- Email: [email protected]
Overexpression of p34 cdc2 Protein Kinase in Epithelial Ovarian Carcinoma
C5J
Overexpression of p34 cdc2 Protein Kinase in Epithelial Ovarian Carcinoma BRIGITTE GARY
L.
A. BARRETTE, M.D., PREETI J. KEENEY, M.D., VERA
SRIVATSA, M.B.,B.S., WILLIAM
A. CLIBY, M.D.,
J. SUMAN, PH.D., KARL C. PODRATZ, M.D., PH.D.,
AND PATRICK C. ROCHE, PH.D.
• Objective: To investigate the role of expression of p34cdC2 protein kinase in normal, benign, and malignant ovarian epithelium. • Material and Methods: Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34 cdc2 protein kinase, followed by detection with use of a standard peroxidaselabeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. • Results: Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34 cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity Epithelial ovarian carcinoma (EOC) is one of the most common gynecologic malignant lesions and usually becomes clinically evident late in its natural evolution. Despite recent advances in surgical and cytotoxic therapy, up to 80% of patients with stage III or IV EOC will die of their disease within 5 years after diagnosis. 1.2 The pathogenesis of EOC is an ill-defined presumptive multistep process, resulting in uncontrolled cell proliferation of the epithelial lining of the ovary. Genetic alterations in the regulatory genes of the cell cycle are acquired in cancer cells.' The growth-regulating factors, both stimulating and inhibiting, are multiple and integrated in a complex framework. DNA replication and chromosome segregation are completed in orderly fashion through the cell cycle transitions, which are controlled by regulatory pathways called checkpoints. Protein kinases and From the Department of Obstetrics and Gynecology (RA.R, P.I.S.), Section of Gynecologic Surgery (W.A.C., K.c.P.), Section of Biostatistics (V,J.S.), and Division of Anatomic Pathology (G.L.K., P.C.R.), Mayo Clinic Rochester, Rochester, Minnesota. Address reprint requests to Dr. B. A. Barrette, Department of Obstetrics and Gynecology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905. Mayo Clin Proc 1997;72:925-929
was not associated with the histologic grade or surgical stage• • Conclusion: Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in carcinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdC2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34 cdc2 protein kinase in other malignant lesions suggests a common mechanism. (Mayo Clin Proc 1997;72:925-929) CDK =cyelin-dependent kinase; EOC =epithelial ovarian carcinoma; FIGO = International Federation of Gynecology and Obstetrics
cyclins are sentinel components that modulate progression through the cell cycle. They form a complex, cyclin-cyclindependent kinase (cyclin-CDK), which is inhibited or stimulated by phosphorylation. In higher eukaryotes, the cyclins D complex to CDK4 or CDK6 to regulate the progression through the G 1 phase; the cyclin E-CDK2 complex regulates entry into the S phase; cyclin A-CDK2 regulates progression through the S phase; and cyclins A and B-CDKI regulate entry into the M phase. Inappropriate progression through the cell cycle seems to be a common feature of many human cancers. G 1 regulatory genes have been shown to be important in oncogenesis.' For example, p53 and retinoblastoma tumor suppressor genes, as well as cyclin Dl, are all critical for proper transition from G 1 to S phase. The highly conserved protein kinase p34"dc2 is a key component in the regulation of entry into mitosis"!" in many eukaryotic systems. The potential for this component of the regulation of the cell cycle to be important in carcinogenesis has been confirmed by several investigations of colorectal, gastric, and oral squamous cell carcinomas.!':" We hypothesize that p34cdc2 protein kinase, a key regulatory enzyme that governs the transition from G2 to M phase 925
© 1997 Mayo Foundationfor Medical Education and Research
926
OVARIAN CARCINOGENESIS
and the induction of mitosis, may be redundantly overexpressed in uncontrolled cellular proliferation. The purpose of this study was to investigate p34 cdc2protein kinase expression in normal, benign, and malignant ovarian epithelium. Additionally, we sought to evaluate any correlation between p34cdc2 expression and clinicopathologic variables in ovarian cancer specimens.
