- Email: [email protected]
Osmotic lysis of tumor spill in ovarian cancer: A murine model
Osmotic lysis of tumor spill in ovarian cancer: A murine model Peter C. Morris, MD, and Valerie Scholten, MD Omaha, Nebraska OBJECTIVE: Our purpose was to determine, in the murine model, whether human ovarian cancer cells injected intraperitoneally are subject to osmotic lysis by peritoneal lavage with sterile water, thereby decreasing the establishment of peritoneal implants. STUDY DESIGN: Preliminary experiments on six nude mice determined that the injection of 20 million cells of the SKOV-3 cell line reliably leads to the establishment of intraperitoneal tumor xenografts in the mice within 60 days. Four other nude mice functioned as sham controls undergoing peritoneal lavage with 3 to 4 ml of saline solution or sterile water to determine any adverse effects from the lavage alone. Subsequently, 36 nude (nu/nu) mice were injected intraperitoneally with 1 ml of the SKOV-3 cell line at a concentration of 20 million cells per milliliter. Alternate mice then underwent intraperitoneal lavage with either 3 to 4 ml of normal saline solution (control group) or sterile water (study group). The mice were followed up until tumor growth caused a moribund status or until 60 days after injection and then were killed. At necropsy the number and size of tumor nodules were recorded, and each mouse was assigned a composite tumor score. Statistical comparison used the x 2 or Fisher's exact test for discrete variables. Time to failure analysis used the Kaplan-Meier method. RESULTS: Tumor growth occurred in 35 of 36 (97%) of the mice during the study period. In the first 30 days 89% of the saline solution group grew clinically visible tumor compared with 55% of the water group (/3 = 0.03). Ascites developed more frequently in the water group than in the saline solution group. The median tumor scores at death were significantly higher for the water group versus the saline solution group. Survival time, as determined by the time from injection until moribund status, was worse for the water group (p = 0.002). CONCLUSIONS: Intraperitoneal lavage with sterile water did not offer protection against the establishment of xenografts after the intraperitoneal injection of human ovarian cancer cells in the nude mouse model. (Am J Obstet Gynecol 1996;175:1489-92.)
Key words: Ovarian cancer, t u m o r spill, osmotic lysis
Ovarian cancer is the second most c o m m o n gynecologic malignancy, with an estimated 26,600 new cases expected in 1995 leading to 14,500 d e a t h s ) It is the fourth leading cause of cancer deaths in women. Early diagnosis is the exception rather than the rule. Approximately 15% to 25% of patients with epithelial ovarian cancer have disease c o n f i n e d to o n e or both ovaries. Epithelial ovarian cancer c o n f i n e d to the ovary at diagnosis has a reported 5-year survival of u p to 90%. There is significant controversy about prognostic factors in early-stage disease. T u m o r grade has b e e n shown to be an i m p o r t a n t prognostic factor. 2 The significance of t u m o r r u p t u r e at surgery remains controversial a n d unanswered. I n t e r n a t i o n a l Federation of Gynecology a n d Obstetrics (FIGO) staging criteria for ovarian cancer From the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Nebraska Medical Center. Presented at the Twenty-second Annual Meeting of the Society of Gynecologic Surgeons, Albuquerque, New Mexico, March 4-6, 1996. Reprint requests: Peter C. Morris, MD, Texas Oncology, 918 Eighth Ave., Fort Worth, TX 76104. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00 + 0 6/6/76166
imply that cyst r u p t u r e is an adverse prognostic factor by up-staging r u p t u r e d m a l i g n a n t tumors apparently confined to one or both ovaries to stage IC equated with similar tumors with m a l i g n a n t ascites. ~ The 1990 FIGO report quotes a 5-year survival of 82.3%, 74.9%, a n d 67.7% for stages IA, IB, a n d IC, respectively. 4 Some studies have shown a worse prognosis for intraoperative r u p t u r e of m a l i g n a n t cysts, 5 whereas others have failed to show a negative impact of cyst rupture. 2' 6-s Some authors r e c o m m e n d lavage of the peritoneal cavity with sterile water in the event of cyst r u p t u r e to affect osmotic lysis of t u m o r ceils, thus decreasing the chance of viable t u m o r implantation. 9 Although theoretically sound, the usefulness of osmotic lysis of t u m o r cells has n o t b e e n investigated. The purpose of this study was to determine whether viable h u m a n ovarian cancer cells injected or "spilled" intraperitoneally in the n u d e mouse m o d e l are subject to osmotic lysis by peritoneal lavage with sterile water. Methods
The experimental protocol was approved by the University Institutional A n i m a l Care a n d Use Committee. 1489
