Increased activity of lysosomal enzymes in the peritoneal fluid of patients with gynecologic cancers and pelvic inflammatory disease.

Purpose To investigate whether the activity of lysosomal enzymes is increased in the peritoneal fluid of patients with gynecologic cancers compared to activity in the peritoneal fluid from normal subjects and those with pelvic inflammatory disease, and fluid from benign ovarian cysts. Patients and methods β-glucuronidase, β-galactosidase, and α-mannosidase activity was measured in the peritoneal fluid from patients with gynecologic cancer, pelvic inflammatory disease, and normal subjects, and fluid from benign ovarian cysts.

The mean±SD of β-glucuronidase, β-galactosidase, and α-mannosidase activity in the gynecologic cancers was 120±50 nmol, 203±86 nmol, and 240±119 nmol 4-methylumbelliferone/ml/h, respectively; in the normal control subjects it was 22±9 nmol, 46±10 nmol, and 80±23 nmol, respectively (P=0.00003, 0.0001, and 0.0001, respectively). The activity was increased even in cases without malignant cells in the peritoneal fluid. In pelvic inflammatory disease it was 148±82 nmol, 278±112 nmol, and 291±140 nmol, respectively. The activity in the fluid of the ovarian cysts was similar to that of the normal peritoneal fluid. There was a significant positive correlation between enzyme activity and stage of cancer, that was stronger for β-glucuronidase (r=0.889, P=0.003).

The increased lysosomal enzyme activity in gynecologic cancers, without overlapping between patients and normal subjects or benign ovarian cyst fluid, indicates that such measurements might be applied for diagnostic purposes.

Benign ovarian cyst, Endometrial carcinoma, Lysosomal enzymes, Ovarian cancer, Pelvic inflammatory disease

The mature lysosomal enzymes are glycoproteins packaged into single lipoprotein membrane organelles, the lysosomes. The precursors of these enzymes are synthesized on membrane-bound polysomes in the rough endoplasmic reticulum (Kornfeld 1986). Subsequently, the sorting and targeting of lysosomal enzymes to their final destination is directed by a series of protein and carbohydrate recognition signals located on the enzymes (Erickson and Blodel 1979; Rosenfeld et al. 1982; Proia and Neufeld 1982). A portion, up to 20%, of the mature lysosomal enzymes is first secreted into the extracellular fluid, and subsequently it is internalized by binding to mannose 6-phosphate receptors on the surface of the cells and stored in the lysosomes (Vladutiu and Rattazzi 1979). In earlier reports our group has shown that the activity of several lysosomal enzymes is increased in infected body fluids, such as the cerebrospinal fluid of bacterial meningitis (Beratis et al. 1997; Beratis et al. 2003) and the peritoneal fluid of patients with bacterial peritonitis (Beratis et al. 2002). In addition, an increased activity of β-glucuronidase was found in the cerebrospinal fluid of children with acute lymphoblastic leukemia receiving high-dose methotrexate, particularly in those with neurotoxicity (Vlacha et al. 2004). The increased lysosomal enzyme activity in extracellular fluids of such patients is attributed to a leakage of these enzymes through the cell membranes facilitated by the inflammation process (Beratis et al. 1997; Beratis et al. 2002; Beratis et al. 2003) or the cellular toxicity induced by chemotherapy (Vlacha et al. 2004). However, there are no published data regarding the activity of lysosomal enzymes in body fluids of patients with malignant diseases. In benign and malignant disorders of the ovaries and oviducts, in malignant disorders of the uterine corpus, and in pelvic inflammatory disease fluid may be encysted or accumulated in the cul-de-sac. Because malignancies cause increased cellular death, lysosomal enzymes released from the lysosomes would cause elevated activities of these enzymes in adjacent extracellular fluids. Malignancies may also increase the rate of synthesis or disturb the packaging of the lysosomal enzymes in the lysosomes and, thus, contribute to increased extracellular enzyme activities. These events are similar to those suggested regarding the body fluids of patients with bacterial infections (Beratis et al. 1997; Beratis et al. 2002; Beratis et al. 2003) or drug-induced cellular toxicity (Vlacha et al. 2004). We hypothesize that the lysosomal enzyme activity in the cell-free peritoneal fluid of patients with gynecologic cancer might be similar to that measured in patients with peritoneal inflammation (Beratis et al. 2002). The purpose of this study was to determine the activities of three lysosomal enzymes, β-glucuronidase, β-galactosidase, and α-mannosidase, in cell-free pelvic peritoneal fluid from patients with ovarian cancer and endometrial carcinoma and compare them with the activities in peritoneal fluid from pelvic inflammatory disease and from subjects free of inflammatory or malignant gynecologic disorders as well as with the activities in fluid from benign ovarian neoplastic cysts.

