日本産科婦人科学会香川地方部会雑誌 vol.7
，
No.1，
pp.35 - 39，
2005 (平 17，
9月 35 - Original Article-E
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Department ofPerinatology and Gynecology
，
Kagawa University School ofMedicine Masayuki Ohno，
Kenji Kanenishi，
Tatsuya Igarashi，
Atsuko Shiota，
Toshiyuki HataAbstract
OBJECTIVE: The aim of this study was to evalu -ate whether telomerase activity in peritoneal washing fluids can be used as novel means for early detection of the recurrence of ovarian can -cer.
METHODS : Twenty patients with ovarian can -cer (stage 1 c in 2
，
II a in 2，
II c in 2，
illa in 2，
illb in 1
，
illc in 4，
andN
in 7) were recruited for the study. These patients received maximal debu-Iking as a primary treatment，
and were followed up using telomerase activity measurement σRAP assay) and cytological examination in peritoneal washing fluids.RESULTS: In 8 patients with telomerase nega -tive and negative peritoneal washing cytology
，
no recurrence was ascertained. Of the 6 peritoneal washing cytology negative cases，
3 were telomer -ase positive. When these 3 were reevaluated for peritoneal cytology，
malignant ascites were iden -tified in all3 patients later. Of the 12 patients with telomerase positive，
9 died，
whereas none died in 8 patients with telomerase negative (p<
O
.
0
0
5
)
.
Patients with telomerase positive had significan -tly poorer outcomes than those with telomerase negative (p<
O
.
0
0
5
)
.
CONCLUSIONS: Our preliminary results reveal that the telomerase test in peritoneal washing flu -ids can be used as an adjuvant to cytopathological methods in early detection of the recurrence of ovarian cancer. Our results suggest that positive telomerase activity in peritoneal washing fluids may indicate the poor prognostic factor in patients with ovarian cancer.KEY WORDS: Ovarian canceηrecurrence
，
tel -omerase，
peritoneal washing fluids，
cytology Introduction Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends using an RNA component as a template.1.2)Extension oftelomeric re -peats by telomeras巴preventstelomere shortening with cell divisions and contributes to chromosomal stabiliザ， possibly leading to immortalization ofthe cells.3，4)Tel -omerase activity has been found in a variety of malig -nant tumors but only rarely in benign tumors or normal tissues. Specifically，
telomerase activiザispresent in 95% of gynecologic malignancies and in 88% of epithel -ial ovarian carcinomas， but it is undetectable in most be -nign tissue.5)Therefore， telomerase activation might be common in gynecologic malignant tumors，
and be a valuable diagnostic parameter that could help to identifシ
potentially progressive 1巳sions.6，7) Malignant ascitic effusion is a common presentation of ovarian cancer，
and reflects peritoneal dissemination. Transcelomic seeding of malignant cells antedates the development of ascites，
so its det巴ctionhas prognostic significance.8)The diagnosis ofmalignant ascitic fluid and the di首巴rentiationamong malignant，
paramalignant and nonmalignant effusions by conventional diagnostic methods are sometimes difficult，
and usually only36 Early Detection of Recurrence of Ovarian Cancer using Telomerase Assay in Peritoneal Washing Fluids 産婦香川会誌7巻 l号 Table 1 Clinical characteristics and telom巴raseactivity in each ovarian cancer Case Age Stage Histology Telomerase activity Follow up Cmonths) Outcome 36 2c serous (一) 112 NED 2 43 1c endometrioid (一) 100 NED 3 46 1c endometrioid (ー) 99 NED 4 53 3b serous C+) 62 AWD 5 46 4 serous C+) 35 000 6 50 4 clear cell C+) 20 000 7 48 3a muclnous (ー) 48 NED 8 52 2c serous C+) 34 000 9 63 4 serous C+) 28 000 10 77 3c serous C+) 24 000 11 51 3a clear cell C+) 23 000 12 60 4 serous C+) 22 000 13 42 2a endometrioid (一) 37 NED 14 56 3c muclnous C+) 4 000 15 58 2a serous (一) 26 NED 16 67 3c serous (ー) 24 NED 17 72 4 serous C+) 6 000 18 73 4 serous C+) 20 AWD 19 74 4 serous C+) 20 AWD 20 61 3c undifferentiated (ー) 17 NED NED
=
no evidence of disease; AWDごalivewith disease; 000=
death of disease. 48-60% of malignant peritoneal fluid in patients with ovarian cancer could be diagnosed by cytological exam-ination of peritoneal fluid.民10)Tseng et al.11)revealed a high sensitivity and specifici勿ofbothtelom巴:ras巴tes -ting and conventional cytology in periton巴alfluids. The-se authors also suggested that the telomerase test in per -itoneal fluids can be used as an adjuvant to cytopathol -ogical methods in the diagnosis of malignant peritoneal ascites，
particularly in cases of negative cytology. These findings encouraged us to evaluat巴 the potential usefulness of telomerase test in washings or ascitic flu -ids from th巴peritonealcavity as a means of detecting re -sidual or recurrent canc巴rcells in patients仕eatedforovarian canc巴r. The aim ofthis study was to deterrnine
whether telomerase test is a useful indicator of recur -rence risk and ultimate outcome.
