Breast cancer is one of the most frequently diagnosed malignancies among women 1 in which death rates have largely decreased by utilizing screening programs. Mammography is a widely accepted screening modality even though it is limited by low detection rates. 12 Mammographic density, referred as the percentage of dense tissue associated with stromal and epithelial proliferation of the entire breast tissue, is considered a known risk factor for the development of breast cancer. Accordingly, women who possess mammographically dense breasts are at a three-to-five-fold heightened risk of developing breast cancer than that of women with mammographically fatty breasts. 345 This phenomenon may be related to microenvironmental factors of the tumor and stroma. 6 In addition, sensitivity in detecting malignant lesions and microcalcifications is reduced in mammograms of dense breasts 17 , causing delayed diagnoses, and as described in a number of studies, revealing cancer at more advanced stages, e.i., larger tumors and nodal involvement. 68910 Her2 amplification which is present in about 15% of breast cancer patients, is a negative prognostic factor for breast cancer. Lack of Her2 overexpression is detected by immunohistochemistry score of either 0 or 1+, considered as negative. However, Her2 0 tumors may be biologically distinct from 1+ tumors since new Her2 targets have shown efficacy in low positivity of 1+ or 2+ unamplified Her2. 11 Ongoing studies are exploring the biology of Her2 expressing tumors.

It is suspected that there may be differences in clinicopathological reports of categories of breast density according to hormone receptor status and Her2 IHC score. This study aims to investigate potential associations between breast density and clinicopathological features of breast cancer.

This cross-sectional study was conducted on all women who underwent diagnostic mammography at the Shafa Imaging Center, Isfahan, Iran between 2016 to 2019. We identified and reviewed medical records of all patients who had undergone diagnostic mammography with a documented diagnosis of a malignant breast mass based on their pathological reports. Women with a second breast cancer, or those with previous breast surgery or radiotherapy were excluded.

Mammography was performed using a digital system in the craniocaudal (CC) and mediolateral oblique (MLO) positions using Hologic, Selenia mammography system with similar settings. We evaluated breast density using the American College of Radiology (ACR) breast composition classification, with breast density categorized as follows: a) almost entirely fatty; b) scattered areas of fibro-glandular density; c) heterogeneously dense; and d) extremely dense. Breast malignancies are typically irregular hypoechoic nodular lesions with ill-defined or spiculated borders and microcalcifications; however, the radiologist will not be able to distinguish all malignant from benign lesions exclusively by the mammogram or ultrasonogram. 112 Margins of the tumor were documented in mammograms to be ill-defined, spiculated, or otherwise (partially obscured, micro lobulated, lobulated, or normal), and mass density was documented as iso dense or highly dense. Based on the ultrasound reports performed at the time of diagnosis, the reported information regarding mass dimension in millimeters, vascularity (positive or negative), and morphology (irregular or oval) was extracted.

Medical records and pathology reports were studied to obtain information regarding histological tumor type, histological grade, and ER, PR, and HER2 status. Positive results for ER and PR were defined as the presence of stained nuclei in 1% or more of the tumor cells. Regarding Her2 status, tumors were scored as 0, 1+, 2+, and 3+ using the immunohistochemical stain. The ER, PR, Her2, Ki67 kits used included rabbit anti-human monoclonal antibody (Master diagnostica, Granada, Spain). Due to biological differences in the amount of Her2 expression 11 , negative tumors were grouped as negative: IHC 0 and compared to otherwise (IHC 1+, 2+, 3+), and again grouped as not overexpressed: IHC 0 or 1+ and compared to the rest. The results of in situ hybridization of equivocal cases (2+) were not adopted. The tumor subtypes were classified into four distinct categories: luminal A, luminal B, HER2 enriched, and basal. Luminal A and luminal B breast cancers were explained as follows: Luminal A: ER and/or PR positive, ki67 low (< 14%). Luminal B: ER and/or PR positive with ki67 high (>14%). HER2-enriched tumors demonstrated overexpression of HER2 which did not express estrogen and progesterone receptors. Basal tumors were identified by the absence of estrogen, progesterone and HER2 receptors.

The data in the study were presented in terms of frequencies and percentages for categorical variables and mean and standard deviation (SD) for continuous variables. The Kolmogorov-Smirnov test was employed to assess the normality of all continuous variables. Clinicopathologic variables were analyzed for differences among the four density groups using the chi-square test for categorical variables and the one-way ANOVA or Kruskal-Wallis test for continuous variables. The four density groups were aggregated into two groups as follows: high density group, composed of heterogeneously dense and extremely dense (ACR density c and d), and lowdensity group, as totally fatty and scattered densities (ACR density a and b). The relationship between breast density and ER, PR, and HER-2 status was investigated using binary logistic regression. Similarly, binary logistic regression was used to examine the associations between high or low breast density and each radiological and pathological variable. Next, a multivariable logistic regression was run for predictors of mammographic high-density categories that were related to breast density with a predetermined P-value of 0.2 or less. Statistical significance was defined as P-value ≤ 0.05. IBM SPSS Statistics, version (ver. 26.0 IBM Corp., Armonk, NY, USA) was used for all statistical analyses.

Among 187 patients with a newly diagnosed breast cancer in diagnostic mammography, 58 patients were excluded due to lack of adequate radiological or pathological information, and finally a total of 129 cases fulfilled the eligibility criteria and were included in the study. The mean age of the participants was 52.86 (10.7), and 12.4% reported a family history of breast cancer. The most frequent histological type of breast cancer was Invasive Ductal Carcinoma (IDC) (91.4%), and the majority of patients, 48.3%, had a grade II tumor. A binary logistic regression was performed to evaluate the effect of age on the likelihood of breast density when categorized as high versus low that was statistically significant (β = -0.085, OR= 0.92. P<0.0001).

