Determination of telomerase activity for differential analysis of multifocal renal cell carcinomas. Secondary tumors are found in approximately 12 to 22% of all renal cell carcinoma, and their origin is currently unknown. To determine their potential for malignancy, we examined the telomerase activity of primary tumors and secondary lesions, and found that 86% of the lesions had an identical telomerase status as the related primary tumors, and thus probably share their malignancy potential

Claussen, Uwe

Dahse, Regine

Ernst, Guenther

Fiedler, Wolfgang

Junker, Kerstin

Schlichter, Andreas

Schubert, Joerg

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Determination of telomerase activity for differential analysis of multifocal renal cell carcinomas 

Kidney International, Vol. 56 (1999), pp. 1286–1288
Determination of telomerase activity for differential analysis of
multifocal renal cell carcinomas
WOLFGANG FIEDLER, KERSTIN JUNKER, ANDREAS SCHLICHTER, JOERG SCHUBERT,
GUENTHER ERNST, REGINE DAHSE, and UWE CLAUSSEN
Institute of Human Genetics and Anthropology, and Department of Urology, Friedrich-Schiller-University Jena, Jena, Germany
Determination of telomerase activity for differential analysis of RCCs exhibiting multifocal lesions in more detail, we
multifocal renal cell carcinomas. Secondary tumors are found in analyzed telomerase status in secondary lesions and cor-
approximately 12 to 22% of all renal cell carcinoma, and their responding primary tumors from 20 different patientsorigin is currently unknown. To determine their potential for
using a polymerase chain reaction (PCR)-based, highlymalignancy, we examined the telomerase activity of primary
sensitive nonradioactive detection method.tumors and secondary lesions, and found that 86% of the le-
sions had an identical telomerase status as the related primary
tumors, and thus probably share their malignancy potential.
METHODS
Samples
Renal cell carcinoma (RCC) constitutes approxi- The study includes 20 patients exhibiting primary
mately 90% of all kidney tumors. Twelve to 20% of cases RCCs and at least one secondary lesion. All tumor
contain multifocal tumor areas that are detected after probes were obtained by surgical resection. The tissue
histopathological examination. Whether these secondary probes were snap frozen in liquid nitrogen immediately
tumors are derived from the primary tumor or have an after resection and were stored at 2808C.
heterogeneous origin is not known. Also, the malignant
potential of the secondary tumors and whether or not Histopathological examination
they characterize a higher risk for metastases are not yet Primary tumors and secondary lesions were analyzed
clear. Therefore, a clearer differentiation of the nature histologically according to the classification of Thoenes.
of multifocality in RCCs is important especially with
Tumor stage was defined according to the TNM system.
respect to a nephron-sparing surgery.
Recently, the determination of the activity of the en- Cell culture and chromosome analysis
zyme telomerase has proved to have diagnostic and prog-
All tissue samples were cultured as described pre-nostic value in a wide range of tumors. As the mainte-
viously by Junker et al [2]. Briefly, the minced and colla-nance of telomere length is elementary for indefinite cell
genase/trypsin treated specimens were resuspended inproliferation, telomerase activity is directly linked to the
Amniomax 100 (GIBCO BRL, Grand Island, NY, USA)potential immortal status of these cells. Furthermore,
and incubated at 378C and 5% CO2. Cytogenetic analysistelomerase activity is closely associated with the malig-
was carried out on cultured cells using standard cytoge-nancy of tumors, as enzyme activity is often present
netic techniques (GTG banding).in malignant tumors but is not present in their benign
counterparts. Telomerase assay
We have previously shown that telomerase is active
For the telomeric repeat amplification protocolin approximately 93% of all tested primary RCCs inde-
(TRAP) assay, the S100 protein extract was isolatedpendent of histopathological classification or grading and
