclusive for cytologic diagnoses were not utilized for the final telomerase / cytology correlation but were utilized for analysis of whether CSF results were influenced by CSF protein or white cell count. Most (.90%) CSF samples were 3–4 ml in volume and were stored at 48C for 2–72 h before processing for telomerase. Specimens were taken from a variety of patients, including those with primary central nervous system (CNS) neoplasms, metastatic tumors to the CNS, carcinomas without known metastatic involvement of the CNS, and leukemias or lymphomas (see Table 1). In addition, a wide variety of non-neoplastic conditions, including inflammatory diseases, viral syndromes, demyelinating disease and neuropathies were represented (selected cases listed in Table 2). The majority of CSF specimens were consecutively collected and, hence, depicted the range of disorders encountered in common clinical practice for which CSF is available. No single malignant or non-malignant disease was overly represented. A total of 281 CSF specimens from 165 patients were included in the study. These included 50 CSF specimens from patients with biopsy, autopsy and / or cytology evidence of meningeal tumor cells (meningeal carcinomatosis), 26 CSF specimens from patients with documented primary or metastatic parenchymal CNS malignancies but no meningeal involvement, 71 CSF specimens from patients with biopsy proven non-CNS malignancies. The remaining 134 CSF specimens were from patients with no known history of malignant disease. Patient-specific data, including CSF parameters (protein count, white blood cell count, and differential), and cytologic, histologic and clinical diagnoses, were ascertained from the files of the Departments of the Pathology and Neurosurgery at UCHSC, as well as from Medical Records when necessary. The presence or absence of gross blood contamination was noted, although actual red blood cell counts were not undertaken. CSF values were recorded from the same sample that was analyzed for telomerase expression whenever possible. When the CSF specimen drawn at the same time could not be clearly identified, the protein and white blood cell count were taken from samples drawn within three days of the CSF sample analyzed for telomerase expression. All CSF samples in the study were evaluated by cytospin preparations for cytologic examination within 2–72 h post-collection. Most were examined within 24 h; 72-h times represent specimens drawn over the weekend that were examined on the next business day. Hemocytometer evaluation was utilized for differential counts.
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
126
Table 1 Correlation of CSF parameters, clinical and cytological diagnoses with telomerase expression Log number
Patient information
Clinical diagnosis
Cases with diagnostic malignancy involving CNS and positive telomerase expression 141a M/31 HIV1, acute lymphocytic leukemia b c d 152a F/49 Periventricular dissemination, anaplastic oligodendroglioma b (positive by MRI) c d e f g h i j k l m n o p q 161a M/59 Meningeal carcinomatosis, lung adenocarcinoma 246a F/59 Meningeal carcinomatosis, lung adenocarcinoma b c d 256a M/76 Meningeal carcinomatosis, lung adenocarcinoma 266a F/30 Meningeal carcinomatosis, melanoma b c d e 278a M/68 Meningeal carcinomatosis, transitional cell carcinoma b c d e f g h i j 308a M/24 Meningeal dissemination, glioblastoma multiforme b (positive by MRI) c d e 322a F/62 Meningeal carcinomatosis, lung adenocarcinoma (biopsy-proven meningeal involvement) 326a M/62 Cerebellar lesion (autopsy-proven meningeal, dural and intravascular CNS lymphoma) 328a F/35 Metastatic breast carcinoma, 1MRI for metastasis with meningeal extension (biopsy-proven meningeal/dural metastasis) Cases with MRI1 lesions and positive telomerase expression 12a F/74 Gastroesophogeal mass carcinoma vs. lymphoma (not biopsied), b (positive by MRI for diffuse, extensive meningeal disease) c
CSF WBC 10 6 /l
Cytologic diagnosis
Telomerase expression
Differential %
Protein mg/dl
ND ND 0 0 8 8 1 1 1 1 1 1 4 8 1 1 8 8 8 8 8 ND 143 ND 28 12 13 210 210 210 210 3 ND 4 4 10 6 3 4 3 9 9 71 113 94 ND ND 36
ND ND 0 0 90 90 99 99 99 99 99 99 100 90 99 99 99 99 99 99 93 ND 99 ND ND 99 96 42 42 42 42 ND ND 13 9 ND ND ND 40 75 ND ND 91 90 99 ND ND 87
ND ND 2 14 276 276 67 67 67 67 67 67 276 276 67 67 72 1220 1220 1220 470 ND 135 ND 59 52 84 140 140 140 140 22 ND 8 9 10 13 9 5 3 8 8 536 296 ND ND ND 73
Atyp Pos Neg Unsat Neg Neg Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Neg Susp Susp Susp Susp Susp Pos Pos Atyp Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Susp Atyp Pos Pos Atyp Neg Atyp Susp Susp Neg
2 2 11 2 2 2 2 2 2 2 2 2 2 2 2 2 2 21 21 21 2 11 2 21 2 2 11 2 21 2 2 21 11 21 2 2 21 11 11 2 11 2 21 2 2 11 21 11
10
100
ND
Neg
11
2
99
34
Neg
11
580 580 1270
5 5 7
550 550 550
Neg Neg Neg
11 2 2
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Table 1. (Continued) Log number
Patient information
Clinical diagnosis
44a b 212a b c d
M/59
Systemic sarcoidosis, brain abscess (by biopsy)
M/52
Eighth nerve lesion (not biopsied)
CSF
Cytologic diagnosis
Telomerase expression
35 35 116 116 116 81
Neg Neg Atyp Atyp Atyp Neg
11 2 21 21 21 2
Cases with systemic extracranial malignancy but no documented CNS involvement with positive telomerase expression 8a M/22 Hodgkin’s disease, possible bacterial meningitis 7 55 b ND ND 103a F/38 Breast carcinoma 2 42 104a F/53 Acute promyelocytic leukemia 2 27 b 11 31 c 11 31 113a F/47 Metastatic fallopian tube adenocarcinoma, AIDP 1 80 173a M/3l HIV1, lymphoma 2 100 b 6 98 179a F/59 Metastatic adenocarcinoma, unknown primary 2 ND 198a M/52 Acute myelocytic leukemia 10 93 203a M/50 Acute myelocytic leukemia 1 98 b 0 0 c 1 98 d ND ND e ND ND f ND ND g ND ND h ND ND i 1460 6 j 2140 13 258a F/43 Metastatic melanoma 2 100 306a F/53 Chronic lymphocytic leukemia 11 100 b 11 100 329a F/59 Metastatic breast carcinoma 12 31
ND ND 36 51 ND ND 60 29 109 43 143 ND ND 140 ND ND ND ND ND 105 266 31 103 103 95
Neg Unsat Neg Atyp Neg Neg Unsat Neg Atyp Neg Unsat Unsat Unsat Neg Unsat Neg Unsat Neg Neg Neg Unsat Neg Atyp Atyp Unsat
11 2 21 11 21 21 21 2 11 11 21 2 2 2 2 2 2 2 2 2 11 11 11 2 11
Cases with no evidence of malignancy with positive telomerase expression 7a F/63 Dementia b c 10a F/30 Headache b 11a F/78 Diabetes mellitus, mental status changes b 26a M/25 Mental status chances b c d 28a F/34 Questionable viral meningitis b 42a F/35 Ataxia b b 105a F/22 Headache 114a M/5 mo Not determined b 122a M/39 HIV1 b 132a F/64 Mental status changes b 138a M/75 Intracranial hematoma, mental status changes 200a M/18 Questionable viral meningitis
31 31 31 28 28 ND ND 46 46 82 82 20 20 27 27 35 31 26 26 27 27 40 40 111 83
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
11 2 2 21 2 2 11 2 2 11 2 2 11 2 11 2 21 2 21 11 2 2 11 11 21
WBC 10 6 /l 8 8 230 230 230 127
0 0 0 6 6 ND ND 12 12 13 13 6 6 2 2 8 0 0 0 2 2 4 4 20 870
Differential % 99 99 97 97 97 95
0 0 0 100 100 ND ND 85 85 98 98 46 46 100 100 99 0 0 0 100 100 73 73 66 99
Protein mg/dl
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
128 Table 1. (Continued) Log number
Patient information
Clinical diagnosis
CSF WBC 10 6 /l
Differential %
Protein mg/dl
269a b 279a 314a 323a
F/39
Hydrocephalus
F/69 F/32 F/66
Demyelinating disease Chronic meningitis Cranial neuropathy
37 6 4 111 77
92 69 99 98 98
21 29 89 67 57
Cytologic diagnosis
Telomerase expression
Atyp Unsat Atyp Neg Neg
21 11 21 21 11
Telomerase expression: 2, no telomerase expression; 11, no telomerase expression on overnight exposure but positive telomerase expression on exposure for one week; 21, telomerase expression on overnight exposure and after exposure for one week. 1MRI, positive magnetic resonance imaging findings; AIDP, acute inflammatory demyelinating polyneuropathy; Atyp, atypical cells present; CSF, cerebrospinal fluid; F, female; HIV1, human immunodeficiency virus; M, male; mo, months; ND, no data available; Neg, negative for malignancy; Pos, positive for malignancy; Susp, suspicious for malignancy; Unsat, unsatisfactory for diagnosis; WBC, white blood cell count.