Patients and control subjects We studied pelvic peritoneal fluid obtained from 12 patients with malignant gynecologic disorders (nine with primary ovarian cancer and three with endometrial carcinoma), from five patients with pelvic inflammatory disease (four with acute salpingitis and one with a ruptured tubo-ovarian abscess), and from ten women with infertility used as normal controls (six primary and four secondary). We also studied the fluid from eight benign ovarian neoplastic cysts (serous fibroadenomas). None of the women included in the infertility control group had endometriosis or any other macroscopically identifiable anomaly. Peritoneal fluid from the patients with acute salpingitis and infertility was obtained during diagnostic laparoscopy and from the patients with cancer and the patient with the tubo-ovarian abscess during laparotomy at surgery. In addition, the fluid from the ovarian cysts was collected after laparotomy. Of the nine patients with ovarian cancer, one was stage I, one was stage IIc, three stage IIIa, two Stage IIIb, and two stage IV by FIGO (International Federation of Gynecology and Obstetrics 1991). Histologically, of the nine patients with primary ovarian cancer, seven had serous cysto-adenocarcinoma, one mucinous carcinoma, and one clear-cell carcinoma. Of the three patients with endometrial carcinoma, one was stage Ic, one stage IIa, and one stage IIIa. Two of the patients had adenocarcinoma and one clear-cell carcinoma. In the peritoneal fluid of the patient with the tubo-ovarian abscess both Escherichia coli and Bacteroides spp. were isolated. In one of the patients with salpingitis the etiologic agent was E. coli and in another patient Chlamydia trachomatis. Cultures from the other two such patients were negative. The study was approved by the Ethics Committee of the University Hospital. Informed consent was obtained from the patients and the control subjects. Enzyme assays Peritoneal and ovarian cyst fluid was centrifuged within 20 min, and the supernatant cell-free fraction was stored at −70 °C until assayed. The activity of β-glucuronidase, β-galactosidase, and α-mannosidase was determined as reported [7]. In brief, the cell-free peritoneal or ovarian cyst fluid was incubated with the appropriate substrate for 30 min at 37°C and the reactions were stopped with 5 ml of 85 mM glycine-carbonate buffer, pH 10.5. β-Glucuronidase was assayed by incubating 50 μl of peritoneal or cyst fluid with 150 μl of 1 mM 4-methylumbelliferyl(4-MU)-β-D-glucuronide in 100 mM acetate buffer, pH 4.0. The intra-assay coefficient of variation was 2.8%. β-Galactosidase activity was measured by incubating 25 μl of test fluid with 75 μl οf 0.5 mM 4-MU-β-D-galactoside in 100 mM citrate-phosphate buffer, pH 4.35, with 400 mM NaCl. The intra-assay coefficient of variation was 2.5%. α-Mannosidase activity was determined by incubating 20 μl of peritoneal or cyst fluid with 200 μl of 4-MU-α-D-mannopyranoside in 100 mM citrate-phosphate buffer, pH 4.0. The intra-assay coefficient of variation was 1.8%. Statistical analyses Assuming a lysosomal enzyme activity in the peritoneal fluid of patients with gynecologic cancers within the range of activities measured in the peritoneal fluid of patients with bacterial peritonitis (Beratis et al. 2002), we calculated that the required sample size for the β-glucuronidase (95% power, two-sided significance level of 0.05) was five patients and ten control subjects; β-glucuronidase is the enzyme that has provided the best distinction between patients with bacterial infections and controls in all previous studies (Beratis et al. 1997; Beratis et al. 2002; Beratis et al. 2003; Vlacha et al. 2004). The required sample size for α-mannosidase was eight patients and ten controls, and for β-galactosidase 12 patients and ten controls. Values were compared with unpaired two-tailed t-test. The enzyme activities were correlated between themselves and to the stage of the tumor by Pearson correlation. Normality was tested by the Kolmogorov-Smirnov method. For the analyses StatMate and Prism statistical software were used (StatMate 2 for Windows and Prism 3.02 for Windows, GraphPad Software, San Diego, Calif., USA). Data are expressed as mean±SD. Significance was set at 0.05. The activities of the enzymes measured are expressed in nmol 4-methylumbelliferone (MU) per ml of tested fluid per hour.

