Epidermal growth factor receptor expression in normal ovarian epithelium and ovarian cancer I. Correlation of receptor expression with prognostic factors In patients with ovarian cancer Andrew Berchuck, MD,'" Gustavo C. Rodriguez, MD:" Ahmed Kamel, MD,-" Richard K. Dodge, MS,c" John T. Soper, MD,'" Daniel L. Clarke-Pearson, MD,'" and Robert C. Bast, Jr., MD b • d •• Durham, North Carolina Previous studies in breast and bladder cancer have suggested that epidermal growth factor receptor is expressed by only a proportion of cancers and is associated with poor clinical outcome. We used a monoclonal antibody specifically reactive with the extracellular domain of the epidermal growth factor receptor to localize this receptor immunohistochemically in frozen sections of normal ovary and epithelial ovarian cancer. Normal ovarian epithelium was found to express epidermal growth factor receptor in all cases. Among 87 ovarian cancers, however, 23% did not express immunohistochemically detectable receptor. Epidermal growth factor receptor expression was not related to histologic grade or stage, but was associated with poo~ survival (p < 0.05). The median length of survival of patients with tumors that did not express epidermal growth factor receptor was 40 months compared with 26 months in patients with tumors that did express epidermal growth factor receptor. As in breast and bladder cancer, expression of epidermal growth factor receptor in ovarian cancer appears to be a poor prognostic factor. (AM J OSSTET GVNECOL
1991 ;164:669-74.)
Key words: Epidermal growth factor receptor, epidermal growth factor, ovarian cancer
The epidermal growth factor receptor is a cell surface molecule that is widely distributed in normal human tissues. In vitro, after binding of one of its ligands, either epidermal growth factor or transforming growth factor-a, to the extracellular domain of the receptor, the tyrosine kinase of the intracytoplasmic domain is activated. This results in the generation of second messengers that transmit the mitogenic signal to the nucleus. 1 In vivo, the epidermal growth factor receptor and its ligands are thought to play a role in normal growth and differentiation although the precise nature of this involvement is not understood. Abnormal expression of the epidermal growth factor receptor and its ligands has been associated with ma-
From the Departments of Obstetrics and Gynecology, Division of Gynecologic Oncology: Medicine,' Biostatistics,' and MicrobiologyImmunology! Duke University, and the Duke Comprehensive Cancer Center.' Supported in part by Grant No. CA 39930 from the National Cancer Institute (A. K. and R. C. B.) and the Kennedy-Dannreuther Fellowship of the American Gynecological and Obstetrical Society (A. B.).
Received for publication July 12, 1990; accepted September 19, 1990. Reprint requests: Andrew Berchuck, MD, Duke University Medical Center, P.O. Box 3079, Durham, NC 27710. 611125555
lignant transformation. Initially, it was noted that viral transformation of normal rat fibroblasts is associated with expression of transforming growth factor-a, which acts to stimulate growth by binding to epidermal growth factor receptors that are present on the same cells. 2 This phenomenon has been referred to as autocrine growth stimulation. Abnormal expression of epidermal growth factor receptor and its ligands also has been noted in human cancers. In breast cancer it has been shown that only one third of cases express detectable epidermal growth factor receptor, and that epidermal growth factor receptor expression is inversely related to estrogen receptor expression and associated with poor prognosis." 4 Conversely, squamous cell cancers frequently overexpress epidermal growth factor receptor, although a direct relationship between overexpression and biologic behavior has not been demonstrated convincingly.5.6 Previously, we examined the relationship between epidermal growth factor receptor expression and prognostic factors in endometrial cancer.7 We found that most, but not all, endometrial cancers express epidermal growth factor receptor, but no relationship to steroid receptor status or prognosis was noted. Although epithelial ovarian cancer causes more deaths than all other gynecologic cancers combined,
669
670
Berchuck et al.
few studies have been performed to examine the relationship between epidermal growth factor receptor expression and the biologic behavior of ovarian cancer. In the present study, we used a monoclonal antibody reactive with the extracellular domain of the epidermal growth factor receptor to examine expression of this receptor in frozen sections of normal ovary and epithelial ovarian cancer. Epidermal growth factor receptor expression in the cancers was contrasted to that of normal epithelium, and the relationship between receptor expression and prognostic factors and survival in ovarian cancer was examined. Material and methods
Patients. All the patients in this study with ovarian cancer underwent exploratory laparotomy in conjunction with treatment for epithelial ovarian cancer at Duke University between 1985 and 1989. Frozen tumor samples from 87 patients were found to be adequate for immunohistochemical analysis. Tumor was obtained at the initial operation in 57 cases (7 stage I or II, 50 stage III or IV). Among these 57 patients, five also had tumor samples frozen at second-look or subsequent laparotomy. In an additional 30 patients, tumor samples were obtained only at second-look laparotomy or at the time of recurrence (7 stage I or II, 23 stage III or IV). In addition, samples of normal ovary were frozen from six patients undergoing hysterectomy and bilateral salpingo-oophorectomy for benign gynecologic diseases. All histologic material from the 57 patients in whom tumor samples were obtained at initial surgery was reviewed by a single pathologist (A.K.) and the histologic type (serous, mucinous, clear cell, endometrioid, undifferentiated) and histologic grade (well differentiated, moderately differentiated, poorly differentiated) were determined with the use of the World Health Organization criteria. 8 Most of the other 30 patients from whom tumor samples were obtained at a subsequent operation had undergone initial surgery elsewhere and slides were not available for review. However, in these 30 patients the diagnosis of epithelial ovarian cancer was confirmed by review of material obtained at a subsequent surgical procedure. Immunohistochemistry. All tissue samples were snap-frozen and stored at -70 0 C until analyzed. Tissue samples were frozen in tissue Tek O.C.T. compound (Ames Division, Miles Laboratories, Elkhart, Ind.) and 4 to 6 /Jom thick cryosections were mounted on gelatin-coated slides. The slides were air dried overnight at room temperature to enhance the cellular morphology. The slides were fixed in acetone for 10 minutes at room temperature and then air dried again for 30 minutes. Then the slides were washed in phosphatebuffered saline solution three times for 5 minutes. Immunohistochemical staining was performed with