MATERIAL AND METHODS Study Population.-A retrospective review was conducted of 26 patients chosen at random with the diagnosis of EOC who underwent a primary cytoreductive surgical procedure for International Federation of Gynecology and Obstetrics (FIGO) stages I to IV disease between 1990 and 1992 at Mayo Clinic Rochester and had tumor tissue available from the primary operation. Six normal, six benign serous cystadenoma, and six benign mucinous cystadenoma specimens randomly chosen were also evaluated. Each specimen was histologically reviewed to confirm the grade and histologic subtype. All histologic analyses were performed by an experienced pathologist (G.L.K.), who was unaware of the patient's clinical course. After histologic review, two patients were excluded from the study--one had a borderline ovarian tumor and the other had pancreatic carcinoma metastatic to the ovary. The study cohort consisted of the other 24 patients. All patients underwent follow-up until death or for a minimum of 4.5 months (median, 4.3 years; maximum, 5.6 years). Study F actors.-Inpatient and outpatient medical records were abstracted for the patients with EOC. Demographic information collected included age at primary cytoreductive surgical procedure, FIGO stage, amount of residual disease after primary cytoreduction, results at "second-look" laparotomy, and survival. Analysis of p34cdc2 Protein Kinase.-Only tumor tissue obtained at cytoreduction or oophorectomy (benign specimens) was used for immunohistochemical analysis. Immunohistochemical staining for p34 cdc2 protein kinase was performed with a peroxidase-labeled streptavidin-biotin technique. Formalin-fixed paraffin-embedded tissue was used. Paraffin tissue sections (5 11m) were mounted on glass slides that had been treated with aminoalkyl-saline. Endogenous peroxidase activity was blocked by incubation with hydrogen peroxide-methanol, and sections were proteolytically digested to enhance antigenicity. The immunoperoxidase technique involved the sequential application of diluted (1 in 25) mouse IgG monoclonal antibody to human p34
Mayo Clio Proc, October 1997, Vol 72
Antigen was visualized by incubation of tissue sections with the chromogen 3-amino-9-ethylcarbazole. Sections were counterstained with 3% hematoxylin and mounted with coverslips. Normal human tonsil tissue was used as a positive control because of the high proliferation and mitotic activity in the germinal centers. The normal ovarian tissue (from six patients) was used as a negative control. Evaluation.-The hematoxylin-eosin-stained sections were initially examined for histologic grade and subtype. The immunohistochemical stainings were evaluated for both the pattern and the intensity of staining in the tumor cells. The pattern of staining was described as either focal or diffuse. "Focal" was defined as scattered positive cells; "diffuse" was defined as 75% or more of the tumor cells positive. The intensity of staining was graded as follows: "negative" = no staining; "weak" = slight staining of tumor cells; and "strong" = intense staining, equivalent to normal proliferating lymphocytes in the germinal centers of tonsillar tissue. Statistical Analysis.-Fisher's exact test was used to assess whether p34cdc2 protein kinase staining pattern or intensity was associated with either histologic grade or FIGO stage. The Kaplan-Meier product-limit method was used to estimate the overall survival. When the survival distribution was estimated, patients found to be alive at the point of last contact (by any means) were censored from the analysis on that date.
RESULTS Study Cohort.-The median age of the 24 patients with EOC was 64 years (range, 15 to 81); 4 patients were younger than 50 years of age, and 7 patients were older than 70 years. Surgical management included definitive staging and aggressive cytoreduction in all patients, the latter process necessitating bowel resection in six patients (25%). Surgical staging (FIGO) identified 9 stage I or II tumors (38%) and 15 stage III or IV tumors (62%). The amount of residual disease at the completion of the initial operation was none or microscopic in 13 patients (54%), macroscopic but 2 em or less in 7 (29%), and more than 2 cm in 4 (17%). Adjuvant cisplatin-based cytotoxic therapy was administered to 19 patients (79%), 12 of whom received 6 or more cycles. A second-look laparotomy was performed in nine patients (38%); at completion, six of these patients had microscopic or no residual disease, and three had macroscopic disease. At last follow-up, 13 patients were alive with no disease, 3 were alive with disease, and 8 had died. The 3-year survival rate was 69% (95% confidence interval, 52 to 91%). Histology.-The nonmalignant specimens consisted of six normal surface epithelial ovarian tissues, six mucinous
Mayo Clio Proc, October 1997, voln
OVARIAN CARCINOGENESIS
927
Fig. 1. Low-power view of normal human tonsil, showing strong p34cdC2 proteinkinase immunostaining (counterstained with hematoxylin). (Original magnification, x25.)
