ABSTRACT Background: Breast Cancer Is the Most Frequent Cancer in the World Among Women

PB-051 HAND2 expression in breast cancer

Q. Lopes¹, E. Guedes Neto², L. Gross4, J. L. Pedrini³, M.C. Schorr³, R. Savaris4 1 Programa de Pós-Graduação em Ginecologia e Obstetrícia - Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil 2 Hospital Moinhos de Vento, Porto Alegre, Rio Grande do Sul, Brazil 3 Grupo Hospitalar Conceição, Mastology, Porto Alegre, Rio Grande do Sul, Brazil 4 Obstetrics and Gynecology Department, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. ABSTRACT Background: Breast cancer is the most frequent cancer in the World among women. The search for new prognostic markers has been sought by many authors. Heart and Neural Crest Derivatives Expressed Transcript 2 (HAND2) is a protein that gene belongs to the basic helix-loop-helix family of transcription factors and plays an important role in tumor suppressor. Its expression is reduced in endometrial cancer. However, little is known about its expression in breast cancer. Material and methods. In this case-control study, 19 formalin–fixed, paraffin-embedded tissues were obtained for pathological archives analysis. Tissue sections of breast tissue derived from benign conditions (n=10) and breast cancer (n=9) were analyzed for the intensity of the staining with 3,3'-diaminobenzidine using rabbit polyclonal antibody against HAND2 (Ab60037), at dilution 1:50 at pH 9. ImageJ software with "color deconvolution" was used for analysis of the expression of this protein. Statistical analysis was performed using unpaired t-test. The sample size was calculated in order to have a power of 95%, an alpha error of 1% to identify an increase in the primary outcome measure from 15 in the control group to 44 in the experimental group. Results: HAND2 expression (mean ± SD) in the breast tissue was 15.5 ± 1.9 (cancer) versus 44.9 ± 7 (controls) (P=0.002). Its expression was present in the nuclear and cytoplasmic compartments of the cells. Conclusion: The immunoexpression of HAND2 is reduced in breast cancer, compared with normal breast tissue. This finding may guarantee further research as a potential prognostic biomarker. keywords: breast cancer, HAND2, immunohistochemistry RESULTS MATERIAL AND METHODS INTRODUCTION A total 19 breast samples (10 benign; 9 cancer) were obtained for analysis. Details of the Patients sampled population is depicted in Table 1. All DAB values passed the D'Agostino & Breast cancer is the most common cancer in women in both developed and developing Women aged between 30 and 70 year-old, with diagnosis of invasive ductal carcinoma with or without ductal carcinoma in situ and Pearson omnibus normality test. The expression of HAND2 protein was mainly in the countries and the second most common cancer in the world.1 Breast cancer is definitely those submitted to breast surgery for benign conditions were included in the sample. cytoplasm. A significant reduction of HAND2 protein expression was observed tissue not a single disease, but rather a group of diseases characterized by clinical, section with breast cancer (Figure 1). The mean (SD) was 15.5(6.1) and 44.9(21.1) in morphological, and molecular heterogeneity; the description of the subtypes of breast Immunohistochemistry st th cancer and control groups, respectively (Figure 2).ANCOVA analysis was conducted to tumors has become more and more sophisticated.2 Four molecular subtypes are Paraffin blocks from subjects with inclusion criteria, dated between January 1 , 2015 and December 30 , 2016, were obtained from identify whether age between groups had influence on HAND2 expression. After running commonly recognized in the current practice: luminal A, luminal B, HER2-enriched, and pathological archive of Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil. Slides were reviewed by a certified board ANCOVA analysis, HAND2 expression was adjusted for age, and the P value between basal-like (also known as triple-negative breast cancer - TNBC) to which the categories of pathologist to confirm the diagnosis. 9 groups was 0.004, confirming that HAND2 expression was significantly different, despite the claudin-low and normal-like can also be added. Also, integrated analysis of copy number Sections 4 µm thick were submitted to standard immunohistochemistry technique. Briefly, paraffin sections were deparaffinized, age difference between groups. alterations with gene expression analysis further extended the number of subtypes to ten rehydrated, and rinsed with phosphate-buffered saline solution (PBS). Slides were incubated in sodium citrate solution, pH=9 at 95ºC