February 1991 Am J Obstet Gynecol
the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.). Slides were placed in a humidified chamber and incubated for 15 minutes with several drops of a 1 : 200 dilution of horse serum in phosphatebuffered saline solution with 2% bovine serum albumin. Then, 70 /Jol of primary antibody was applied over the tissue sections for 60 minutes. After washing with phosphate-buffered saline solution, biotinylated goat antimouse immunoglobulin G (IgG) antibody was added for 30 minutes followed by the avidinperoxidase complex. Finally, the slides were developed for 4 minutes with the enzyme substrate diaminobenzidine (0.5% diaminobenzidine in 0.05% Tris hydrochloride buffer, 0.6% hydrogen peroxide). The slides then were rinsed for 10 minutes in running tap water, counterstained with 1% methyl green (Sigma Chemical Co., St. Louis), dehydrated, and mounted. Monoclonal antibodies. Purified 528 is a murine monoclonal antibody that is specifically reactive with the extracellular domain of the epidermal growth factor receptor. This antibody was generated by immunization of mice with A431 cells, which expressed markedly increased numbers of epidermal growth factor receptors 9 (528 was the generous gift of John Mendelsohn, Memorial Sloan-Kettering Cancer Center, New York). We tested 528 at concentrations of 0.01 to 5.0 /Jog/ml and determined that maximal immunohistochemical staining of the epidermal growth factor receptor was obtained with a concentration of 1.0 /Jog/ml. This concentration of antibody subsequently was used to stain all the tissue samples in this study. We used purified mouse IgG specific for nonhuman tissue (Coulter Immunology, Hialeah, Fla.) at a dilution of 1 : 100 as a negative control. Anticytokeratin AE 1 / AE3 antibodies (Boehringer-Mannheim Biochemicals, Indianapolis) were used as a positive control. Immunohistochemical staining of the epidermal growth factor receptor was evaluated with serial sections. First, a section stained with hematoxylin and eosin was examined to evaluate the histology. Then a negative control slide stained with nonspecific mouse IgG was examined to assess nonspecific staining. A positive control slide stained with anticytokeratin antibodies was examined to confirm the presence of viable tumor cells in the section. Cases in which the negative or positive controls were not adequate were repeated. Finally, the slide in which 528 was used as the primary antibody was examined. The intensity of staining for epidermal growth factor receptor was graded as 0 (staining not greater than negative control), 1 + (light staining), 2 + (moderate staining), or 3 + (heavy staining). The intensity of staining recorded was the consensus of two observers who used a double-headed microscope. The identity of the patients was not known to the observers while the slides were being scored. Statistics. Tables were analyzed with Fisher's two-
Epidermal growth factor receptor in ovarian cancer
Volume 164 Number 2
671
A
A
I
B Fig. 1. Immunohistochemical staining of epidermal growth factor receptor in normal ovarian epithelium, A, negative control; B, epidermal growth factor receptor.
Fig. 2. Immunohistochemical staining of epidermal growth factor receptor in ovarian cancer, A, negative control; B, epidermal growth factor receptor.
tailed exact test. Survival estimates were calculated with the Kaplan-Meier life table method. lo Differences In survival were tested with the log rank statistic. 11
growth factor receptor was absent in 11 (19%), light in 18 (32%), moderate in 22 (38%), and heavy in 6 patients (11 %). In addition to staining in the malignant epithelial tissue component, staining frequently was seen in the accompanying stroma (Fig. 2, A and B). In 11 patients with advanced-stage disease, tumor samples were obtained from multiple sites at the time of initial cytoreductive surgery. In these patients, epidermal growth factor receptor expression was evaluated in both the primary tumor and in one to three metastases. In all cases, the intensity of staining for epidermal growth factor receptor within a single patient was similar in the primary tumor and metastases. In addition, in five patients epidermal growth factor receptor expression was evaluated in tumor samples obtained both at initial surgery and at a subsequent laparotomy. In all cases, the intensity of staining for epidermal growth factor receptor was unchanged after therapy. Because epidermal growth factor receptor expression did not appear to be altered during the course of treatment, we also analyzed epidermal growth factor receptor expression in an additional 30 tumor samples
Results
Immunohistochemically detectable epidermal growth factor receptor was seen in normal ovarian epithelium in all cases examined. Homogeneous staining of all epithelial cells was noted in each case. In three cases, light staining was noted, and in three cases moderate staining was seen (Fig. 1, A and B). In all cases, lighter staining of the underlying stroma also was observed. Among the 57 patients with ovarian cancer in whom tumor was frozen at initial surgery, 41 patients had serous tumors, 6 patients had mucinous tumors, 3 patients had endometrioid tumors, 2 patients had clear cell tumors, 4 patients had undifferentiated tumors, and 1 patient had a mixed serous-clear cell tumor. Among these 57 patients, cancer was welldifferentiated in 6, moderately differentiated in 23, and poorly differentiated in 28. Staining for epidermal
672
Berchuck et al.