1490
Morris and Scholten
December 1996 Am J Obstet Gynecol
Table I. Assigned t u m o r score
Table II. Summary of study results
No. of points
Criterion
0
No evidence of tumor growth Tumor at injection site, no intraperitoneal growth Intraperitoneal nodules --<1 mm greatest dimension Intraperitoneal nodules 1-5 mm greatest dimension Intraperitoneal nodules >5 mm greatest dimension, confluent plaques, ascites, or diffuse carcinomatosis
1
2 3 4
H u m a n ovarian epithelial cancer cells (SKOV-3) were obtained from an established h u m a n ovarian cancer tissue culture from the A m e r i c a n Type Culture Collection (Rockville, Md.). Cells were grown in Dulbecco's modified Eagle's m e d i u m with 15% fetal calf serum and 1% L-glutamine. At harvest trypsinized cells were diluted with Dulbecco's modified Eagle's m e d i u m , centrifuged, and c o u n t e d with a h e m o c y t o m e t e r and r e s u s p e n d e d in a c o n c e n t r a t i o n of 2 × 10 7 cells per 1 ml of Dulbecco's m o d i f i e d Eagle's m e d i u m . Athymic ( n u / n u ) n u d e mice (Harlan Sprague) at 6 to 8 weeks old were housed in a pathogen-free e n v i r o n m e n t with controlled 12-hour light-dark cycles with free access to food and water. Preliminary experiments were perf o r m e d on six mice to d e t e r m i n e the optimal n u m b e r of h u m a n ovarian cancer cells to be inoculated to p r o d u c e implants and to d e t e r m i n e t h e time to d e v e l o p m e n t of the clinical ovarian cancer syndrome in the mice. Mice receiving 15 to 20 million cells had clinically detectable intraabdominal t u m o r by 60 days. Four o t h e r mice f u n c t i o n e d as sham controls, u n d e r g o i n g peritoneal lavage with 3 to 4 ml of n o r m a l saline solution or sterile water until the a b d o m e n distended to d e t e r m i n e any adverse effects f r o m the lavage alone. The study g r o u p consisted of 36 mice injected intraperitoneally with 1 ml of the SKOV-3 cell line at a c o n c e n t r a t i o n of 2 × 10 7 c e l l s / m l with a 21-gauge butterfly needle. After this injection alternate mice u n d e r w e n t intraperitoneal lavage with either 3 to 4 ml of n o r m a l saline solution (control group) or 3 to 4 ml of sterile water (study group). This volume of lavage solution was sufficient to distend the a b d o m e n . Aspiration of lavage solution p r o v e d difficult; only 0.5 to 1 ml was o b t a i n e d in return. T h e mice were m o n i t o r e d daily for general condition and t u m o r growth. Mice showing clinical signs of ascites, massive intraabdominal t u m o r growth, or o t h e r clinical behavioral signs of pain, sickness, or morbidity were killed according to protocol. R e m a i n i n g mice were killed 60 days after intraperitoneal injection. All mice had necroscopy performed. The a b d o m e n was o p e n e d and all peritoneal surfaces, bowel, liver, spleen, and dia-
Saline solution group
Water group
16 (89%)
10 (55%)
p = 0.03*
4 (22%) 2
13 (72%) 4
p = 0.003* p = 0.05j-
Parameter
Tumor growth at 30 days Ascites Median tumor score
Significance
*Fisher's exact test. tX 2 test.
p h r a g m were inspected for gross tumor. T u m o r was c o n f i r m e d histologically. T h e presence of ascites was noted. T h e n u m b e r of t u m o r nodules was noted, and the size of the largest t u m o r n o d u l e was d e t e r m i n e d with calipers. T h e dimensions of any needle-site t u m o r growth were also determined. T u m o r volumes were calculated by the formula Length × Width 2. Each mouse was assigned a composite t u m o r score (Table I). Statistical comparison between the groups used the X2 for discrete variables and Student t test for continuous variables. Time-to-failure analysis used the Kaplan-Meier method. E i g h t e e n mice in the study and control groups would yield a statistical power of 80% ([3 0.2) to detect a 30% decrease in the establishment of t u m o r metastasis in the study group, with an ct of 0.05.
Results Thirty-five of 36 mice (97%) had clinically detectable t u m o r growth during the study period. T h e study results are summarized in Table II. Within the first 4 weeks after innoculation, 16 (89%) of the saline solution group had clinically detectable t u m o r growth, primarily at the innoculation sites, c o m p a r e d with only 10 (55%) of the water group. T h e water group had ascites m o r e frequently than the saline solution group, 72% versus 22%. T h e m e d i a n t u m o r scores at death were significantly h i g h e r for the water group than for the saline solution g r o u p (Table II). T i m e to failure ( m o r i b u n d status) is shown in Fig. 1. T h e group of mice that u n d e r w e n t lavage with sterile water fared worse than the saline solution group, requiring euthanasia e a r l i e r than the saline solution group.