2.2. Specimens
was loaded into each reaction for analysis of telomerase expression.
CSF specimens were centrifuged and rinsed with wash buffer (10 mM HEPES–KOH, pH 7.5, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM dithiothreitol), pelleted by centrifugation at 90003g for 10 min at 48C, and the dry pellets were stored at 2708C. Each CSF cell pellet was resuspended in 10 ml of lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM b-mercaptoethanol, 0.5% CHAPS, 10% glycerol) containing 40 units / ml of RNase inhibitor (Ambion, Austin, TX, USA) and agitated on ice for 30 min in an orbital shaker. A 5-ml volume of the resulting cell lysates
2.3. Telomeric repeat amplification protocol The TRAP assay was performed on the cell lysates, standardized to volume, according to Kim et al. [23]. Determination of the protein concentration of the cell lysates was not attempted, to maximize the volume of lysate that could be included in the TRAP assay. Coamplification of an internal control (internal telomerase amplification standard) [36] was not performed, in order to maximize the theoretical sensitivity of the assay. This was
Table 2 Selected patients with concordant negative telomerase expression / negative cytology results Log number
Patient information
Clinical diagnosis
CSF WBC 10 6 / l
Differential %
Protein mg / dl
1a 166a 169a 188a 199a 216a 222a 226a 229a 234a 239a 240a 243a 245a 247a 255a 262a 263a 265a 274a 289a 291a 300a 302a
F / 60 M / 47 M / 34 M / 34 F / 48 M / 42 M / 26 F / 42 M / 63 M / 49 M / 40 M / 46 M / 58 M / 50 F / 25 F / 63 F / 35 F / 36 M / 44 M / 37 M / 62 F / 53 M / 56 F / 26
Primary central nervous system lymphoma Acute myelocytic leukemia HIV1, acute demyelination Lymphoma, systemic Medulloblastoma Brain cyst Questionable demyelination Questionable multiple sclerosis, 1 MRI Glioblastoma multiforme, spinal cord Transverse myelitis, rheumatoid arthritis HIV1, lymphoma HIV1, questionable promyelocytic leukemia Neuropathy Acute myelomonocytic leukemia Acute lymphocytic leukemia Acute myelocytic leukemia, meningitis Hydrocephalus Fever Craniopharngioma Cranial neuropathy Ependymoma Lymphoma, systemic Probable multiple sclerosis Seventh nerve palsy
9 0 2 0 0 1 0 38 2 6 ND 0 ND 0 1 7 127 2 0 2 4 1 7 7
95 0 100 0 0 ND 0 99 ND 100 ND 0 ND 0 83 99 99 72 0 ND 31 80 92 100
67 16 32 29 12 186 19 42 164 31 ND 50 ND 16 57 65 166 15 65 21 80 27 81 ND
See Table 1 footnote for abbreviations.
Cytologic diagnosis
Telomerase expression
Neg Neg Neg Unsat Neg Neg Neg Neg Neg Neg Neg Neg Neg Unsat Unsat Neg Neg Unsat Neg Neg Neg Neg Neg Unsat
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
done since the semi-competitive co-amplification of the internal control could slightly decrease the sensitivity of the assay. Positive controls included HeLa cells (a cervical carcinoma cell line, American Type Culture Collection, Rockville, MD, USA), and telomerase-positive cells (10 4 cell equivalents) provided by ONCOR (Gaithersburg, MD, USA). Negative controls included reactions containing lysis buffer. The polymerase chain reaction (PCR) products were electrophoresed on nondenaturing 12% polyacrylamide gels in 0.53 TBE buffer, pH 8.4 (0.045 M Tris–borate, 0.001 M EDTA) for 2 h at 2000 V. The gels were then dried for 1 h with heat under vacuum and were exposed to Kodak X-OMAT AR film overnight (15–20 h) and then again for seven days at 2708C. Two reviewers (BKD and KRS) scored films for telomerase expression in a blinded fashion, without knowledge of the corresponding cytologic results or CSF parameters. Positive telomerase expression was defined as amplification of five or more discrete bands including the 40, 46, 52, 58 and 64 base pair bands after a seven day exposure. Amplification of the smallest molecular weight, 40 base pair, band alone could represent a primer–dimer artifact and, thus, amplification of the 40 base pair band without amplification of the 46, 52, 58 and 64 base pair bands was not included in the definition of a positive specimen. Telomerase expression was scored as negative (2), positive only after a seven day exposure (11), or positive after an overnight exposure (12). After scoring the telomerase expression results from the films and correlating these results with cytologic diagnosis (see below), patients with discordant cytology / histology / CSF parameters and TRAP results had a review of their medical records in an attempt to explain the discordance.
2.4. Quantitation of sensitivity of telomeric repeat amplification protocol Titration studies were conducted to determine the number of malignant cells that were detectable by the TRAP assay utilizing HeLa cells. Positive controls were standardized to 5310 4 cells / ml, from which dilutions were taken in order to assay 10, 100 and 1000 cells for telomerase expression. As few as ten cells could be detected easily on the gel using a seven day exposure (Fig. 1).
2.5. Statistical analysis The sensitivity and specificity of telomerase expression versus cytologic diagnosis were performed on 225 specimens. The remaining 56 specimens were not used due to the inconclusive cytologic diagnosis of either ‘unsatisfactory’ or ‘atypical’. The statistical analysis used to analyze the relationship between telomerase expression and CSF protein levels utilized 230 specimens. For the remaining 51 specimens, the clinician had not ordered protein data on
129
Fig. 1. TRAP assay polyacrylamide gel electrophoresis of PCR products. Note the characteristic six base-pair ladders that are diagnostic of positive telomerase expression; this sensitive assay gives positive results with preparations containing as few as ten HeLa cells, a well-characterized cervical carcinoma cell line (far left lane). Telomerase expression in cell preparations of 100 (middle lane) or 1000 (far right lane) HeLa cells is shown to result in proportionally stronger bands.
the specimen. The statistical analysis comparing white blood cell (WBC) count with telomerase expression included 245 specimens that had WBC counts. For the remaining 36 specimens, the clinician had not ordered WBC counts on the specimen. The statistical significance of telomerase expression relative to the determination of final patient diagnosis, protein counts, white blood cell counts and days of storage was determined using the generalized estimating equation (GEE) for CSF specimen data [27].
3. Results Table 1 shows the correlation of telomerase expression with CSF parameters and clinical and cytological diagnoses. Only patients with at least one positive telomerase sample are listed in this table. Specimens are grouped by patient, with several patients having multiple CSF specimens. Results are organized by patient categories, including patients with diagnostic malignancies involving the CNS, those with radiographic abnormalities and clinical pictures that were strongly suggestive of malignancies involving the CNS but who were not biopsied, patients with malignancies that were not known to involve the CNS, and patients with no evidence of malignancy. Table 2 lists selected patients with inflammatory diseases, de-
130
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myelinating diseases, neuropathies or viral syndromes who had negative telomerase results. Table 2 represents a subset of our telomerase-negative patients, selected to illustrate the variety of neurologic disorders included in our study. In addition, Table 2 shows that such disorders usually do not result in false positive telomerase expression. Fig. 2 illustrates the results of telomerase analysis in representative cases that were positive for telomerase expression. The detection of telomerase expression showed a summary relationship with cytologic diagnosis (P50.001). When CSF samples with a definitive cytologic diagnosis were analyzed individually, i.e. by specimen, correlation of the original cytologic diagnosis with the results of analysis of telomerase expression showed a specificity of 87% and a sensitivity of 60% for telomerase detection of malignant disease. Subsequent biopsy or autopsy studies, however, clarified discordant, positive telomerase / negative cytology results in three patients. Two patients with positive telomerase results but negative cytology had malignant meningeal (specimen 322a) and meningeal / dural (specimen 328a) involvement, which was identified on sub-
Fig. 2. TRAP assay from several patients in our study with meningeal carcinomatosis and both positive cytology and positive telomerase results in their CSF samples. Positive telomerase expression is defined as identifying at least the four bottom six base-pair repeats. Strongly positive signals are seen in specimens from a patient with disseminated anaplastic oligodendroglioma (lanes 152 n, o, p), a patient with disseminated glioblastoma multiforme (lanes 308 a, d, e) and in a patient with chronic meningitis of unknown etiology (lane 314a). Weaker, but still clearly positive, signals are seen in a patient with meningeal carcinomatosis from transitional cell carcinoma of the bladder (lanes 278 f, g). Remaining lanes on the gel show either completely negative signal or non-specific (irregularly migrating) band patterns that are lacking one or more of the four bottom bands in the characteristic six base-pair ladder (278h, 300a, 302a, 303a, 304a, 307a, 309a, 311a, 312a and 313a).