February 1991 Am J Obstet Gynecol
Table I. Relationship between epidermal growth factor receptor expression and prognostic factors in advanced epithelial ovarian cancer EGF receptor-negative
I
No. Histologic grade Well differentiated Moderately differentiated Poorly differentiated Cytoreduction Optimal «1 cm) Suboptimal (> 1 cm)
o
EGF receptor-positive
No.
o
4 17 20
49
6 31
84
33
3
I
%
6
66
3 10
23
77
% 10
41
16
EGF, Epidermal growth factor.
Table II. Relationship between epidermal growth factor receptor expression and findings at second-look laparotomy Second-look positive EGF receptor
Absent Present
Unevaluable patients
Progression of disease
4 13
9
o
Gross
4
23
I
Microscopic
Second-look negative
2 5
5 8
EGF, Epidermal growth factor.
obtained from patients at the time of second-look or other subsequent laparotomy. Among these 30 patients, staining for epidermal growth factor receptor was absent in 9 (30%), light in 8 (27%), moderate in 13 (43%), and heavy in none (0%). Overall, among all the patients in this study, epidermal growth factor receptor was absent in 20 of 87 cases of ovarian cancers studied (23%). Epidermal growth factor receptor was absent in 5 of 14 patients who had stage I or II disease (36%) compared with 15 of 73 patients who had stage III or IV disease (21 %). In Table I, the relationship between epidermal growth factor receptor expression and other prognostic factors is presented for the 50 patients with stage III or IV disease in whom tumor samples were obtained at primary surgery. There was no significant relationship between epidermal growth factor receptor expression and histologic grade or the ability to perform an optimal cytoreductive operation that left no tumor nodule> 1 cm in diameter. Table II examines the relationship between epidermal growth factor receptor expression and response to primary therapy in the 73 patients with advanced-stage disease. Among the 15 patients whose tumors did not express epidermal growth factor receptor, 4 patients (27%) did not undergo second-look laparotomy because of patient refusal or poor medical condition. Among the 58 patients with tumors in which staining for epidermal growth factor receptor was seen, 13 (22%) did not undergo second-look laparotomy. Although 5 of 15 patients whose tumors did not express
epidermal growth factor receptor achieved a negative second-look laparotomy (33%) compared with only 8 of 49 evaluable patients whose tumors did express epidermal growth facta):' receptor (16%), this difference was not statistically significant (p = 0.1). In Fig. 3, the relationship between epidermal growth factor receptor expression and survival is shown. Among all 87 patients, survival of patients whose tumors did not express epidermal growth factor receptor was significantly better than that of patients whose tumors did express epidermal growth factor receptor (p < 0.05). The median survival of patients whose tumors did not express epidermal growth factor receptor was 40 months compared with 26 months in patients whose tumors did express epidermal growth factor receptor. If only the 73 patients with stage III or IV disease are considered, the survival advantage for epidermal growth factor receptor-negative cases was at the limit of statistical significance (p = 0.06). The median survival of patients whose tumors did not express epidermal growth factor receptor was 37 months compared with 21 months in patients whose tumors did express epidermal growth factor receptor.
Comment Previously, it was shown that epidermal growth factor receptor is expressed by a wide range of normal human cells of both epithelial and mesenchymal origin. For example, in the uterus, we and others have shown that epidermal growth factor receptor is expressed by endometrial glands, endometrial stromal cells, and myo-
Epidermal growth factor receptor in ovarian cancer
Volume 164 Kumber 2
metrium. 7. !2 Similarly, in this study both the surface epithelium and underlying stroma of the ovary were found to express epidermal growth factor receptor. The physiologic role of epidermal growth factor receptor in the ovary remains unknown. Studies of epidermal growth factor receptor expression in human cancers generally have found that epidermal growth factor receptor levels are increased in some squamous cell cancers, whereas a proportion of adenocarcinomas appear to have lost epidermal growth factor receptor relative to normal glandular epithelium. In some but not all studies, alterations in epidermal growth factor receptor expression were found to correlate with clinical outcome. For example, a group from Newcastle, England, found that absence of epidermal growth factor receptor is associated with favorable clinical outcome in patients with breast' 4 and bladder cancer.!3 Conversely, in lung!4 and endometrial' adenocarcinomas, no such relationship was noted. In squamous cell cancers of the lung, one group reported that those with the highest levels of epidermal growth factor receptor (greater than fivefold increased) have poor survival rates.!5 However, other studies of squamous cell cancers arising in the head and neck6 and other sites have not confirmed these findings. Although ovarian cancer causes more deaths than all other gynecologic cancers combined, little data have been reported with regard to epidermal growth factor receptor expression in ovarian cancer. A single study reported that high epidermal growth factor receptor expression in ovarian tumors correlated with a favorable prognosis.!6 However, in this study all adnexal tumors including epithelial, germ cell, and stromal tumors were analyzed together. In the present study, we examined epidermal growth factor receptor expression in a group of patients with epithelial ovarian cancer with the use of an immunohistochemical technique. Immunohistochemically detectable epidermal growth factor receptor was not seen in 23% of cases. This incidence of epidermal growth factor receptor negativity is similar to that found in other immunohistochemical studies of epidermal growth factor receptor in lung!1 and endometrial cancers.7 On the other hand, studies that assess epidermal growth factor receptor expression by measuring binding of radio labeled epidermal growth factor to tissue homogenates usually report a higher incidence of epidermal growth factor receptor negativity, for example, one half to two thirds in breast cancer. 3 . 4 Some of the differences in conclusions between different studies of the same type of cancer may relate to these methodologic differences. Whereas binding assays are quantitative, it is clear that benign stromal elements in homogenized tumor samples contribute to epidermal growth factor receptor content. On the other hand, although immunohistochemical
673
A 1.0 0.8
c:: •52
\~ ...