Fig. 2. High-power view of normal human tonsil, depicting strong specific cytoplasmic and perinuclear p34cdC2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x150.)
cystadenomas, and six serous cystadenomas. The distribution of the histologic cellular subtypes for the 24 EOe specimens was as follows: 9 serous (38%), 5 mucinous (21%), 2 clear cell (8%), 5 endometrioid (21%), and 3 mixed variety (12%). Assessment of histologic grade for the 24 tumors demonstrated 8 grade 1 (33%),3 grade 2 (12%), 10 grade 3 (42%), and 3 grade 4 (12%). Immunohistochemistry.-The positive tissue control (normal human tonsil) yielded uniformly strong cytoplasmic staining, with occasional cells showing nuclear positivity. A low-power view of normal human tonsil with strong p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is shown in Figure l. All immunostaining was preponderantly cytoplasmic and perinuclear, but occasional scattered cells also displayed nuclear positivity. A highpower view of normal human tonsil with strong specific cytoplasmic and perinuclear p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is presented in Figure 2.
Surface epithelial cells from six normal ovaries were used as control specimens and failed to stain. Of six benign mucinous ovarian cystadenomas, five failed to stain, and one showed a weak, focal pattern. Of six benign serous ovarian cystadenomas, five also failed to stain, and one demonstrated a weak, focal pattern. A low-power view of EOe with diffuse p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is depicted in Figure 3, and a high-power view of EOe with large pleomorphic cells showing diffuse p3¥dc2 protein kinase immunostaining (counterstained with hematoxylin) is displayed in Figure 4. Positive immunostaining was observed in 19 of 24 tumors (79%), and 16 of the 19 (84%) demonstrated strong expression of the p34 cdc2 protein kinase. Of the 19 tumors that showed positive staining, 10 (53%) had a diffuse and 9 (47%) had a focal pattern. The stage of the EOe versus the intensity and pattern of p34 protein kinase immunostaining is summarized in Table 1. No evidence suggested that surgical stage (high versus low) was associated with the
Fig. 3. Low-power view of epithelial ovarian carcinoma, demonstrating diffuse p34cdc2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x25.)
Fig. 4. High-power view of epithelial ovarian carcinoma with large pleomorphic cells, showing diffuse p34cdC2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x150.)
928
Mayo Clin Proc, October 1997, Vol 72
OVARIAN CARCINOGENESIS
Table 1.-Stage of Epithelial Ovarian Carcinoma Versus Pattern (Focal or Diffuse) and Intensity (Negative or Weak Versus Strong) of p34 Protein Kinase Immunostaining Intensity
Pattern Focal Stage
No.
%of total
I
2
10.5
II III
7
36.8
Total
9
47.4
IV
Negative or weak
Diffuse No.
%of total
3 1 5 1 10
Strong
No.
%of total
15.8 5.3 26.3 5.3
2 2 4
52.6
8
pattern of p34 staining (focal versus diffuse: P = 0.628) or intensity (negative or weak versus strong: P = 0.412). The grade of EOC versus the pattern and intensity of the p34 cdc2 protein kinase irnrnunostaining is shown in Table 2. We found no evidence to suggest that histologic grade (low versus high) was associated with the pattern of p34 staining (focal versus diffuse: P = 0.370) or intensity (negative or weak versus strong: P = 0.999). DISCUSSION
p34"dc2 protein kinase is an important CDK for the transition from G 2 to M phase and the induction of mitosis. cdc2 kinase is the active subunit of the M-phase promoting factor. The p34 cdc that we used was a mouse monoclonal IgG2a antibody. It exhibits broad interspecies reactivity recognizing p34 cdc2 from humans and mice but with no cross-reactivity with any other protein tested to date. The highly conserved protein kinase p34 cdc2 is a key player in the regulation and induction of the M phase."!" In the fission yeast Schizosaccharomyces pombe, this kinase is the product of the cdc" gene and is required both for progression from G\ to S phase and as a checkpoint in the G 2
No.
