Expression of Tp53, p21, and HDM2 in ovarian tumors
Expression and mutations of Tp53 have been investigated by many laboratories and the prevalence has been well established for ovarian cancer [11
]. Our goal was to investigate in more details of the alteration of the Tp53 pathway and its impact on epithelial cell organization, and we investigated the expression of Tp53, p21, and HDM2 in ovarian cancer tumor tissue microarrays and selective tissue sections. The cell cycle inhibitor p21WAF1/CIP1
is induced by Tp53 and may be used as an indicator of the transcriptional activity of Tp53 [23
]; HDM2 is a negative regulator of Tp53 protein expression and the expression of HDM2 may be a mechanism for the suppression of the Tp53 activity [18
The staining protocols were first refined and both positive and negative controls were established in our laboratory. Adjacent sections were stained for Tp53, p21, and HDM2. After excluding defective cores, we obtained informative results from a total of 83 cases from the initial 170 cores derived from 85 individual ovarian tumor cases (Supplemental Table 1
). Tp53 is positive in 75.9% and p21 is positive in 12.0% of the cases. Of these 83 cases, only one case, a Grade 1–2 ovarian adenocarcinoma, showed HDM2 positive staining (1.2%). We examined the relationship between the expression of Tp53 and p21 (). In 9 out of 10 cases in which p21 is positive, Tp53 is also positive. In only one case of p21 positive, Tp53 negative tumor was observed. In the majority (56/65) of Tp53 positive tumors, p21 is negative. Examples of the staining are shown in for a Tp53-positive, p21-negative (), and a Tp53-positive, p21-positive () ovarian carcinoma. The Tp53-positive, p21-negative cases indicate the inability of the Tp53 to induce p21, suggesting the presence of Tp53 mutation in these tumors. These correlations are consistent with a regulation of p21 by Tp53, and Tp53 mutations that abolish the transcriptional activation of the protein are prevalent in ovarian cancer.
Correlation between immunostaining of Tp53 and p21.
Fig. 1 Immunostaining of ovarian cancer for Tp53 and p21. Adjacent sections of 83 ovarian carcinomas were stained with Tp53 and p21. Representative examples are shown. (A) An ovarian carcinoma stained positive for Tp53 but negative for p21. (B) An ovarian carcinoma (more ...)
There are 17 cases (20.5%) of ovarian carcinomas in which both Tp53 and p21 are negative in immunostaining (). It is possible that the Tp53 pathway is not activated in these tumors. However, these p53 and p21 negative tumors may still have p53 inactivation/mutation that precluded expression of the antigens recognized by the p53 antibody used. Expression of p21 may be also induced by a Tp53-independent mechanism as suggested by one case of Tp53 negative but p21-positive tumor ().
Further examination of Tp53 and p21 staining indicates that though positive p21 staining often correlates with positivity for p53 staining, the percentage of cells with a positive p21 staining varies (Supplemental Table 1
). For example, in an ovarian tumor where 80% of the tumor cells stained positive for Tp53, only 40% of the cells were found to be p21-positive in an adjacent section. In general, the percentage of cells positive for p21 is significantly less than that positive for Tp53.
Contiguous epithelia linking benign and neoplastic cells
We reviewed our collection of ovarian cancer samples to select interesting cases that contained contiguous epithelia linking morphological normal monolayer with multilayered neoplastic cells. These types of transitional epithelia may be informative for genetic and epigenetic changes that can determine or be associated with transition from monolayer epithelial cells to neoplastic cells of the transformed epithelia. Presumably, the genetic and epigenetic states of the cells around the histological transition are similar since they are derived from a relatively recent precursor cell, and the differences in gene expression and genetic mutation(s) may be critical in the formation of the morphological changes.
Over the years, we collected both malignant and benign ovarian tumors that contain both normal and adjacent tumor areas that may be informative for insight in changes in markers associated with epithelial transformation. Of the 38 cases of ovarian carcinomas collected and studied by our lab, 28 were malignant including 18 serous adenocarcinomas, 5 mucinous adenocarcinomas, 3 endometrioid adenocarcinomas, and 3 clear-cell carcinomas; 9 LMP tumors including 6 serous and 2 mucinous subtypes; the remaining one case was benign fibrous cystadenoma. Areas of contiguous epithelia with histological transition from benign to malignant epithelium were identified in 10 out of these 38 ovarian tumors. Although most of the cases we investigated are serous carcinomas, such histological transitions were found in all histological subtypes. As shown in for an example of a serous ovarian carcinoma, the lesion (, indicated by a “*”) contains morphological normal monolayer epithelial cells (, arrowhead), which are contiguously linked with neoplastic, multilayer epithelial cells (, arrows) with some gradual transition from normal to malignant. The monolayer epithelial cells appear non-neoplastic, showing no nuclear atypia or mitotic figures. Herein, we refer to these monolayer epithelia linking contiguously with multilayered overt neoplastic cells as histological transition areas, or transition epithelia. We reason such epithelial histological transitions may be informative for studying of genetic or epigenetic changes associated with neoplastic transformation.