Comment T h e prognostic c o n s e q u e n c e of cyst rupture and tum o r spill at surgical removal of ovarian cancer apparently c o n f i n e d to the ovaries is controversial. T h e FIGO staging criteria equate cyst rupture with malignant ascites and assign these cases to stage IC, implying a worse prognosis. Many Of the early studies on the prognostic significance of cyst rupture are difficult to interpret because in some of these studies the patients were n o t completely
Volume 175, Number 6 AmJ Obstet Gynecol
Morris and Scholten
1491
Cumulative Percent Surviving 1 .9
"~ WATER I SALINE
:
.7 ,5 .4
.3
p=0.022"
l_.
.2 ,1
i
30
35
i
i
i
i
i
f
i
40 45 50 55 Days until moribund
t
i
60
I
65
Fig. 1. Time to failure (survival), water group versus saline solution group. Asterisk, Log rank test.
staged according to today's criteria. Up to 40% of patients with disease a p p a r e n t l y c o n f i n e d to o n e o r b o t h ovaries will be up-staged w h e n a p p r o p r i a t e l y m p h n o d e a n d u p p e r a b d o m i n a l biopsy s p e c i m e n s are obtained.10. 11 O t h e r factors such as a d h e s i o n s to pelvic structures, capsule e x c r e s c e n c e s , a n d h i g h t u m o r g r a d e may be m o r e often associated with cyst r u p t u r e a n d i m p a r t a worse p r o g n o s i s i n d e p e n d e n t o f cyst rupture. 2 Cyst r u p t u r e with t u m o r spill alone may not be sufficient to establish intraperitoneal metastases. T h e t u m o r cells may require a genetic predisposition to metastasize and grow. It may be that these m o r e aggressive tumors are m o r e likely to r u p t u r e and disseminate. Nonetheless, it seems p r u d e n t to r e m o v e ovarian cysts intact w h e n e v e r possible. In the event of cyst rupture it seems reasonable to a t t e m p t to lower the "inoculum" of viable t u m o r cells by copious irrigation of the a b d o m i n a l and pelvic cavities. DiSaia and Creasman 9 have r e c o m m e n d e d sterile water to cause osmotic lysis of free-floating t u m o r cells. 9 T h e r e a r e no prior studies to d e t e r m i n e w h e t h e r sterile water or saline solution lavage improves the prognosis in early-stage ovarian cancer in the .event of cyst rupture. Copious amounts of sterile water lavaged intraperitoneally could lead to hypervolemia and hyponatremia, and its use should be considered only i f a beneficial effect can be established. In our study the water lavage g r o u p a p p e a r e d to do worse. We postulated that this may be due to growth factors being released f r o m lysed cells, stimulating the r e m a i n i n g cells to proliferate. T h e n u m b e r of ceils in the i n o c u l u m along with the v o l u m e and dwell time of the sterile water may have an effect on the usefulness of sterile water lavage. In our study the n u m b e r of cells infused may have b e e n too great, and the dwell time of the water too short to affect significant cell lysis. We also f o u n d it technically difficult
to r e m o v e the lavage solution. We did not relavage either, as m i g h t be d o n e at laparotomy. Goldstein et al. 19 studied peritoneal t u m o r cell implantation in the m u r i n e m o d e l with a bladder t u m o r cell line and irrigation with either heparin or GRGDS (the p e n t a peptide Gly-Arg-Gly-Asp-Ser) to p r e v e n t t u m o r cell adh e r e n c e and growth. A l t h o u g h their results were not statistically significant, they f o u n d a trend toward a decreased n u m b e r and size of t u m o r implants in the g r o u p that u n d e r w e n t irrigation. They r e c o m m e n d prophylactic irrigation with a heparin solution or GRGDS to decrease t u m o r cell adherence. The increasing use of laparoscopic surgery in the diagnosis and m a n a g e m e n t of ovarian neoplasms has b r o u g h t with it a serious c o n c e r n about cyst rupture, t u m o r spillage, and seeding of the peritoneal cavity. A b d o m i n a l wall seeding from paracentesis and laparoscopy is now a well-documented occurrence. ~3' 14 T h e first sign of t u m o r growth in our mice was in the a b d o m i n a l wall n e e d l e tract. These n e e d l e tract tumors were invariably larger than the intraabdominal t u m o r masses. T h e c o n s e q u e n c e of cyst rupture and seeding of the trocar site should n o t be minimized. In summary, lavage with sterile water after t u m o r spill in the m u r i n e m o d e l had no protective effect against the establishment of intraperitoneal t u m o r metastasis and in fact s e e m e d to p r o m o t e t u m o r growth. We postulate that this u n e x p e c t e d o u t c o m e may occur as a result of lysing the "less aggressive" t u m o r ceils with the release of growth factors, supporting the growth of the m o r e virulent cell lines. O t h e r factors that may affect the usefulness of the lavage are the n u m b e r of cells in the inoculum, the dwell time, and a m o u n t of lavage. We thank Michael Perry and Ewa Rakowicz-Szulczynska, PhD, for supplying the ovarian cancer cell line.
