No abstract

462 Background: The activity of the receptor activator of nuclear factor-kβ RANK/RANKL in cancer cells has been correlated with tumor progression and poor prognosis in solid tumors including bladder cancer. Regulatory T cells (Tregs), often identified by FOXP3 biomarker, suppress the anti-tumor response and allow immune tolerance through suppression of T cells. Immunomodulator OncoTherad is an inorganic phosphate nanocomplex associated with glycosidic protein, developed by the University of Campinas/Brazil, with antitumor effects. Previous reports have demonstrated immune activation and antitumor effects of Platelet Rich Plasma (PRP). We evaluated the effects of OncoTherad associated with PRP in the RANK/RANKL system and Tregs in a mouse model of non-muscle invasive bladder cancer (NMIBC). Methods: C57BL/6J mice were assigned to groups (n = 42): Control; Cancer (N-ethyl-N-nitrosourea carcinogen, 50 mg/ml); PRP (0.1 ml); OncoTherad (20 mg/ml); OncoTherad+PRP 10 mg/ml and OncoTherad+PRP 20 mg/ml. The intravesical doses (0.1 ml) were instilled once a week for 6 consecutive weeks after induction. Results: After NMIBC induction, all animals in the Cancer group showed flat carcinoma in situ (pTis) and both percentages of RANK, RANKL, OPG, and FOXP3 positive cells and the intensity of immunoreaction for these antigens were significantly higher in comparison with healthy animals. In addition to ensuring this NMIBC model, these results indicated the involvement of RANK/RANKL in urothelial carcinogenesis and the presence of Tregs in a suppressed immune tumor microenvironment. Mice treated with PRP only showed a 28.6% rate of tumor progression inhibition (TPI) and exhibited papillary urothelial carcinoma (pTa) and pTis. In this group, the intensity of the RANKL and FOXP3 immunoreaction was weaker when compared to the Cancer group. Thus, PRP showed immunomodulatory effects, reducing Tregs that are sources of RANKL. Oncotherad immunotherapy led to an TPI of 85.7%, and benign flat hyperplasia was the most frequent diagnosis. Oncotherad reduced the total RANK and RANKL immunoreactivities and decreased the intensity of RANKL immunostaining in comparison to the Cancer. In the OncoTherad+PRP 10 mg/ml or 20 mg/ml group, TPI was 71.4%, with a predominance of non-malignant lesions such as flat hyperplasia, low-grade intraurothelial neoplasia, and reactive atypia. Treatments with Oncotherad and Oncotherad plus PRP decreased the percentage of FOXP3+ cells and reduced the intensity of FOXP3 immunoreaction compared to the Cancer and PRP groups. Conclusions: The tumor inhibition obtained with Oncotherad plus PRP was related to the alteration of the immune profile of the tumor microenvironment by decreasing RANK/RANKL expression and Tregs, resulting in an effective immune response against the tumor.

e17560 Background: Ovarian cancer (OC) ranks fifth among cancer deaths in women and remains the deadliest of all gynecological cancers. OncoTherad is a nanostructured complex developed by University of Campinas/Brazil, which exhibits immunomodulatory and antitumor properties. Erythropoietin (EPO) can exert non-hematopoietic functions and the association of immunotherapy and EPO is a reality in anemic patients due to cancer or chemotherapy who may use immunotherapies. We evaluated the effects of OncoTherad and EPO, alone or in combination, on PD-1/PDL-1 and CTLA-4 checkpoints, pro-angiogenic (VEGF) and anti-angiogenic (Endostatin) markers, and microvessel density (CD31-labeled capillaries) in chemically induced ovarian cancer in rats. Methods: Thirty-five Fischer rats were distributed into groups: Control (Sham surgery); Cancer (7,12-dimethylbenzoanthracene – DMBA injection in the ovarian bursa, 1.25 mg/kg); OncoTherad (20mg/kg); EPO (8.4 µg/kg); and OncoTherad+EPO (same doses of the isolated treatments). Intraperitoneal doses were administered twice a week for 4 weeks. Immunohistochemistry was analyzed as total immunoreactivity and intensity of immunoreaction. Results: The Cancer group showed an increase (p < 0.05) in microvascular density and histopathological lesions, predominantly high-grade dysplasias with non-metastatic phenotype, which was related to the little involvement of PD-1/PD-L1 and CTLA-4 checkpoints in this carcinogenesis stage. The treatments altered the frequency of ovarian lesions, especially OncoTherad promoted a significant reduction of cystic lesions, while in the EPO group there was marked hyperplasia of the interstitial tissue and the associated treatment led to intermediate results. OncoTherad immunotherapy was effective in reducing angiogenesis, promoting a decrease (p < 0.05) in VEGF immunostaining and microvessel count, and an increase (p < 0.05) in endostatin immunoreactivity. EPO treatment decreased (p < 0.05) endostatin levels in comparison with OncoTherad group and led to a weaker (p < 0.05) immunoreaction intensity of PD-1/PD-L1 and CTLA-4 compared to the Cancer group. These effects might be related to the modification of the tumor microenvironment modulated by EPO with a decrease in the population of immune cells with immunosuppressive phenotype as well as histopathological alterations including reduction of folliculogenesis and luteogenesis. Conclusions: OncoTherad was able to prevent histopathological lesions and reduce the angiogenesis involved in the ovarian carcinogenesis. EPO treatment decreased endostatin, which possibly had effects on angiogenic activity. However, EPO also modulated the OC microenvironment by reducing PD-1/PD-L1 and CTLA-4. The association of OncoTherad+EPO might have triggered a compensatory effect between the isolated treatments.