Fig. 2 Examples of epithelial histological transitions. Reviewing a large collection of ovarian tumors identified cases containing epithelial histological transitions from benign to neoplastic. (A) An example shows an ovarian serous carcinoma containing an epithelial (more ...)
Epithelial proliferation correlates with transformation
In all the histological transitions, a striking correlation we found is the association of cell proliferation, as indicated by Ki-67 staining, with morphological transformation from monolayer to multilayers (). The epithelial cells residing in the monolayer regions show no mitotic figures and lack nuclear atypia. Consistently, few cells within the monolayer of ovarian surface epithelium (arrowhead, ), including the epithelia of the histological transition, are positive for ki67. However, a high percentage of the neoplastic cells immediately adjacent to the histological transition are Ki-67-positive (arrow, ). Additionally, the monolayer epithelia that are negative for ki67 are positive for calretinin (), and the calretinin negative cancer cells show obvious nuclear atypia. Calretinin was determined to be a marker for ovarian surface epithelial cells but its expression is lost upon neoplastic transformation [26
]. Thus, histological transition from monolayer to neoplastic cells associates with a dysregulation of cell cycle progression and proliferation. We reasoned that cell growth contributes to the formation of multilayer epithelial neoplastic cells.
Fig. 3 Analysis of histological transitions. (A) Four examples of Ki-67 staining as a marker for cell proliferation are presented. A striking correlation between positive Ki-67 staining and morphological transformation from monolayer (arrowhead) to multilayers (more ...)
Correlation of Tp53 and p21 expression with morphological transformation of ovarian surface epithelia
We further examined the epithelial histological transitions by staining Tp53 and p21. In most of the cases, the staining of Tp53 and p21 is complementary: Tp53 is negative in the monolayer region of the epithelial histological transition, where p21 is positive; Tp53 is positive in the neoplastic cells, in which p21 is negative, as shown in an example (). In this carcinoma, a Tp53 positive staining suggests the presence of Tp53 mutation, and the mutant protein lacks activity to induce p21 expression.
Using larger captured microdissection (LCM), we dissected ovarian surface epithelial, hyperplastic epithelial, and tumor cells () for analysis of Tp53 mutations by PCR-sequencing. Following gaining of experience in some failed initial cases, we were able to obtain sequence information for all 8 exons (exons 4 to 11) and to identify Tp53 mutation from LCM dissected materials including ovarian surface epithelia, hyperplastic lesions, and invasive carcinoma cells from a single slide (). In all 4 cases of tumors in which Tp53 is positive and p21 is negative, Tp53 point mutations were found, as shown by an example (). We did not find mutations in 4 cases in which p21 is positive. Thus, the results suggest that p21 expression is a very good indicator of Tp53 transcriptional activity, and positive staining for Tp53 with negative p21 suggests a mutant Tp53, and positive staining for Tp53 with positive p21 suggests a wild type Tp53.
Fig. 4 Examples showing laser capture microdissection (LCM) to dissect cells for mutation analysis. (A) Using LCM, normal ovarian surface epithelial, hyperplastic epithelial, and tumor cells were dissected for PCR and sequencing analysis. H&E stainings (more ...)
Progressive mutation of Tp53 in epithelial transformation
In 2 cases, we were able to obtain sufficient cells from the epithelial monolayer at the site of histological transition to produce informative mutation analysis of Tp53. In the first case, no Tp53 mutation was identified in the dissected epithelial cells of the monolayer, though a point mutation (base #14487, G to T, GenBank accession U94788) was identified in the tumor, resulting in a CGT to CTT, and Arg to Leu conversion at exon 8. In the second case, following LCM and PCR-sequencing of exons 4 to 11 of Tp53 from the dissected materials (), we identified a heterozygous Tp53 mutation in the transitional epithelial cells and a homozygous mutation (GTG:Val to ATG:Met) in the tumor cells (). The base change occurs at exon 6 of the p53 gene at base 13406 from “G” to “A” (GenBank accession U94788), resulting in a conversion of Val to Met in the mutant protein. We estimated that the monolayer epithelial cells dissected contained less than 10% of stromal cells, and thus the heterozygous Tp53 mutation signal was not due to an artifact of the potential contamination of stromal cells in the dissected sample. A homozygous Tp53 mutation of the same base was identified in the adjacent tumor cells (). Also, materials scraped from the slides that contain largely stromal cells show a wild type Tp53 sequence (), suggesting that the mutation is associated with tumorigenesis.
Fig. 5 Laser capture microdissection (LCM) and mutation analysis of cells at the histological transitions. (A) Ovarian carcinomas containing histological transitions were subjected to LCM to isolate surface epithelial cells and adjacent tumor cells on a contiguous (more ...)
Thus, we have found that the monolayer epithelial cells at the histological transition exhibited a heterozygous state of Tp53 mutation, which became homozygous state in the adjacent tumor cells. We reason that these morphological normal epithelial cells in the histological transitions can be considered as precursor lesions of ovarian cancer.