1492
Morris and Scholten
REFERENCES
1. Wingo PA, Tong T, Bolden S. Cancer statistics 1995. CA Cancer J Clin 1995;45:8-30. 2. Dembo AJ, Davy M, Stenwig AE, Berle EJ, Bush RS, Cjorstad K. Prognostic factors in patients with stage I epithelial ovarian cancer. Obstet Gynecol 1990;75:263-72. 3. International Federation of Gynecology and Obstetrics. Annual report on the results of treatment in gynecologic cancer. IntJ Gynecol Obstet 1989;28:189-90. 4. International Federation of Gynecology and Obstetrics. Annual report on the results of treatment in gynecologic cancer. IntJ Gynecol Obstet 1991;36(Suppl 1):238-40. 5. Sainz de la Cuesta R, Goff BA, Fuller AF, Nikrui N, Eichorn JH, Rice LW. Prognostic importance of intraoperative rupture of malignant ovarian epithelial neoplasms. Obstet Gynecol 1994;84:1-7. 6. Sevelda P, Vavra N, Schemper M, Salzer H. Prognostic factors for survival in stage I epithelial ovarian carcinoma. Cancer 1990;65:2549-52. 7. Grogan RH. Accidental rupture of malignant ovarian cysts during surgical removal. Obstet Gynecol 1967;30:716-20. 8. Sevelda P, Dittrich C, Salzer H. Prognostic value of rupture of the capsule in stage I epithelial ovarian cancer. Gynecol Oncol 1989;35:321-2. 9. DiSaia PJ, Creasman WT. Clinical gynecologic oncology. 4th ed. St. Louis: Mosby-Year Book, 1993. 10. Young RC, Decker DG, Wharton JT. Staging laparotomy in early ovarian cancer. JAMA 1983;250:3072-6. 11. McGowan L, Lesher LP, Norris HJ, Barnett M. Misstaging of ovarian cancer. Obstet Gynecol 1985;65:568-72. 12. Goldstein DS, Lu ML, Hattori T, Ratliff TL, Loughlin K, Kavoussi LR. Inhibition of peritoneal tumor-cell implantation: model for laparoscopic cancer surgery. J Endourol 1993;7:237-41. 13. Hsiu J, Given FT, Kemp GM. Tumor implantation after diagnostic laparoscopic biopsy of serous ovarian tumors of low malignant potential. Obstet Gynecol 1986;68(Suppl): 90-3. 14. Gleeson NC, Nicosia SV, Mark JE, Hoffman MS, Cavanagh D. Abdominal wall metastases from ovarian cancer after laparoscopy. Am J Obstet Gynecol 1993;169:522-3. Discussion
DR. GREGORY SUTTON, Indianapolis, Indiana. T h e authors q u o t e the fourth edition of Creasman and DiSaia's Clinical Gynecologic Oncology, 1 "If rupture does occur, lavage of the peritoneal cavity with sterile water has b e e n r e c o m m e n d e d to cause lysis of the cells, a n d this may be helpful. T h e peritoneal surfaces will absorb the water, so care should be taken to prevent delays in retrieving the fluid." They correctly challenged the idea of sterile water lavage a n d t h e n c o n d u c t e d a well-conceived animal e x p e r i m e n t that appears to refute the hypothesis that sterile water lavage inhibits the growth of intraperitoneal ovarian cancer. This a p p r o a c h to a clinical p r o b l e m b r o u g h t to m i n d Grimes' r e c e n t article. 2 Grimes suggests that our current m o d e l of clinical p r o b l e m solving, one of autocracy, be replaced by an evidence-based system that places value
December 1996 Am J Obstet Gynecol
on individual opinions only insofar as they are supported by scientific evidence. Grimes' article is inspirational reading and suggests that a shift to evidence-based med-icine supported by resources such as MEDLINE, ACOGNET, T h e A m e r i c a n College of Obstetricians and Gynecologists Resource Center, and o t h e r accessible data sources provides an ideal opportunity for medical student, resident, and faculty education. Such a shift in emphasis will interface handily with the restructuring of medical school curricula toward problem-based learning. I have three questions. (1) Was survival better in the saline solution g r o u p or was survival worse a m o n g the animals treated with sterile water? To restate t h e question, what would survival have b e e n in an u n t r e a t e d control population? Perhaps b o t h types of lavage impaired or i m p r o v e d survival. (2) Do you have an explanation for the relative effects of the two lavage fluids on growth of ovarian cancer cells in this animal model? (3) What e x p e r i m e n t would you propose to test this hypothesis?