Most colorectal cancers (CRC) arise from precursor lesions. We aimed to characterize the mutation profile of CRC precursor lesions in a Brazilian population. In total, 90 FFPE lesions, including 67 adenomas, 7 sessile serrated lesions, and 16 hyperplastic polyps, were analyzed by next-generation sequencing. The genetic ancestry of the patients was estimated. Somatic driver mutations were identified in 66.7% of cases, including alterations in APC (32.2%), TP53 (20.0%), KRAS (18.9%), BRAF (13.3%) and EGFR (7.8%). Adenomas displayed a higher number of mutations, mainly in APC, compared to serrated polyps (73.1% vs. 47.8%, p = 0.039). Advanced adenomas had a higher frequency of mutation in KRAS and GNAS and a high overall mutation rate than early adenomas (92.9% vs. 59%, p = 0.002). Concerning the serrated pathway, a higher frequency of mutations, mainly in BRAF, was observed in sessile serrated lesions (85.7%) compared to hyperplastic polyps (31.3%, p = 0.027). A high degree of ancestry admixture was observed in the population, with a predominance of European followed by African components. The mutation profile of Brazilian colorectal precursor lesions exhibits a similar landscape to other populations. These results bestow the knowledge of CRC's biological history and may contribute to a molecular screening approach.

e17589 Background: The term ovarian carcinoma (OC) refers to a heterogeneous collection of five distinct diseases known as histotypes. While histotype-specific treatment is still a clinical challenge, well-characterized models are required for testing new therapies. OncoTherad is a nanoimmunotherapy developed by University of Campinas/Brazil, which exhibits antitumor properties. Erythropoietin (EPO) has cytoprotective effects including in the ovaries. We assessed whether a chemically-induced animal OC model was representative of human disease by evaluating which histotype it best represents. We evaluated both mutational and immunohistochemical (IHC) biomarkers routinely used for human OC and used the observed features to provide context in the evaluation of OncoTherad and EPO effects. Methods: Thirty-five Fischer rats were distributed into 5 groups: Control (Sham surgery); Cancer (7,12-dimethylbenzoanthracene – DMBA injection in the ovarian bursa, 1.25 mg/kg); OncoTherad (20mg/kg IP); EPO (8.4 µg/kg IP); and OncoTherad+EPO (same doses). Ovary specimens were formalin-fixed into paraffin-embedded donor blocks. After DNA extraction and tissue microarray construction, we assessed typical gene mutations directly by Sanger sequencing (Pik3ca, Ctnnb1, and Kras) or indirectly using IHC surrogates (Arid1a and p53). Finally, we examined biomarkers typical of different OC histotypes (Wt1, Pr, Hnf1β) as well as lymphocyte density (CD3) by IHC. Results: The results were consistent across the cancer-induced animals. The majority of abnormal epithelial cells were Wt1+, Pr-, and Hnf1b-. Therefore, our rat model of DMBA-induced ovarian cancer was most likely serous type ovarian carcinoma, in agreement with the histopathological analysis. The ovarian lesions were molecularly similar to low-grade serous ovarian carcinoma as abnormal pattern of p53 staining was rarely observed. Loss of immunoreactivity for Arid1a protein was seen in some abnormal epithelium. However, the interpretability of mutation surrogates in rat tissues may not always be consistent with IHC analysis systems applied for human tissues. Furthermore, DNA sequencing did not reveal driver mutations in any of the sampled specimens. The treatments, especially OncoTherad+EPO, increased the number of CD3 positive immune cells. After EPO treatment, the tumor and abnormal areas showed a higher number of CD3+ lymphocytes than the normal regions; following a previous work, this could be due to substantial presence of Foxp3 positive cells. Conclusions: The features analyzed favored a low-grade serous carcinoma model in which treatments with OncoTherad and EPO showed immunomodulatory properties related to the reduction of ovarian lesions seen in previous work. The overall data highlight the importance of further characterizations of animal models to provide a complete and specific panel for testing new drugs.