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Clinical Investigations |
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada [G. M. Y., M. Z., A. C., M. D., W. J. H., E. P. D.]; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada [G. M. Y., L. G. K., M. Z., A. C., E. P. D.]; National Center of Scientific Research "Demokritos," JPC, 153 10 Athens, Greece [A. S.]; Department of Obstetrics and Gynecology, Gynecologic Oncology Unit, University of Turin, 10126 Turin, Italy [S. F., M. M., D. K.]; and Departments of Pathology [B. G.] and Breast and Gynecological Oncology [G. G., G. R.], S. Anna Hospital, 10126 Turin, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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23,000 new cases of ovarian cancer and 14,000 deaths from the disease were expected for the year 2000 (2)
, giving it the highest mortality rate of all gynecological malignancies. Currently, the only tumor marker that has a well-defined and validated role in the management of ovarian cancer is CA125. Serum CA125 has been evaluated in the screening for ovarian cancer, differentiation between benign and malignant ovarian masses, and prognosis (3, 4, 5, 6) . However, it does not yet have a clear place in diagnosis, prognosis, or in making treatment decisions (7 , 8) . In addition to ovarian cancer, high levels of CA125 were found in 1% of the normal population, 6% of patients with benign disease, and 28% of patients with nongynecological malignancies (9) .
Many potential new serum markers have been evaluated, either alone or in combination with CA125, including CA15-3, CA19-9, OVX1, lysophosphatidic acid (LPA), and carcinoembryonic antigen (CEA; Refs. 7 , 10 , 11 ). These new markers do not have a well-defined contribution at present, and only the combination of CA125 with untrasonography yields the highest available sensitivity and specificity (8) .
Kallikreins are serine proteases with diverse physiological functions. We, and others, have recently identified 12 new members of the KLK2 gene family on chromosome 19q13.3-q13.4 (12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . Several groups have shown that many KLK genes are differentially expressed in various malignancies (reviewed in Ref. 22 ). PSA is the best marker for prostate cancer (23) . hK2 (encoded by the KLK2 gene) is a useful marker for certain subgroups of patients (24, 25, 26, 27) . KLK10 [(normal epithelial cell-specific gene 1 (NES1)] was found to be a tumor suppressor gene (28) . The human stratum corneum chymotryptic enzyme (HSCCE) has been shown to be expressed at abnormally high levels in ovarian cancer (29) , and KLK5 is a poor prognostic marker for ovarian cancer (30) . Two new kallikrein proteins, hK6 and hK10, appear to be novel serological markers of ovarian carcinoma (31 , 32) .
KLK9 (formerly known as KLK-L3) is a newly identified member of the KLK gene family (14 , 33) , expressed in many tissues including cerebellum, spinal cord, testis, prostate, ovary, and skin. KLK9 was also found to be under steroid hormonal regulation in cancer cell lines (14) . Interestingly, KLK8 [tumor-associated differentially expressed gene-14 (TADG-14)/neuropsin] and KLK10, the two genes flanking KLK9, were found to be differentially expressed in ovarian cancer (34, 35, 36) . In addition, a very closely localized gene, KLK6, is also differentially expressed in primary ovarian tumors (19 , 31) . We thus hypothesized that KLK9 may be another member of this group of genes that are differentially expressed in ovarian cancer, and that it may represent a novel diagnostic and/or prognostic marker.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patient ages ranged from 25 to 82 years, with a median of 59 years. The sizes of residual tumors after surgery ranged from 0 to 9 cm, with a median of 2 cm. Follow-up information (median follow-up period, 62 months) was available from 166 patients, among whom 91 (55%) had relapsed and 56 (34%) had died. With respect to histological type, 82 tumors were serous papillary, 31 were endometrioid, 27 were undifferentiated, 13 were mucinous, and 14 were clear cell. The size of the residual tumors ranged from 0 to 9 cm, with a median of 1.0 cm.
Classification of histological types followed the WHO criteria (37) . All of the patients were staged according to the International Federation of Gynecology and Obstetrics staging system (38) . Grading information was available for 167 patients; 59 (35%) had grade 1 or 2 and 108 (65%) had grade 3 ovarian carcinoma. Grading was established for each ovarian tumor according to the criteria of Day et al. (39) . All of the patients were treated with postoperative platinum-based regimen chemotherapy. The first-line chemotherapy regimens included cisplatin in 94 (56%) patients, carboplatin in 50 (30%), cyclophosphamide in 69 (41%), doxorubicin in 12 (7%), epirubicin in 20 (12%), paclitaxel in 27 (16%), and methotrexate in 2 (1%). Grade 1 and stage I patients received no further treatment. Response to chemotherapy was assessed as follows: complete response was defined as a resolution of all evidence of disease for at least 1 month; a decrease (lasting at least 1 month) of at least 50% in the diameters of all measurable lesions without the development of new lesions was termed partial response. Stable disease was defined as a decrease of <25% in the product of the diameters of all measurable lesions, an increase of [mteq]25% was termed as a progressive disease. Investigations were performed in accordance with the Helsinki declaration and were approved by the Institute of Obstetrics and Gynecology, Turin, Italy. Tumor specimens were snap-frozen in liquid nitrogen immediately after surgery. Histological examination, performed during intrasurgery frozen-section analysis, allowed representative portions of each tumor containing >80% tumor cells to be selected for storage until analysis.