Gynecologic cancers The activity of β-glucuronidase, β-galactosidase, and α-mannosidase in the peritoneal fluid of the patients with endometrial and ovarian carcinoma was 120±50 nmol 4-MU/ml/h, 203±86 nmol 4-MU/ml/h, and 240±119 nmol 4-MU/ml/h, respectively. In the peritoneal fluid of the women with infertility, who were free of inflammatory disease or malignancy, the activity of the enzymes measured was 22±9 nmol (range 12 nmol to 42 nmol), 46±10 nmol (range 33 nmol to 66 nmol), and 80±23 nmol (range 44 nmol to 114 nmol), respectively. The difference in the activity of the three enzymes between the cancer patients and the normal control subjects is significant (t=6.568, P<0.00003; t=5.223, P=0.0001; and t=4.165, P=0.0001, respectively). The location, the histologic type, and the FIGO staging of the gynecologic cancers, as well as the activity of the enzymes measured in the peritoneal fluid of each case are listed in Table 1. There was a significant positive correlation between the cancer stage and the activity of β-glucuronidase (r=0.889, P=0.001), β-galactosidase (r=0.778, P=0.013), and α-manosidase (r=0.668, P=0.049). A significant positive correlation was also observed between the activities of β-glucuronidase and β-galactosidase (r=0.745, P=0.005), β-glucuronidase and α-mannosidase (r=0.728, P=0.007) and β-galactosidase and α-mannosidase (r=0.904, P<0.0001) in the peritoneal fluid of the gynecologic cancers. Table 1. | Location | Histology | Staging | β-Gluc | β-Gal | α-Mann | |---|---|---|---|---|---| | Endometrium | Adenocarcinoma | Ic | 67 | 163 | 172 | | Endometrium | Adenocarcinoma | IIa | 105 | 214 | 250 | | Endometrium | Clear-cell carcinoma | IIIaa | 101 | 179 | 218 | | Ovary | Serious cysto-adenocarcinoma | I | 65 | 72 | 189 | | Ovary | Serous cysto-adenocarcinoma | IIc | 74 | 163 | 156 | | Ovary | Serous cysto-adenocarcinoma | IIIa | 95 | 163 | 156 | | Ovary | Serous cysto-adenocarcinoma | IIIa | 122 | 214 | 244 | | Ovary | Serous cysto-adenocarcinoma | IIIa | 126 | 214 | 264 | | Ovary | Serous cysto-adenocarcinoma | IIIb | 116 | 163 | 156 | | Ovary | Mucinous carcinoma | IIIb | 137 | 194 | 260 | | Ovary | Clear-cell carcinoma | IV | 189 | 434 | 572 | | Ovary | Serous cysto-adenocarcinoma | IV | 237 | 264 | 346 | aWithout malignant cells in the peritoneal fluid Pelvic inflammatory disease In the peritoneal fluid of the patients with pelvic inflammatory disease the β-glucuronidase activity was 148±82 nmol (range 95 nmol to 294 nmol), the β-galactosidase activity was 278±112 nmol (range 199 nmol to 459 nmol), and the α-mannosidase activity was 291±140 nmol (range 166 nmol to 520 nmol). The difference from the activities measured in the peritoneal fluid of the control subjects with infertility is significant (t=4.956, P=0.0003; t=6.796, P=0.00001; and t=4.834, P=0.0003, respectively). However, there was no significant difference from the enzyme activities observed in the patients with gynecologic cancer (t=0.877, P=0.4; t=1.197, P=0.2; t=0.989, P=0.3, respectively). The greatest activity of the enzymes studied was measured in the peritoneal fluid of the patient with ruptured tubo-ovarian abscess (294 nmol, 459 nmol, and 520 nmol, respectively) as compared to the activities found in the patients with acute salpingitis (86±44 nmol, 234±65 nmol, and 233±56 nmol, respectively). Benign ovarian neoplastic cysts In the fluid derived from the ovarian cysts, the activity of β-glucuronidase was 20±9 nmol (range 11 nmol to 38 nmol), the activity of β-galactosidase was 42±12 nmol (range 18 nmol to 61 nmol), and the activity of α-mannosidase was 62±13 nmol (range 44 nmol to 78 nmol). These activities did not differ significantly from those measured in the peritoneal fluid of the women with infertility, used as control subjects. However, the activity of β-glucuronidase, β-galactosidase, and α-mannosidase activity in the peritoneal fluid of the patients with cancer was significantly greater than in the fluid of the benign ovarian neoplastic cysts (t=6.687, P=0.00002; t=6.402, P=0.00004; t=5.446, P=0.0002, respectively). Figure 1 illustrates the lysosomal enzyme activity in the peritoneal fluid from gynecologic cancers, pelvic inflammatory disease, infertility and from benign ovarian neoplastic cysts.