sequent brain biopsies. A third patient was found to have CNS lymphoma with intravascular, focal meningeal and dural involvement at autopsy (specimen 326a), underscoring the superiority of the telomerase result over the cytology result in these three patients. Six more specimens from which positive telomerase expression / negative cytology results were obtained were possibly explainable by visible, gross peripheral blood contamination of the CSF. These six specimens (103a; 104a, b, c; 198a and 329) were taken from patients with acute leukemias and widely metastatic breast carcinomas, who may have had circulating tumor cells that may have accounted for false positive telomerase results when peripheral blood contaminated the CSF. By contrast, 25 of 167 of our large, concordant, negative telomerase expression / negative cytological group also had blood contamination of CSF, but none of these patients had underlying malignancies. Therefore, it appeared that peripheral blood contamination might have spuriously affected the telomerase assay in the setting of underlying malignancies. However, since both acute leukemias or metastatic breast cancers have a known propensity to involve meninges, we cannot completely rule out the possibility that these six specimens had small numbers of tumor cells in their CSF specimens that were undetectable by cytology but detectable by telomerase expression. Since this issue was also unresolved following medical chart review, we chose to eliminate these six specimens from further analysis. After accounting for the three patients in whom subsequent pathology studies showed meningeal involvement despite negative cytology and excluding these six cancer patients with peripheral blood contamination of their CSF, an adjusted specificity of 90% and a sensitivity of 64% were achieved for the detection of CSF tumor cells by telomerase expression (see Table 3). An even higher true detection rate of telomerase expression for CSF malignant cells probably exists. Ten of our discordant specimens came from patients with documented meningeal carcinomatosis. At least one CSF sample from each patient had shown concordant, positive telomerase / positive cytology results, but other CSF specimens taken from approximately the same time period had yielded discordant, negative telomerase / positive cytology findings. Had we calculated our sensitivity by patient, and not by CSF specimen, and considered any combination of positive telomerase and positive cytology results for a single patient to be concordant regardless of whether they came from the identical CSF sample, our sensitivity would have been near 100%. For our statistical analysis, however, we did not adjust the sensitivity and specificity for these latter ten specimens. CSF protein levels were also recorded, with 19 of 167 specimens from our large concordant negative telomerase expression / negative cytological group having CSF protein levels greater than 100 mg / dl [representing at least a two-fold elevation over normal (15–45 mg / dl)]. Only
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131
Table 3 Final diagnosis versus TRAP results on CSF specimens Positive / probable malignancy
Negative malignancy
Total number of CSF specimens analyzed
Positive for telomerase expression
18 a
20
38
Negative for telomerase expression
10 b
177
187
Total CSF specimens analyzed
28
197
225
All results based on cytology correlation except as noted below. a Includes three specimens adjusted to this column after radiologic and pathologic correlation showed meningeal involvement by tumor. b All ten CSF specimens in this category are taken from patients with known meningeal carcinomatosis; patients have other CSF specimens with concordant positive telomerase / positive cytology results that are included in the group of 18 specimens above. Sensitivity, 64%; specificity, 90%. 1. The sensitivity and specificity of telomerase expression versus cytologic diagnosis were performed on 225 specimens. The remaining 56 specimens were not used due to inconclusive cytologic diagnosis of either ‘unsatisfactory’ or ‘atypical’. 2. The statistical analysis used to analyze the relationship between telomerase expression and CSF protein levels utilized 230 specimens. For the remaining 51 specimens, the clinician had not ordered protein data on the specimen. 3. The statistical analysis comparing WBC count with telomerase expression included 245 specimens that had WBC counts. For the remaining 36 specimens, the clinician had not ordered WBC counts on the specimen.
three of 19 in our discordant positive telomerase expression / negative cytological group had these elevated levels. CSF protein levels showed no statistical correlation with the detection of telomerase expression (P50.321). Finally, we addressed the possibility that high CSF white counts, especially high CSF lymphocyte counts, as assessed from the differential count of lymphocytes and neutrophils, could contribute to spurious positive telomerase results. There was no statistical correlation between the CSF mononuclear cell count and the detection of telomerase activity (P50.196). However, the group of patients in whom positive telomerase / negative cytology results were obtained but were otherwise unexplained, included several with diseases that were likely to have activated mononuclear cells in CSF. These unexplained positive telomerase / negative cytology cases included case 323a (cranial neuropathy), case 44a (systemic sarcoidosis and a brain abscess by biopsy), case 28a and case 114a (possible viral meningitis but no CSF white count elevations), and case 113a (acute inflammatory demyelinating polyneuropathy).
4. Discussion Standard cytologic preparations of CSF specimens, processed by cytocentrifuging and staining with Papinicolou stain, yield an overall sensitivity of only about 15% in primary cerebral malignancies and only 20% in metastatic cerebral tumors [2,13]. Often only ‘suspicious’ or ‘atypical’ diagnoses can be rendered, and unsatisfactory specimens are common in the everyday practice of pathology. The sensitivity of cytologic examination of the CSF for the diagnosis of malignancy is dependent on a variety of
factors, including tumor type, anatomic location and how readily malignant cells exfoliate into the CSF. A positive diagnosis is most likely to occur in cases with diffuse infiltration of the leptomeninges by malignant cells with poor cell-to-cell cohesion. By contrast, tumors that are located deep within the parenchyma of the brain, or those that involve the extradural space but do not involve the leptomeninges, are unlikely to shed tumor cells and, thus, are unlikely to be detected by microscopic evaluation of the CSF [3,13]. Except for actual brain biopsy, the only other way to document tumor cell involvement of leptomeninges pre-mortem is by sensitive radiographic studies such as magnetic resonance imaging. Given the relatively low sensitivity and specificity of traditional cytologic examination of CSF, identifying a molecular cancer marker in CSF that might enhance the detection of tumor cells would be welcomed. Telomerase has proven to be a nearly ubiquitous marker for cancers of many different types and from many different sites, especially late-stage malignancies [4,5,7,17–19,23,33]. Previous work from our laboratory has demonstrated the high percentage of telomerase positivity in primary cerebral malignancies [10,11] and neoplasms metastatic to the brain [12], laying the groundwork for exploring its expression for detection of tumor cells in CSF. The fact that CSF is one of the most difficult cytologic fluids to analyze made the use of an objective, molecular assay, such as that for telomerase, especially attractive. In order to simulate an actual clinical setting, we utilized virtually all of the CSF samples available to us over the 15-month study period. The specimens that we analyzed represented residual fluid following the performance of the cytologic, cell count and / or protein CSF analysis ordered
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by the patient’s physician. CSF samples were drawn for the purpose of patient care and not specifically for our study. In order to accurately define the sensitivity and specificity of the telomerase assay, we applied strict criteria to determine which of the 281 samples could be included in the final analyses. Cases with an inconclusive cytologic diagnosis (‘unsatisfactory’ or ‘atypical’) were not included in the calculation of sensitivity and specificity. Utilizing the 225 specimens with a definitive cytologic diagnosis, specificity was 87% and the sensitivity was 60% for the correlation of telomerase expression with cytologic positivity in individual CSF specimens. After adjusting for three patients with subsequent brain biopsy or autopsy which showed meningeal involvement and excluding six patients with leukemia or widely metastatic carcinoma and known peripheral blood contamination of CSF, an adjusted specificity of 90% and a sensitivity of 64% were achieved (see Table 3). From our statistical analyses, neither elevated CSF protein levels nor the CSF white blood cell counts adversely affected sensitivity or specificity for telomerase detection. Positive telomerase results were found in several patients without known CNS or systemic malignancies. Review of medical records of these patients corroborated the clinical impression that they were unlikely to harbor occult malignancies. Several of these patients had been clinically diagnosed with viral syndromes or demyelinating diseases (see Tables 1 and 2). These few patients with discordant positive telomerase / negative cytology results represented a small proportion (9%) of our 281 cases (see Table 3). Many of our patients with biopsy- or cytology-proven meningeal carcinomatosis provided multiple CSF specimens (see Table 1). In a few of these samples, positive cytology / negative telomerase results on one or more of the CSF specimens were noted. This finding may represent varying numbers of tumor cells in the portion of the CSF specimen used for cytology versus the portion used for the telomerase assay. We cannot exclude the possibility, however, that false negative results in some of our CSF samples may be due to degradation of telomerase activity in what should have been a positive sample. At present, there is no internal control system to evaluate for the stability of telomerase activity. The development of such a system will be crucial for the development of telomerase expression as a diagnostic adjunct in either tissue or body fluids. It is useful to note, however, that we found no apparent loss of activity in telomerase expression with storage times in the refrigerator of up to 72 h. In this study, we chose to use the original TRAP protocol [23] rather than the modified quantitation protocol [36], since the latter contains an internal standard that could slightly inhibit the co-amplification of the telomerase extension product. Use of the original TRAP protocol in our laboratory allows reliable and reproducible detection of telomerase expression in as few as ten carcinoma cells by
