Epidermal growth factor receptor gene and c-erbB-2 gene amplification in ovarian cyst fluid

Epidermal Growth Factor Receptor Gene and c-e&B-2 Gene Amplification in Ovarian Cyst Fluid TORALF REIMER, MD, KARSTA LUETTICH, Objective:

To test for

tional and neoplastic growth factor (EGF) amplification rates.

a significant ovarian receptor

difference

between

Methods: We determined amplification and c-erbB-2 genes by differential polymerase (PCR) on 138 ovarian-cyst aspirates. The differential of a target

PCR gene

is based on simultaneous and a reference gene.

were detected by densitometry amide gels and were scored numbers. coefficient, evaluate predict

those with significantly

in

Low-copy (twogene amplification neoplastic cysts,

to

women with functional fn = 59) or malignant

(n

to fourfold copy numbers) was found in 22 of 67 (33%) but in only eight (11%) of histology differed in correlation to

amplification difference

to functional

Conclusions: Low-copy seems to be a marker

EGF-receptor neoplastic

this gene Amplification

to

and

amplification with respect tology (r = .083, P = .32).

growth factor in proliferation esis. However,

polyacrylor high-copy

amplification

Neoplastic histology

low-copy gene was no significant

for

co-amplification Amplification rates

oncogene

functional cysts. from functional

EGF-receptor .OOl). There

EGF-receptor chain reaction semiquantitative

test, Pearson correlation regression were used

Results: There were 71 (51.5%) cysts, whereas 67 (48.5%) had benign = 8) tumors. EGF-receptor women with

of

of silver-stained as single-, low-,

Wilcoxon ranked sum and multiple logistic the differences histology.

func-

cysts with respect to epidermal and c-erbB-2 proto-oncogene

in

(Y = .279, c-erbB-2

P < gene

neoplastic

his-

and gene histology.

amplification Epidermal

receptor gene amplification may be involved and growth in an early stage of tumorigenfurther studies are required to investigate

structure abnormality on of c-erbB-2 proto-oncogene

VOL.

College

88, NO.

of Obstetricians

6, DECEMBER

Iyyh

and

In the past few years, oncogenes have been found to play a significant role in human tumorigenesis. The mode of oncogene activation is reported to vary according to tumor type. To date, the abnormalities in ovarian tumors reported most often involve the c-myc, H-ras, K-ras, and c-e&B-2 (human epidermal growth factor receptor-2 [HER-2/ PZU]) oncogenes.’ Epidermal growth factor (EGF) receptor and HER-2/ liebl are growth factor receptors expressed in elevated levels on the surface of malignant cells due to multiple gene copy number. Both have tvrosine kinase activity linking them to cell replication. -2 The EGF-receptor gene is a proto-oncogene on chromosome 7 that encodes the 175-kilodalton (kD) EGF-receptor,3 and expression of this protein has been shown to indicate poor prognosis in several types of malignancy, particularly in breast cancer.’ The c-erbB-2 gene on chromosome 17 encodes a 185-kD transmembrane protein that is closely related to the EGFrecept0r.s Slamon et al’ detected an amplification and overexpression of the c-erbB-2 proto-oncogene in 2530% of human breast and ovarian cancers. The aim of this investigation is to use a quantitative polymerase chain reaction (PCR) system for the estimation of EGF-receptor and c-erbB-2 gene amplification in frozen samples of ovarian cyst contents. Although prior studies have analyzed copy numbers for these genes in tumor tissue, DNA from ovarian cyst fluid has not yet been analyzed. Gene amplification rates are correlated with functional and neoplastic histology.

a gene function level. does not appear to

be a common factor in the development of ovarian tumors. (Obstet Gynecol 1996;88:967-72. Copyright 0 1996 by The American

AND BERND GERBER, PhD

Gynecologists.)

Materials and Methods From June 1994 to May 1995, 172 consecutive ovarian cyst aspirates were obtained intraoperatively (laparoscopy or laparotomy) and stored at -8OC, for between 1 week and 6 months, until DNA extraction. Women with cysts caused by iatrogenic ovulation induction were excluded, as were women with more than one cyst. A

002~~3844/96/515.00 PI1 SW29-7844(96)00360-2

967

Figure chain molecular c-e&B-Z

1. Detection reaction with

of gene amplification by differential interferon-gamma as reference gene.

weight marker gene amplification

V; lanes rates;

2-4, 2.0.fold, lane 5-7,

2.5.fold, 2.0-fold,

3.0-fold epidermal growth factor receptor gene lane 8, DNA molecular weight marker VIII.