In the BI-RADS mammographic density categories a, b, c, and d, there were 7 (5.4%), 47 (36.4%), 41 (31.8%) and 34 (26.4%) patients, respectively. Significant differences in mass density, mass morphology and HER2 status were observed in the four groups ( Table 1) which shows that categories of mass density (χ 2 ,=11.6), mass morphology (χ 2 ,=8.5), and Her2 stain (χ 2 ,= 13.1) were significantly different across breast density categories.

Binary logistic regression showed that dense breast versus fatty breast was associated with HER-2 expression in breast cancer (OR 5.56, 95% CI 1.57-20.23, P<0.008). The association remained for Her2 score 0 versus otherwise expression when entered in the multivariate model. Age was associated with breast density; however, there was no significant relationship between breast density and other features of tumor such as grade, size, molecular subtypes, and mass margin (Table 2).

Finally, predictors which were related to breast density with a predetermined p value of 0.2 or less were used in a multivariable regression. These variables were age, Her2 status, mass border, mass vascularity, and mass density. The results are demonstrated in table 2.

In this retrospective study, our results suggest that there might be a positive association between breast density and HER2 expressing breast tumors. Patients who had higher breast density categories demonstrated greater likelihood of manifesting tumors with positive scores of Her2 IHC as opposed to those demonstrating lower breast density. In particular, this significance remained when the data was controlled in the multivariate model.

Apart from age, which is a known negative confounding factor affecting breast density 6 , results of this study showed that regarding the ER and PR status, no difference was observed between the groups categorized as low-density and high-density in contrast to studies showing a positive relation. 13141516 These results are on the basis of evidence which suggests that the precursors of the dense composition of the breast may be driven by environmental factors and epi-genetics similar to risk factors for ERpositive breast cancers. These include low parity, use of exogenous estrogen in combination with progestin, and postponed menopause. Genetics may also have a large impact on many unidentified inherited factors. 14 However, the results of the relatedness of receptor markers and dense breasts are contradictory. 10 Consistent with some studies, this study found no obvious relation between density and the development of a particular intrinsic molecular subtype of breast cancer, or ER, PR, and Ki67 171819 , while there are other studies which show different results. 202122 Inconsistency in findings may reflect differences in research populations, assessment methods, screening patterns, different adjustments for covariates, or the presence of possible biases. 23 Above all, breast density is a risk factor of all breast cancer subtypes and should be considered for screening and monitoring subjects at risk. 2314 Regarding our findings of the Her2 receptor positivity and higher breast density, there is evidence in the literature associating the risk of HER2-Breast Cancer and higher mammographic breast density in a study on a Canadian screening population. 24 Breast density and distribution of receptor status are known to vary by race. In particular, a study conducted on the Asian population showed that women with BI-RADS D were more likely to be diagnosed with HER2-enriched tumors. The Asian decent is known for lower breast cancer incidence but a higher proportion of dense breasts poses more frequent Her2 enriched tumors. 25 A recent systematic review and metaanalysis have showed that higher breast density might contribute to the heightened incidence of HER2-positive subtypes among Asian women. 26 Another study has reported that individuals with high breast density and HER2-positive breast cancers show an increase in STAT3 signaling pathway which promotes tumorigenesis of breast cancer 27 , suggesting a connection between HER2 and the molecular pathways of breast density. 28 Notably, the earliest trials on the Her2 receptor investigated the population who benefited most from the first Her2 targeted therapy. Those with 3+ (complete, intense circumferential membranous staining in > 10% of tumor cells) or 2+ (weak to moderate complete membrane staining in >10% of tumor cells) with a positive FISH test, have long been considered the only Her2 positive subgroup and eligible to receive trastuzumab. Now, weak expressions of Her2 are also of interest because newer targets of this receptor have shown efficacy in this population. The results of the later studies reflect biological and clinical differences between Her2 non-expressing tumors and those with Her2 expression. 1129 Our investigation did not discover any association between breast density and typical tumor prognostic features, such as size and grade. The relation is inconsistent among different studies but some 3031 have suggested a link between high breast density and adverse breast tumor clinical characteristics, such as larger tumor size, nodal involvement, and advanced stage at diagnosis, possibly due to screening and early detection limitations in dense breasts.

This study was strengthened by use of a multivariable model method which allows for controlling the effect of a number of potential process factors simultaneously, but was methodologically restricted as causality and risk cannot be recognized. The number of the cases included was not large enough to make the study sufficiently rigorous for detecting a difference in breast density across tumor hormone receptors. Furthermore, no documented data related to BMI or menopausal state of patients at diagnosis was available. Also, the analysis may have been best interpreted if the results of in situ hybridization of Her2 2+ cases were incorporated. However, as discussed about population-based studies, the results of this single-center crosssectional study may inspire the evaluation of histopathological characteristics of an Iranian cohort. We suggest future research on the possible shared underlying mechanisms of breast density and incidence of Her2 positive breast cancers.

In conclusion, this study suggests considering breast density as a plausible risk factor for HER2-positive breast cancer but this possibility needs to be proven in population-based studies. While treatment decisions are currently based on pathology, mammographic features of different cancer subtypes could provide additional information to refine a patient's risk profile. The knowledge of dominant subtypes of cancers across breast compositions may have distinct implications. Firstly, in terms of the biological aspects, shared underlying etiologies and molecular pathways which contribute to the linkage of stromal-epithelial proliferation and receptor expression can be explored. Secondly, from a clinical point of view this can result in setting different thresholds of tumor detection for mammogram density categories in women susceptible to certain subtypes of breast cancer. Therefore, further investigation is required to validate the results and to discover the underlying mechanisms.

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