titration assay (see Fig. 1). Also, the finding of a four-fold logarithmic variation in telomerase expression between different types of brain metastases (Part 1, companion paper), some of which showed weak expression, suggested that the most sensitive assay method for telomerase should be utilized for this CSF study. Studies in our laboratory are underway to determine if co-amplification of an internal standard on CSF shows the same utility as the original TRAP protocol. Based on this CSF study, our companion Part 1 study, and our previous experience with both protocols, we would predict that the protocol using coamplification of an internal standard would yield less sensitivity than the original protocol for the detection of telomerase expression from small numbers of tumor cells metastatic to CSF. This hypothesis is strengthened by the finding in this study that, even with the use of the original sensitive TRAP protocol [23], some of our CSF specimens with positive cytology showed only weak telomerase expression that was visible after a one-week gel exposure. We envision the use of CSF telomerase analysis as an adjunct to, not as a replacement for, traditional CSF cytologic methods. In clinical practice, the use of the two methods together may reassure the diagnosis of malignancy when a positive telomerase result is obtained in conjunction with a ‘suspicious’ or ‘atypical’ traditional cytology result. A positive telomerase result on CSF that is ‘unsatisfactory’ or negative by traditional cytology might mandate analysis of an additional CSF specimen. For neoplastic diseases, our study suggests that the detection of telomerase may be more sensitive than traditional cytology, a finding in accordance with the few other non-CSF telomerase / cytology correlation reports in the literature [6,9,14,20,24,25,35,37–40]. Studies conducted on the utility of telomerase as an adjunct to cytology in extracranial malignancies have had the advantage of readily accessible biopsy or resection material with which to compare results. This made the calculation of sensitivity of telomerase for the detection of malignant cells much easier than in our CSF study. Unlike patients with cytologically abnormal cervical smears, who all eventually come to biopsy or tissue resection, patients with CNS meningeal disease are usually not biopsied once diagnosis is made from cytology. In our study, CNS tissue with which to compare telomerase results was not always available from patients. Our ‘gold standard’ with which to compare telomerase results was the traditional CSF cytologies on these patients, a test with known low sensitivity [2,13]. Hence, in this CSF telomerase study, we attempted to supplement the study by undertaking clinico-pathologic correlation from medical chart reviews to determine the sensitivity and specificity of the assay. Our adjusted sensitivity of 64% and specificity of 90% are comparable with those reported for cytology / telomerase correlation from extracranial malignancies [6,20,24,25,35,37–40]. Three studies on urine or bladder washings for the detection of transitional cell carcinoma performed on 23,
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
109 and 87 specimens, showed sensitivities of 95.7, 62 and 67.8% and specificities of 100, 96.4 and 100%, respectively [24,37,39]. Colonic luminal washings conducted for the detection of colonic adenocarcinoma on 24 patients showed a sensitivity of 60% and a specificity of 100%. There was no evidence of telomerase expression in nine of 24 washings taken from inflammatory bowel disease patients, a possible confounding, non-neoplastic inflammatory condition [39]. Sixty-six oral rinses from head and neck squamous cell carcinoma patients analyzed for telomerase expression showed a relatively low sensitivity, but a high specificity of 95% [6]. Likewise, fine needle aspirates of breast lesions have generally exhibited a moderate sensitivity and a high specificity for the detection of malignant cells [35]. Telomerase detection of carcinoma in cervical cytology specimens in two studies demonstrated a high level of sensitivity for the detection of high grade squamous intraepithelial lesions and squamous cell carcinoma [25,40]. However, a third study was less encouraging and showed telomerase positivity in only one of 22 cases of high-grade squamous intraepithelial lesions [14]. The most promising telomerase / cytology correlation study to date is that of Hiyama et al. [20]. In their study, twelve brush samples from pancreatic duct for the detection of ductal adenocarcinoma yielded both a sensitivity and specificity of 100%. In summary, telomerase activity was detected in a high proportion of CSF specimens from patients with malignant involvement of the meninges as proven by definitive CSF cytology or subsequent biopsies. Telomerase was usually not detected in patients with negative CSF cytology. Further studies and refinement of the technique are warranted to see if the TRAP assay for telomerase can be adapted to the clinical practice of CSF cytology on a widespread basis.
Acknowledgements
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15] [16]
[17] [18]
Drs B.K. Kleinschmidt-DeMasters and K.R. Shroyer are members of the Colorado Cancer Center. Dr A. Laurie Shroyer was funded and sponsored, in part, by the Department of Veterans Affairs Office of Performance and Program for Cooperative Studies. The authors gratefully acknowledge the assistance of Ms Monique Paques in the Clinical Laboratory, Ms Mary Wilfawn in Medical Records, and Ms Ginger Woodward and Ms Velma Parker for assistance with the preparation of the manuscript.
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[26] Nakatani K, Yoshimi N, Mori H, Yoshimura S, Sakai H, Shinoda J, et al. The significant role of telomerase activity in human brain tumors. Cancer 1997;80:471–6. [27] Nelder JA, Wedderbum RWM. Generalized linear models. J R Stat Soc 1972;135:370–84. [28] Nouso K, Urabe Y, Higashi T, et al. Telomerase as a tool for the differential diagnosis of human hepatocellular carcinoma. Cancer 1996;78:232–6. [29] Parkinson EK. Do telomerase antagonists represent a novel anticancer strategy? Br J Cancer 1996;73:1–4. [30] Sharma HW, Maltese JY, Zhu X, Kaiser HE, Narayanan R. Telomeres, telomerase and cancer: Is the magic bullet real? Anticancer Res 1996;16:511–6. [31] Sharma HW, Sokoloski JA, Perez JR, Maltese JY, Sartorelli AC, Stein CA, et al. Differentiation of immortal cells inhibits telomerase activity. Proc Natl Acad Sci USA 1996;92:12343–6. [32] Shay J, Gazdar A. Telomerase in the early detection of cancer. J Clin Pathol 1997;50:106–9. [33] Shroyer KR, Stephens JK, Silverberg SG, Markham N, Shroyer AL, Wilson M, et al. Telomerase expression in normal endometrium, endometrial hyperplasia, and endometrial adenocarcinoma. Int J Gynecol Pathol 1997;16:225–32.
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Part II. Telomerase expression in cerebrospinal fluid specimens as an adjunct to cytologic diagnosis 1 B.K. Kleinschmidt-DeMasters
a ,b ,
*, Lynnette C. Evans a , Mitchell A. Bitter a ,b , A. Laurie Shroyer c , Kenneth R. Shroyer a
a
University of Colorado Health Sciences Center, Department of Pathology, 4200 East Ninth Avenue, Denver, CO 80262, USA University of Colorado Health Sciences Center, Department of Neurology, 4200 East Ninth Avenue, Denver, CO 80262, USA c Department of Cardiac Research, Veterans Affairs Medical Center, 1055 Clermont Street, Denver, CO 80220, USA
b
Received 18 March 1998; received in revised form 25 June 1998; accepted 26 June 1998
Abstract The diagnosis of meningeal carcinomatosis hinges on the cytologic examination of cerebrospinal fluid (CSF), which has a known low sensitivity for the identification of malignant cells. Often only ‘suspicious’ or ‘atypical’ diagnoses can be rendered, and specimens are commonly unsatisfactory for evaluation due to poor morphologic preservation. Telomerase is widely expressed in most brain metastases, medulloblastomas, lymphomas, oligodendrogliomas, and is expressed focally in glioblastomas. Little is known about the level of telomerase expression in these tumors, except for brain metastases, where a four-fold variation in telomerase levels exists. In our laboratory, as few as ten carcinoma cells can be detected by a sensitive polymerase chain reaction-based assay, the telomeric repeat amplification protocol (TRAP), for telomerase, but it was unclear whether varying levels of telomerase expressed by different types of metastases would influence detection. Using the TRAP protocol, we studied 281 CSF samples from a wide variety of patients with neurologic and non-neurologic conditions for telomerase expression. An adjusted specificity of 90% and a sensitivity of 64% were achieved for detection of malignant cells in CSF by telomerase expression. The TRAP assay for telomerase detection may serve as an adjunct to the traditional examination of CSF. Neither previously documented four-fold variation in the levels of telomerase expression in brain metastases, high CSF protein levels nor high white blood cell counts precluded detection of malignant cells in CSF. 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Cerebrospinal fluid; Cytology; Meningeal carcinomatosis; Metastases; Telomerase; Central nervous system; Brain; Prognosis
1. Introduction Telomerase is a ribonucleoprotein enzyme complex that synthesizes G-rich, six base pair sequences onto the ends of chromosomes, known as telomeres [1]. Telomeric reduction signals the cessation of cellular division and may serve as a biological clock to regulate the lifespan of the cell [15,22,30]. Telomerase is down-regulated in cells as they undergo senescence or differentiation, but continues *Corresponding author. Tel.: 1303-315-7298; fax: 1303-315-4792. 1 Presented in abstract form at the 74th annual meeting of the American Association of Neuropathologists, Minneapolis, MN, USA, on June 19 1998, where it was the recipient of the Rubinstein award for the best paper in Neuro-oncology for 1998.