polymerase Lane 1, DNA and 3.0-fold 2.5.fold, and

amplification

rates;

minimum of 1 mL of cyst fluid was required for analysis. Cyst fluid samples were digested with proteinase K, and DNA was extracted by the phenolchloroform method, precipitated with ethanol, and resuspended in sterile water. The DNA content was estimated by measuring the optical density at 260 nm (OD,,,). The DNA quality was tested by a 35-cycle PCR run, using primers for the glutaraldehydedehydrogenase housekeeping gene. Differential PCR allows for simultaneous co-amplification of a target gene and a reference gene. If the target gene copy number is greater than that of the reference gene, the PCR product of the target gene will be overrepresented, and the PCR amplification of the reference gene will be suppressed.7 Approximately 200400 ng of DNA per sample was amplified according to Neubauer et al8 in a final volume of 25 PL using a thermocycler (MJ Research, Watertown, MA). The sequences of oligonucleotide primers used in this study were published previously: c-erhB-2 (98 base pairs),5 EGF-receptor (110 base pairs),3 and interferon-gamma as reference gene (150 base pairs).’ Human placental DNA served as single-copy control and a highly amplified tumor sample as high-copy control. DNA was omitted in the negative control. The PCR products were separated by polyacrylamide gel electrophoresis, using a 12% gel, and then visualized by silver staining. DNA molecular weight markers V and VIII (Boehringer, Mannheim, Germany) were used to document amplification of appropriately sized fragments (Figure 1). A Bio-Rad model 670 imaging densitometer (Bio-Rad Laboratories, Hercules, CA) was used to scan the polyacrylamide gels, and the resulting images were analyzed with the associated Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA).

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Oncogencs

in 07wim

Cysts

Rem1 ts In a total of 172 consecutive samples, sufficient DNA for PCR analysis was extracted from only 138 (80%). Ovarian cysts with mutinous and endometrioid content yielded a very low rate of usable DNA. There was no correlation between DNA extraction output and cyst size. Of the 138 subjects, 71 (51.5%) had functional cysts, and 67 (48.5%) had benign or malignant tumors (Table 1). The median age at surgery was 38 years (range, 12-83 years). The functional cysts showed a premenopausal and the neoplastic cysts a postmenopausal maximum (Table 2). The pattern of body mass index (BMI) with respect to age and histology is presented in Figure 2. A majority of functional cysts were aspirated at laparoscopy (94.4”/ O, n = 67), whereas only 5.6% (M = 4) were aspirated at laparotomy. Of the neoplastic cysts, 52.2% (IZ = 35) were laparoscopically obtained; 47.8% (n = 32) were collected during laparotomy. In each case, the diagnosis was supported by pelvic examination and ultrasound. Two-thirds of women with functional cysts were admitted with lower abdominal pain (67.6%, IZ = 48). In the functional histology

Table

1. Histologic (N

Types

of Ovarian

Cyst

Aspirates

= 138)

Histologic

n

%

cysts

71

51.5

luteum rubrum

2x 4 10

20.3 2.9 7.2

Parovarian

6

4.4

Tuba-ovarian Ruptured

8 15

5.8 10.9

67 43 3

48.5 31.2 2.2

5 6

3.6 4.3

2 3

1.4 2.2

5

3.6

Functional Corpus Corpus Follicular

Neoplastic Serous Mutinous

Type

cysts cystadenoma cystadenoma

Teratoma Cystadenofibroma Cystadenoma Borderline Serous

and tumor

teratoma

cvstadenocarcinoma

Obstetrics

& Gynecolog/

2. Age Distribution Histology

Table

According

to Cyst

Fluid

36)

I

Histology Functional

Neoplastic

02 = 71) Age

W

(n = 67)