This is the first diagnostic approach to gynecologic cancers or any other malignancies by utilizing the activity of lysosomal enzymes in any extracellular fluid. The enzyme activities were increased in the peritoneal fluid of all cancer cases studied when compared to normal controls, without overlapping between the two groups. Similarly, the activities were increased in the peritoneal fluid of the patients with pelvic inflammatory disease and, therefore, lysosomal enzyme measurements cannot differentiate between these conditions. Thus, for the differential diagnosis between these conditions, clinical findings and additional laboratory procedures should be employed. The increased activity of the 3 enzymes measured, β-glucuronidase, β-galactosidase, and α-mannosidase, in the peritoneal fluid of patients with pelvic inflammatory disease is in accordance with previous reports of our group showing increased activity of lysosomal enzymes in the cerebrospinal fluid of bacterial meningitis (Beratis et al. 1997; Beratis et al. 2003) and the peritoneal fluid of bacterial peritonitis (Beratis et al. 2002). However, the activity of the enzymes found in the patients with acute salpingitis was at the lower range of the activities measured in the peritoneal fluid of patients with bacterial peritonitis (Beratis et al. 2002). The higher activity of the enzymes studied, that was found in the peritoneal fluid of the patient with peritonitis due to a ruptured tubo-ovarian abscess, might indicate that the enzyme activity in such cases is greater than in acute salpingitis. It should be noted that in the peritoneal fluid of the patients with acute salpingitis the enzyme activities were elevated even if these patients did not have peritonitis, a factor that may explain the smaller increment of the activities in salpingitis. However, the small number of patients with pelvic inflammatory disease studied does not permit a definite answer on this matter and additional studies are indicated. The increased activity of the lysosomal enzymes assayed in the peritoneal fluid of patients with cancer of the ovaries and the endometrium seems to have the potential of a clinical application for the diagnosis of gynecologic malignancies since aspiration of peritoneal fluid at laparoscopy in women suspected of having a gynecologic malignancy is technically easy. Measurement of the activity of these enzymes might prove to be not only a complementary diagnostic test of the cytology of free or encysted body fluids, but even an earlier index of malignancy because the elevation of the enzyme activity seems to precede the presence of malignant cells in the fluid. This is supported by the observation that the activity of the lysosomal enzymes studied was increased in the peritoneal fluid of cancer patients with negative peritoneal fluid cytology. The presence of enzyme activity in aspirated fluid of benign ovarian neoplastic cysts, that did not differ significantly from that found in normal peritoneal fluid, suggests that measurement of these enzymes in encysted ovarian fluid, if such fluid is aspirated, might be helpful in the early diagnosis of malignancy. Actually, the activity of β-glucuronidase and β-galactosidase was almost identical in the fluid of benign ovarian cysts and the normal peritoneal fluid; the α-mannosidase activity was slightly greater in the peritoneal fluid, but the difference did not reach the level of significance. These findings are useful in future studies employing encysted fluids in other organs, such as in the breasts, for which diagnostic aspiration is the standard procedure. This aspect warrants investigation with parallel study of the enzyme activity and the cytology in aspirated fluids, for establishing the sensitivity and specificity of the two diagnostic approaches. The cause of the increased activity of the lysosomal enzymes in the cell-free peritoneal fluid of patients with gynecologic cancer is uncertain. The increased activity of these enzymes in the cerebrospinal fluid of patients with bacterial meningitis (Beratis et al. 1997; Beratis et al. 2003) and the peritoneal fluid of patients with bacterial peritonitis (Beratis et al. 2002) has been attributed to an increased enzyme leakage through the cell membranes to the extracellular fluids. This could be due to a disturbance of the final packaging of the enzymes in the lysosomes or of the