P
a
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i
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t
s
and Methods
A total of20 patients (aged 36-77 years
，
mean 56.7 years) with histologically confinned primarγepithelial ovarian cancer were studied between September 1992 and August 2000 at Kagawa Medical University Hospi -tal，
Miki，
Japan (Table 1). These patients received maximal debulking as a primary treatment，
and chemo-therapy was not given before surgery. The patients were staged according to criteria recommended by the Inter -national Federation of Obstetricians and Gynecologists (FIGO).12)There were 2 stage 1 patients，
4 stagen
patients
，
7 stageIllpatients，
and 7 stage Npatients. The staging system defined by FIGO assumes that an ad -equate staging operation has been perforrned.13)Tumorswer巴classifiedhistologically according to World Health
Organization (WHO) criterial2) as serous (n=12)， mucinous (n=2)
，巴
ndometrioid (n=3)，
clear cell2005年9月 100
g
80E
と コ帥 60 VI 40 占 帽』由 20 111111t 一1 1 1 1 1 4 S E E 一l E t J i -干 4 2 ) 一 -一 一 8 一 ω -一 同 一 一 尚 一 一 n H -m -( 一 ω 叩 -e 一 一 一 一 町 一 ↑ ↑ 一 t “ 一 守 一 ﹄ l t 翁 u l み 白 ﹃ i t -9 ・ τill--ltY1111103a ﹄ l g b “ 一 -一 e 司 Z -ザ 勾 n H 一 七 回 れ 一 一 e 一 川 一 一 一 s-niuι 一 ι 百 一 O-一 い 剛 一 戸 一 一 一 月 一 ! 一 ) 一 町 -ω 叩 2 -' 1 1 1 ト ' ' ' ' ' ト 1 1 1 1 3﹁ l F ' I T -匂e
"
-=
一 1 m 一 ザ n -占 一 ザ ( 一 一 一 一 e 一 } 一 ↑ v 一 一 -N 比 一 一 一 一 s -o -一 一 一 n V ャ 一 句 一 一 一 ﹂ 111 ド 1 1 1 1 r ﹂ HF ﹁ -ザ 句 S -一 一 ω a M -ω 一 -r 一 “ 一 句 。 切 一 一 一 一 m 一 一 一 周 回 一 -一 一 e -一 一 一 T 一•
O O 20 40 Ohno. et a.l 37 60 80 100 120 Months after surgery Figure 1 Kaplan-Meier survival curves for patients with ovarian cancer cording to telom巴ras巴activity. Table 2 Patient outcome according to telomerase activity Telomerase activity Outcome Group n At surgery After treatment Final testduring follow up NED AWD DOD
6 し) (-) (-) 6
。 。
E 3 (旬) し) (十)
。
2E 2 (+) (+ or -) (ー) 2
。 。
N 9 (+) (+ or -) (十)
。
2 7NED
=
no evidence of disease; AWD=
alive with disease; DOD=
death of disease.(n=2)， and undiffe1'entiated (n=1). A
1
I
20 patients1'e-ceived cisplatin-and taxan-based1'egimens afte1'frrst
surgery. The study was app1'oved by the 10cal ethica1
committe巴ofKagawaMedical Unive1'sity， and standa1'
-dized informed consent was obtained from each patient.
