Vinícius Milani Budel

Growth inhibition of human in vitro and mouse in vitro and in vivo mammary tumor models by retinoids in comparison with tamoxifen and the RU-486 anti-progestagen

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (α, β, and γ) and the three RXR isotypes (α, β, and γ) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 μM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-senstive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcin oma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TTNPB was markedly more efficient than tamoxifen in terms of both inhibiting the cell proliferation level (measured by means of computer-assisted microscopy applied to Feulgen-stained nuclei, a method which enables the percentage of cells in the S phase of the cell cycle to be determined) and triggering cell death (measured by means of the determination of the transglutaminase activity) in both the MXT-HI and MXT-HS models. The very significant TTNPB-induced inhibition of the macroscopic MXT-HS growth rate relates to the triggering of cell death (apoptosis) rather than to an inhibition of cell proliferation. All these results clearly indicate that retinoids are very efficient agents against breast cancer, at least as efficient as tamoxifen.

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model

PURPOSE: To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization. METHODS: In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells. RESULTS: A concordance analysis using Kappas statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p<0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells. CONCLUSIONS: Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects.

Quimioterapia neoadjuvante em câncer localmente avançado do colo do útero

ABSTRACT Purpose: to evaluate neoadjuvant chemotherapy in locallyadvanced cervical cancer as to its acceptability, tolerability,toxicity, surgical complications, operability, response rate,and overall survival in 5 years. Methods: sixty women with locally advanced cervical cancer(stages IIB and IIIB), who were submitted to neoadjuvantchemotherapy, were included. All patients were treated withdoxorubicin-bleomycin-cisplatin. Those who had a goodresponse, allowing a surgical approach, underwent theWertheim-Meigs procedure. After surgery, they were submittedto pelvic radiotherapy. Those that could not be submitted tosurgery after chemotherapy underwent total radiotherapy. Results: the average follow-up was 108 months, and 80% ofthe patients had an overall response to neoadjuvantchemotherapy. In the IIB group, the response rate was 100%,and in the IIIB group it was 60%. The operability rate afterneoadjuvant chemotherapy was 65%. The overall survivalin 5 years was 62%. Comparing the operated group (n=34)with the nonoperated group (n=18), the overall survival in5 years was 82.14 and 16.67%, respectively.

Ploidy level determination and quantitative chromatin pattern description in pregnancy-associated breast cancers

The present study deals with the characterization of hormone-sensitivity in pregnancy-associated breast cancers (PBCs). Thischaracterization was carried out in 22 PBCs as opposed to 88non-pregnancy-associated breast cancers (NPBCs). For this study, we used thedigital cell image analysis of Feulgen-stained nuclei to assess the type ofhormone-sensitivity. In a previous study it was demonstrated that thechromatin pattern in breast cancers is related to the amounts of estrogenreceptors they contain. Our results demonstrated that the quantitative description of the chromatin pattern by means of 15 parameters (relating tomorphometric, densitometric, and textural features) made it possible toidentify typical cell nuclei populations in the PBC and NPBC groups. Theuse of specific statistical analyses (principal-components anddiscriminant) made it possible to quantify the proportion of each cellnucleus type in the PBCs. Furthermore, of the 22 PBCs under study, 13contained a large majority of cell nuclei whose chromatin pattern wascharacteristic of hormone-sensitive cells, while 5 cases contained a largemajority of typically hormone-insensitive ones. The remaining 4 casescontained a relatively similar proportion of typically hormone-sensitive and insensitive cell nuclei. The quantitative chromatin pattern descriptionthus made it possible to characterize the hormone-sensitivity level inPBCs, whereas DNA ploidy level determination did not enable any suchcharacterization to be carried out. The chromatin pattern assay describedhere, which enables hormone-sensitive pregnancy-associated breast cancersto be identified from hormone-insensitive ones independently frombiochemical assays, should help the physician regarding therapy adaptation.

Characterization of chemotherapy-induced morphonuclear modifications in the P388 leukaemia and the MXT mammary tumour models of the mouse

Chemotherapy-induced morphonuclear modifications were monitored in vivo by means of the digital cell image analysis of Feulgen-stained nuclei. Two experimental models were used, i.e. the P388 mouse leukaemia and the MXT mouse mammary carcinoma. The drugs used were doxorubicin, etoposide and cyclophosphamide. The results indicate that the chemotherrapy induced a significant decrease in the MXT tumour growth and a significant increase in the survival of the P388 leukaemic mice. These effects were accompanied at the morphonuclear level by an increase in the nuclear area, by modifications in the DNA content in accordance with the effects of the drugs on the cells cycle and by several modifications in the chromatin texture in accordance with the effects of the drugs on the cells cycle and by several modifications in the chromatin texture in accordance with the model or drugs studied. While there were neither homogeneous morphonuclear changes in all treatment groups nor clearcut correlations between the morphonuclear changes and tumour growth or the survival of the animals, the present study nertherless shows that it is possible, at least partly, to monitor in vivo certain chemotherapy-induced effects occurring at the morphonuclear level, and subsequently to obtain information on the mode of action of the drugs.

Abstract P6-02-03: The Use of Charcoal Suspension Labeling as an Adjunct in the Detection of Non-Palpable Breast Lesions

Introduction and Objectives: Inert charcoal suspension is used as a tissue marker, which provides innumerous advantages over other labeling methods and techniques, such as dyes and metallic needles. In addition, the use of charcoal does not present problems with regards to its diffusion, labeling the trajectory from the lesion to the skin, being easily identified by both the surgeon and pathologist. The occurrence of morphological alterations due to the use of charcoal is very rare. In contrast, there are still doubts and questions concerning the interference of charcoal labeling with regards to the anatomopathological diagnosis accuracy. The main objectives in this study are to analyze the efficiency of labeling impalpable breast lesions with inert charcoal suspension; to evaluate the morphological alterations associated with its use and to determine whether the use of charcoal labeling hampers with the diagnostic interpretation of the pathologist. Materials and Methods: The study evaluated a total of 135 cases of impalpable breast lesions, previously labeled with charcoal suspension. Histological H/E stained slides containing charcoal pigments were analyzed using optical microscopy, by which both quantitative and qualitative evaluations with regards to inflammatory response and interference in diagnosis were performed. Lymphocyte, giant cells and neutrophils were evaluated and quantified. Moreover, the distribution of the charcoal suspension present in the lesions was evaluated. Results: As to the quantitative and qualitative evaluation of the inflammatory response caused by the use of charcoal labeling, it was observed that granulomas were present in all samples, regardless of the quantity of injected charcoal. Lymphocytic inflammatory response was absent in 5.19% of the samples only, were 82.22% demonstrated discrete intensity and 12.59% was moderate. With regards to acute inflammatory response, 42.96% showed total absence of neutrophilic exudate, were as 42.22% demonstrated discrete and 11.11% moderate, and only 3.7% of cases were intense. Conclusion: This study corroborates the utility and easiness of the charcoal method as a means of efficient labeling of impalpable breast lesions. In addition, this technique is easy to use, has a low cost, high efficiency and does not interfere with the histological analysis. Moreover, it is comfortable for the patient and is of great help in finding and localizing the lesions to both the surgeon and pathologist. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-02-03.