REFERENCES
1. Tile adnexal mass and early ovarian cancer. In: DiSaia PJ, Creasman WT, editors. Clinical gynecologic oncology. 4th ed. St. Louis: Mosby-Year Book, 1993. 2. Grimes DA. Introducing evidence-based medicine into a department of obstetrics and gynecology'. Obstet Gynecol 1995;86:451-7. DR. MORRIS (Closing). T h e design of our e x p e r i m e n t makes it impossible to d e t e r m i n e whether the survival was better in the saline solution group or worse in the group treated with sterile water c o m p a r e d with an untreated control group. We did n o t have e n o u g h funds to obtain 18 mice to keep for observation only. We did, however, have two mice that did n o t u n d e r g o any experimental treatments who were alive and well at the completion of the 60-day study. C o n c e r n i n g the relative effects of the two lavage fluids on growth of ovarian cancer cells, because the results that we obtained were the opposite of what we expected, we postulate that the sterile water may preferentially lyse less virulent t u m o r cells, releasing growth factors that may stimulate the m o r e virulent cells. This hypothesis could be tested in vitro with cell cultures or in the n u d e mouse m o d e l again. Ovarian t u m o r cells could be lysed with sterile water in vitro with the supernatant of these lysed cells a d d e d to aliquots of t u m o r cells to be injected into cohorts of n u d e mice. Obviously o u r study is not the definitive answer to the question posed, and m o r e study in the matter is warranted.
Key words: Ovarian cancer, t u m o r spill, osmotic lysis
Ovarian cancer is the second most c o m m o n gynecologic malignancy, with an estimated 26,600 new cases expected in 1995 leading to 14,500 d e a t h s ) It is the fourth leading cause of cancer deaths in women. Early diagnosis is the exception rather than the rule. Approximately 15% to 25% of patients with epithelial ovarian cancer have disease c o n f i n e d to o n e or both ovaries. Epithelial ovarian cancer c o n f i n e d to the ovary at diagnosis has a reported 5-year survival of u p to 90%. There is significant controversy about prognostic factors in early-stage disease. T u m o r grade has b e e n shown to be an i m p o r t a n t prognostic factor. 2 The significance of t u m o r r u p t u r e at surgery remains controversial a n d unanswered. I n t e r n a t i o n a l Federation of Gynecology a n d Obstetrics (FIGO) staging criteria for ovarian cancer From the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Nebraska Medical Center. Presented at the Twenty-second Annual Meeting of the Society of Gynecologic Surgeons, Albuquerque, New Mexico, March 4-6, 1996. Reprint requests: Peter C. Morris, MD, Texas Oncology, 918 Eighth Ave., Fort Worth, TX 76104. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00 + 0 6/6/76166
imply that cyst r u p t u r e is an adverse prognostic factor by up-staging r u p t u r e d m a l i g n a n t tumors apparently confined to one or both ovaries to stage IC equated with similar tumors with m a l i g n a n t ascites. ~ The 1990 FIGO report quotes a 5-year survival of 82.3%, 74.9%, a n d 67.7% for stages IA, IB, a n d IC, respectively. 4 Some studies have shown a worse prognosis for intraoperative r u p t u r e of m a l i g n a n t cysts, 5 whereas others have failed to show a negative impact of cyst rupture. 2' 6-s Some authors r e c o m m e n d lavage of the peritoneal cavity with sterile water in the event of cyst r u p t u r e to affect osmotic lysis of t u m o r ceils, thus decreasing the chance of viable t u m o r implantation. 9 Although theoretically sound, the usefulness of osmotic lysis of t u m o r cells has n o t b e e n investigated. The purpose of this study was to determine whether viable h u m a n ovarian cancer cells injected or "spilled" intraperitoneally in the n u d e mouse m o d e l are subject to osmotic lysis by peritoneal lavage with sterile water. Methods
The experimental protocol was approved by the University Institutional A n i m a l Care a n d Use Committee. 1489
1490
Morris and Scholten
December 1996 Am J Obstet Gynecol
Table I. Assigned t u m o r score
Table II. Summary of study results
No. of points
Criterion
0
No evidence of tumor growth Tumor at injection site, no intraperitoneal growth Intraperitoneal nodules --<1 mm greatest dimension Intraperitoneal nodules 1-5 mm greatest dimension Intraperitoneal nodules >5 mm greatest dimension, confluent plaques, ascites, or diffuse carcinomatosis
1
2 3 4