from 1 million cells of cultured secondary tumors orstaging [1]. Thus, telomerase reactivation seems to be a
approximately 50 mg of the homogenized frozen tissuecritical and necessary step in the tumorigenesis of all
samples (all tested primary tumors and secondary lesionskinds of primary RCCs. In order to characterize the
of cases 175, 186, 238, and 251) as described by Kim et
al [3]. The concentration of protein was measured using
Key words: telomerase, tumor, metastases, cancer. the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA,
USA). An aliquot of the extract representing 1.5 mg of 1999 by the International Society of Nephrology
1286
Fiedler et al: Determination of telomerase activity 1287
Table 1. Telomerase activity in primary and secondary tumors ofprotein solution or a 10 mm cryostat section (in cases
renal cell carcinoma (RCC)
238, 321, 348, 361, 363, and primary and secondary tumor
Histopathologic Staging/ Telomerase#1 from case 180) was mixed with 75 mm Tris-HCl (pH
Case no/area feature grading activity Karyotype8.3), 1.5 mm MgCl2, 20 mm (NH4)2SO4, 0.1 mm phenyl-
118/pr. tumor clear cell pT2/G1 nt 1methylsulfonyl fluoride, 50 mm dNTPs, 0.05 mg TS
sec. tumor G1 2
oligonucleotide (59-AATCCGTCGAGCAGAGTT-39, 132/pr. tumor clear cell pT3b/G1 nt 3
sec. tumor G2 1labeled with infrared dye IRD 800; Boehringer-Mann-
141/pr. tumor clear cell pT3a/G2 nt 3heim, Mannheim, Germany), and 0.5 mg T4 gene 32 sec. tumor adenoma G2 2
protein (Pharmacia, Uppsala, Sweden) in a total volume 151/pr. tumor chromophobe pT2/G2 2 1
sec. tumor G2 2of 25 ml. The reaction mixture was incubated for 10 min-
152/pr. tumor clear cell pT1/G1 1 1utes at 238C for telomerase-mediated extension of the /sec. tumor 1 G1 1
TS primer and was then heated at 948C for 90 seconds sec. tumor 2 1
159/pr. tumor chromophobe pT2/G2 nt 1for inactivating the telomerase. After adding 0.05 mg CX
sec. tumor G2 1primer (59-CCCTTACCCTTACCCTTACCCTTA-39) 163/pr. tumor chromophobe pT2/G2 2 2
sec. tumor chromophobe G2 2and 1 unit of Taq DNA polymerase (Eurogentec, Se-
175/pr. tumor clear cell pT3a/G2 1 ntraing, Belgium), the probes were subjected to 31 PCR
sec. tumor 1
cycles of 948C for 30 seconds, 528C for 30 seconds, and 180/pr. tumor clear cell nt 1 2
sec. tumor 1 clear cell 1 2728C for 45 seconds. For control experiments, protein
sec. tumor 2 clear cell 2 3extracts were RNAse pretreated to a final concentration
sec. tumor 3 clear cell 1 2
of 0.05 mg/ml before performing the TRAP assay. Fi- sec. tumor 4 clear cell 2 3
181/pr. tumor clear cell pT2 1 2nally, 4 ml of each PCR product were separated by elec-
sec. tumor clear cell 1trophoresis on a 6% denaturing polyacrylamide gel using 185/pr. tumor clear cell pT2/G1 nt 3
a LI-COR DNA sequencer. The laser detection system sec. tumor G1 2
186/pr. tumor clear cell pT3a/G2 1 1of the LI-COR makes visible the extended and amplifi-
sec. tumor G2 1
cated PCR products as an increasing DNA ladder. 198/pr. tumor eosinophil pT3a/G3 1 2
sec. tumor G3 2
202/pr. tumor clear cell G2 nt 3
sec. tumor adenoma G2 2RESULTS
238/pr. tumor clear cell pT3b/G2 1 nt
There was no correlation of multifocality or telomerase sec. tumor 1
251/pr. tumor nt nt 1 ntactivity with a specific cell type or grading and staging
sec. tumor 1of the RCCs (Table 1). Nine out of 14 (64%) tested 321/pr. tumor clear cell pT3a/G2 2 nt
primary tumors displayed telomerase activity. In total, 24 sec. tumor clear cell 2
348/pr. tumor clear cell pT2/G3 1 ntdifferent secondary lesions from 20 patients with primary
sec. tumor clear cell 1
RCC were analyzed for telomerase activity. Thirteen 361/pr. tumor chromophil pT2/G3 2
sec. tumor chromophil 2 nt(54%) of these secondary lesions were telomerase posi-
363/pr. tumor chromophil pT2/G2 2 3tive (Fig. 1 and Table 1). With the exception of case 198,
sec. tumor clear cell G1 1
all telomerase-positive primary tumors exhibited at least
Numbers signify: (1) concordant karyotypes of primary and secondary tumors;
one secondary lesion with active telomerase. Twelve out (2) concordant clonal chromosomal aberrations: (3) different karyotypes; nt, not
tested. Abbreviations are: pr., primary; sec., secondary.of 14 (86%) cases with primary and secondary tumor
