Affiliations of authors: B. A. Werness, P. Parvatiyar (Department of Pathology), J. Wiest (Department of Environmental Health), University of Cincinnati Medical Center, OH; S. J. Ramus, B. A. J. Ponder, Department of Oncology, University of Cambridge, U.K.; A. S. Whittemore, K. Garlinghouse-Jones, I. Oakley-Girvan, Department of Health Research and Policy, Stanford University Medical Center, CA; R. A. DiCioccio (Department of Cancer Genetics), M. S. Piver (The Gilda Radner Familial Ovarian Cancer Registry), Roswell Park Cancer Institute, Buffalo, NY; Y. Tsukada, Sisters Hospital, Buffalo.
Correspondence to: Bruce A. Werness, M.D., Department of Pathology, University of Cincinnati Medical Center, 231 Bethesda Ave., Cincinnati, OH 45267 (e-mail: bruce.werness{at}uc.edu).
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ABSTRACT |
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INTRODUCTION |
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SUBJECTS AND METHODS |
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Mutation analysis. BRCA1 was amplified from the DNA of peripheral white blood cells in 40 fragments and was screened for mutations by use of combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA). Mutations in samples showing alterations by SSCP or HA were identified by sequencing an independent DNA amplification product (4).
Microdissection of tissue. Microdissection of abnormal epithelial cells and stromal cells from fixed, paraffin-embedded sections of ovary was done by use of an Arcturus PixCell II Laser Capture microdissecting microscope (Arcturus Engineering, Inc., Mountain View, CA). This system utilizes a transparent thermoplastic film applied to the surface of the tissue section on standard histopathology slides. The ovarian epithelial or stromal cells to be microdissected were identified and targeted through a microscope, and a narrow (~15 µM) carbon dioxide laser-beam pulse specifically activated the film above these cells. The resulting strong focal adhesion allowed selective procurement of only the targeted cells.
p53 immunohistochemistry.
Tissue sections of 4 µm were cut, deparaffinized, and stained with a monoclonal antibody to p53 (clone DO-1, Oncogene Research, Cambridge, MA) at a dilution of 1 : 300. Sections were subjected to heat-induced antigen retrieval overnight at 75 °C in citrate buffer prior to addition of the primary antibody. All staining was carried out by use of the avidinbiotin method on a Ventana 320 Automated System (Ventana Medical System, Tucson, AZ). Diaminobenzidine was used as the chromogen. Negative controls consisted of substituting an isotypic mouse immunoglobulin G (IgG) (MOPC-21, IgG1 ; Sigma Immunochemicals, St. Louis, MO) for the primary antibody. The positive control consisted of a breast carcinoma from the surgical pathology files of the University of Cincinnati that was known from previous analysis to overexpress p53 protein.
LOH analysis. Two microsatellite markers linked to BRCA1 (D17S786 and D17S579) and to TP53 (TP53 and D17S855) were analyzed for LOH. DNA from microdissected tissue was extracted in 50 µL of solution containing 0.04% proteinase K, 0.5% Tween, 50 mM Tris (pH 8.5), and 1 mM EDTA (pH 8.0) at 37 °C for 24 hours followed by heat inactivation at 95 °C for 5 minutes. One microliter of the solution containing DNA was added to 13-µL polymerase chain reaction (PCR) mixtures described as follows: For D17S786, TP53, and D17S579, these contained 2.5 mM of each deoxynucleoside triphosphate (dNTP) (Panvera, Madison, WI), 7 µM of each primer, 1x ExTaq buffer, and 1.25 U of TaKaRa ExTaq DNA polymerase (Panvera). For D17S855, the amplification mixture contained 10 mM of each dNTP, 7 µM of each primer, 1.25 U of MasterTaq, 1x TaqMaster buffer, and 1x TaqMaster PCR enhancer (Eppendorf, Westbury, NY). Primers with the following sequences were synthesized by the University of Cincinnati Core Biopolymer Facility: D17S786forward 5`-TACAGGGATAGGTAGCCGAG-3` and reverse 5`-GGATTTGGGCTCTTTTGTAA-3`; D17S855forward 5`-ACACAGACTTGTCCTACTGCC-3` and reverse 5`-GGATGGCCTTTTAGAAAGTGG-3`; D17S579forward 5`-AGTCCTGTAGACAAAACCTG-3` and reverse 5`-CAGTTTCATACCAAGTTCCT-3`; and TP53forward 5`-AGGGATACTATTCAGCCCGAGGTG3` and reverse 5`-ACTGCCACTCCTTGCCCCATTC- 3`. (Note: TP53 is used to designate this specific microsatellite polymorphism as well as the gene, TP53.)