H u m a n ovarian epithelial cancer cells (SKOV-3) were obtained from an established h u m a n ovarian cancer tissue culture from the A m e r i c a n Type Culture Collection (Rockville, Md.). Cells were grown in Dulbecco's modified Eagle's m e d i u m with 15% fetal calf serum and 1% L-glutamine. At harvest trypsinized cells were diluted with Dulbecco's modified Eagle's m e d i u m , centrifuged, and c o u n t e d with a h e m o c y t o m e t e r and r e s u s p e n d e d in a c o n c e n t r a t i o n of 2 × 10 7 cells per 1 ml of Dulbecco's m o d i f i e d Eagle's m e d i u m . Athymic ( n u / n u ) n u d e mice (Harlan Sprague) at 6 to 8 weeks old were housed in a pathogen-free e n v i r o n m e n t with controlled 12-hour light-dark cycles with free access to food and water. Preliminary experiments were perf o r m e d on six mice to d e t e r m i n e the optimal n u m b e r of h u m a n ovarian cancer cells to be inoculated to p r o d u c e implants and to d e t e r m i n e t h e time to d e v e l o p m e n t of the clinical ovarian cancer syndrome in the mice. Mice receiving 15 to 20 million cells had clinically detectable intraabdominal t u m o r by 60 days. Four o t h e r mice f u n c t i o n e d as sham controls, u n d e r g o i n g peritoneal lavage with 3 to 4 ml of n o r m a l saline solution or sterile water until the a b d o m e n distended to d e t e r m i n e any adverse effects f r o m the lavage alone. The study g r o u p consisted of 36 mice injected intraperitoneally with 1 ml of the SKOV-3 cell line at a c o n c e n t r a t i o n of 2 × 10 7 c e l l s / m l with a 21-gauge butterfly needle. After this injection alternate mice u n d e r w e n t intraperitoneal lavage with either 3 to 4 ml of n o r m a l saline solution (control group) or 3 to 4 ml of sterile water (study group). This volume of lavage solution was sufficient to distend the a b d o m e n . Aspiration of lavage solution p r o v e d difficult; only 0.5 to 1 ml was o b t a i n e d in return. T h e mice were m o n i t o r e d daily for general condition and t u m o r growth. Mice showing clinical signs of ascites, massive intraabdominal t u m o r growth, or o t h e r clinical behavioral signs of pain, sickness, or morbidity were killed according to protocol. R e m a i n i n g mice were killed 60 days after intraperitoneal injection. All mice had necroscopy performed. The a b d o m e n was o p e n e d and all peritoneal surfaces, bowel, liver, spleen, and dia-
Saline solution group
Water group
16 (89%)
10 (55%)
p = 0.03*
4 (22%) 2
13 (72%) 4
p = 0.003* p = 0.05j-
Parameter
Tumor growth at 30 days Ascites Median tumor score
Significance
*Fisher's exact test. tX 2 test.
p h r a g m were inspected for gross tumor. T u m o r was c o n f i r m e d histologically. T h e presence of ascites was noted. T h e n u m b e r of t u m o r nodules was noted, and the size of the largest t u m o r n o d u l e was d e t e r m i n e d with calipers. T h e dimensions of any needle-site t u m o r growth were also determined. T u m o r volumes were calculated by the formula Length × Width 2. Each mouse was assigned a composite t u m o r score (Table I). Statistical comparison between the groups used the X2 for discrete variables and Student t test for continuous variables. Time-to-failure analysis used the Kaplan-Meier method. E i g h t e e n mice in the study and control groups would yield a statistical power of 80% ([3 0.2) to detect a 30% decrease in the establishment of t u m o r metastasis in the study group, with an ct of 0.05.
Results Thirty-five of 36 mice (97%) had clinically detectable t u m o r growth during the study period. T h e study results are summarized in Table II. Within the first 4 weeks after innoculation, 16 (89%) of the saline solution group had clinically detectable t u m o r growth, primarily at the innoculation sites, c o m p a r e d with only 10 (55%) of the water group. T h e water group had ascites m o r e frequently than the saline solution group, 72% versus 22%. T h e m e d i a n t u m o r scores at death were significantly h i g h e r for the water group than for the saline solution g r o u p (Table II). T i m e to failure ( m o r i b u n d status) is shown in Fig. 1. T h e group of mice that u n d e r w e n t lavage with sterile water fared worse than the saline solution group, requiring euthanasia e a r l i e r than the saline solution group.
Comment T h e prognostic c o n s e q u e n c e of cyst rupture and tum o r spill at surgical removal of ovarian cancer apparently c o n f i n e d to the ovaries is controversial. T h e FIGO staging criteria equate cyst rupture with malignant ascites and assign these cases to stage IC, implying a worse prognosis. Many Of the early studies on the prognostic significance of cyst rupture are difficult to interpret because in some of these studies the patients were n o t completely
Volume 175, Number 6 AmJ Obstet Gynecol
Morris and Scholten
1491
Cumulative Percent Surviving 1 .9
"~ WATER I SALINE
:
.7 ,5 .4
.3
p=0.022"
l_.
