Analysis of Ki-67 expression in women with breast cancer: Comparative evaluation of two different methodologies by immunophenotyping.

The Ki-67 antigen is a nuclear protein with proven prognostic value in different neoplasms and recognizes the predictive value in breast cancer (BC). No consensus exists on the ideal cutoff point. In this study, Ki-67 expression was evaluated in samples of BC by flow cytometry (FC) and compared with immunohistochemical (IHC) examination. For this, the BC tissue samples were sectioned, macerated, filtered, and marked with anti-Ki-67 FITC and anti-CD45 V500 antibodies. We selected the neoplastic cells according to CD45 expression and size and internal complexity (FSC × SSC) using the Infinicity 1.7 software. Lymphocytes were negative control. We compared the results with IHC analyses carried out in parallel and independently. The expression of Ki-67 was evaluated in both methodologies through Bland-Altman analysis. Among the 44 samples analyzed, only three showed bias higher than the established confidence interval (mean bias 2.1%, p = 0.62), with no significant difference for the perfect mean bias (0%). Therefore, one can state that FC provides results equivalent to IHC analysis and possibly analyzes more cells simultaneously. The results obtained in this study show the absence of observational bias through software analysis in a larger number of tumor cell populations. We can conclude that FC may be a promising alternative method for investigating Ki-67 in solid tumours.

Evaluation of ER, PR and HER2 markers by flow cytometry for breast cancer diagnosis and prognosis.

BACKGROUND AND AIMS: Laboratory diagnosis of breast cancer (BC) is done by morphological analysis and immunohistochemistry (IHC). However, this methodology still has some limitations. The aim of this study is to validate flow cytometry (FC) immunophenotyping to investigate diagnostic and prognostic markers of BC. METHODS: Tumor samples from surgical specimens of patients previously diagnosed with BC, were first sliced and then macerated together with PBS. Then, sample was filtered and the single cell suspension obtained was labeled with antibodies against estrogen (ERα), progesterone (PR) and HER2 receptors and CD45. The results were compared, in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), with reference methods. RESULTS: Results obtained comparing FC with reference methods were: ERα detection (sensitivity: 75%; specificity: 90%; PPV: 96.7%; NPV: 47.4%); PR detection (sensitivity: 72%; specificity: 70%; PPV: 79.3%; NPV: 60.8%); HER2 detection (sensitivity: 80%; specificity: 90.2%; PPV: 66.7%; NPV: 94.9%). CONCLUSION: The results obtained show the capacity of this methodology on BC markers differentiation. FC, together with morphological analysis and IHC can overcome individual limitations of each methodology and provide reliable results on a faster and efficient manner, resulting in improvements on BC diagnosis and prognosis.

Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity.

Malignant neoplasms are one of the leading causes of death worldwide and hematologic malignancies, including acute leukemia (AL) is one of the most relevant cancer types. Current available chemotherapeutics are associated with high morbidity and mortality rates, therefore, the search for new molecules with antitumor activity, specific and selective for neoplastic cells, became a great challenge for researchers in the oncology field. As pyrazolines stand out in the literature for their great variety of biological activities, the aim of this study was to synthesize and evaluate the antileukemic activity of five new pyrazoline derivatives. All pyrazolines showed adequate physicochemical properties for a good oral bioavailability. The two unpublished and most effective pyrazoline derivatives have been selected for further experiments. These compounds are highly selective for leukemic cells when compared to non-neoplastic cells and did not cause lysis on human red blood cells. Additionally, selected pyrazolines induced cell cycle arrest at G0/G1 phase and decreased cell proliferation marker KI67. Apoptotic cell death induced by selected pyrazolines was confirmed by morphological analysis, assessment of phosphatidylserine residue exposure and DNA fragmentation. Several factors indicate that both intrinsic and extrinsic apoptosis occurred. These were: increased FasR expression; the predominance of Bax in relation to Bcl-2; the loss of mitochondrial membrane potential; AIF release; decreased expression of survivin (an antiapoptotic protein); and the activation of caspase-3. The selected pyrazolines were also found to be cytotoxic against neoplastic cells collected from the peripheral blood and bone marrow of patients with different subtypes of acute leukemia.