J:: f
;"';""':
;
..
~
" "'\.~ ,
0.2 l 0.0
~----'--------'----'---_'-
o
20
40
60
80
_ _-' 100
Months
B 1.0
~--,
"
'~\.t.,
0.8
c::
.g...
0.6
:::
0.4
'"
0..
'. '\ .
op.
' ... ~.
'''\ '''~
0.2 0.0
~
o
_ _L -_ _- ' - -_ _- , - -_ _~_ _--, 20 40 60 80 100
Months Fig. 3. Relationship between epidermal growth factor receptor expression and survival of patients with ovarian cancer (solid line, epidermal growth factor receptor negative; dashed line, epidermal growth factor receptor positive), A, all 87 patients (p < 0.05); B, 73 patients with advanced-stage disease (p = 0.06).
techniques allow direct assessment of epidermal growth factor receptor expression by cancer cells, evaluation of the intensity of staining is subjective. In this initial study, with the use of univariate analysis, we found a statistically significant relationship between epidermal growth factor receptor expression and survival in patients with epithelial ovarian cancer. Similar to breast and bladder cancer, absence of detectable epidermal growth factor receptor was associated with better long-term survival. Furthermore, although not statistically significant because of the small number of early stage patients, we found that absence of epidermal growth factor receptor was more common in stage I or II tumors (36%) compared with stage III or IV tumors (21 %). This also is suggestive that absence of epidermal growth factor receptor is associated with cancers of lower biologic aggressiveness. Larger studies in which multivariate analysis can be used are needed to determine whether epidermal growth factor receptor status is an independent prognostic factor in ovarian cancer. More recently, two other membrane-spanning
674
Berchuck et al.
receptor-like molecules were discovered that have a high degree of homology to the epidermal growth factor receptor. The first, HER-2/neu (c-erb B2),17 has been reported by Slamon 18 to be overexpressed in approximately 30% of breast and ovarian cancers. We confirmed the association between HER-2/neu overexpression and poor prognosis in ovarian cancer. I9 Both Slamon's study and ours found that the prognostic value of HER-2/neu overexpression in ovarian cancer was much stronger than that observed for epidermal growth factor receptor status in the present study. In addition, more recently, a third member of the erb B family (c-erb B3) has been identified!O It has been noted that c-erb B3, like c-erb B2, is overexpressed in some breast cancers. No studies of c-erb B3 expression in ovarian cancer have been performed to date. In the future, in addition to studies such as the present one that examine the prognostic significance of receptor expression, it will be critical to determine the actual biomolecular role of the c-erb B family of receptors in the process of malignant transformation of normal ovarian epithelium. We thank Regina Whitaker and Bennie Penny for their expert technical assistance. REFERENCES 1. Carpenter G. Properties of the receptor for epidermal growth factor. Cell 1984;37:357-8. 2. Todaro Gj, Fryling C, DeLarco J. Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Nat! Acad Sci 1980:77:5258-62. 3. Sainsbury jRC, Farndon jR, Needham GK, Malcolm Aj, Harris AL. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985; 1:364-6. 4. Sainsbury jR, Farndon jR, Needham GK, Malcolm Aj, Harris AL. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1987;1:1398-402. 5. Ozanne B, Richards CS, Hendler F, Burns D, Gusterson B. Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas. j Pathol 1986; 194:9-14.
February 1991 Am J Obstet Gynecol
6. Ishitoya j, Toriyama M, Oguchi N, et al. Gene amplification and overexpression of EGF receptor in squamous cell carcinomas of the head and neck. Br j Cancer 1989;59:559-62. 7. Berchuck A, Soisson AP, Olt Gj, et al. Epidermal growth factor receptor expression in normal and malignant endometrium. AM j OBSTET GYNECOL 1989;161:1247-52. 8. Serov SF, Scully RE. Histological typing of ovarian tumors. In: International Histological Classification of Tumors, No.9. Geneva: World Health Organization, 1973. 9. Kawamoto T, Sato jD, Le A, Polikoff j, Sato GH, Mendelsohn J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Nat! Acad Sci USA 1983;80: 1337 -41. 10. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. j Am Stat Assoc 1958;53:457-81. II. Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep 1966;50:163-70. 12. Lin TH, Mukku VR, Verner G, KirklandjL, Stancel GM. Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types. Bioi Reprod 1988;38:403-11. 13. Neal DE, Sharples L, Smith K, Fennelly j, Hall RR, Harris AL. The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer 1990;65: 1619-25. 14. Sobol RE, Astarita RW, Hofeditz C, et al. Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. jNCI 1987; 79:403-7. 15. Hendler Fj, Shum-Siu A, Oechsli M, Nanu L, Richards CS, Ozanne BW. Increased EGF-Rl binding predicts a poor survival in squamous tumors. Cancer Cells 1989; 7:347-51. 16. Bauknecht T, Runge M, Schwall M, Pfeiderer A. Occurrence of epidermal growth factor receptors in human adnexal tumors and their prognostic value in advanced ovarian carcinomas. Gynecol OncoI1988;29:147-57. 17. Schechter AL, Stern DF, Vaidyanathan L, et al. The neu oncogene: an erb B-related gene encoding a 185,000-M tumor antigen. Nature 1984;312:513-6. 18. Siamon Dj, Godolphin W,jones LA, etal. Studies of HER2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707-12. 19. Berchuck A, Kamel A, Whitaker R, et al. Overexpression ofHER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res 1990;50:4087-91. 20. Kraus MH, Issing W, Miki T, Popescu NC, Aaronson SA. Isolation and characterization of ERB B3, a third member of the ERB B/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Nat! Acad Sci 1989;86:9193-7.