%of total
8.3 8.3 16.7
4 1 10 1
16.7 4.2 41.7 4.2
33.3
16
66.7
phase that determines when the cell initiates mitosis. 14-16 The equivalent protein kinase in the budding yeast Saccharomyces cerevisiae is encoded by the gene cdc28 17 and has a role in the transition from G 1 to S phase and in mitosis.v" The p34 cdc2 protein kinase is also implicated in the induction of mitosis in other organisms, including mammals.v-" The human homologue of cdc2+ and cdc28 is cdc2 Hs (or CDKl), which encodes for the threonine-serine protein kinase p34 cdc2 and is required for mitosis," whereas initiation of the S phase requires CDK2, a cdc2-related protein kinase. 22-25 As many as eight other human CDK proteins have been identified." Our study demonstrated negative immunostaining of normal epithelial ovarian tissue and most benign epithelial tissue (10 of 12), in comparison with strong up-regulation in most cases of EOe. Several investigators have reported overexpression of cdc2 in solid tumors. Yasui and coworkers" found a higher p34 cdc2 protein kinase activity in 91.7% of gastric carcinoma specimens in comparison with the corresponding nonneoplastic mucosa, but they also failed to demonstrate any relationship between the protein expression and the clinical features. All colonic carcinoma cell lines
Table 2.-Grade of Epithelial Ovarian Carcinoma Versus Pattern (Focal or Diffuse) and Intensity (Negative or Weak Versus Strong) of p34 Protein Kinase Immunostaining Intensity
Pattern Focal Grade
No.
%of total
1 2 3 4
5
26.3
4
21.1
Total
9
47.4
Diffuse No.
%of total
1 2 4 3 10
Negative or weak
Strong No.
%of total
12.5 4.2 16.7
5 2 6 3
20.8 8.3 25.0 12.5
33.3
16
66.7
No.
%of total
5.3 10.5 21.1 15.8
3 1 4
52.6
8
Mayo Clin Proc, October 1997, Vol 72
OVARIAN CARCINOGENESIS
929
studied had higher p34
Overexpression of p34 cdc2 Protein Kinase in Epithelial Ovarian Carcinoma BRIGITTE GARY
L.
A. BARRETTE, M.D., PREETI J. KEENEY, M.D., VERA
SRIVATSA, M.B.,B.S., WILLIAM
A. CLIBY, M.D.,
J. SUMAN, PH.D., KARL C. PODRATZ, M.D., PH.D.,
AND PATRICK C. ROCHE, PH.D.
• Objective: To investigate the role of expression of p34cdC2 protein kinase in normal, benign, and malignant ovarian epithelium. • Material and Methods: Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34 cdc2 protein kinase, followed by detection with use of a standard peroxidaselabeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. • Results: Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34 cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity Epithelial ovarian carcinoma (EOC) is one of the most common gynecologic malignant lesions and usually becomes clinically evident late in its natural evolution. Despite recent advances in surgical and cytotoxic therapy, up to 80% of patients with stage III or IV EOC will die of their disease within 5 years after diagnosis. 1.2 The pathogenesis of EOC is an ill-defined presumptive multistep process, resulting in uncontrolled cell proliferation of the epithelial lining of the ovary. Genetic alterations in the regulatory genes of the cell cycle are acquired in cancer cells.' The growth-regulating factors, both stimulating and inhibiting, are multiple and integrated in a complex framework. DNA replication and chromosome segregation are completed in orderly fashion through the cell cycle transitions, which are controlled by regulatory pathways called checkpoints. Protein kinases and From the Department of Obstetrics and Gynecology (RA.R, P.I.S.), Section of Gynecologic Surgery (W.A.C., K.c.P.), Section of Biostatistics (V,J.S.), and Division of Anatomic Pathology (G.L.K., P.C.R.), Mayo Clinic Rochester, Rochester, Minnesota. Address reprint requests to Dr. B. A. Barrette, Department of Obstetrics and Gynecology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905. Mayo Clin Proc 1997;72:925-929
was not associated with the histologic grade or surgical stage• • Conclusion: Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in carcinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdC2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34 cdc2 protein kinase in other malignant lesions suggests a common mechanism. (Mayo Clin Proc 1997;72:925-929) CDK =cyelin-dependent kinase; EOC =epithelial ovarian carcinoma; FIGO = International Federation of Gynecology and Obstetrics