secretion-recapture mechanism of the lysosomal enzymes (Vladutiu et al. 1979). In addition, the increased extracellular concentration of lysosomal enzymes in patients with gynecologic cancers could result from an augmented enzyme synthesis by the malignant cells or from an increased rate of cellular death. The latter seems as the most prominent factor causing the increased enzyme activity in these cases and it is in accordance with the observed strong positive correlation between enzyme activity and cancer stage. The anatomy and physiology of the peritoneum may explain the elevated enzyme activity in the peritoneal fluid of patients with gynecologic cancer, particularly, when the tumor is limited to the organ of origin, without peritoneal metastases. The peritoneal surface layer has stomata (spaces) between the surface mesothelial cells that allow transfer of molecules from the underlying stroma to the peritoneal cavity. Since the peritoneal and serosal membranes lie in proximity to the primary tumor or its metastases, it is plausible to assume that the lysosomal enzymes released from malfunctioning or dead cells penetrate the subjacent stromal tissues and basement membrane and through the stomata of the surface epithelium are transferred to the peritoneal cavity. The events associated with the progression of gynecologic cancers are complex and still poorly understood. An aspect that needs investigation is the possibility that a wide number of lysosomal enzymes may participate in malignant progression. There are several reports indicating that the lysosomal proteolytic enzymes cathepsins play a key role in tumor progress and in metastatic spread by promoting the destruction of normal tissue architecture (Mathieu et al. 1990; Mikkelsen et al. 1995; Vetvicka et al. 1997). Cathepsin D plays an important role at many tumor progression steps by affecting proliferation, angiogenesis, and apoptosis (Berchem et al. 2002). A prognostic value and correlation with p53 expression of cathepsin D in ovarian cancer has been reported (Lösch et al. 2004). Whether or not there is an association between tumor progression and the increased activity of β-glucuronidase, β-galactosidase, and α-mannosidase remains unknown; the present study was not planned to investigate this issue. The pelvic peritoneal fluid from the patients with gynecologic cancer was obtained at surgery immediately after laparotomy. Although no aspirated peritoneal fluids from women with gynecologic malignancy were studied, there is no reason to assume that the activity of the lysosomal enzymes will be affected by the collection procedure. This is supported by the observation that the enzyme activities measured in the aspirated peritoneal fluid at laparoscopy from women with infertility, used as normal controls in this study, were similar to the activities found in the peritoneal fluid obtained at surgery from children operated on for anatomic abnormalities (Beratis et al. 2002). It is noteworthy that similar lysosomal enzyme activities were measured in body fluids from subjects without microbial inflammation or malignancy that have been studied, namely cerebrospinal (Beratis et al. 1997; Beratis et al. 2003), peritoneal (Beratis et al. 2002) and benign ovarian cysts. This observation seems to simplify the application of measurements of lysosomal enzyme activities in body fluids for diagnostic purposes. However, the removal of the free-floating cells from fluids to be studied is essential because the lysosomal enzymes are primarily intracellular enzymes packaged within the lysosomes. Cells, therefore, lysed before or during the assay process would release the enzymes into the test fluid or the reaction mixture and, thus, greatly affect the outcome of the measurements. A major question generated by the observed increased activity of lysosomal enzymes in the peritoneal fluid of patients with malignancies is how early in the disease process is the activity of these enzymes elevated. Another question that needs to be answered, is whether the increased activity of lysosomal enzymes in the peritoneal fluid is an invariable finding in cases of gynecologic cancers. Additional large studies for the determination of the activity of lysosomal enzymes in peritoneal fluid are needed to answer these clinically important questions.