1991 ;164:669-74.)
Key words: Epidermal growth factor receptor, epidermal growth factor, ovarian cancer
The epidermal growth factor receptor is a cell surface molecule that is widely distributed in normal human tissues. In vitro, after binding of one of its ligands, either epidermal growth factor or transforming growth factor-a, to the extracellular domain of the receptor, the tyrosine kinase of the intracytoplasmic domain is activated. This results in the generation of second messengers that transmit the mitogenic signal to the nucleus. 1 In vivo, the epidermal growth factor receptor and its ligands are thought to play a role in normal growth and differentiation although the precise nature of this involvement is not understood. Abnormal expression of the epidermal growth factor receptor and its ligands has been associated with ma-
From the Departments of Obstetrics and Gynecology, Division of Gynecologic Oncology: Medicine,' Biostatistics,' and MicrobiologyImmunology! Duke University, and the Duke Comprehensive Cancer Center.' Supported in part by Grant No. CA 39930 from the National Cancer Institute (A. K. and R. C. B.) and the Kennedy-Dannreuther Fellowship of the American Gynecological and Obstetrical Society (A. B.).
Received for publication July 12, 1990; accepted September 19, 1990. Reprint requests: Andrew Berchuck, MD, Duke University Medical Center, P.O. Box 3079, Durham, NC 27710. 611125555
lignant transformation. Initially, it was noted that viral transformation of normal rat fibroblasts is associated with expression of transforming growth factor-a, which acts to stimulate growth by binding to epidermal growth factor receptors that are present on the same cells. 2 This phenomenon has been referred to as autocrine growth stimulation. Abnormal expression of epidermal growth factor receptor and its ligands also has been noted in human cancers. In breast cancer it has been shown that only one third of cases express detectable epidermal growth factor receptor, and that epidermal growth factor receptor expression is inversely related to estrogen receptor expression and associated with poor prognosis." 4 Conversely, squamous cell cancers frequently overexpress epidermal growth factor receptor, although a direct relationship between overexpression and biologic behavior has not been demonstrated convincingly.5.6 Previously, we examined the relationship between epidermal growth factor receptor expression and prognostic factors in endometrial cancer.7 We found that most, but not all, endometrial cancers express epidermal growth factor receptor, but no relationship to steroid receptor status or prognosis was noted. Although epithelial ovarian cancer causes more deaths than all other gynecologic cancers combined,
669
670
Berchuck et al.
few studies have been performed to examine the relationship between epidermal growth factor receptor expression and the biologic behavior of ovarian cancer. In the present study, we used a monoclonal antibody reactive with the extracellular domain of the epidermal growth factor receptor to examine expression of this receptor in frozen sections of normal ovary and epithelial ovarian cancer. Epidermal growth factor receptor expression in the cancers was contrasted to that of normal epithelium, and the relationship between receptor expression and prognostic factors and survival in ovarian cancer was examined. Material and methods
Patients. All the patients in this study with ovarian cancer underwent exploratory laparotomy in conjunction with treatment for epithelial ovarian cancer at Duke University between 1985 and 1989. Frozen tumor samples from 87 patients were found to be adequate for immunohistochemical analysis. Tumor was obtained at the initial operation in 57 cases (7 stage I or II, 50 stage III or IV). Among these 57 patients, five also had tumor samples frozen at second-look or subsequent laparotomy. In an additional 30 patients, tumor samples were obtained only at second-look laparotomy or at the time of recurrence (7 stage I or II, 23 stage III or IV). In addition, samples of normal ovary were frozen from six patients undergoing hysterectomy and bilateral salpingo-oophorectomy for benign gynecologic diseases. All histologic material from the 57 patients in whom tumor samples were obtained at initial surgery was reviewed by a single pathologist (A.K.) and the histologic type (serous, mucinous, clear cell, endometrioid, undifferentiated) and histologic grade (well differentiated, moderately differentiated, poorly differentiated) were determined with the use of the World Health Organization criteria. 8 Most of the other 30 patients from whom tumor samples were obtained at a subsequent operation had undergone initial surgery elsewhere and slides were not available for review. However, in these 30 patients the diagnosis of epithelial ovarian cancer was confirmed by review of material obtained at a subsequent surgical procedure. Immunohistochemistry. All tissue samples were snap-frozen and stored at -70 0 C until analyzed. Tissue samples were frozen in tissue Tek O.C.T. compound (Ames Division, Miles Laboratories, Elkhart, Ind.) and 4 to 6 /Jom thick cryosections were mounted on gelatin-coated slides. The slides were air dried overnight at room temperature to enhance the cellular morphology. The slides were fixed in acetone for 10 minutes at room temperature and then air dried again for 30 minutes. Then the slides were washed in phosphatebuffered saline solution three times for 5 minutes. Immunohistochemical staining was performed with
February 1991 Am J Obstet Gynecol