3. Nevertheless, new markers for breast cancer have been sought in order to have a better for 20 minutes for antigen retrieval. Endogenous peroxidase was blocked with hydrogen peroxide 5% in methanol for 20 min. After characterization of the tumor and to choose the best possible treatment.4 Among the new blocking nonspecific sites with skimmed powder milk 5% diluted in phosphate buffer (PBS), the slides were rinsed with distilled water Table 1. Demographics of the sample tumor markers in general, the Heart and Neural Crest Derivatives Expressed Transcript 2 2 x 5 min in PBS. Primary antibodies were incubated for 12 hours at 4ºC in a humid dark chamber using rabbit polyclonal antibody 5 against HAND2 (Ab60037- Anti-HAND2 antibody - Carboxyterminal end; Abcam Cambridge, MA 02139-1517, USA), at dilution 1:50 Breast cancer Control (HAND2) called our attention. This protein is encoded by 3727 base pairs located in the Characteristics of the sample P value (n=10) (n=9) 6 long arm of the chromosome 4. HAND2 protein expression is located mainly in the at pH 9. After 2 x 5 min in rinse PBS, secondary antibody (AP132P - Goat anti-rabbit IgG; Merck KGaA, Darmstadt, Germany) was Age (yrs) mean (SD) 49.7(12.5) 37.1(10.5) 0.02* 7 nuclear compartment, but it is also found in the cytosol. incubated for 90 minutes at 22ºC in the same chamber using a dilution of 1:200. Detection of the primary antibody was obtained using Ethnicity the Liquid DAB+Substrate Chromogen System (K3468, Dako, DK-2600 Glostrup, Denmark) according to the manufacture instructions caucasian 8 7

1** Li et al found that the inhibitory effects of progesterone under endometrial proliferation using 3,3'-diaminobenzidine (DAB) as chromogen. The slides were counterstained with hematoxylin, mounted and analyzed with non caucasian 2 2 FIGURE 2. Graphic representation of HAND2 protein expression in controls and in breast cancer. Bars present the mean expression were mediated by HAND2. These authors verified that in a knockout mice model for optical microscope. Hepatocarcinoma was used as positive external control. Negative controls were obtained by replacing primary in each group. Unpaired Student t-test with Welch's correction. * Unpaired Student t-test HAND2, an increased endometrial proliferation was observed.8 In women, Buell-gutbrod et antibody with mouse IgG2a, kappa monoclonal - Isotype Control (ab170191 - Abcam). ** Fisher's exact test al. found that biopsies derived from endometrial cancer had a lower expression of HAND2, Stained sections were analyzed under optical microscope (Olympus BX51 microscope; Olympus Optical Co., Tokyo, Japan) compared to normal controls The immunohistochemical expression of HAND2 is connected to a digital color camera/Q-Color 5 (Olympus). Images were obtained with a 20x objective UPLanFI (resolution: 2.75 mm), A C CONCLUSION accentuated in benign endometrial diseases but is significantly reduced or absent in cases at a size of 2560 x 1920 pixels (resolution: 1 mm = 3000 pixels), under standard conditions. Pictures were taken from the whole slide of endometrial hyperplasia or carcinoma.5 to perform image analysis. The immunoexpression of HAND2 is reduced in breast cancer, compared with normal The absence of HAND2 expression in endometrial cancer, this protein expression may be breast tissue. a useful biomarker in the cases of hyperplasia and endometrial cancer.5 Image Analysis with ImageJ Photomicrographs were coded and blindly analyzed with a specific software (ImageJ - v1.43j; National Institutes of Health, Bethesda, 10 REFERENCES The proliferative effects of estrogen on the endometrium, the absence of inhibitory action MD, available at http://rsbweb.nih.gov/ij/) using a built-in "color deconvolution plugin" for hematoxylin and DAB built-in vector. 11,12 of progesterone due to low expression of HAND2 and the presence of estrogen as risk Analysis was performed as previously described using Digital HSCORE (D-HSCORE). Briefly, only the glandular and tumoral 1. Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide. IARC CancerBase No. 11. Lyon, France: International Agency for Research on Cancer. sites of the tissue sections were selected as region of interest (ROI). After selecting the ROI, images were submitted "color (2013). Available at: http://globocan.iarc.fr. (Accessed: 16th February 2018) factor for breast cancer led us to verify whether the expression of HAND2 would be 2. Cappelletti, V. et al. Metabolic Footprints and Molecular Subtypes in Breast Cancer. Dis. Markers 2017, 7687851 (2017). reduced in breast cancer. deconvolution" analysis. From the 3 images obtained from “color deconvolution, the average DAB intensity was calculated according 3. Curtis, C. et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352 (2012). 4. Kim, R. N., Moon, H.-G., Han, W. & Noh, D.-Y. Perspective Insight into Future Potential Fusion Gene Transcript Biomarker Candidates in Breast Cancer. Int. J. Mol. Sci. 19, (2018). to the formula: ƒ= 255- i, where ƒ= final DAB intensity, i= mean DAB intensity obtained from the software. The final DAB intensity 5. Buell-Gutbrod, R., Cavallo, A., Lee, N., Montag, A. & Gwin, K. Heart and Neural Crest Derivatives Expressed Transcript 2 (HAND2): a novel biomarker for the identification of atypical hyperplasia B D varied from 0 (white, no expression) to 255 (dark brown, highest expression). and Type I endometrial carcinoma. Int. J. Gynecol. Pathol. 34, 65–73 (2015). 6. Ota, T. et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40–45 (2004). 7. Database, G. H. G. HAND2 Gene - GeneCards | HAND2 Protein | HAND2 Antibody. Available at: http://www.genecards.org/cgi-bin/carddisp.pl?gene=HAND2&keywords=p61296#localization. (Accessed: 16th February 2018) Ethics, Sample Size Calculation, and Statistical Analysis 8. Li, Q. et al. The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science 331, 912–916 (2011). Ethical approval was obtained from local Hospital de Clínicas de Porto Alegre Ethical Review Board (15-0327). Sample size was 9. Rasmussen, O. F. & Rudbeck, L. Immunohistochemistry: A Dako Perspective. in Handbook of Practical Immunohistochemistry 57–67 (2015). 10. Ruifrok, A. C. & Johnston, D. A. Quantification of histochemical staining by color deconvolution. Anal. Quant. Cytol. Histol. 23, 291–299 (2001). 12 calculated according to the literature in order to have a power of 95%, an alpha error of 1% to identify an increase in the primary 11. Grudzinski, M., Fuhrich, D. G. & Savaris, R. F. Expression of elafin in fallopian tubes of ectopic pregnancies is reduced. Appl. Immunohistochem. Mol. Morphol. 23, 349–354 (2015). 12. Fuhrich, D. G., Lessey, B. A. & Savaris, R. F. Comparison of HSCORE assessment of endometrial beta3 integrin subunit expression with digital HSCORE using computerized image analysis outcome measure from 15 in the control group to 40 in the experimental group, having a standard deviation of 10. Standard deviation (ImageJ). Anal Quant Cytopathol Histpathol 35, 210–216 (2013). was obtained from a pilot study with 5 normal samples. These figures yielded a minimum of 6 cases in each group. GraphPad Prism 13. Julious, S. A. Sample Sizes for Clinical Trials. (CRC Press, 2009). version 6 for Macintosh (GraphPad Software Inc., San Diego, CA) was used for statistical analysis, using the unpaired Student t test with Welch correction to compare the expression of HAND2 (final DAB intensity) in the breast tissue between the 2 groups, if data ACKNOWLEDGEMENT had a Gaussian distribution and different SDs. Gaussian distribution was calculated with the D’Agostino & Pearson omnibus normality test. ANCOVA analysis (http://vassarstats.net/ancova2L.html) was conducted to identify whether age between groups had influence FIGURE 1. HAND2 immunohistochemistry expression in breast cancer (A: InSitu; B: infiltrative) and normal breast (C). Staining is present FIPE -Fundação Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre. (Grant in cytoplasm and in some nuclei (D - higher power). Positive and negative external controls (hepatocarcinoma), in the lower right corner. Bars 15-0327) on HAND2 expression. HAND2 expression was the dependent variable; fixed factors were the groups, and age was the concomitant represent 0.05 mm. variable.