tested in parallel displayed identical telomerase status.
Eleven secondary lesions derived from 10 different pa-
tients showed no telomerase activity. In four of these DISCUSSION
cases (151, 163, 321, and 361), the corresponding primary A comparison of biological parameters of secondary
tumors were also telomerase negative. Only one of the and respective primary tumors was carried out to more
primary tumors (case 363) tested to be telomerase nega- clearly define the nature of multifocality in RCCs. The
tive showed a secondary tumor that was telomerase posi- identical telomerase status in primary and secondary le-
tive. In this case, the histopathological feature and the sions in 86% of analyzed cases indicates an clonal origin
karyotype also were different between the primary and and analogous biological behavior of these secondary
secondary tumors. Two cases (180 and 198) showed te- lesions. This hypothesis of clonal conformity of primary
lomerase-positive primary tumors, whereas the corre- and secondary tumors is confirmed by the concordance
sponding secondary tumors were telomerase negative. of the cytogenetic and telomerase data, as all primary
Case 180 exhibited four different secondary lesions. Two tumors and corresponding secondary lesions displaying
of them displayed telomerase activity, whereas the other identical telomerase status also have concordant chro-
mosomal alterations or concordant karyotypes. In thetwo lacked telomerase activity.
Fiedler et al: Determination of telomerase activity1288
Fig. 1. Detection of telomerase activity in renal cell carcinoma (RCC). Lanes 1 through 20 are the results of the telomeric repeat amplification
protocol (TRAP) assay from primary and secondary tumors. Active telomerase is visible as an increasing DNA ladder starting at 40 bp. Abbreviations
are: C, control assay (TRAP with RNAse treated protein extract); S, molecular weight standard.
two cases (180 and 198) with a telomerase-negative sec- mary and secondary) tumors may be proposed, as only
ondary lesion but a telomerase-active primary tumor, telomerase-active primary tumor exhibited telomerase-
the secondary tumors may have a different clonal origin. active secondary tumors (with the exception of case 363),
It is also possible that these results are the consequence and most secondary tumors are telomerase positive.
of artifacts from analyzing cell cultures. Because of the Also, the absence of telomerase activity in secondary
usually small size of the secondary tumors, in most cases, tumors of cases 141 and 202 both classified as benign
cultured cells of the RCCs were used for the telomerase adenomas may underscore this presumption.
assay. This implies a risk of cultivating and analyzing
contaminating normal cells, and may be true especially Reprint requests to Wolfgang Fiedler, M.D., Institute of Human Ge-
netics and Anthropology, Friedrich-Schiller–University of Jena, Kolleg-for tumors without telomerase activity and normal
iengasse 10, D-07740 Jena, Germany.karyotypes (secondary tumors of cases 118, 141, 151, 180,
E-mail: WFIE@mti-n.uni-jena.de
185, and 202). Thus, more secondary tumors with active
telomerase may exist in reality. For a better assessment
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9:1227–1235, 1996As a consequence of our data, it is obvious that most 2. Junker K, Schlichter A, Junker U, Knofel B, Kosmehl H,
secondary lesions are more likely the result of intrarenal Schubert J, Claussen U: Cytogenetic, histopathologic, and immu-
nologic studies of multifocal renal cell carcinoma. Cancer 79:975–metastasis than the result of multifocal genesis. We con-
981, 1997clude by the concordance of cytogenetic and enzymatic
3. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Hodata that secondary tumors may have malignant poten- PL, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific
tial similar to that of the related primary tumors. A association of human telomerase activity with immortal cells and
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Determination of telomerase activity for differential analysis of multifocal renal cell carcinomas

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