the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.). Slides were placed in a humidified chamber and incubated for 15 minutes with several drops of a 1 : 200 dilution of horse serum in phosphatebuffered saline solution with 2% bovine serum albumin. Then, 70 /Jol of primary antibody was applied over the tissue sections for 60 minutes. After washing with phosphate-buffered saline solution, biotinylated goat antimouse immunoglobulin G (IgG) antibody was added for 30 minutes followed by the avidinperoxidase complex. Finally, the slides were developed for 4 minutes with the enzyme substrate diaminobenzidine (0.5% diaminobenzidine in 0.05% Tris hydrochloride buffer, 0.6% hydrogen peroxide). The slides then were rinsed for 10 minutes in running tap water, counterstained with 1% methyl green (Sigma Chemical Co., St. Louis), dehydrated, and mounted. Monoclonal antibodies. Purified 528 is a murine monoclonal antibody that is specifically reactive with the extracellular domain of the epidermal growth factor receptor. This antibody was generated by immunization of mice with A431 cells, which expressed markedly increased numbers of epidermal growth factor receptors 9 (528 was the generous gift of John Mendelsohn, Memorial Sloan-Kettering Cancer Center, New York). We tested 528 at concentrations of 0.01 to 5.0 /Jog/ml and determined that maximal immunohistochemical staining of the epidermal growth factor receptor was obtained with a concentration of 1.0 /Jog/ml. This concentration of antibody subsequently was used to stain all the tissue samples in this study. We used purified mouse IgG specific for nonhuman tissue (Coulter Immunology, Hialeah, Fla.) at a dilution of 1 : 100 as a negative control. Anticytokeratin AE 1 / AE3 antibodies (Boehringer-Mannheim Biochemicals, Indianapolis) were used as a positive control. Immunohistochemical staining of the epidermal growth factor receptor was evaluated with serial sections. First, a section stained with hematoxylin and eosin was examined to evaluate the histology. Then a negative control slide stained with nonspecific mouse IgG was examined to assess nonspecific staining. A positive control slide stained with anticytokeratin antibodies was examined to confirm the presence of viable tumor cells in the section. Cases in which the negative or positive controls were not adequate were repeated. Finally, the slide in which 528 was used as the primary antibody was examined. The intensity of staining for epidermal growth factor receptor was graded as 0 (staining not greater than negative control), 1 + (light staining), 2 + (moderate staining), or 3 + (heavy staining). The intensity of staining recorded was the consensus of two observers who used a double-headed microscope. The identity of the patients was not known to the observers while the slides were being scored. Statistics. Tables were analyzed with Fisher's two-
Epidermal growth factor receptor in ovarian cancer
Volume 164 Number 2
671
A
A
I
B Fig. 1. Immunohistochemical staining of epidermal growth factor receptor in normal ovarian epithelium, A, negative control; B, epidermal growth factor receptor.
Fig. 2. Immunohistochemical staining of epidermal growth factor receptor in ovarian cancer, A, negative control; B, epidermal growth factor receptor.
tailed exact test. Survival estimates were calculated with the Kaplan-Meier life table method. lo Differences In survival were tested with the log rank statistic. 11
growth factor receptor was absent in 11 (19%), light in 18 (32%), moderate in 22 (38%), and heavy in 6 patients (11 %). In addition to staining in the malignant epithelial tissue component, staining frequently was seen in the accompanying stroma (Fig. 2, A and B). In 11 patients with advanced-stage disease, tumor samples were obtained from multiple sites at the time of initial cytoreductive surgery. In these patients, epidermal growth factor receptor expression was evaluated in both the primary tumor and in one to three metastases. In all cases, the intensity of staining for epidermal growth factor receptor within a single patient was similar in the primary tumor and metastases. In addition, in five patients epidermal growth factor receptor expression was evaluated in tumor samples obtained both at initial surgery and at a subsequent laparotomy. In all cases, the intensity of staining for epidermal growth factor receptor was unchanged after therapy. Because epidermal growth factor receptor expression did not appear to be altered during the course of treatment, we also analyzed epidermal growth factor receptor expression in an additional 30 tumor samples
Results
Immunohistochemically detectable epidermal growth factor receptor was seen in normal ovarian epithelium in all cases examined. Homogeneous staining of all epithelial cells was noted in each case. In three cases, light staining was noted, and in three cases moderate staining was seen (Fig. 1, A and B). In all cases, lighter staining of the underlying stroma also was observed. Among the 57 patients with ovarian cancer in whom tumor was frozen at initial surgery, 41 patients had serous tumors, 6 patients had mucinous tumors, 3 patients had endometrioid tumors, 2 patients had clear cell tumors, 4 patients had undifferentiated tumors, and 1 patient had a mixed serous-clear cell tumor. Among these 57 patients, cancer was welldifferentiated in 6, moderately differentiated in 23, and poorly differentiated in 28. Staining for epidermal
672
Berchuck et al.
February 1991 Am J Obstet Gynecol
Table I. Relationship between epidermal growth factor receptor expression and prognostic factors in advanced epithelial ovarian cancer EGF receptor-negative
I
No. Histologic grade Well differentiated Moderately differentiated Poorly differentiated Cytoreduction Optimal «1 cm) Suboptimal (> 1 cm)
o
EGF receptor-positive
No.
o
4 17 20
49
6 31
84
33
3
I
%
6
66
3 10
23
77
% 10
41
16
EGF, Epidermal growth factor.