to be expressed at low levels in proliferative tissues, including germ cells, endometrium, basal keratinocytes and lymphoid cells [22,30,31]. High telomerase expression, by contrast, is characteristic of immortalized cells and is found in the vast majority of all cancers [23]. While the grade and stage at which telomerase is expressed differs for each given tumor type and site, high telomerase levels are virtually always found in late-stage malignancies [4,5,7,17,18,21,23,33]. This nearly ubiquitous expression of telomerase in a wide variety of human neoplasms has prompted a plethora of reports addressing telomerase as a universal cancer marker or therapeutic target [4,15,16,28,29,31,32,35, 37,39]. Most studies have concentrated on analyzing tissue
0022-510X / 98 / $ – see front matter 1998 Published by Elsevier Science B.V. All rights reserved. PII: S0022-510X( 98 )00254-8
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
extracts of primary tumors [8,11,17,19,21,26,34], although several have specifically addressed telomerase expression in metastases [16,17,12]. Nakatani et al. [26] found positive telomerase expression in three of three tumors metastatic to the brain. In a study preliminary to the current investigation, our laboratory investigated telomerase expression in 35 brain metastases of different types, correlating telomerase expression levels with prognosis and interval to patient demise [12]. From this previous work, we found that at least 32 of 35 advanced-staged malignancies metastatic to the brain demonstrated positive telomerase expression, albeit at widely varying levels. It was unclear from that study whether the four-fold variation in telomerase levels would influence detection of metastatic cells in cerebrospinal fluid (CSF). Previous work by our group had also shown that primary cerebral malignancies that were likely to disseminate to the meninges, i.e. medulloblastomas [11], oligodendrogliomas and glioblastoma multiformes [10], were all telomerase-positive, at least focally. These findings prompted us to explore the utility of testing CSF for telomerase expression, as a potential adjunct to the diagnosis of tumors with metastases and dissemination to the meninges. Several papers had confirmed the potential for utilizing telomerase detection as an adjunct to the cytologic diagnosis of malignancy for a variety of body fluids, exfoliative cells, or body cavity washings [6,9,14,17,20,24,25,35,37–40]. Most of these studies had shown encouraging results, with good sensitivity and specificity of telomerase expression for detecting cytologically positive tumors. However, in most of these studies, sample numbers have been small, and only modest attempts have been made to explain discrepancies between telomerase expression and cytologic diagnosis. Also, few have included patients with possible confounding inflammatory or other non-neoplastic diseases for comparison. The current study is the first, to our knowledge, that addresses the utility of the telomeric repeat amplification protocol (TRAP) assay for telomerase expression as an adjunct to the cytologic diagnosis of CSF, and is the largest, to date, to study telomerase expression in fluids of any kind. In this study of 281 specimens, we paid particular attention to possible influential parameters, such as time interval between the clinical procedure and processing of the fluid, CSF protein levels and white blood cell counts. We also undertook thorough medical chart review to investigate all the differences between telomerase expression results and cytologic diagnoses. 2. Materials and methods
2.1. Patient information All available CSF specimens were collected from pa-
125
tients seen at the University of Colorado Health Sciences Center (UCHSC) between September 1996 to December 1997 for clinically indicated reasons. No patient had CSF fluid drawn specifically and solely for this study. Two hundred and eighty-one specimens from 165 patients were available for the TRAP analysis. CSF specimens that were unsatisfactory or inconclusive for cytologic diagnoses were not utilized for the final telomerase / cytology correlation but were utilized for analysis of whether CSF results were influenced by CSF protein or white cell count. Most (.90%) CSF samples were 3–4 ml in volume and were stored at 48C for 2–72 h before processing for telomerase. Specimens were taken from a variety of patients, including those with primary central nervous system (CNS) neoplasms, metastatic tumors to the CNS, carcinomas without known metastatic involvement of the CNS, and leukemias or lymphomas (see Table 1). In addition, a wide variety of non-neoplastic conditions, including inflammatory diseases, viral syndromes, demyelinating disease and neuropathies were represented (selected cases listed in Table 2). The majority of CSF specimens were consecutively collected and, hence, depicted the range of disorders encountered in common clinical practice for which CSF is available. No single malignant or non-malignant disease was overly represented. A total of 281 CSF specimens from 165 patients were included in the study. These included 50 CSF specimens from patients with biopsy, autopsy and / or cytology evidence of meningeal tumor cells (meningeal carcinomatosis), 26 CSF specimens from patients with documented primary or metastatic parenchymal CNS malignancies but no meningeal involvement, 71 CSF specimens from patients with biopsy proven non-CNS malignancies. The remaining 134 CSF specimens were from patients with no known history of malignant disease. Patient-specific data, including CSF parameters (protein count, white blood cell count, and differential), and cytologic, histologic and clinical diagnoses, were ascertained from the files of the Departments of the Pathology and Neurosurgery at UCHSC, as well as from Medical Records when necessary. The presence or absence of gross blood contamination was noted, although actual red blood cell counts were not undertaken. CSF values were recorded from the same sample that was analyzed for telomerase expression whenever possible. When the CSF specimen drawn at the same time could not be clearly identified, the protein and white blood cell count were taken from samples drawn within three days of the CSF sample analyzed for telomerase expression. All CSF samples in the study were evaluated by cytospin preparations for cytologic examination within 2–72 h post-collection. Most were examined within 24 h; 72-h times represent specimens drawn over the weekend that were examined on the next business day. Hemocytometer evaluation was utilized for differential counts.
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
126
Table 1 Correlation of CSF parameters, clinical and cytological diagnoses with telomerase expression Log number
Patient information
Clinical diagnosis
Cases with diagnostic malignancy involving CNS and positive telomerase expression 141a M/31 HIV1, acute lymphocytic leukemia b c d 152a F/49 Periventricular dissemination, anaplastic oligodendroglioma b (positive by MRI) c d e f g h i j k l m n o p q 161a M/59 Meningeal carcinomatosis, lung adenocarcinoma 246a F/59 Meningeal carcinomatosis, lung adenocarcinoma b c d 256a M/76 Meningeal carcinomatosis, lung adenocarcinoma 266a F/30 Meningeal carcinomatosis, melanoma b c d e 278a M/68 Meningeal carcinomatosis, transitional cell carcinoma b c d e f g h i j 308a M/24 Meningeal dissemination, glioblastoma multiforme b (positive by MRI) c d e 322a F/62 Meningeal carcinomatosis, lung adenocarcinoma (biopsy-proven meningeal involvement) 326a M/62 Cerebellar lesion (autopsy-proven meningeal, dural and intravascular CNS lymphoma) 328a F/35 Metastatic breast carcinoma, 1MRI for metastasis with meningeal extension (biopsy-proven meningeal/dural metastasis) Cases with MRI1 lesions and positive telomerase expression 12a F/74 Gastroesophogeal mass carcinoma vs. lymphoma (not biopsied), b (positive by MRI for diffuse, extensive meningeal disease) c
CSF WBC 10 6 /l
Cytologic diagnosis
Telomerase expression
Differential %
Protein mg/dl
ND ND 0 0 8 8 1 1 1 1 1 1 4 8 1 1 8 8 8 8 8 ND 143 ND 28 12 13 210 210 210 210 3 ND 4 4 10 6 3 4 3 9 9 71 113 94 ND ND 36
ND ND 0 0 90 90 99 99 99 99 99 99 100 90 99 99 99 99 99 99 93 ND 99 ND ND 99 96 42 42 42 42 ND ND 13 9 ND ND ND 40 75 ND ND 91 90 99 ND ND 87
