Heart and Neural Crest Derivatives Expressed Transcript 2 (HAND2) is Reduced in Women with Breast Cancer. A Case-Control Study

Abstract

Background: The search for new markers for breast cancer (BC) has been sought in order to better understand this type of cancer. The Heart and Neural Crest Derivatives Expressed Transcript 2 (HAND2) has been related to endometrial cancer but there are scant data related to BC. The aim of this study is to compare the immunohistochemical expression of HßAND2 in normal breast tissue vs. BC and to correlate with the estrogen receptor (ERα). Patients and Methods: In this case-control study, 19 formalin–fixed, paraffin-embedded tissues were obtained from pathological archives. Benign (n=9; control) and cancer (n=10) breast tissue were analyzed with immunohistochemistry for HAND2 (Ab60037), at dilution 1:50 at pH 9 and ERα (SP1). ImageJ software with "color deconvolution" was used for analysis of the expression of these proteins. The sample size was calculated (power=95%, α error= 1%) to identify an increase from mean 15 DAB units (control) to 40 DAB units in cancer. Results: HAND2 expression (mean ± SD) was 15.5 ± 6.1 (cancer) versus 44.8 ± 21.1 (control) (P=0.002, Student t-test). Its expression was mainly present in the cytosol of the cells. No correlation was observed between ERα and HAND2 (Pearson r = -0.28 (95%CI=-0.6 to 0.22; P=0.2). Conclusions: The protein expression of HAND2, using Ab60037 antibody, is reduced in breast cancer, compared with normal breast tissue. The expression of HAND2 is not correlated with ERα expression.

Deep learning neural network image analysis of immunohistochemical protein expression reveals a significantly reduced expression of biglycan in breast cancer

“…After 2 x 5 minutes in a PBS rinse, secondary antibody anti-mouse IgG (whole molecule), namely peroxidase antibody produced in rabbit (A9044, Sigma-Aldrich, Darmstadt, Germany), was incubated for 90 minutes at 22°C in the same chamber using a dilution of 1:1000. The primary antibody visualization and counterstained were performed as previously reported [ 22 ]. Negative controls were obtained by replacing the primary antibody with mouse IgG2a, kappa monoclonal [18C8BC7AD10]—Isotype Control (ab170191)—Abcam, Cambridge, UK).…”

“…Immunohistochemistry. Immunohistochemistry methodology was performed according to standard technique [21] and as previously reported by our group with minor modifications PLOS ONE [22]. Modifications included deparaffinized at 75˚C for two hours, followed by xylol rinse, rehydrated in successive steps of ethanol, water, and phosphate-buffered saline solution (PBS); slide incubation was done in sodium citrate solution, pH 6 at 90˚C for 45 min.…”