Expressão de citocinas no estroma cervical de pacientes com neoplasia intra-epitelial cervical de alto grau após tratamento com Interferon α-2b intralesional

Abstract

Introduction: Uterine cervix cancer is the second most common type of cancer among women, being responsible for the death of, approximately 250 thousand women a year, and its incidence twice higher in less developed countries. Nowadays, HPV role is very well defined as a causative factor for the appearance of cervical cancer and its precursor lesions, being the excised methods used a lot in the treatment of these lesions. HPV infection is becoming more and more frequent in young patients. This fact expresses the need for conservative therapies such as interferon, which does not alter the anatomy of the cervix and preserves the reproductive future of the patients. Objectives: Evaluate the expression of messenger RNA of the cytokines (IFN- , TNF- , IL-2, IL-12, IL-4, IL-10, TGF- 1, TGF- 2 e TGF- 3) at the stoma of the uterine cervix of patients with high level of CIN, before and after the treatment with IFN -2b intralesional and evaluate the presence of HPV of high risk in responsive patients and therapeutical failure, before and after the treatment. Casuistry and Methods: Study group was composed by 10 patients ranging from 18 to 50 years with a diagnosis of high-grade cervical intraepithelial neoplasia who did not underwent previous treatment. In this work it was used Intralesional Human recombinant Interferon -2b 3.000.000 U (Blauferon B R-Blausiegel). Applications were done using a 1,0 ml syringe and 13 x 0,45 needle three times a week on alternate days (Mondays, Wednesdays and Fridays), for 6 consecutive weeks, resulting in 18 applications. Biopsy collection was done before the first application and after the 18th one of IFN -2b. For analyzing the RNAm expression of the cytokines it was used the PCR technique, using specific primers for each sequence of cytokines studied. After the PCR reactions, amplified products were submitted to polyacrylamide gel electro-1 phoresis at 10% and stained in silver. Evaluation of the viral load of HPV was done by hybrid capture. Results: At initial diagnosis 60% (n=6) of the patients were CIN II and 40% (n=4) of them were CIN III. Related to the treatment response: 60% (n=6) had an answer while 40% (n=4) had therapeuthical failure. According to the habits and life conditions questioned in the initial protocol (parity, smoking and number of sexual partners) its was seen that 70% (n=7) were multiparas, 60% (n=6) were smokers and 50% (n=5) have already had 3 or more sexual partners and the mean age of sexual intercourse was of 16,6 years (minimum of 14 and maximum of 18 years). About the expression of the cytokines before and after the treatment with IFN -2b we observed: lack of expression of cytokines Th1 (IFN- , TNF- , IL-2 e IL-12), before the treatment with IFN -2b in patients who answered and also in those who failed the treatment. However, after the treatment with IFN -2b, we observed that responsive patients expressed IFN- , IL-2, TNF- suggesting a Th1 potential related to the healing. IL-10 expression (Th2) was absent in all patients, before and after the treatment with IFN -2b. IL-4 (Th2) was expressed before the treatment with IFN -2b, in 2 responsive patients and also in 2 patients who failed the treatment. IL-4 expression, before the treatment with IFN -2b, in patients with therapeutical failure, suggests that the Th2 immune response might have contributed to the persistence of the high level lesion. Yet, concomitant expression of IL-4 and TGF- 3 in patient with therapeutical failure after the treatment with IFN -2b, suggests that the immune response of Th3 may have modulated the Th2 immune response, avoiding a satisfactory answer to the treatment. Related to the Messenger RNA expression of cytokines TGF- 1, TGF- 2 e TGF- 3, it was not possible to correlate the results with the clinical response to the treatment with IFN -2b, since one responsive patient presented concomitant expression of TGF- 1 and TGF- 2 after the treatment with IFN -2b, while other two patients who did not answer to the treatment, expressed 1 TGF- 2 and TGF- 3 after the treatment with IFN -2b. We also observed that these patients with therapeutical failure had concomitant expression of IL-12/TGF- 2 and IL-4/TGF- 3, suggesting that the immune response of Th3 may had modulated the immune response of Th1 and Th2 during the treatment with IFN and, because of that, there was a therapeutical failure. Controversial results are found in works that analyze the presence of Th1, Th2 and Th3 cytokines in premalignant cervical lesions. It was observed a significant fall of the viral load of high8 risk HPV in responsive patients (p=0,0313, Wilcoxon’s test), after the treatment with IFN -2b. Conclusions: Treatment with Intralesional IFN -2b in patients with High11 level CIN had satisfactory clinical response in 60% of the patients. Th1 immune response (IFN- , TNF- , IL-2) seems to be related to the decrease of the level of CIN, after the treatment with IFN -2b in responsive patients. TGF- expression after treatment with IFN -2b in patients with therapeutical failure suggests an immunomodulatory role of this cytokine inhibiting the Th1 protector response. There was a significant decrease on the HPV viral load of high risk in patients who answered the treatment with IFN -2b.