Table II. Relationship between epidermal growth factor receptor expression and findings at second-look laparotomy Second-look positive EGF receptor
Absent Present
Unevaluable patients
Progression of disease
4 13
9
o
Gross
4
23
I
Microscopic
Second-look negative
2 5
5 8
EGF, Epidermal growth factor.
obtained from patients at the time of second-look or other subsequent laparotomy. Among these 30 patients, staining for epidermal growth factor receptor was absent in 9 (30%), light in 8 (27%), moderate in 13 (43%), and heavy in none (0%). Overall, among all the patients in this study, epidermal growth factor receptor was absent in 20 of 87 cases of ovarian cancers studied (23%). Epidermal growth factor receptor was absent in 5 of 14 patients who had stage I or II disease (36%) compared with 15 of 73 patients who had stage III or IV disease (21 %). In Table I, the relationship between epidermal growth factor receptor expression and other prognostic factors is presented for the 50 patients with stage III or IV disease in whom tumor samples were obtained at primary surgery. There was no significant relationship between epidermal growth factor receptor expression and histologic grade or the ability to perform an optimal cytoreductive operation that left no tumor nodule> 1 cm in diameter. Table II examines the relationship between epidermal growth factor receptor expression and response to primary therapy in the 73 patients with advanced-stage disease. Among the 15 patients whose tumors did not express epidermal growth factor receptor, 4 patients (27%) did not undergo second-look laparotomy because of patient refusal or poor medical condition. Among the 58 patients with tumors in which staining for epidermal growth factor receptor was seen, 13 (22%) did not undergo second-look laparotomy. Although 5 of 15 patients whose tumors did not express
epidermal growth factor receptor achieved a negative second-look laparotomy (33%) compared with only 8 of 49 evaluable patients whose tumors did express epidermal growth facta):' receptor (16%), this difference was not statistically significant (p = 0.1). In Fig. 3, the relationship between epidermal growth factor receptor expression and survival is shown. Among all 87 patients, survival of patients whose tumors did not express epidermal growth factor receptor was significantly better than that of patients whose tumors did express epidermal growth factor receptor (p < 0.05). The median survival of patients whose tumors did not express epidermal growth factor receptor was 40 months compared with 26 months in patients whose tumors did express epidermal growth factor receptor. If only the 73 patients with stage III or IV disease are considered, the survival advantage for epidermal growth factor receptor-negative cases was at the limit of statistical significance (p = 0.06). The median survival of patients whose tumors did not express epidermal growth factor receptor was 37 months compared with 21 months in patients whose tumors did express epidermal growth factor receptor.
Comment Previously, it was shown that epidermal growth factor receptor is expressed by a wide range of normal human cells of both epithelial and mesenchymal origin. For example, in the uterus, we and others have shown that epidermal growth factor receptor is expressed by endometrial glands, endometrial stromal cells, and myo-
Epidermal growth factor receptor in ovarian cancer
Volume 164 Kumber 2
metrium. 7. !2 Similarly, in this study both the surface epithelium and underlying stroma of the ovary were found to express epidermal growth factor receptor. The physiologic role of epidermal growth factor receptor in the ovary remains unknown. Studies of epidermal growth factor receptor expression in human cancers generally have found that epidermal growth factor receptor levels are increased in some squamous cell cancers, whereas a proportion of adenocarcinomas appear to have lost epidermal growth factor receptor relative to normal glandular epithelium. In some but not all studies, alterations in epidermal growth factor receptor expression were found to correlate with clinical outcome. For example, a group from Newcastle, England, found that absence of epidermal growth factor receptor is associated with favorable clinical outcome in patients with breast' 4 and bladder cancer.!3 Conversely, in lung!4 and endometrial' adenocarcinomas, no such relationship was noted. In squamous cell cancers of the lung, one group reported that those with the highest levels of epidermal growth factor receptor (greater than fivefold increased) have poor survival rates.!5 However, other studies of squamous cell cancers arising in the head and neck6 and other sites have not confirmed these findings. Although ovarian cancer causes more deaths than all other gynecologic cancers combined, little data have been reported with regard to epidermal growth factor receptor expression in ovarian cancer. A single study reported that high epidermal growth factor receptor expression in ovarian tumors correlated with a favorable prognosis.!6 However, in this study all adnexal tumors including epithelial, germ cell, and stromal tumors were analyzed together. In the present study, we examined epidermal growth factor receptor expression in a group of patients with epithelial ovarian cancer with the use of an immunohistochemical technique. Immunohistochemically detectable epidermal growth factor receptor was not seen in 23% of cases. This incidence of epidermal growth factor receptor negativity is similar to that found in other immunohistochemical studies of epidermal growth factor receptor in lung!1 and endometrial cancers.7 On the other hand, studies that assess epidermal growth factor receptor expression by measuring binding of radio labeled epidermal growth factor to tissue homogenates usually report a higher incidence of epidermal growth factor receptor negativity, for example, one half to two thirds in breast cancer. 3 . 4 Some of the differences in conclusions between different studies of the same type of cancer may relate to these methodologic differences. Whereas binding assays are quantitative, it is clear that benign stromal elements in homogenized tumor samples contribute to epidermal growth factor receptor content. On the other hand, although immunohistochemical
673
A 1.0 0.8
c:: •52
\~ ...
J:: f
;"';""':
;
..
~
" "'\.~ ,
0.2 l 0.0
~----'--------'----'---_'-
o
20
40
60
80
_ _-' 100
Months
B 1.0
~--,
"
'~\.t.,
0.8
c::
.g...