ND ND 2 14 276 276 67 67 67 67 67 67 276 276 67 67 72 1220 1220 1220 470 ND 135 ND 59 52 84 140 140 140 140 22 ND 8 9 10 13 9 5 3 8 8 536 296 ND ND ND 73
Atyp Pos Neg Unsat Neg Neg Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Atyp Neg Susp Susp Susp Susp Susp Pos Pos Atyp Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Susp Atyp Pos Pos Atyp Neg Atyp Susp Susp Neg
2 2 11 2 2 2 2 2 2 2 2 2 2 2 2 2 2 21 21 21 2 11 2 21 2 2 11 2 21 2 2 21 11 21 2 2 21 11 11 2 11 2 21 2 2 11 21 11
10
100
ND
Neg
11
2
99
34
Neg
11
580 580 1270
5 5 7
550 550 550
Neg Neg Neg
11 2 2
B.K. Kleinschmidt-DeMasters et al. / Journal of the Neurological Sciences 161 (1998) 124 – 134
127
Table 1. (Continued) Log number
Patient information
Clinical diagnosis
44a b 212a b c d
M/59
Systemic sarcoidosis, brain abscess (by biopsy)
M/52
Eighth nerve lesion (not biopsied)
CSF
Cytologic diagnosis
Telomerase expression
35 35 116 116 116 81
Neg Neg Atyp Atyp Atyp Neg
11 2 21 21 21 2
Cases with systemic extracranial malignancy but no documented CNS involvement with positive telomerase expression 8a M/22 Hodgkin’s disease, possible bacterial meningitis 7 55 b ND ND 103a F/38 Breast carcinoma 2 42 104a F/53 Acute promyelocytic leukemia 2 27 b 11 31 c 11 31 113a F/47 Metastatic fallopian tube adenocarcinoma, AIDP 1 80 173a M/3l HIV1, lymphoma 2 100 b 6 98 179a F/59 Metastatic adenocarcinoma, unknown primary 2 ND 198a M/52 Acute myelocytic leukemia 10 93 203a M/50 Acute myelocytic leukemia 1 98 b 0 0 c 1 98 d ND ND e ND ND f ND ND g ND ND h ND ND i 1460 6 j 2140 13 258a F/43 Metastatic melanoma 2 100 306a F/53 Chronic lymphocytic leukemia 11 100 b 11 100 329a F/59 Metastatic breast carcinoma 12 31
ND ND 36 51 ND ND 60 29 109 43 143 ND ND 140 ND ND ND ND ND 105 266 31 103 103 95
Neg Unsat Neg Atyp Neg Neg Unsat Neg Atyp Neg Unsat Unsat Unsat Neg Unsat Neg Unsat Neg Neg Neg Unsat Neg Atyp Atyp Unsat
11 2 21 11 21 21 21 2 11 11 21 2 2 2 2 2 2 2 2 2 11 11 11 2 11
Cases with no evidence of malignancy with positive telomerase expression 7a F/63 Dementia b c 10a F/30 Headache b 11a F/78 Diabetes mellitus, mental status changes b 26a M/25 Mental status chances b c d 28a F/34 Questionable viral meningitis b 42a F/35 Ataxia b b 105a F/22 Headache 114a M/5 mo Not determined b 122a M/39 HIV1 b 132a F/64 Mental status changes b 138a M/75 Intracranial hematoma, mental status changes 200a M/18 Questionable viral meningitis
31 31 31 28 28 ND ND 46 46 82 82 20 20 27 27 35 31 26 26 27 27 40 40 111 83
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
11 2 2 21 2 2 11 2 2 11 2 2 11 2 11 2 21 2 21 11 2 2 11 11 21
WBC 10 6 /l 8 8 230 230 230 127
0 0 0 6 6 ND ND 12 12 13 13 6 6 2 2 8 0 0 0 2 2 4 4 20 870
Differential % 99 99 97 97 97 95
0 0 0 100 100 ND ND 85 85 98 98 46 46 100 100 99 0 0 0 100 100 73 73 66 99
Protein mg/dl
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128 Table 1. (Continued) Log number
Patient information
Clinical diagnosis
CSF WBC 10 6 /l
Differential %
Protein mg/dl
269a b 279a 314a 323a
F/39
Hydrocephalus
F/69 F/32 F/66
Demyelinating disease Chronic meningitis Cranial neuropathy
37 6 4 111 77
92 69 99 98 98
21 29 89 67 57
Cytologic diagnosis
Telomerase expression
Atyp Unsat Atyp Neg Neg
21 11 21 21 11
Telomerase expression: 2, no telomerase expression; 11, no telomerase expression on overnight exposure but positive telomerase expression on exposure for one week; 21, telomerase expression on overnight exposure and after exposure for one week. 1MRI, positive magnetic resonance imaging findings; AIDP, acute inflammatory demyelinating polyneuropathy; Atyp, atypical cells present; CSF, cerebrospinal fluid; F, female; HIV1, human immunodeficiency virus; M, male; mo, months; ND, no data available; Neg, negative for malignancy; Pos, positive for malignancy; Susp, suspicious for malignancy; Unsat, unsatisfactory for diagnosis; WBC, white blood cell count.
2.2. Specimens
was loaded into each reaction for analysis of telomerase expression.
CSF specimens were centrifuged and rinsed with wash buffer (10 mM HEPES–KOH, pH 7.5, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM dithiothreitol), pelleted by centrifugation at 90003g for 10 min at 48C, and the dry pellets were stored at 2708C. Each CSF cell pellet was resuspended in 10 ml of lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM b-mercaptoethanol, 0.5% CHAPS, 10% glycerol) containing 40 units / ml of RNase inhibitor (Ambion, Austin, TX, USA) and agitated on ice for 30 min in an orbital shaker. A 5-ml volume of the resulting cell lysates
2.3. Telomeric repeat amplification protocol The TRAP assay was performed on the cell lysates, standardized to volume, according to Kim et al. [23]. Determination of the protein concentration of the cell lysates was not attempted, to maximize the volume of lysate that could be included in the TRAP assay. Coamplification of an internal control (internal telomerase amplification standard) [36] was not performed, in order to maximize the theoretical sensitivity of the assay. This was
Table 2 Selected patients with concordant negative telomerase expression / negative cytology results Log number
Patient information
Clinical diagnosis
CSF WBC 10 6 / l
Differential %
Protein mg / dl
1a 166a 169a 188a 199a 216a 222a 226a 229a 234a 239a 240a 243a 245a 247a 255a 262a 263a 265a 274a 289a 291a 300a 302a
F / 60 M / 47 M / 34 M / 34 F / 48 M / 42 M / 26 F / 42 M / 63 M / 49 M / 40 M / 46 M / 58 M / 50 F / 25 F / 63 F / 35 F / 36 M / 44 M / 37 M / 62 F / 53 M / 56 F / 26
Primary central nervous system lymphoma Acute myelocytic leukemia HIV1, acute demyelination Lymphoma, systemic Medulloblastoma Brain cyst Questionable demyelination Questionable multiple sclerosis, 1 MRI Glioblastoma multiforme, spinal cord Transverse myelitis, rheumatoid arthritis HIV1, lymphoma HIV1, questionable promyelocytic leukemia Neuropathy Acute myelomonocytic leukemia Acute lymphocytic leukemia Acute myelocytic leukemia, meningitis Hydrocephalus Fever Craniopharngioma Cranial neuropathy Ependymoma Lymphoma, systemic Probable multiple sclerosis Seventh nerve palsy
9 0 2 0 0 1 0 38 2 6 ND 0 ND 0 1 7 127 2 0 2 4 1 7 7
95 0 100 0 0 ND 0 99 ND 100 ND 0 ND 0 83 99 99 72 0 ND 31 80 92 100
67 16 32 29 12 186 19 42 164 31 ND 50 ND 16 57 65 166 15 65 21 80 27 81 ND
See Table 1 footnote for abbreviations.
Cytologic diagnosis
Telomerase expression
Neg Neg Neg Unsat Neg Neg Neg Neg Neg Neg Neg Neg Neg Unsat Unsat Neg Neg Unsat Neg Neg Neg Neg Neg Unsat
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
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done since the semi-competitive co-amplification of the internal control could slightly decrease the sensitivity of the assay. Positive controls included HeLa cells (a cervical carcinoma cell line, American Type Culture Collection, Rockville, MD, USA), and telomerase-positive cells (10 4 cell equivalents) provided by ONCOR (Gaithersburg, MD, USA). Negative controls included reactions containing lysis buffer. The polymerase chain reaction (PCR) products were electrophoresed on nondenaturing 12% polyacrylamide gels in 0.53 TBE buffer, pH 8.4 (0.045 M Tris–borate, 0.001 M EDTA) for 2 h at 2000 V. The gels were then dried for 1 h with heat under vacuum and were exposed to Kodak X-OMAT AR film overnight (15–20 h) and then again for seven days at 2708C. Two reviewers (BKD and KRS) scored films for telomerase expression in a blinded fashion, without knowledge of the corresponding cytologic results or CSF parameters. Positive telomerase expression was defined as amplification of five or more discrete bands including the 40, 46, 52, 58 and 64 base pair bands after a seven day exposure. Amplification of the smallest molecular weight, 40 base pair, band alone could represent a primer–dimer artifact and, thus, amplification of the 40 base pair band without amplification of the 46, 52, 58 and 64 base pair bands was not included in the definition of a positive specimen. Telomerase expression was scored as negative (2), positive only after a seven day exposure (11), or positive after an overnight exposure (12). After scoring the telomerase expression results from the films and correlating these results with cytologic diagnosis (see below), patients with discordant cytology / histology / CSF parameters and TRAP results had a review of their medical records in an attempt to explain the discordance.