“I

%

11

% 4.5 16.4

12-20

11

15.5

3

21-30 31-40

14 26

19.7 36.6

11 11

41-50 51-60 61-70

16 3 1

22.5 4.2 1.4

8 13 13

>70

0

0

8

Median

36 years

16.4 11.9 19.4 19.4 11.9 51 years

21-30 group, 35.2% (25 of 71) were nulligravidas, 8.5% (n = 6) had previous pelvic inflammatory disease (PID), and 4.2% (n = 3) had a history of ovarian cyst. The corresponding data for women with neoplastic histology (n = 67) were 53.8% (n = 36) for lower abdominal pain, 14.9% (n = 10) for nulligravidas, 6.0% (n = 4) for previous PID, and 7.5% (11 = 5) for history of ovarian cyst. The distribution of EGF-receptor and c-erbB-2 amplification rates according to histologic category are shown in Figures 3 and 4. Low-copy (twofold to fourfold copy numbers) EGF-receptor gene amplification were seen in 22 subjects with neoplastic cysts (33%), but in only eight (11%) with functional cysts. With respect to low-copy EGF-receptor gene amplification, neoplastic histology was significantly different from functional histology in correlation (Y = ,279, P < .OOl). Of all ovarian cyst aspirates, 108 (78.3%) showed a single-copy EGFreceptor gene, 30 (21.7%) of the cases were found to have a low-copy EGF-receptor gene, and no high-copy (more than fourfold) EGF-receptor sample could be detected. In the neoplastic group, one borderline tumor and two carcinomas yielded low-copy EGF-receptor gene amplification. There was no significant correlation between c-eubB-2 amplification and neoplastic histology (Y = .083, P = .32). Only 11 neoplastic tumors (16%) showed low-copy (n = 8) or high-copy (12 = 3) numbers of the c-eubB-2 gene. One borderline tumor and two carcinomas had a more than fourfold amplified c-e&B-2 gene. One International Federation of Gynecology and Obstetrics stage III carcinoma sample revealed an eightfold amplification rate. Using the multiple logistic regression model with EGF-receptor amplification rate and age as covariates, 81.7% (n = 58) of all observed functional and 68.7% (IZ = 46) of the neoplastic cysts could be predicted exactly. The overall correct prediction of histologic group was

VOL.

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41-50

61-70

Age (years) Figure 2. Median body histology (N = 138).

mass index

(BMI)

with

respect

to age and

75.4%. Including the c-eubB-2 amplification rate in this analysis, we found a deterioration of this value (73.9%).

Discussion For this study, we used a fast and PCR-based method for the determination

c

nonradioactive of oncogene

7

.&0

I

23

G .M z 2 5

dd 1. s N=

71

67

functional

neoplastic

Histology Figure 3. Box-plot diagram of epidermal growth factor receptor (EGF-R) gene amplification rate in ovarian cyst fluid (1’4 = 138) for functional and neoplastic histology. The box represents the range between the 25th and 75th percentiles with a horizontal line at the median. The bars delineate the 5th and 95th percentiles. The circle (0) and asterisk indicate extreme values (0 = more than 1.5 box lengths from 75th percentile and * = more than 3.0 box lengths from 75th percentile).

Reimer

et al

Oncogenes

in Ovarian

Cysts

969

01

N=

71

67

functional

neoplastic

Histology Figure 4. Box-plot of c-erbB-2 gene amplification rate in ovarian cyst samples (N = 138) for functional and neoplastic histology, The circle (0) and asterisk indicate extreme values (9 = more than 1.5 box lengths from 75th percentile and * = more than 3.0 box lengths from 75th percentile).

amplification. The need for much less sample material than for classical methods such as Southern transfer and slot blot is a great advantage in the analysis of a fluid with a known low cell and DNA content. Previous investigations by Hruza et al” and Hunter et ali* showed that differential PCR is a valuable technique for gene amplification detection compared with conventional hybridization methods. However, the nonlinearity of PCR amplification techniques does limit the power to detect differences in the oncogene levels. Comparative genomic hybridization needs to be considered in further studies as an alternative. We know of no previous study that specifically addressed growth factor receptors in ovarian cyst fluid by PCR technique. The typical cytologic findings of the different histologic categories as the source of DNAbased methods have been reviewed recently by Gerber et al.‘” Prior studies of ovarian cyst fluid for cytology, steroid hormones, and tumor markers have not revealed an accurate marker for discrimination between functional and neoplastic cysts. Only Andolf et al” have shown a strong correlation between high urokinase concentration and neoplastic histology, as determined by radioimmunoassay. The issue of oncogene amplification in human tumors has been investigated extensively by various molecular biologic methods. However, the role of EGF-receptor in tumorigenesis is still controversial. Specifically, it has been shown that the EGF-receptor gene is amplified in some squamous cancers, glioblastomas, and esophageal

970

Reimer

et al

ofICO<~C’Z”