.2 ,1
i
30
35
i
i
i
i
i
f
i
40 45 50 55 Days until moribund
t
i
60
I
65
Fig. 1. Time to failure (survival), water group versus saline solution group. Asterisk, Log rank test.
staged according to today's criteria. Up to 40% of patients with disease a p p a r e n t l y c o n f i n e d to o n e o r b o t h ovaries will be up-staged w h e n a p p r o p r i a t e l y m p h n o d e a n d u p p e r a b d o m i n a l biopsy s p e c i m e n s are obtained.10. 11 O t h e r factors such as a d h e s i o n s to pelvic structures, capsule e x c r e s c e n c e s , a n d h i g h t u m o r g r a d e may be m o r e often associated with cyst r u p t u r e a n d i m p a r t a worse p r o g n o s i s i n d e p e n d e n t o f cyst rupture. 2 Cyst r u p t u r e with t u m o r spill alone may not be sufficient to establish intraperitoneal metastases. T h e t u m o r cells may require a genetic predisposition to metastasize and grow. It may be that these m o r e aggressive tumors are m o r e likely to r u p t u r e and disseminate. Nonetheless, it seems p r u d e n t to r e m o v e ovarian cysts intact w h e n e v e r possible. In the event of cyst rupture it seems reasonable to a t t e m p t to lower the "inoculum" of viable t u m o r cells by copious irrigation of the a b d o m i n a l and pelvic cavities. DiSaia and Creasman 9 have r e c o m m e n d e d sterile water to cause osmotic lysis of free-floating t u m o r cells. 9 T h e r e a r e no prior studies to d e t e r m i n e w h e t h e r sterile water or saline solution lavage improves the prognosis in early-stage ovarian cancer in the .event of cyst rupture. Copious amounts of sterile water lavaged intraperitoneally could lead to hypervolemia and hyponatremia, and its use should be considered only i f a beneficial effect can be established. In our study the water lavage g r o u p a p p e a r e d to do worse. We postulated that this may be due to growth factors being released f r o m lysed cells, stimulating the r e m a i n i n g cells to proliferate. T h e n u m b e r of ceils in the i n o c u l u m along with the v o l u m e and dwell time of the sterile water may have an effect on the usefulness of sterile water lavage. In our study the n u m b e r of cells infused may have b e e n too great, and the dwell time of the water too short to affect significant cell lysis. We also f o u n d it technically difficult
to r e m o v e the lavage solution. We did not relavage either, as m i g h t be d o n e at laparotomy. Goldstein et al. 19 studied peritoneal t u m o r cell implantation in the m u r i n e m o d e l with a bladder t u m o r cell line and irrigation with either heparin or GRGDS (the p e n t a peptide Gly-Arg-Gly-Asp-Ser) to p r e v e n t t u m o r cell adh e r e n c e and growth. A l t h o u g h their results were not statistically significant, they f o u n d a trend toward a decreased n u m b e r and size of t u m o r implants in the g r o u p that u n d e r w e n t irrigation. They r e c o m m e n d prophylactic irrigation with a heparin solution or GRGDS to decrease t u m o r cell adherence. The increasing use of laparoscopic surgery in the diagnosis and m a n a g e m e n t of ovarian neoplasms has b r o u g h t with it a serious c o n c e r n about cyst rupture, t u m o r spillage, and seeding of the peritoneal cavity. A b d o m i n a l wall seeding from paracentesis and laparoscopy is now a well-documented occurrence. ~3' 14 T h e first sign of t u m o r growth in our mice was in the a b d o m i n a l wall n e e d l e tract. These n e e d l e tract tumors were invariably larger than the intraabdominal t u m o r masses. T h e c o n s e q u e n c e of cyst rupture and seeding of the trocar site should n o t be minimized. In summary, lavage with sterile water after t u m o r spill in the m u r i n e m o d e l had no protective effect against the establishment of intraperitoneal t u m o r metastasis and in fact s e e m e d to p r o m o t e t u m o r growth. We postulate that this u n e x p e c t e d o u t c o m e may occur as a result of lysing the "less aggressive" t u m o r ceils with the release of growth factors, supporting the growth of the m o r e virulent cell lines. O t h e r factors that may affect the usefulness of the lavage are the n u m b e r of cells in the inoculum, the dwell time, and a m o u n t of lavage. We thank Michael Perry and Ewa Rakowicz-Szulczynska, PhD, for supplying the ovarian cancer cell line.