- Beratis NG, Mavrommatis T, Hatiris I, Kavaliotis J, Tsagaropoulou-Stiga H, Syrogiannopoulos GA (1997) Increased activity of lysosomal acid hydrolases in the cell-free cerebrospinal fluid of bacterial meningitis. Pediatr Res 41:235–241 [DOI] [PubMed] [Google Scholar] - Beratis NG, Georgiou G, Eliopoulou M (2002) Increased activity of lysosomal enzymes in the peritoneal fluid of bacterial peritonitis. Pediatrics 109:E44 [DOI] [PubMed] [Google Scholar] - Beratis NG, Eliopoulou MI, Syrogiannopoulos GA (2003) β-Glucuronidase in the diagnosis of bacterial meningitis and responce to treatment. Acta Paediatr 92:1272–1276 [PubMed] [Google Scholar] - Berchem G, Glondu M, Gleizes M, Brouillet JP, Vignon F, Garcia M, Liaudet-Coopman E (2002) Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene 21:5951–5955 [DOI] [PubMed] [Google Scholar] - Erickson AH, Blobel G (1979) Early events in the biosynthesis of the lysosomal enzyme cathepsin D. J Biol Chem 254:11771–11774 [PubMed] [Google Scholar] - International Federation of Gynecology and Obstetrics (1991) Annual report on the results of treatment in gynecological cancer. Int J Gynecol Obstet: 36[Supp]:132–240 - Kornfeld S (1986) Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest 77:1–6 [DOI] [PMC free article] [PubMed] [Google Scholar] - Lösch A, Schindl M, Kohlberger P, Lahodny J, Breitenecker G, Horvat R, Birner P (2004) Cathepsin D in ovarian cancer: prognostic value and correlation with p53 expression and microvessel density. Gynecol Oncol. 92:545–552. [DOI] [PubMed] [Google Scholar] - Mathieu M, Rochefort H, Barenton B, Prébois C, Vignon F (1990) Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II. Mol Endocrinol 4:1327-1335 [DOI] [PubMed] [Google Scholar] - Mikkelsen T, Yan PS, Ho KL, Sameni M, Sloane BF, Rosenblum ML (1995) Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis. J Neurosurg 83:285-290 [DOI] [PubMed] [Google Scholar] - Proia RL, Neufeld EF (1982) Synthesis of β-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor α chain in a rare form of Tay-Sachs disease. Proc Natl Acad Sci USA 79:6360–6364 [DOI] [PMC free article] [PubMed] [Google Scholar] - Rosenfeld MG, Kreibich G, Popov D, Kato K, Sabatini DD (1982) Biosynthesis of lysosomal hydrolyses: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution. J Cell Biol 93:135–143 [DOI] [PMC free article] [PubMed] [Google Scholar] - Vetvicka V, Vetvickova J, Hilgert I, Voburka Z, Fusek M (1997) Analysis of the interaction of procathepsin D activation peptide with breast cancer cells. Int J Cancer 73:403–409 [DOI] [PubMed] [Google Scholar] - Vlacha V, Eliopoulou M, Haidas S, Beratis NG (2004) Correlation of cerebrospinal fluid β-glucuronidase activity with plasma methotrexate concentrations in leukemic children receiving high dose methotrexate. Pediatr Blood Cancer 42:350–356 [DOI] [PubMed] [Google Scholar] - Vladutiu GD, Rattazzi M (1979) Excretion-reuptake route of β-hexosaminidase in normal and I-cell disease cultured fibroblasts. J Clin Invest 63:595–601 [DOI] [PMC free article] [PubMed] [Google Scholar]