cyclins are sentinel components that modulate progression through the cell cycle. They form a complex, cyclin-cyclindependent kinase (cyclin-CDK), which is inhibited or stimulated by phosphorylation. In higher eukaryotes, the cyclins D complex to CDK4 or CDK6 to regulate the progression through the G 1 phase; the cyclin E-CDK2 complex regulates entry into the S phase; cyclin A-CDK2 regulates progression through the S phase; and cyclins A and B-CDKI regulate entry into the M phase. Inappropriate progression through the cell cycle seems to be a common feature of many human cancers. G 1 regulatory genes have been shown to be important in oncogenesis.' For example, p53 and retinoblastoma tumor suppressor genes, as well as cyclin Dl, are all critical for proper transition from G 1 to S phase. The highly conserved protein kinase p34"dc2 is a key component in the regulation of entry into mitosis"!" in many eukaryotic systems. The potential for this component of the regulation of the cell cycle to be important in carcinogenesis has been confirmed by several investigations of colorectal, gastric, and oral squamous cell carcinomas.!':" We hypothesize that p34cdc2 protein kinase, a key regulatory enzyme that governs the transition from G2 to M phase 925
© 1997 Mayo Foundationfor Medical Education and Research
926
OVARIAN CARCINOGENESIS
and the induction of mitosis, may be redundantly overexpressed in uncontrolled cellular proliferation. The purpose of this study was to investigate p34 cdc2protein kinase expression in normal, benign, and malignant ovarian epithelium. Additionally, we sought to evaluate any correlation between p34cdc2 expression and clinicopathologic variables in ovarian cancer specimens.
MATERIAL AND METHODS Study Population.-A retrospective review was conducted of 26 patients chosen at random with the diagnosis of EOC who underwent a primary cytoreductive surgical procedure for International Federation of Gynecology and Obstetrics (FIGO) stages I to IV disease between 1990 and 1992 at Mayo Clinic Rochester and had tumor tissue available from the primary operation. Six normal, six benign serous cystadenoma, and six benign mucinous cystadenoma specimens randomly chosen were also evaluated. Each specimen was histologically reviewed to confirm the grade and histologic subtype. All histologic analyses were performed by an experienced pathologist (G.L.K.), who was unaware of the patient's clinical course. After histologic review, two patients were excluded from the study--one had a borderline ovarian tumor and the other had pancreatic carcinoma metastatic to the ovary. The study cohort consisted of the other 24 patients. All patients underwent follow-up until death or for a minimum of 4.5 months (median, 4.3 years; maximum, 5.6 years). Study F actors.-Inpatient and outpatient medical records were abstracted for the patients with EOC. Demographic information collected included age at primary cytoreductive surgical procedure, FIGO stage, amount of residual disease after primary cytoreduction, results at "second-look" laparotomy, and survival. Analysis of p34cdc2 Protein Kinase.-Only tumor tissue obtained at cytoreduction or oophorectomy (benign specimens) was used for immunohistochemical analysis. Immunohistochemical staining for p34 cdc2 protein kinase was performed with a peroxidase-labeled streptavidin-biotin technique. Formalin-fixed paraffin-embedded tissue was used. Paraffin tissue sections (5 11m) were mounted on glass slides that had been treated with aminoalkyl-saline. Endogenous peroxidase activity was blocked by incubation with hydrogen peroxide-methanol, and sections were proteolytically digested to enhance antigenicity. The immunoperoxidase technique involved the sequential application of diluted (1 in 25) mouse IgG monoclonal antibody to human p34
Mayo Clio Proc, October 1997, Vol 72
Antigen was visualized by incubation of tissue sections with the chromogen 3-amino-9-ethylcarbazole. Sections were counterstained with 3% hematoxylin and mounted with coverslips. Normal human tonsil tissue was used as a positive control because of the high proliferation and mitotic activity in the germinal centers. The normal ovarian tissue (from six patients) was used as a negative control. Evaluation.-The hematoxylin-eosin-stained sections were initially examined for histologic grade and subtype. The immunohistochemical stainings were evaluated for both the pattern and the intensity of staining in the tumor cells. The pattern of staining was described as either focal or diffuse. "Focal" was defined as scattered positive cells; "diffuse" was defined as 75% or more of the tumor cells positive. The intensity of staining was graded as follows: "negative" = no staining; "weak" = slight staining of tumor cells; and "strong" = intense staining, equivalent to normal proliferating lymphocytes in the germinal centers of tonsillar tissue. Statistical Analysis.-Fisher's exact test was used to assess whether p34cdc2 protein kinase staining pattern or intensity was associated with either histologic grade or FIGO stage. The Kaplan-Meier product-limit method was used to estimate the overall survival. When the survival distribution was estimated, patients found to be alive at the point of last contact (by any means) were censored from the analysis on that date.