1492
Morris and Scholten
REFERENCES
1. Wingo PA, Tong T, Bolden S. Cancer statistics 1995. CA Cancer J Clin 1995;45:8-30. 2. Dembo AJ, Davy M, Stenwig AE, Berle EJ, Bush RS, Cjorstad K. Prognostic factors in patients with stage I epithelial ovarian cancer. Obstet Gynecol 1990;75:263-72. 3. International Federation of Gynecology and Obstetrics. Annual report on the results of treatment in gynecologic cancer. IntJ Gynecol Obstet 1989;28:189-90. 4. International Federation of Gynecology and Obstetrics. Annual report on the results of treatment in gynecologic cancer. IntJ Gynecol Obstet 1991;36(Suppl 1):238-40. 5. Sainz de la Cuesta R, Goff BA, Fuller AF, Nikrui N, Eichorn JH, Rice LW. Prognostic importance of intraoperative rupture of malignant ovarian epithelial neoplasms. Obstet Gynecol 1994;84:1-7. 6. Sevelda P, Vavra N, Schemper M, Salzer H. Prognostic factors for survival in stage I epithelial ovarian carcinoma. Cancer 1990;65:2549-52. 7. Grogan RH. Accidental rupture of malignant ovarian cysts during surgical removal. Obstet Gynecol 1967;30:716-20. 8. Sevelda P, Dittrich C, Salzer H. Prognostic value of rupture of the capsule in stage I epithelial ovarian cancer. Gynecol Oncol 1989;35:321-2. 9. DiSaia PJ, Creasman WT. Clinical gynecologic oncology. 4th ed. St. Louis: Mosby-Year Book, 1993. 10. Young RC, Decker DG, Wharton JT. Staging laparotomy in early ovarian cancer. JAMA 1983;250:3072-6. 11. McGowan L, Lesher LP, Norris HJ, Barnett M. Misstaging of ovarian cancer. Obstet Gynecol 1985;65:568-72. 12. Goldstein DS, Lu ML, Hattori T, Ratliff TL, Loughlin K, Kavoussi LR. Inhibition of peritoneal tumor-cell implantation: model for laparoscopic cancer surgery. J Endourol 1993;7:237-41. 13. Hsiu J, Given FT, Kemp GM. Tumor implantation after diagnostic laparoscopic biopsy of serous ovarian tumors of low malignant potential. Obstet Gynecol 1986;68(Suppl): 90-3. 14. Gleeson NC, Nicosia SV, Mark JE, Hoffman MS, Cavanagh D. Abdominal wall metastases from ovarian cancer after laparoscopy. Am J Obstet Gynecol 1993;169:522-3. Discussion
DR. GREGORY SUTTON, Indianapolis, Indiana. T h e authors q u o t e the fourth edition of Creasman and DiSaia's Clinical Gynecologic Oncology, 1 "If rupture does occur, lavage of the peritoneal cavity with sterile water has b e e n r e c o m m e n d e d to cause lysis of the cells, a n d this may be helpful. T h e peritoneal surfaces will absorb the water, so care should be taken to prevent delays in retrieving the fluid." They correctly challenged the idea of sterile water lavage a n d t h e n c o n d u c t e d a well-conceived animal e x p e r i m e n t that appears to refute the hypothesis that sterile water lavage inhibits the growth of intraperitoneal ovarian cancer. This a p p r o a c h to a clinical p r o b l e m b r o u g h t to m i n d Grimes' r e c e n t article. 2 Grimes suggests that our current m o d e l of clinical p r o b l e m solving, one of autocracy, be replaced by an evidence-based system that places value
December 1996 Am J Obstet Gynecol
on individual opinions only insofar as they are supported by scientific evidence. Grimes' article is inspirational reading and suggests that a shift to evidence-based med-icine supported by resources such as MEDLINE, ACOGNET, T h e A m e r i c a n College of Obstetricians and Gynecologists Resource Center, and o t h e r accessible data sources provides an ideal opportunity for medical student, resident, and faculty education. Such a shift in emphasis will interface handily with the restructuring of medical school curricula toward problem-based learning. I have three questions. (1) Was survival better in the saline solution g r o u p or was survival worse a m o n g the animals treated with sterile water? To restate t h e question, what would survival have b e e n in an u n t r e a t e d control population? Perhaps b o t h types of lavage impaired or i m p r o v e d survival. (2) Do you have an explanation for the relative effects of the two lavage fluids on growth of ovarian cancer cells in this animal model? (3) What e x p e r i m e n t would you propose to test this hypothesis?
REFERENCES
1. Tile adnexal mass and early ovarian cancer. In: DiSaia PJ, Creasman WT, editors. Clinical gynecologic oncology. 4th ed. St. Louis: Mosby-Year Book, 1993. 2. Grimes DA. Introducing evidence-based medicine into a department of obstetrics and gynecology'. Obstet Gynecol 1995;86:451-7. DR. MORRIS (Closing). T h e design of our e x p e r i m e n t makes it impossible to d e t e r m i n e whether the survival was better in the saline solution group or worse in the group treated with sterile water c o m p a r e d with an untreated control group. We did n o t have e n o u g h funds to obtain 18 mice to keep for observation only. We did, however, have two mice that did n o t u n d e r g o any experimental treatments who were alive and well at the completion of the 60-day study. C o n c e r n i n g the relative effects of the two lavage fluids on growth of ovarian cancer cells, because the results that we obtained were the opposite of what we expected, we postulate that the sterile water may preferentially lyse less virulent t u m o r cells, releasing growth factors that may stimulate the m o r e virulent cells. This hypothesis could be tested in vitro with cell cultures or in the n u d e mouse m o d e l again. Ovarian t u m o r cells could be lysed with sterile water in vitro with the supernatant of these lysed cells a d d e d to aliquots of t u m o r cells to be injected into cohorts of n u d e mice. Obviously o u r study is not the definitive answer to the question posed, and m o r e study in the matter is warranted.