Ascitic fluid was withdrawn from all 20 patients at the time of laparotomy just afte1'opening the pe1'itone
-um. About 500 cc of normal ste1'ile saline was used to
wash the wh01巴pelvisifno ascites was noted within the pe1'itoneal cavity. Along with1'outine investigations11)， the ascitic fluid was submitted fo1'cytologica1 examin -ation and telomerase assay. Data acqui1'ed企omcytology and te10me1'ase assay we1'e collected in a doub1e-blind fashion until analysis. Reservoir was placedjust befo1'e closing the p巴1'itoneum，and used fo1'the1'oute fo1'intrap -巴1'itonea1chemothe1'apy and 1'et1'ieval of ascitic fluids during follow-up p巴1'iod. Telomerase activi勿wasdete1'
-mined using the TRAPeze telome1'as巴detectionkit (On回
co，'1 Inc.， Gaith巴:1'sbu1'g，M D，)as desc1'ibed p1'evious1y. 14)The diffe1'ence between te10me1'ase status in perito
-neal ascites was determined using chi-squa1'巴test.Su1'
-vival curves we1'e plotted using the method of Kaplan
-Meie，'1 and the log-1'ank test was used to determine the
diffe1'ence between life tables. A value ofp<0.05was conside1'ed statistically significant.
R
e
s
u
l
t
s
Patient outcome acco1'ding to telome1'ase activity was shown in Table 2. In 8 patients with te10merase negative and negative peritoneal washing cytology at fi・38 Early Detection of Recurrence of Ovarian Cancer using Telomerase Assay in Peritoneal Washing Fluids 産婦香川会誌7巻1号 nal test during follow up
，
no recurrence was ascertained. Of the 6 peritoneal washing cytology negative cases at final test during follow up，
3 were t巴lomerasepositive. U弓lenthese 3 were reevaluated for peritoneal cytology，
malignant ascites were identifi巴din all 3 patients later. Ofthe 12patients with telomerase positive at final test during follow up，
9 died，
whereas none died in 8 patients with telomerase negative(pく0.005). Out of 11 alivepatients
，
8 showed no evidence of disease，
and 3 were aliv巳withdisease (Table1).Patients with telomerasepositive had significan
t
1
y poorer outcomes than those with telomerase negative(pく0.005)(Figur巴1).D
i
s
c
u
s
s
i
o
n
Duggan et a.l15)reported that TRAP assay is more
sensitive than cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian car -cinoma. These authors suggested that the presence of telomerase activity in abdominal fluids and washings from patients treated for ovarian carcinoma may be a strong indicator of residual disease and improve the abil -ity to detect early disease recurrences. In this study
，
3 cases were histologically negative but telomerase posi -tive during follow-up period (Cases 4，
5 and 8 in Table 1). When histology was repeated in thes巴cases，
a1l3showed positive histology later (3 months later in Case 4
，
3 months later in Case 5，
and 15months lat巴rinCase8). Several factors supportth巴meritofthis approach for
the detection of ovarian carcinoma. The high sensitivity of the TRAP assay ensures the detection of仕aceam同
ounts of tumor cells in the presence oflarge excesses of normal cells.15)For example
，
TRAP assay is extremelysensitive
，
enough to detect telomerase activity in ex -仕actsequivalent to 100 immortal cancer cells，
whereas the conventional peritoneal assessment is difficult if there are less than 1000 cells.11)In this study，
res巴rvoir was placed just beforec
1
0sing the peritoneum，
and used for the route for intraperitoneal chemotherapy and retri -eval of ascitic fluids during follow-up period. Peritoneal washings could easily obtained from patients with ovarian carcinoma by use of lavage through long-term abdominal catheters. Therefore，
the telomerase test in peritoneal washing fluids could be used as an adjuvant to cytopathological methods for early detection ofresidual disease of ovarian carcinoma in patient follow-up pro -tocols. Telomerase activity has been found in a variety of malignant tumors but only rarely in benign tumors or normal tissues. Telomerase activation might therefore be a valuable diagnostic parameter that could help to identifシ
potentiallyprogressive lesions.7)Telomerase activity is associated with development and extension of epithelial ovarian cancer.16)The decreas巴oftelomerase activity levels parallel cell growth impairment，
and the observed telomerase activity remaining after treatment with antineoplastic agents is most likely to reflect activ -ity企omthe remaining viable cells.17，18)Therefore， the disappearance oftelomerase activity might be a reliable marker oftumor cell killing. Moreover， the diagnostic and therapeutic implications of telomerase activation need to bec
1
arified in clinical trials. In this study，
of th巴12patients with t巴lomerasepositive at final test dur -ing follow up，
9 died，
whereas none died in 8 pati巴nts with telomeras巴negative.Patients with telomerase posi -tive had significant
1
y poorer outcomes than those with telomerase negative. These results suggest that the tel司 omerase test might be a novel clinical prognostic indica -tor of human ovarian carcinoma.. However，
the data and its interpretation should be taken with some degree of caution because of the small number of subjects stu -died. Further study is needed toc
1
arifシ
theclinical useおlnessoftelomerase assay as a prognostic indicator m ovanan carcmoma.References
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