Ki67 and E-cadherin Are Independent Predictors of Long-term Survival in Endometrial Carcinoma

Patients and Methods

In all, 106 patients were followed until their demise, or for a median of 120 months in the case of survival (range=84-240 months). At the end of the study, 38 patients had died, and 68 were alive. Our whole, larger series of endometrial carcinoma patients was purged of those surviving patients with a follow-up period of less than 72 months, because this was the period in which the bulk (33/38, 86.8%) of death events occurred, with five outliers scattered among the rest of the follow-up period. The series was also purged of those cases not submitted to a complete surgical staging at our center, with an unclear histology, or with two or more missing immunohistochemical data.

Histologically, 89 [84%] were endometrioid carcinomas, followed by the papillary serous [9], mucinous [3], clear cell [2], solid [2] and mixed clear cell/solid variety [1].

Immunohistochemistry. The expression of estrogen and progesterone receptors (ER and PR), Ki67, c-erb-B2, p53, Bcl-2, and E-cadherin were studied by means of immunohistochemistry.

The immunohistochemical technique employed and antibodies used have been described extensively elsewhere (1-3).

Briefly, 5 μM sections were obtained from the corresponding paraffin blocks, and subsequently processed in an automatic Dako autostainer, using the dedicated Dako EnVision system (Dako, Glostrup, Denmark). The antibodies used were also purchased from Dako in prediluted, ready-to-use form, with the only exception of the c-erb-B2 antibody, which was diluted 1:50 in our laboratory prior to use.

All specimens were diagnosed by the same pathologist (IGR), thus ensuring uniformity of interpretation. A final score was obtained from two parameters: number of stained cells (1: 0-10%; 2: 11-50%; 3: >50%), and intensity of staining (absent: 0; weaker than the positive control: 1; similar to the positive control: 2; stronger than the positive control: 3). The addition of both partial scores gave a final score which ranged from 0 to 6.

We used as cutoff levels for positivity the same ones obtained from our previous studies on the biological relevance of the different markers tested (1-3), with the only exception of p53. Thus, only cases in the very upper range (scores 5 & 6) were considered positive in the case of ER, PR, c-erb-B2, and E-cadherin, whereas Bcl-2 was considered positive from score 2 upwards. This is very practical for clinical use, since a strongly positive immunohistochemical reaction in most tumor cells and any degree of positivity in any number of tumor cells are relatively easy to interpret. Ki67 was treated as a continuous variable, expressed as the percentage of positive cells. The case of p53, as mentioned, is particular. In our previous studies (1-3), we had applied a cutoff upwards of 10% staining nuclei, often used in practice for the identification of the mutant protein. However, recent evidence (4) points towards the fact that, in the particular case of endometrial carcinoma, as addressed here, only very strong nuclear staining in virtually all tumor cells represents mutant p53 protein. We have thus shifted our cutoff correspondingly to the upper tertile of the distribution (scores 5 & 6), at variance with our previous reports.

Ethics. The study, involving data and specimens from actual patients treated at the institution, was approved by the Ethics Committee (code 2021.405) of Hospital Universitario Valdecilla, Santander, Spain.

Statistics. The statistical analysis was carried out after blinding all personal data pertaining to the individual patients studied. Differences in survival were calculated by means of the Kaplan–Meier method and visualized by the corresponding curves.

Hazard ratios were calculated by means of a Weibull regression model (5). Successive restricted models, eliminating confounding variables yielded a final restricted model best adapted to practical use in the clinical setting. Values were considered significant when p<0.05.