Total RNA Extraction and cDNA Synthesis.
Samples were shipped and stored at -80°C. They were then minced with a scalpel, on dry ice, and transferred immediately to 2-ml polypropylene tubes. They were then homogenized, and total RNA was extracted using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically. Total RNA (2 µg) was reverse transcribed into first-strand cDNA using the Superscript preamplification system (Life Technologies, Inc.). The final volume was 20 µl.
Quantitative Real-Time Reverse Transcription-PCR Analysis.
On the basis of the published genomic sequence of KLK9 (GenBank accession no. AF135026), two gene-specific primers were designed (L2–3: 5'-CAA GAC CCC CCT GGA TGT GG-3' and 5L2: 5'-AGT TTT CAG AGT CCG TCT CGG-3'). These primers spanned more than two exons to avoid contamination by genomic DNA.
Real-time monitoring of PCR reactions was performed using the LightCycler system (Roche Molecular Systems, Indianapolis, IN) and the SYBR Green I dye, which binds preferentially to double-stranded DNA. Fluorescence signals, which were proportional to the concentration of the PCR product, were measured at the end of each cycle and immediately displayed on a computer screen, permitting real-time monitoring of the PCR reaction (40) . The reaction was characterized by the point during cycling, when amplification of PCR products are first detected, rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting quantity of the template, the earlier a significant increase in fluorescence was observed (41) . The threshold cycle was defined as the fractional cycle number at which fluorescence passes a fixed threshold above baseline (42) .
Endogenous Control.
For each sample, the amount of the target and of an endogenous control (ß actin, a housekeeping gene) were determined using a calibration curve (see "Calibration Curves" below). The amount of the target molecule was then divided by the amount of the endogenous reference, to obtain a normalized target value (41)
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Calibration Curves.
Separate calibration (standard) curves for actin and KLK9 were constructed using serial dilutions of total cDNA from healthy human ovarian tissue (purchased from Clontech, Palo Alto, CA), as described previously (41
, 43)
. The standard curve calibrators were included in each run. The LightCycler software automatically calculated the standard curve by plotting the starting dilution of each standard sample versus the threshold cycle, and the sample concentrations were then calculated accordingly (Fig. 1)
. Standards for both KLK9 and actin RNAs were defined to contain an arbitrary starting concentration, and serial dilutions (with concentrations defined according to the dilution factor) were used to construct the standard curve.
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Melting Curve.
For distinguishing specific from nonspecific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 70°C for 30 s, followed by a gradual increase in temperature to 99°C at a rate of 0.1°C/s, with the signal acquisition mode set at step, as described previously (Ref. 44
; Fig. 1
). To verify the melting curve results, representative samples of the PCR products were run on 1.5% agarose gels, purified, and cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The inserts were sequenced using vector-specific primers, with an automated DNA sequencer.
Statistical Analysis.
Associations between clinicopathological parameters such as stage, grade, histotype, and residual tumor, and KLK9 expression were analyzed by the 
2 test or the Fisher’s exact test, when appropriate. For survival analysis, two different end points, cancer relapse (either local recurrence or distant metastasis) and death, were used to calculate PFS and OS, respectively. PFS was defined as the time interval between the date of surgery and the date of identification of recurrent or metastatic disease. OS was defined as the time interval between the date of surgery and the date of death.
The Cox univariate and multivariate proportional hazard regression model (45) was used to evaluate the HR (relative risk of relapse or death in the KLK9-positive group). In the multivariate analysis, the models were adjusted for KLK9 expression, clinical stage, histological grade, residual tumor, and age.
Kaplan-Meier survival curves (46) were constructed for KLK9-positive and KLK9-negative patients. For further analysis, patients were divided into two groups, either by the tumor grade (grade 1–2 versus grade 3), tumor stage (stage I-II versus stage III-IV), or by the success of debulking (optimal versus suboptimal debulking group). In each category, survival rates (disease-free survival and OS) were compared between KLK9-positive and KLK9-negative groups. The differences between the survival curves between groups were tested for statistical significance by the log-rank test (47) .
Immunohistochemistry.