2.4. Quantitation of sensitivity of telomeric repeat amplification protocol Titration studies were conducted to determine the number of malignant cells that were detectable by the TRAP assay utilizing HeLa cells. Positive controls were standardized to 5310 4 cells / ml, from which dilutions were taken in order to assay 10, 100 and 1000 cells for telomerase expression. As few as ten cells could be detected easily on the gel using a seven day exposure (Fig. 1).
2.5. Statistical analysis The sensitivity and specificity of telomerase expression versus cytologic diagnosis were performed on 225 specimens. The remaining 56 specimens were not used due to the inconclusive cytologic diagnosis of either ‘unsatisfactory’ or ‘atypical’. The statistical analysis used to analyze the relationship between telomerase expression and CSF protein levels utilized 230 specimens. For the remaining 51 specimens, the clinician had not ordered protein data on
129
Fig. 1. TRAP assay polyacrylamide gel electrophoresis of PCR products. Note the characteristic six base-pair ladders that are diagnostic of positive telomerase expression; this sensitive assay gives positive results with preparations containing as few as ten HeLa cells, a well-characterized cervical carcinoma cell line (far left lane). Telomerase expression in cell preparations of 100 (middle lane) or 1000 (far right lane) HeLa cells is shown to result in proportionally stronger bands.
the specimen. The statistical analysis comparing white blood cell (WBC) count with telomerase expression included 245 specimens that had WBC counts. For the remaining 36 specimens, the clinician had not ordered WBC counts on the specimen. The statistical significance of telomerase expression relative to the determination of final patient diagnosis, protein counts, white blood cell counts and days of storage was determined using the generalized estimating equation (GEE) for CSF specimen data [27].
3. Results Table 1 shows the correlation of telomerase expression with CSF parameters and clinical and cytological diagnoses. Only patients with at least one positive telomerase sample are listed in this table. Specimens are grouped by patient, with several patients having multiple CSF specimens. Results are organized by patient categories, including patients with diagnostic malignancies involving the CNS, those with radiographic abnormalities and clinical pictures that were strongly suggestive of malignancies involving the CNS but who were not biopsied, patients with malignancies that were not known to involve the CNS, and patients with no evidence of malignancy. Table 2 lists selected patients with inflammatory diseases, de-
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myelinating diseases, neuropathies or viral syndromes who had negative telomerase results. Table 2 represents a subset of our telomerase-negative patients, selected to illustrate the variety of neurologic disorders included in our study. In addition, Table 2 shows that such disorders usually do not result in false positive telomerase expression. Fig. 2 illustrates the results of telomerase analysis in representative cases that were positive for telomerase expression. The detection of telomerase expression showed a summary relationship with cytologic diagnosis (P50.001). When CSF samples with a definitive cytologic diagnosis were analyzed individually, i.e. by specimen, correlation of the original cytologic diagnosis with the results of analysis of telomerase expression showed a specificity of 87% and a sensitivity of 60% for telomerase detection of malignant disease. Subsequent biopsy or autopsy studies, however, clarified discordant, positive telomerase / negative cytology results in three patients. Two patients with positive telomerase results but negative cytology had malignant meningeal (specimen 322a) and meningeal / dural (specimen 328a) involvement, which was identified on sub-
Fig. 2. TRAP assay from several patients in our study with meningeal carcinomatosis and both positive cytology and positive telomerase results in their CSF samples. Positive telomerase expression is defined as identifying at least the four bottom six base-pair repeats. Strongly positive signals are seen in specimens from a patient with disseminated anaplastic oligodendroglioma (lanes 152 n, o, p), a patient with disseminated glioblastoma multiforme (lanes 308 a, d, e) and in a patient with chronic meningitis of unknown etiology (lane 314a). Weaker, but still clearly positive, signals are seen in a patient with meningeal carcinomatosis from transitional cell carcinoma of the bladder (lanes 278 f, g). Remaining lanes on the gel show either completely negative signal or non-specific (irregularly migrating) band patterns that are lacking one or more of the four bottom bands in the characteristic six base-pair ladder (278h, 300a, 302a, 303a, 304a, 307a, 309a, 311a, 312a and 313a).
sequent brain biopsies. A third patient was found to have CNS lymphoma with intravascular, focal meningeal and dural involvement at autopsy (specimen 326a), underscoring the superiority of the telomerase result over the cytology result in these three patients. Six more specimens from which positive telomerase expression / negative cytology results were obtained were possibly explainable by visible, gross peripheral blood contamination of the CSF. These six specimens (103a; 104a, b, c; 198a and 329) were taken from patients with acute leukemias and widely metastatic breast carcinomas, who may have had circulating tumor cells that may have accounted for false positive telomerase results when peripheral blood contaminated the CSF. By contrast, 25 of 167 of our large, concordant, negative telomerase expression / negative cytological group also had blood contamination of CSF, but none of these patients had underlying malignancies. Therefore, it appeared that peripheral blood contamination might have spuriously affected the telomerase assay in the setting of underlying malignancies. However, since both acute leukemias or metastatic breast cancers have a known propensity to involve meninges, we cannot completely rule out the possibility that these six specimens had small numbers of tumor cells in their CSF specimens that were undetectable by cytology but detectable by telomerase expression. Since this issue was also unresolved following medical chart review, we chose to eliminate these six specimens from further analysis. After accounting for the three patients in whom subsequent pathology studies showed meningeal involvement despite negative cytology and excluding these six cancer patients with peripheral blood contamination of their CSF, an adjusted specificity of 90% and a sensitivity of 64% were achieved for the detection of CSF tumor cells by telomerase expression (see Table 3). An even higher true detection rate of telomerase expression for CSF malignant cells probably exists. Ten of our discordant specimens came from patients with documented meningeal carcinomatosis. At least one CSF sample from each patient had shown concordant, positive telomerase / positive cytology results, but other CSF specimens taken from approximately the same time period had yielded discordant, negative telomerase / positive cytology findings. Had we calculated our sensitivity by patient, and not by CSF specimen, and considered any combination of positive telomerase and positive cytology results for a single patient to be concordant regardless of whether they came from the identical CSF sample, our sensitivity would have been near 100%. For our statistical analysis, however, we did not adjust the sensitivity and specificity for these latter ten specimens. CSF protein levels were also recorded, with 19 of 167 specimens from our large concordant negative telomerase expression / negative cytological group having CSF protein levels greater than 100 mg / dl [representing at least a two-fold elevation over normal (15–45 mg / dl)]. Only
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Table 3 Final diagnosis versus TRAP results on CSF specimens Positive / probable malignancy
Negative malignancy
Total number of CSF specimens analyzed
Positive for telomerase expression
18 a
20
38
Negative for telomerase expression
10 b
177
187
Total CSF specimens analyzed
28
197
225
All results based on cytology correlation except as noted below. a Includes three specimens adjusted to this column after radiologic and pathologic correlation showed meningeal involvement by tumor. b All ten CSF specimens in this category are taken from patients with known meningeal carcinomatosis; patients have other CSF specimens with concordant positive telomerase / positive cytology results that are included in the group of 18 specimens above. Sensitivity, 64%; specificity, 90%. 1. The sensitivity and specificity of telomerase expression versus cytologic diagnosis were performed on 225 specimens. The remaining 56 specimens were not used due to inconclusive cytologic diagnosis of either ‘unsatisfactory’ or ‘atypical’. 2. The statistical analysis used to analyze the relationship between telomerase expression and CSF protein levels utilized 230 specimens. For the remaining 51 specimens, the clinician had not ordered protein data on the specimen. 3. The statistical analysis comparing WBC count with telomerase expression included 245 specimens that had WBC counts. For the remaining 36 specimens, the clinician had not ordered WBC counts on the specimen.
three of 19 in our discordant positive telomerase expression / negative cytological group had these elevated levels. CSF protein levels showed no statistical correlation with the detection of telomerase expression (P50.321). Finally, we addressed the possibility that high CSF white counts, especially high CSF lymphocyte counts, as assessed from the differential count of lymphocytes and neutrophils, could contribute to spurious positive telomerase results. There was no statistical correlation between the CSF mononuclear cell count and the detection of telomerase activity (P50.196). However, the group of patients in whom positive telomerase / negative cytology results were obtained but were otherwise unexplained, included several with diseases that were likely to have activated mononuclear cells in CSF. These unexplained positive telomerase / negative cytology cases included case 323a (cranial neuropathy), case 44a (systemic sarcoidosis and a brain abscess by biopsy), case 28a and case 114a (possible viral meningitis but no CSF white count elevations), and case 113a (acute inflammatory demyelinating polyneuropathy).
