EXPLORATION OF PROTEIN PCP4 AS A POTENTIAL TUMOR MARKER IN UTERINE LEIOMYOMA

Objective: Evaluation of the possible role of Purkinje cell protein 4 (PCP4) as a potential tumor marker of uterine leiomyoma by measurement of antibody titer against this protein in blood serum and the applicability of this technique for evaluation of proteins as potential vaccine antigens. Materials and methods: cDNA fragment clone was derived from PCP4 gene in leiomyoma nodule. After that the soluble PCP4-GFP chimera based on E. coli was constructed, and the recombinant protein was purified. This product was used to evaluate the upper limit of titer to detect antibodies in blood serum in patients using indirect enzyme immunoassay. Serum samples (24) were collected from donors among the patients undergoing treatment at the Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, who gave informed consent to participate in the study. Four groups of patients were formed to study the immunological activity of sera against the 6His-PCP4hs-GFP protein. Group 1 was the control group and was composed of men. Group 2 consisted of women without diagnosed fibroids who had five or more successful pregnancies in history. Group 3 included women with fibroids, who also had five or more successful pregnancies in history. Group 4 included women with recurrent myomas. Results: The pRSET-EmGFP expression vector containing the PCP4 gene and the reporter gene of the green fluorescent protein EmGFP was derived from the cDNA of the myoma nodule with a driver mutation in the MED12 gene. Human PCP4 in preparative amounts was obtained using this construct embedded in the E. coli genome, that was sufficient for analysis of its reaction with antibodies from blood serum in different groups of patients. Blood sera in the control group of men showed high immunological reactivity against the 6His-PCP4hs-GFP protein, whereas the difference between the groups of women was insignificant. Minimum difference between sera was found in groups 2 and 4. Moreover, the reaction of sera in women with recurrent fibroids was higher compared with women without fibroids, who had five or more successful pregnancies. Comparison of women in groups 3 and 4 showed statistically significant differences in the optical density values obtained in dilution of sera 1:1600, 1:3200 and 1:6400. At the same time the reaction of sera of women with recurrent fibroids was higher compared with sera of women with fibroids, who had five or more successful pregnancies. Based on the level of activity, sera were divided into 3 categories. It was found that reaction of sera of men against the 6His-PCP4hs-GFP antigen was most pronounced. The proportion of high activity in sera was 60%. In the group of women without fibroids, who had five or more successful pregnancies, the proportion of low activity in sera was 44%, that is the maximum value of indicator compared with the other groups. The group of women with fibroids, who had five or more successful pregnancies, is characterized by the moderate level of reactivity with the 6His-PCP4hs-GFP antigen. This category accounts for 75% of sera, that is the highest value of indicator among all groups. Finally, the group of women with recurrent fibroids is characterized by equal number of sera with high and moderate activity, that shows a greater tendency for production of antibodies against PCP4/PEP19 in this group of patients compared with multiparous women. Conclusion: For the first time, human PCP4 was in preparative amounts sufficient for analysis of its reaction with antibodies from blood serum in different groups of patients. Testing showed that most samples in all groups had significant titers of antibodies against PCP4. The activity of these antibodies varies widely both in sera of men and women. Antibodies against PCP4 are most common in women with recurrent fibroids compared with multiparous women, especially without leiomyomas. Thus, it is unlikely that PCP4 immunization сarries the risk of any pathology. The titers of antibodies to PCP4 in the general group of multiparous women were significantly lower compared with men and patients with recurrent fibroids. Therefore, using PCP4 as the basis to develop a preventive vaccine against leiomyomas is not advisable. © 2025 Elsevier B.V., All rights reserved.