Rabbit polyclonal antibody was raised against hK9 peptide sequence: N2H-CPHPGFNKDLSANDHN-CONH2 according to standard procedures. Immunohistochemical staining for hK9 was performed according to a standard immmunoperoxidase method. Briefly, paraffin-embedded tissue sections (4 µm) were fixed and dewaxed. Endogenous peroxidase activity was blocked with 3% aqueous hydrogen peroxide for 15 min. Sections were then treated with 0.4% pepsin at pH 2.0 for 5 min at 42°C and blocked with 20% protein blocker (Signet Labs) for 10 min. The primary antibody was then added at 1:6000 dilution for 1 h at room temperature. After washing, biotinylated antirabbit antibody (Signet Labs) was added, diluted 4-fold in antibody dilution buffer (Dako). After incubation and washing, streptavidin-tagged horseradish peroxidase was added for 30 min at room temperature. After washing, detection was achieved with amino ethyl carbazol (AEC) for 5–10 min. The slides were then counterstained with hematoxylin and then mounted with coverslips.
Cell Lines and Hormonal Stimulation Experiments.
The epithelial ovarian cancer cell line BG-1 and breast cancer cell lines BT-474 and T-47D, and MCF-7 line were purchased from the American Type Culture Collection (ATCC), Manassas, VA. Cells were cultured in RPMI media (Life Technologies, Inc.) supplemented with glutamine (200 mmol/liter), bovine insulin (10 mg/liter), fetal bovine serum (10%), antibiotics, and antimycotics, in plastic flasks, to near confluency. The cells were then aliquoted into 24-well tissue culture plates and cultured to 50% confluency. Twenty-four h before the experiments, the culture media were changed into Phenol Red-free media containing 10% charcoal-stripped fetal bovine serum. For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added to the culture media at a final concentration of 10-8 mM. Cells stimulated with 100% ethanol were included as controls. The cells were cultured for 24 h, and then were harvested for mRNA extraction.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 analysis, based on the ability of KLK9 values to predict the OS of the study population. As shown in Fig. 2
, a value of 1.84 (this is a unitless ratio) was shown to be the optimal cutoff (2 = 8.54; P = 0.003). This cutoff (54th percentile) identifies 46% of patients as being KLK9 positive.
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depicts the distribution of KLK9 expression (positive or negative) in ovarian tumor tissues, in relation to clinical stage, grade, histological type, size of residual tumor, menopausal status, and chemotherapy response. KLK9 expression was significantly higher in patients with early stages (I or II) compared with advanced stages (III or IV; P = 0.044) and in patients with optimal debulking (P = 0.019). Also, a slightly higher percentage of patients with positive KLK9 expression was found to have grade 1 or 2 (46%) compared with grade 3 (44%); however, this difference was not statistically significant (P = 0.49). Also, KLK9 expression was higher (although not statistically significant) in patients with complete or partial response to chemotherapy, compared with those with no response or with progression of the disease (46% versus 37%, respectively) and in patients with no residual tumors (56%) compared with those with residual tumors (38%). On the other hand, no significant associations were found between KLK9 expression and different histological types, or menopausal status.
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. Stage of disease, histological grade, and residual tumor size showed a strong association with cancer relapse and death (P < 0.001). KLK9 expression was also found to be a significant predictor of PFS and OS (HR of 0.45 and 0.49 and P of < 0.001 and 0.019, respectively). Kaplan-Meier survival curves (Fig. 3)
also demonstrate that patients with KLK9-positive tumors have substantially longer PFS and OS (P < 0.001 and P = 0.016, respectively) compared with those who are KLK9-negative.
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), the stage of disease and residual tumor size retained their prognostic significance. KLK9 expression retained its prognostic significance for PFS, but not the OS, (HR of 0.58 and 0.71 and P of 0.025 and 0.28 for the PFS and OS, respectively).
When the Cox proportional hazard regression analysis was applied to subgroups of patients (Table 3)
, KLK9 expression was found to be a significant predictor of PFS in the subgroup of patients with grade 1 or 2 (HR, 0.13; P = 0.0015; Table 3
), Stage I or II (HR, 0.099; P = 0.045) and those with optimal debulking success (HR, 0.26; P = 0.012). After adjusting for other known prognostic variables, KLK9 retained its independent prognostic value in all of these subgroups of patients. With respect to the OS, KLK9 expression was a favorable prognostic marker for the subgroup of patients with grade 1 or 2 tumors and retained its independent prognostic value after adjusting for other known prognostic variables (adjusted HR, 0.20; P = 0.038; Table 3
).