4. Discussion Standard cytologic preparations of CSF specimens, processed by cytocentrifuging and staining with Papinicolou stain, yield an overall sensitivity of only about 15% in primary cerebral malignancies and only 20% in metastatic cerebral tumors [2,13]. Often only ‘suspicious’ or ‘atypical’ diagnoses can be rendered, and unsatisfactory specimens are common in the everyday practice of pathology. The sensitivity of cytologic examination of the CSF for the diagnosis of malignancy is dependent on a variety of
factors, including tumor type, anatomic location and how readily malignant cells exfoliate into the CSF. A positive diagnosis is most likely to occur in cases with diffuse infiltration of the leptomeninges by malignant cells with poor cell-to-cell cohesion. By contrast, tumors that are located deep within the parenchyma of the brain, or those that involve the extradural space but do not involve the leptomeninges, are unlikely to shed tumor cells and, thus, are unlikely to be detected by microscopic evaluation of the CSF [3,13]. Except for actual brain biopsy, the only other way to document tumor cell involvement of leptomeninges pre-mortem is by sensitive radiographic studies such as magnetic resonance imaging. Given the relatively low sensitivity and specificity of traditional cytologic examination of CSF, identifying a molecular cancer marker in CSF that might enhance the detection of tumor cells would be welcomed. Telomerase has proven to be a nearly ubiquitous marker for cancers of many different types and from many different sites, especially late-stage malignancies [4,5,7,17–19,23,33]. Previous work from our laboratory has demonstrated the high percentage of telomerase positivity in primary cerebral malignancies [10,11] and neoplasms metastatic to the brain [12], laying the groundwork for exploring its expression for detection of tumor cells in CSF. The fact that CSF is one of the most difficult cytologic fluids to analyze made the use of an objective, molecular assay, such as that for telomerase, especially attractive. In order to simulate an actual clinical setting, we utilized virtually all of the CSF samples available to us over the 15-month study period. The specimens that we analyzed represented residual fluid following the performance of the cytologic, cell count and / or protein CSF analysis ordered
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by the patient’s physician. CSF samples were drawn for the purpose of patient care and not specifically for our study. In order to accurately define the sensitivity and specificity of the telomerase assay, we applied strict criteria to determine which of the 281 samples could be included in the final analyses. Cases with an inconclusive cytologic diagnosis (‘unsatisfactory’ or ‘atypical’) were not included in the calculation of sensitivity and specificity. Utilizing the 225 specimens with a definitive cytologic diagnosis, specificity was 87% and the sensitivity was 60% for the correlation of telomerase expression with cytologic positivity in individual CSF specimens. After adjusting for three patients with subsequent brain biopsy or autopsy which showed meningeal involvement and excluding six patients with leukemia or widely metastatic carcinoma and known peripheral blood contamination of CSF, an adjusted specificity of 90% and a sensitivity of 64% were achieved (see Table 3). From our statistical analyses, neither elevated CSF protein levels nor the CSF white blood cell counts adversely affected sensitivity or specificity for telomerase detection. Positive telomerase results were found in several patients without known CNS or systemic malignancies. Review of medical records of these patients corroborated the clinical impression that they were unlikely to harbor occult malignancies. Several of these patients had been clinically diagnosed with viral syndromes or demyelinating diseases (see Tables 1 and 2). These few patients with discordant positive telomerase / negative cytology results represented a small proportion (9%) of our 281 cases (see Table 3). Many of our patients with biopsy- or cytology-proven meningeal carcinomatosis provided multiple CSF specimens (see Table 1). In a few of these samples, positive cytology / negative telomerase results on one or more of the CSF specimens were noted. This finding may represent varying numbers of tumor cells in the portion of the CSF specimen used for cytology versus the portion used for the telomerase assay. We cannot exclude the possibility, however, that false negative results in some of our CSF samples may be due to degradation of telomerase activity in what should have been a positive sample. At present, there is no internal control system to evaluate for the stability of telomerase activity. The development of such a system will be crucial for the development of telomerase expression as a diagnostic adjunct in either tissue or body fluids. It is useful to note, however, that we found no apparent loss of activity in telomerase expression with storage times in the refrigerator of up to 72 h. In this study, we chose to use the original TRAP protocol [23] rather than the modified quantitation protocol [36], since the latter contains an internal standard that could slightly inhibit the co-amplification of the telomerase extension product. Use of the original TRAP protocol in our laboratory allows reliable and reproducible detection of telomerase expression in as few as ten carcinoma cells by
titration assay (see Fig. 1). Also, the finding of a four-fold logarithmic variation in telomerase expression between different types of brain metastases (Part 1, companion paper), some of which showed weak expression, suggested that the most sensitive assay method for telomerase should be utilized for this CSF study. Studies in our laboratory are underway to determine if co-amplification of an internal standard on CSF shows the same utility as the original TRAP protocol. Based on this CSF study, our companion Part 1 study, and our previous experience with both protocols, we would predict that the protocol using coamplification of an internal standard would yield less sensitivity than the original protocol for the detection of telomerase expression from small numbers of tumor cells metastatic to CSF. This hypothesis is strengthened by the finding in this study that, even with the use of the original sensitive TRAP protocol [23], some of our CSF specimens with positive cytology showed only weak telomerase expression that was visible after a one-week gel exposure. We envision the use of CSF telomerase analysis as an adjunct to, not as a replacement for, traditional CSF cytologic methods. In clinical practice, the use of the two methods together may reassure the diagnosis of malignancy when a positive telomerase result is obtained in conjunction with a ‘suspicious’ or ‘atypical’ traditional cytology result. A positive telomerase result on CSF that is ‘unsatisfactory’ or negative by traditional cytology might mandate analysis of an additional CSF specimen. For neoplastic diseases, our study suggests that the detection of telomerase may be more sensitive than traditional cytology, a finding in accordance with the few other non-CSF telomerase / cytology correlation reports in the literature [6,9,14,20,24,25,35,37–40]. Studies conducted on the utility of telomerase as an adjunct to cytology in extracranial malignancies have had the advantage of readily accessible biopsy or resection material with which to compare results. This made the calculation of sensitivity of telomerase for the detection of malignant cells much easier than in our CSF study. Unlike patients with cytologically abnormal cervical smears, who all eventually come to biopsy or tissue resection, patients with CNS meningeal disease are usually not biopsied once diagnosis is made from cytology. In our study, CNS tissue with which to compare telomerase results was not always available from patients. Our ‘gold standard’ with which to compare telomerase results was the traditional CSF cytologies on these patients, a test with known low sensitivity [2,13]. Hence, in this CSF telomerase study, we attempted to supplement the study by undertaking clinico-pathologic correlation from medical chart reviews to determine the sensitivity and specificity of the assay. Our adjusted sensitivity of 64% and specificity of 90% are comparable with those reported for cytology / telomerase correlation from extracranial malignancies [6,20,24,25,35,37–40]. Three studies on urine or bladder washings for the detection of transitional cell carcinoma performed on 23,
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109 and 87 specimens, showed sensitivities of 95.7, 62 and 67.8% and specificities of 100, 96.4 and 100%, respectively [24,37,39]. Colonic luminal washings conducted for the detection of colonic adenocarcinoma on 24 patients showed a sensitivity of 60% and a specificity of 100%. There was no evidence of telomerase expression in nine of 24 washings taken from inflammatory bowel disease patients, a possible confounding, non-neoplastic inflammatory condition [39]. Sixty-six oral rinses from head and neck squamous cell carcinoma patients analyzed for telomerase expression showed a relatively low sensitivity, but a high specificity of 95% [6]. Likewise, fine needle aspirates of breast lesions have generally exhibited a moderate sensitivity and a high specificity for the detection of malignant cells [35]. Telomerase detection of carcinoma in cervical cytology specimens in two studies demonstrated a high level of sensitivity for the detection of high grade squamous intraepithelial lesions and squamous cell carcinoma [25,40]. However, a third study was less encouraging and showed telomerase positivity in only one of 22 cases of high-grade squamous intraepithelial lesions [14]. The most promising telomerase / cytology correlation study to date is that of Hiyama et al. [20]. In their study, twelve brush samples from pancreatic duct for the detection of ductal adenocarcinoma yielded both a sensitivity and specificity of 100%. In summary, telomerase activity was detected in a high proportion of CSF specimens from patients with malignant involvement of the meninges as proven by definitive CSF cytology or subsequent biopsies. Telomerase was usually not detected in patients with negative CSF cytology. Further studies and refinement of the technique are warranted to see if the TRAP assay for telomerase can be adapted to the clinical practice of CSF cytology on a widespread basis.
Acknowledgements
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15] [16]
[17] [18]
Drs B.K. Kleinschmidt-DeMasters and K.R. Shroyer are members of the Colorado Cancer Center. Dr A. Laurie Shroyer was funded and sponsored, in part, by the Department of Veterans Affairs Office of Performance and Program for Cooperative Studies. The authors gratefully acknowledge the assistance of Ms Monique Paques in the Clinical Laboratory, Ms Mary Wilfawn in Medical Records, and Ms Ginger Woodward and Ms Velma Parker for assistance with the preparation of the manuscript.
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