Fluorescent labels were incorporated at the 5` end of the forward primers. Touchdown PCR conditions employed were as follows: denaturation at 95 °C for 5 minutes; 20 cycles of denaturation at 95 °C for 40 seconds, annealing temperature plus 10 °C for 40 seconds (decreased by 0.5 °C per cycle), and extension at 72 °C for 1.5 minutes; 10 cycles of denaturation at 95 °C for 40 seconds, annealing temperature for 40 seconds, and extension at 72 °C for 1.5 minutes; and a final extension of 72 °C for 10 minutes.
The annealing temperatures were 58 °C, 55 °C, 58 °C, and 50 °C for D17S786, D17S855, TP53, and D17S579, respectively. PCR products were detected, and microsatellite allele sizes were determined on an ABI 377 sequencing instrument (Perkin-Elmer Applied Biosystems, Foster City, CA) with the use of 1 µL of amplified product. Visual inspection of electropherograms and Genotyper 2000 software (Perkin-Elmer Applied Biosystems) were used to compare the relative intensities of the two alleles and to determine LOH by use of criteria recommended by the manufacturer. These criteria determine the ratio of the peak heights on the electropherogram corresponding to the tumor and normal alleles; if this ratio is less than 0.67 or greater than 1.35, then LOH is established.
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RESULTS |
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Pathology.
The original pathology report describes the ovaries as grossly unremarkable, yellowtan, bosselated structures appropriate for the patient's age. The greatest measurements from the glass slides are 1.8 cm and 2.3 cm for the right and left ovaries, respectively. Histologically, normal, flat to cuboidal surface epithelium is seen focally on both ovaries, but the left ovary exhibits a small focus of markedly atypical epithelium on the ovarian surface that extends focally into stromal invaginations of the surface epithelium (Fig. 1, A and B). The cells comprising this epithelium are enlarged, stratified, and exhibit loss of polarity (Fig. 1
, C). The cytologic and nuclear features are those of a high-grade carcinoma. These features include marked nuclear pleomorphism, coarse chromatin with irregular chromatin distribution, irregular macronucleoli, and frequent mitoses (Fig. 1
, C and D). Features of stromal invasion, such as an irregular epithelial/stromal interface, single cells or small groups of cells within stroma, and stromal reaction, are not present. The interface between malignant and normal surface epithelium is present in several areas and exhibits an abrupt boundary, without an area of transition (Fig. 1
, E). Strong nuclear immunoreactivity for p53, the protein product of TP53, is present and is confined to the abnormal cells (Fig. 1
, F).
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DISCUSSION |
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Similarly, there have been difficulties in determining the timing of TP53 alterations in ovarian cancer. Attempts to classify a TP53 mutation as an "early" or "late" event in ovarian carcinogenesis based on the relative proportion of patients showing mutations in early- or late-stage ovarian cancers have generated conflicting opinions (1221). However, such classification cannot address the question of whether or not TP53 is mutated prior to invasion. The abnormal ovarian epithelial cells in the patient on whom we report showed both LOH near the TP53 locus and strong immunoreactivity for p53. Normally, cellular p53 has a short half-life, contributing to low steady-state levels of p53 that are not detected by immunohistochemistry. However, TP53 missense mutations alter the structure of p53, resulting in a longer half-life, greater stability, and detection by immunohistochemistry (22). Thus, there is strong evidence for both mutation of TP53 and LOH of the remaining allele. Although TP53 mutations do not appear necessary for ovarian carcinogenesis in women with germline BRCA1 mutations (2,23,24), they appear substantially more often in BRCA1-related ovarian cancers than in sporadic ovarian cancers (23,24).
In summary, we have identified a carcinoma in situ in an ovary removed prophylactically from a woman with a germline BRCA1 mutation. Analysis of DNA from microdissected abnormal cells showed LOH at loci linked to both the BRCA1 and TP53 genes, as well as immunohistochemical evidence for mutation of the TP53 gene. This is the first molecular analysis of this rarely seen lesion and provides insight into the development of carcinoma in these patients.
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NOTES |
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Manuscript received February 16, 2000; revised April 26, 2000; accepted May 1, 2000.
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