RESULTS Study Cohort.-The median age of the 24 patients with EOC was 64 years (range, 15 to 81); 4 patients were younger than 50 years of age, and 7 patients were older than 70 years. Surgical management included definitive staging and aggressive cytoreduction in all patients, the latter process necessitating bowel resection in six patients (25%). Surgical staging (FIGO) identified 9 stage I or II tumors (38%) and 15 stage III or IV tumors (62%). The amount of residual disease at the completion of the initial operation was none or microscopic in 13 patients (54%), macroscopic but 2 em or less in 7 (29%), and more than 2 cm in 4 (17%). Adjuvant cisplatin-based cytotoxic therapy was administered to 19 patients (79%), 12 of whom received 6 or more cycles. A second-look laparotomy was performed in nine patients (38%); at completion, six of these patients had microscopic or no residual disease, and three had macroscopic disease. At last follow-up, 13 patients were alive with no disease, 3 were alive with disease, and 8 had died. The 3-year survival rate was 69% (95% confidence interval, 52 to 91%). Histology.-The nonmalignant specimens consisted of six normal surface epithelial ovarian tissues, six mucinous
Mayo Clio Proc, October 1997, voln
OVARIAN CARCINOGENESIS
927
Fig. 1. Low-power view of normal human tonsil, showing strong p34cdC2 proteinkinase immunostaining (counterstained with hematoxylin). (Original magnification, x25.)
Fig. 2. High-power view of normal human tonsil, depicting strong specific cytoplasmic and perinuclear p34cdC2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x150.)
cystadenomas, and six serous cystadenomas. The distribution of the histologic cellular subtypes for the 24 EOe specimens was as follows: 9 serous (38%), 5 mucinous (21%), 2 clear cell (8%), 5 endometrioid (21%), and 3 mixed variety (12%). Assessment of histologic grade for the 24 tumors demonstrated 8 grade 1 (33%),3 grade 2 (12%), 10 grade 3 (42%), and 3 grade 4 (12%). Immunohistochemistry.-The positive tissue control (normal human tonsil) yielded uniformly strong cytoplasmic staining, with occasional cells showing nuclear positivity. A low-power view of normal human tonsil with strong p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is shown in Figure l. All immunostaining was preponderantly cytoplasmic and perinuclear, but occasional scattered cells also displayed nuclear positivity. A highpower view of normal human tonsil with strong specific cytoplasmic and perinuclear p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is presented in Figure 2.
Surface epithelial cells from six normal ovaries were used as control specimens and failed to stain. Of six benign mucinous ovarian cystadenomas, five failed to stain, and one showed a weak, focal pattern. Of six benign serous ovarian cystadenomas, five also failed to stain, and one demonstrated a weak, focal pattern. A low-power view of EOe with diffuse p34 cdc2 protein kinase immunostaining (counterstained with hematoxylin) is depicted in Figure 3, and a high-power view of EOe with large pleomorphic cells showing diffuse p3¥dc2 protein kinase immunostaining (counterstained with hematoxylin) is displayed in Figure 4. Positive immunostaining was observed in 19 of 24 tumors (79%), and 16 of the 19 (84%) demonstrated strong expression of the p34 cdc2 protein kinase. Of the 19 tumors that showed positive staining, 10 (53%) had a diffuse and 9 (47%) had a focal pattern. The stage of the EOe versus the intensity and pattern of p34 protein kinase immunostaining is summarized in Table 1. No evidence suggested that surgical stage (high versus low) was associated with the
Fig. 3. Low-power view of epithelial ovarian carcinoma, demonstrating diffuse p34cdc2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x25.)