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are the PFS and OS curves for cancer patients with histological grades 1–2 or 3. Patients with KLK9-positive tumors had substantially longer PFS and OS than did patients with KLK9-negative tumors (P < 0.001 and 0.021, respectively). These differences were not seen in patients with grade 3 tumors except in PFS. With respect to stage of the disease, KLK9-positive patients in stage I or II have a significantly better PFS (P = 0.007) but not OS (Fig. 5)
. Similarly, patients with KLK9-positive tumors who had undergone optimal debulking had a higher probability of PFS (but not OS) than did patients who had KLK9-negative tumors (P = 0.013; Fig. 6
). No differences in the PFS or OS were observed when surgical debulking was suboptimal (Fig. 6)
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, hK9 is seen in the cytoplasm but not in the nuclei of the epithelial cells of normal ovarian and ovarian cancer tissues, which confirms the epithelial origin of the protein and is consistent with previous reports indicating that it is a secreted protein. These results are consistent with previous results for other kallikrein proteins which were also localized in the cytoplasm of epithelial cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A recent study suggested that CA125 could be used for prediction of optimal primary tumor cytoreduction, but only in stage III tumors (8)
. Because KLK9 expression levels are significantly different in patients with optimal and suboptimal cytoreduction, and in patients with early and late stages of the disease (Table 3
and Figs. 5
and 6
), it might also be tested for such applications. In addition, the role of CA125 in follow-up and prediction of prognosis is uncertain (7)
. KLK9, being a favorable prognostic factor (Fig. 3)
, may find applicability in this regard.
Our findings indicate that KLK9 is a favorable prognostic factor in ovarian cancer. Interestingly, additional data from other groups and our laboratory indicate that four other kallikrein genes (KLK6, KLK7, KLK8, and KLK10) are all differentially expressed in ovarian cancer (19 , 29 , 31 , 34 , 35 , 50) , and, with the exception of KLK8, all of the genes are found to have higher expression levels in advanced and more aggressive cancer. In view of this data, it will be interesting to examine simultaneously the expression of many kallikreins in ovarian cancer and to determine their function in this tissue.
The mechanism by which KLK9 and other kallikreins might be involved in the pathogenesis or progression of ovarian cancer is not known. We speculate that the enzymatic activity of these serine proteases might initiate or terminate certain biological events, e.g., the onset of angiogenesis, activation or inactivation of growth factors, receptors, cytokines, and so forth. A recent report provided evidence that another closely related kallikrein, hK3 (PSA), has antiangiogenic activity, and that this activity may be related to its action as a serine protease (51) . This study suggested also that other members of the kallikrein multigene family should be evaluated for potential antiangiogenic action. Other studies suggested that PSA inhibits the growth of MCF-7 breast cancer cell lines and prolongs the doubling time of PC-3 prostate cancer cell lines (52 , 53) .
To explore the mechanism by which KLK9 is down-regulated in advanced ovarian cancer, we examined the effect of steroid hormones on KLK9 expression in different ovarian and breast cancer cell lines. Our results indicate that KLK9 is up-regulated by steroid hormones, primarily progesterone and estrogen. Our data show also that KLK9 is a favorable prognostic factor for ovarian cancer. Ovarian cancer is one of the endocrine-related malignancies (54) , and oral contraceptive pill administration decreases the risk of ovarian cancer (1) . Furthermore, the growth of ovarian carcinoma cell lines is sensitive to estrogen (55) . Progesterone promotes cell differentiation and apoptosis, and it has been shown to inhibit DNA synthesis and cell division (56) . Recent studies supported the favorable prognostic value of progesterone receptor and its level of expression in ovarian cancer, and indicated that progesterone receptor-negative status is more abundant in grade 3 ovarian tumors (54 , 57) . Taken together, these data allow us to hypothesize that KLK9 is a candidate downstream target through which progestins and estrogens are involved in the pathogenesis of ovarian cancer.
KLK9 expression levels are negatively correlated with serum CA125 concentration (Fig. 7)
, in agreement with previous studies showing that higher CA125 levels are associated with poor prognosis in ovarian cancer (58)
. High CA125 expression levels were associated with serous and endometrioid tumors (58)
. Here, we found equal levels of KLK9 expression in serous and nonserous tumors (45 versus 42%; P = 0.39; Table 1
). This can be used for assessing prognosis, in the subgroup of patients with nonserous ovarian cancer, in which CA125 is not usually informative.
In conclusion, we here report for the first time that higher KLK9 expression has favorable prognostic value in ovarian cancer. These data add to the growing recent literature, which suggests that many other members of the same gene family (notably KLK6, KLK7, KLK8, and KLK10) also have prognostic value in ovarian cancer. It is conceivable that all of these kallikreins participate in a common pathway that is activated during ovarian cancer initiation and progression.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Mount Sinai Hospital Department of Pathology and Laboratory Medicine, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Phone: (416) 586-8443; Fax: (416) 586-8628; E-mail: ediamandis{at}mtsinai.on.ca 
2 The abbreviations used are: KLK, human kallikrein (gene); hK, human kallikrein (protein); PSA, prostate-specific antigen; PFS, progression-free survival; OS, overall survival; HR, hazard ratio.
Received 5/11/01. Accepted 9/ 4/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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