ill

0iV?i’;l7il

Cysts

adenocarcinomas.‘5 Additionally, the EGF-receptor is overexpressed in approximately 40% of breast carcinomas and is inversely correlated with the expression of the estrogen receptor. Amplification of the EGFreceptor locus, however, occurs infrequently in approximately 5% of all cases.16 Not unexpectedly, normal ovarian surface epithelial cells express EGF-receptor and respond to EGF in vitro.17 In two studies’8,19 of ovarian carcinoma cells, EGF-receptor expression correlated with adverse prognosis. At least one repor?’ suggested that expression of EGF-receptor is indicative of a good prognosis and better response to chemotherapy in ovarian cancer. Our results revealed that low-copy EGF-receptor gene amplification is a marker for indicating abnormality in a third of 67 neoplastic ovarian cysts, whereas the 71 functional cysts showed only sporadic low-copy EGF-receptor amplification, This difference in low-copy EGF-receptor amplification was statistically significant (P < ,001). The neoplastic group consisted mainly of benign histology (88%). According to our findings, EGF-receptor may act as a proliferation and growth index in an early stage of tumorigenesis. However, because we examined only gene structure and not gene function, further research is required to elucidate the role of EGF-receptor in tumorigenesis. There is still debate over the malignant potential of benign ovarian cysts. Benign mutinous cystadenomas have been associated with borderline tumors, but direct transformation to an adenocarcinoma is difficult to documentzl Puls et aI*’ suggested that certain benign serous or mutinous ovarian tumors have the potential for malignant transformation. Histologic findings in 14 cases by Bell et a123 revealed that at least a subset of ovarian epithelial cancers develops de novo. Further studies are needed to evaluate the importance of EGF-receptor gene amplification in the malignant potential of ovarian cysts. The EGF-receptor gene amplifying cysts could be a premalignant stage of ovarian cancer; in particular, it is expected that the EGF-receptor stimulates not only proliferation but vascular endothelial growth factor expression as well.*” Vascular endothelial growth factor, which stimulates the proliferation and migration of endothelial cells, plays a crucial role in the vascularization of human tumors. The c-erbB-2 (HER-2/ netl) oncogene is amplified in several tumors, including bladder, gastric, renal, salivary gland, breast, and ovarian carcinomas.‘5 In the latter two neoplasms, c-e&B-2 amplification seems to be a prognostic index of clinical outcome. Slamon et al6 observed that the oncogene c-e&B-2 was amplified in 31 (26%) of 120 primary epithelial ovarian tumors and that there was a perfect correlation between amplification

Obstetrics ~5 Gy~ccology

and overexpression. In tissue sections and loose cyst fluid cells of benign ovarian tumors, no overexpression of c-erbB-2 proteins was found by Harlozinska et alz5 Hruza et al” tested 196 ovarian cancer samples by competitive PCR; the c-eubB-2 gene was found to be amplified in 32% (low-copy) and 8% (high-copy, greater than fivefold) of all cases. In ovarian carcinomas, cerbB-2 amplification by PCR occurred in 34% of 32 fresh tumor samples, and correlation between amplification and clinical staging was observed.2h These results suggest that amplification of the cerbB-2 gene may play a role in the pathogenesis of advanced ovarian carcinomas. Liu et al’” detected cerbB-2 gene amplification by differential PCR in 48% of in situ carcinomas of the breast and in 21% of stage II breast cancers. This high incidence in noninvasive breast tumors suggests that pertubations of the c-erbB-2 oncogene are among the earliest and most common genetic lesions in human breast cancer. Our results revealed only occasional low-copy cerbB-2 gene amplification (12%) in 67 neoplastic ovarian cysts. High-copy (more than fourfold) amplification rates were seen exclusively in borderline tumors or carcinomas. The c-erbB-2 amplification did not discriminate between functional and neoplastic ovarian cysts (P = .32). This result is probably due to the small number of the advanced ovarian carcinoma aspirates (Z = 5) in the neoplastic group. The amplification of c-erbB-2 gene seems to be unrelated to early ovarian tumorigenesis. The analysis of growth factor receptors in human carcinomas is important for diagnosis, prognosis, and-of late-therapy. Further studies with larger sample sizes are required to determine the molecular mechanism responsible for the association between gene amplification of EGF-receptor and neoplastic histology. In future studies, the observed gene structure abnormalities should be supported by gene function analyses. A second important addition is comparative genomic hybridization, a technique that may provide the same sort of information as an allele imbalance study, but covers the entire genome in one sweep. However, this method has limited sensitivity at present. Finally, the extension of ovarian cyst studies to the tumor suppressor gene p53 could be useful. Kupryjanczyk et a127 suggested that p53 abnormalities may be early events in ovarian cancer, possibly contributing to malignant transformation of some borderline tumors, endometriosis, and other carcinoma precursors.

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CRITICAL

Address

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Universitat Restock PF 10 08 88 D-l 8055 Ros tack Germany

Received February 29, 1996. Received in revised form August 20, 1996. Accepted September 10, 1996. Copyright 0 1996 by The American College of Obstetricians Gynecologists. Published by Elsevier Science Inc.

and

CARE

February 23-26, 1995 The American College of Obstetricians and Gynecologists is sponsoring a course on critical care for obstetric and gynecologic patients, to be held at the Sun Valley Resort, in Sun Valley, Idaho. This course has been approved for 16 cognate hours (Formal Learning) by the American College of Obstetricians and Gynecologists. For further information, contact the Registrar, The American College of Obstetricians and Gynecologists, 409 12th Street SW, Washington, DC 20024-2188;(202) 863-2541.

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Oncogenes in Ovarian Cysts

Obstefvics 6 Gynecology