Fig. 4. High-power view of epithelial ovarian carcinoma with large pleomorphic cells, showing diffuse p34cdC2 protein kinase immunostaining (counterstained with hematoxylin). (Original magnification, x150.)
928
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OVARIAN CARCINOGENESIS
Table 1.-Stage of Epithelial Ovarian Carcinoma Versus Pattern (Focal or Diffuse) and Intensity (Negative or Weak Versus Strong) of p34 Protein Kinase Immunostaining Intensity
Pattern Focal Stage
No.
%of total
I
2
10.5
II III
7
36.8
Total
9
47.4
IV
Negative or weak
Diffuse No.
%of total
3 1 5 1 10
Strong
No.
%of total
15.8 5.3 26.3 5.3
2 2 4
52.6
8
pattern of p34 staining (focal versus diffuse: P = 0.628) or intensity (negative or weak versus strong: P = 0.412). The grade of EOC versus the pattern and intensity of the p34 cdc2 protein kinase irnrnunostaining is shown in Table 2. We found no evidence to suggest that histologic grade (low versus high) was associated with the pattern of p34 staining (focal versus diffuse: P = 0.370) or intensity (negative or weak versus strong: P = 0.999). DISCUSSION
p34"dc2 protein kinase is an important CDK for the transition from G 2 to M phase and the induction of mitosis. cdc2 kinase is the active subunit of the M-phase promoting factor. The p34 cdc that we used was a mouse monoclonal IgG2a antibody. It exhibits broad interspecies reactivity recognizing p34 cdc2 from humans and mice but with no cross-reactivity with any other protein tested to date. The highly conserved protein kinase p34 cdc2 is a key player in the regulation and induction of the M phase."!" In the fission yeast Schizosaccharomyces pombe, this kinase is the product of the cdc" gene and is required both for progression from G\ to S phase and as a checkpoint in the G 2
No.
%of total
8.3 8.3 16.7
4 1 10 1
16.7 4.2 41.7 4.2
33.3
16
66.7
phase that determines when the cell initiates mitosis. 14-16 The equivalent protein kinase in the budding yeast Saccharomyces cerevisiae is encoded by the gene cdc28 17 and has a role in the transition from G 1 to S phase and in mitosis.v" The p34 cdc2 protein kinase is also implicated in the induction of mitosis in other organisms, including mammals.v-" The human homologue of cdc2+ and cdc28 is cdc2 Hs (or CDKl), which encodes for the threonine-serine protein kinase p34 cdc2 and is required for mitosis," whereas initiation of the S phase requires CDK2, a cdc2-related protein kinase. 22-25 As many as eight other human CDK proteins have been identified." Our study demonstrated negative immunostaining of normal epithelial ovarian tissue and most benign epithelial tissue (10 of 12), in comparison with strong up-regulation in most cases of EOe. Several investigators have reported overexpression of cdc2 in solid tumors. Yasui and coworkers" found a higher p34 cdc2 protein kinase activity in 91.7% of gastric carcinoma specimens in comparison with the corresponding nonneoplastic mucosa, but they also failed to demonstrate any relationship between the protein expression and the clinical features. All colonic carcinoma cell lines
Table 2.-Grade of Epithelial Ovarian Carcinoma Versus Pattern (Focal or Diffuse) and Intensity (Negative or Weak Versus Strong) of p34 Protein Kinase Immunostaining Intensity
Pattern Focal Grade
No.
%of total
1 2 3 4
5
26.3
4
21.1
Total
9
47.4
Diffuse No.
%of total
1 2 4 3 10
Negative or weak
Strong No.
%of total
12.5 4.2 16.7
5 2 6 3
20.8 8.3 25.0 12.5
33.3
16
66.7
No.
%of total
5.3 10.5 21.1 15.8
3 1 4
52.6
8
Mayo Clin Proc, October 1997, Vol 72
OVARIAN CARCINOGENESIS
929
studied had higher p34