This exploratory, cross-sectional, analytical casecontrol study was approved by the Ethics Committee of the Cancer Institute of the State of So Paulo (project number 1035/2016) and by the Ethics Committee for Analysis of Research Projects at the Hospital das Clnicas of the USP Medical School (CAPPesq). Potential subjects were informed about the study at the time of a scheduled appointment, and if they agreed to participate and satisfied the inclusion and exclusion criteria, they provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations and were consistent with the Declaration of Helsinki.

Patient selection occurred between September 2017 and December 2018. During this period, all patients diagnosed with breast cancer who were seen at the First Consultation Clinic of the Cancer Institute of the State of So Paulo and who satisfied the inclusion criteria were invited to participate. Patients initially referred to the General Didactic Outpatient Clinic at the Mastology section at the Hospital das Clnicas of the USP Medical School because of abnormalities observed on a mammogram performed elsewhere, and who subsequently underwent a second mammogram with negative results at our center, and who met the inclusion criteria, were recruited for the control group.

The inclusion criteria of the breast cancer group were a histological diagnosis of breast cancer, no previous treatment for breast cancer, age between 25 and 75years, the absence of signs or symptoms of other neoplasias and no previous history of other neoplasms. The exclusion criteria of the breast cancer group were the presence of noncarcinoma breast neoplasia, such as sarcoma or phyllodes tumor, and the presence of other comorbidities, such as nephropathies, liver disease, heart disease, hematopathologies, immunological diseases or other neoplasms. The inclusion criteria for the control group were women between 25 and 75years of age, the absence of current signs or symptoms of other neoplasms and no previous history of neoplasms. The exclusion criteria for the control group were the presence of any neoplasia and the presence of other comorbidities, such as kidney disease, liver disease, heart disease, hematopathologies, immunological diseases or other neoplasms. Race was self-identified by each subject.

During the initial consultation, 10ml of blood was collected in nonheparinized tubes and transported to the Structural and Molecular Research Laboratory in Gynecology at the Faculty of Medicine of the University of So Paulo within 30min of collection. After clot formation, the serum fraction was collected by centrifugation and stored in aliquots at 80C. Thawed serum was diluted 1:200 in phosphate-buffered saline-Tween 20 and tested for the concentration of HSPA1A using a commercial ELISA kit validated for human sera and specific for HSPA1A (R&D Systems, Minneapolis, MN). Each sample was tested in duplicate, and the average values were obtained. Values were converted to pg/ml by reference to a standard curve that was generated for each assay. The lower limit of sensitivity was 156pg/ml. The demographic and clinical data of the patients participating in the study were obtained through consultation of electronic medical records.

Based on histopathological characteristics according to the WHO criteria20, breast cancer was classified as ductal carcinoma in situ, invasive carcinoma of no special type (invasive ductal carcinoma), invasive lobular carcinoma, and invasive mucinous carcinoma. The tumors were also classified into subtypes according to standard immunohistochemistry (IHC) findings. IHC was used to determine the expression of estrogen and progesterone receptors, HER2 expression and the level of Ki6721,22. Ki67 is a marker of cell proliferation and is expressed exclusively during active phases of the cell cycle. Therefore, higher Ki67 values indicate an elevated rate of cell proliferation. Additional characteristics were used to classify the tumors based on histological grade and nuclear grade according to the 8th edition of the TNM classification system23.

In all patients the HSPA1A levels are described using the median value and interquartile range. Values between categories were compared using the MannWhitney test for variables with 2 categories or the Kruskal-Walli tests for variables with more than 2 categories. The Spearman rank correlation test was used to evaluate associations between the HSPA1A level and clinical and demographic characteristics. The generalized linear model (MLG) was used for the variables that presented descriptive levels below 0.2 in the unadjusted analyses (p<0.2) and that had biological plausibility to influence the marker24,25. The present study was designated as exploratory due to the limited number of participants and, thus, was underpowered to assess differences in HSPA1A among subtypes of breast cancer lesions. The analyses were performed using IBM-SPSS for Windows version 22.0 software and tabulated using Microsoft-Excel 2010 software, and all tests were performed with a 5% significance level.

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The circulating 70 kDa heat shock protein (HSPA1A) level is a potential biomarker for breast carcinoma and its progression | Scientific Reports -...

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