Patient and Tumor Specimens
The cohort of three hundred and eight specimens of ovarian carcinoma archived blocks containing formalin-fixed, paraffin-embedded infiltrating ovarian carcinoma was obtained from the Department of Pathology, The University of Texas M.D. Anderson Cancer Center. Patients who had undergone initial surgery between 1990 and 2001 were included in this study. Follow-up was updated till June 2003 by review of medical records and the United States Social Security Index. Demographic and survival data were entered into a comprehensive database created with SPSS (version 15.0). Histopathology diagnosis was based on WHO criteria; the samples were assigned a grade based on Gynecologic Oncology Group criteria [16
] and staged according to the International Federation of Gynecology and Obstetrics system [17
]. The percentage of subjects who survived the disease for a defined period of time identifies disease-specific survival. Survival was calculated from the date of diagnosis to the date of death, and only deaths from the disease were considered. For the entire population of 308 cases, with 282 valid cases, and 26 missing cases, the mean age was 58.36 yr; the median age was 59.83 yr (range: 67). The mean follow-up was 51.4 mo (range: 0.2–262.7 mo).
Construction of Tissue Microarrays
Tissue blocks were stored under ambient conditions at −24°C. Core samples from morphologically representative areas of paraffin-embedded tumor tissues were assembled on a recipient paraffin block to create tissue microarrays. Arrays were constructed with a precision instrument (Beecher Instruments, Silver Spring, MD) that uses two separate core needles for punching the donor and recipient blocks and a micrometer-precise coordinate system for assembling tissue samples on a block. Five-micrometer sections were obtained and stained with H&E to confirm the presence of tumor and to assess tumor histology. Tumor samples were arranged at random.
Sample tracking was based on coordinate positions for each tissue spot in the tissue microarray block. Spots were transferred onto tissue microarray slides for staining. This sample tracking system was linked to a Microsoft access database containing demographic, clinicopathologic, and survival data, thereby allowing rapid links between histologic data and clinical features. The array was read according to the tissue microarray map. Each core was scored individually and the results were presented as the mean of the two replicate core samples.
The immunoperoxidase staining method used in these studies was a modification of the avidin–biotin complex technique as described previously [3
]. The modifications from the standard method were incorporated to ensure high sensitivity and specificity. Tissue microarray sections (5 µm) were deparaffinized, dehydrated, and subjected to antigen retrieval using microwave oven (2 min at 1000 W and 6 min at 200 W) followed by incubation with 0.05% trypsin in phosphate-buffered solution (PBS) for 15 min at room temperature. The endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and the slides were then treated with 10% normal horse or goat serum for 30 min. Incubation with primary antibodies was performed at 4°C overnight. Following washes with PBS, the slides were incubated with biotinylated secondary antibodies and incubated with avidin–horseradish peroxidase complex (Vector Laboratories, Burlingame, CA). Detection was performed with the 0.125% aminoethylcarbazole chromogen substrate solution (AEC; Sigma, St. Louis, MO). After counter-staining with Mayer’s hematoxylin (DakoCytomation, Carpinteria, CA), the slides were mounted. In the study for the correlation of nuclear EGFR expression, a polyclonal antibody (Upstate, Lake Placid, NY) was used to detect EGFR in a total of 308 cases consisted of tumor specimens. Concentrations of the antibodies used were as follows: EGFR, 3.4 mg/L (diluted 1:150 from 510 mg/L); Ki-67, 0.47 mg/L; and EGFR, 1.3 mg/L.
To ensure absolute objectivity of these immunohistochemical studies, these experiments used the ACIS III Automated Cellular Imaging System (from Dako company) to analyze tissues scoring and quantification (nuclear or membrane and cytoplasm applications based on percent and intensity). The percentage of positive tumor cells was used for statistical analysis.
The correlation between the expression level of EGFR, cyclin D1, and Ki-67 in the immunostained tumor specimens was analyzed using the Pearson χ2 test. To correlate the levels of nuclear EGFR, cyclin D1, and Ki-67 with overall patient survival, Kaplan–Meier survival analysis and log-rank test were performed. All statistical analyses were done using SPSS 15.0 software.
Immunofluorescence and Confocal Analysis of Cancer Cells
Following serum starvation for 24 h, OVCA420 cells were treated with or without EGF (50 ng/mL) for 30 min, washed twice with ice-cold PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and permeablized using 0.2% triton X-100. Following treatment with 0.1% normal goat serum for 30 min, cells were incubated with indicated primary antibodies (i.e., monoclonal EGFR antibody COOH terminus, Novocastra, Bannockburn, IL) for 1 h at room temperature. Following washes, cells were further incubated with goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) tagged with fluorescein diluted at 1:500. To delineate the nuclear morphology, nuclear marker ToPro3 was used. Immunostained cells were examined under an OLYMPUS FV300 laser microscope.
Immunofluorescence and Confocal Analysis of Primary Tumors
Ovarian cancer tissue sections were deparaffinized and dehydrated in a graded series of alcohol. Then, they were heated to induce epitope retrieval (HIER) with 10 mM Tris–EDTA buffer, pH 9.0. Then, they were blocked in 3% H2O2 solution for 10 min, and treated with 3% BSA in PBS for 30 min. The slides were incubated overnight at 4°C with RTU-EGFR 384 monoclonal antibody (ready to use, from Novocastra). Slides were extensively washed with PBST (0.05% Tween 20 in PBS), and incubated for 60 min at room temperature with Alexa 633 goat–mouse IgG, work dilution 1:500 (from Invitrogen). After PBST buffer washing, the slides were incubated for 2 h at room temperature with Ki-67 and cyclin D1 polyclonal antibody (ready to use, from Neomarkers, Union City, CA). The slides were then washed with PBST buffer and incubated for 60 min at room temperature with fluorescein goat–rabbit IgG, with a working dilution of 1:500 (from Invitrogen). To delineate the nuclear morphology, the nuclear marker DAPI was used. Immunofluorescent stained tissues were examined under an OLYMPUS FV300 laser microscope.
Nuclear Fractionation and Western Blot Analyses
Nuclear fractions extracted from serum-starved OVCA420, OVCA433, OVCAR3 cells treated with or without 50 ng/mL EGF for 30 min were subjected to SDS–PAGE and Western blot analyses as described previously [20
]. Cellular fractionation was performed as described previously [21
]. Briefly, cells were washed twice with ice-cold PBS, harvested, and lysed in a lysis buffer. After incubation on ice for 10 min, the cells were homogenized by 20 strokes in a tightly fitting Dounce homogenizer. The homogenate was centrifuged at 1500g
for 5 min to sediment the nuclei. The supernatant was then centrifuged for 20 min, and the resulting supernatant formed the non-nuclear fraction. The nuclear pellet was washed (3×) with lysis buffer. To extract nuclear proteins, the isolated nuclei were resuspended in NETN buffer, and sonicated briefly. Nuclear lysates were collected after centrifugation. Samples were subjected to SDS–PAGE, and then transferred to nitrocellulose membranes. Immunoreactive protein bands were detected with an enhanced chemiluminescence reagent (Pierce or Amersham Biosciences, Piscat-away, NJ). The antibodies used in this study were as follows: anti-EGFR (Novocastra Laboratories), anti-lamin B (Calbiochem, San Diego, CA), anti-a tubulin (Sigma). All secondary antibodies were obtained from Vector Laboratories (Burlingame, CA) and Jackson Immunoresearch Laboratories (West Grove, PA).