0.6
:::
0.4
'"
0..
'. '\ .
op.
' ... ~.
'''\ '''~
0.2 0.0
~
o
_ _L -_ _- ' - -_ _- , - -_ _~_ _--, 20 40 60 80 100
Months Fig. 3. Relationship between epidermal growth factor receptor expression and survival of patients with ovarian cancer (solid line, epidermal growth factor receptor negative; dashed line, epidermal growth factor receptor positive), A, all 87 patients (p < 0.05); B, 73 patients with advanced-stage disease (p = 0.06).
techniques allow direct assessment of epidermal growth factor receptor expression by cancer cells, evaluation of the intensity of staining is subjective. In this initial study, with the use of univariate analysis, we found a statistically significant relationship between epidermal growth factor receptor expression and survival in patients with epithelial ovarian cancer. Similar to breast and bladder cancer, absence of detectable epidermal growth factor receptor was associated with better long-term survival. Furthermore, although not statistically significant because of the small number of early stage patients, we found that absence of epidermal growth factor receptor was more common in stage I or II tumors (36%) compared with stage III or IV tumors (21 %). This also is suggestive that absence of epidermal growth factor receptor is associated with cancers of lower biologic aggressiveness. Larger studies in which multivariate analysis can be used are needed to determine whether epidermal growth factor receptor status is an independent prognostic factor in ovarian cancer. More recently, two other membrane-spanning
674
Berchuck et al.
receptor-like molecules were discovered that have a high degree of homology to the epidermal growth factor receptor. The first, HER-2/neu (c-erb B2),17 has been reported by Slamon 18 to be overexpressed in approximately 30% of breast and ovarian cancers. We confirmed the association between HER-2/neu overexpression and poor prognosis in ovarian cancer. I9 Both Slamon's study and ours found that the prognostic value of HER-2/neu overexpression in ovarian cancer was much stronger than that observed for epidermal growth factor receptor status in the present study. In addition, more recently, a third member of the erb B family (c-erb B3) has been identified!O It has been noted that c-erb B3, like c-erb B2, is overexpressed in some breast cancers. No studies of c-erb B3 expression in ovarian cancer have been performed to date. In the future, in addition to studies such as the present one that examine the prognostic significance of receptor expression, it will be critical to determine the actual biomolecular role of the c-erb B family of receptors in the process of malignant transformation of normal ovarian epithelium. We thank Regina Whitaker and Bennie Penny for their expert technical assistance. REFERENCES 1. Carpenter G. Properties of the receptor for epidermal growth factor. Cell 1984;37:357-8. 2. Todaro Gj, Fryling C, DeLarco J. Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Nat! Acad Sci 1980:77:5258-62. 3. Sainsbury jRC, Farndon jR, Needham GK, Malcolm Aj, Harris AL. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985; 1:364-6. 4. Sainsbury jR, Farndon jR, Needham GK, Malcolm Aj, Harris AL. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1987;1:1398-402. 5. Ozanne B, Richards CS, Hendler F, Burns D, Gusterson B. Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas. j Pathol 1986; 194:9-14.
February 1991 Am J Obstet Gynecol
6. Ishitoya j, Toriyama M, Oguchi N, et al. Gene amplification and overexpression of EGF receptor in squamous cell carcinomas of the head and neck. Br j Cancer 1989;59:559-62. 7. Berchuck A, Soisson AP, Olt Gj, et al. Epidermal growth factor receptor expression in normal and malignant endometrium. AM j OBSTET GYNECOL 1989;161:1247-52. 8. Serov SF, Scully RE. Histological typing of ovarian tumors. In: International Histological Classification of Tumors, No.9. Geneva: World Health Organization, 1973. 9. Kawamoto T, Sato jD, Le A, Polikoff j, Sato GH, Mendelsohn J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Nat! Acad Sci USA 1983;80: 1337 -41. 10. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. j Am Stat Assoc 1958;53:457-81. II. Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep 1966;50:163-70. 12. Lin TH, Mukku VR, Verner G, KirklandjL, Stancel GM. Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types. Bioi Reprod 1988;38:403-11. 13. Neal DE, Sharples L, Smith K, Fennelly j, Hall RR, Harris AL. The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer 1990;65: 1619-25. 14. Sobol RE, Astarita RW, Hofeditz C, et al. Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. jNCI 1987; 79:403-7. 15. Hendler Fj, Shum-Siu A, Oechsli M, Nanu L, Richards CS, Ozanne BW. Increased EGF-Rl binding predicts a poor survival in squamous tumors. Cancer Cells 1989; 7:347-51. 16. Bauknecht T, Runge M, Schwall M, Pfeiderer A. Occurrence of epidermal growth factor receptors in human adnexal tumors and their prognostic value in advanced ovarian carcinomas. Gynecol OncoI1988;29:147-57. 17. Schechter AL, Stern DF, Vaidyanathan L, et al. The neu oncogene: an erb B-related gene encoding a 185,000-M tumor antigen. Nature 1984;312:513-6. 18. Siamon Dj, Godolphin W,jones LA, etal. Studies of HER2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707-12. 19. Berchuck A, Kamel A, Whitaker R, et al. Overexpression ofHER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res 1990;50:4087-91. 20. Kraus MH, Issing W, Miki T, Popescu NC, Aaronson SA. Isolation and characterization of ERB B3, a third member of the ERB B/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Nat! Acad Sci 1989;86:9193-7.
