**Contents**


Reprinted from: *J. Pers. Med.* **2021**, *11*, 324, doi:10.3390/jpm11050324 . . . . . . . . . . . . . . . . **97**


Image-Guided Localization Techniques for Surgical Excision of Non-Palpable Breast Lesions: An Overview of Current Literature and Our Experience with Preoperative Skin Tattoo Reprinted from: *J. Pers. Med.* **2021**, *11*, 99, doi:10.3390/jpm11020099 . . . . . . . . . . . . . . . . . **207**


### **About the Editors**

#### **Gianluca Franceschini**

Dr. Gianluca Franceschini has been a top-level senior physician at the Breast Unit, the Department of Women and Children's Health, Catholic University, "Agostino Gemelli" Hospital, Rome, Italy, since 1 June 2006.

He has headed the unit for integrated therapies for breast cancer at the Department of Women and Children's Health, Catholic University, "Agostino Gemelli" Hospital, Rome, since 1st July 2014. He has been an Associate Professor in General Surgery since July 2016 at the Catholic University of Rome.

He is a lecturer at the PhD School "Technological Innovations in the Integrated Therapies of Breast Tumours" (2010–2013) and at the School of Specialization in General Surgery, Plastic Surgery and Oncology at the Catholic University of Rome (2011–2016). He is the Academic Coordinatorforthe University Master's Degree Course "Endocrinology and Breast Pathology" at the Catholic University of Rome (2014–2016).

He is a reviewer and member of the Editorial Boards of several scientific journals.

On the 15th February 2016, he was appointed as a Permanent Member of the College of Italian Breast Surgeons.

He has authored numerous scientific a rticles, b ooks, and b ook c hapters. His p resent interests include therapy for pathologies of the breast and surgical treatment of breast cancer with oncoplastic techniques.

#### **Alejandro Martin Sanchez**

Dr. Alejandro Martin Sanchez is a researcher at Multidisciplinary Breast Center, Dipartimento Scienze della Salute della Donna e del Bambino e di Sanita` Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy, with expertise in breast surgery, surgery, surgical oncology, breast cancer management, breast cancer screening, senology, breast imaging, mammography, breast cancer, and breast cancer stem cells.

#### **Riccardo Masetti**

Riccardo Masetti is an expert breast surgeon. He is the head of the Breast Surgery Unit, Women, Children and Public Health Sciences Department, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma.

### *Editorial* **Innovations in the Integrated Management of Breast Cancer**

**Gianluca Franceschini , Alejandro Martin Sanchez \* , Elena Jane Mason and Riccardo Masetti**

Multidisciplinary Breast Center, Dipartimento Scienze della Salute della Donna e del Bambino e di Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy; gianlucafranceschini70@gmail.com (G.F.); elenajanemason@gmail.com (E.J.M.); riccardo.masetti@policlinicogemelli.it (R.M.)

**\*** Correspondence: martin.sanchez@policlinicogemelli.it; Tel.: +39-06-30156328

Breast cancer is commonly acknowledged as an international priority in healthcare. To date, it is the most common cancer in women worldwide and demographic trends show a steady increase in incidence.

Over the years, increasing efforts and resources have been devoted to a meticulous analysis of risk factors, diagnostic tools and treatment strategies in order to enhance every step of breast cancer management.

Researchers and clinicians strive in search of an optimized, systematic strategy in the diagnosis and treatment of this disease. This effort has led to the creation of the "breast unit model", which is today considered a gold standard to ensure optimal clinical services centered on patients and based on research through multidisciplinary and integrated management [1]. This approach, involving surgical, radiation and medical oncology, allows the optimization of oncological and cosmetic outcomes and the prolonged survival and improvement of patient quality of life; the integrated treatment is tailored to each patient and based on clinical examination, patient status, disease staging, biologic phenotype such as hormone receptor status and human epidermal growth factor receptor 2 (HER2) overexpression, and patient preferences. The decision-making process in the management of breast cancer includes a detailed discussion with the patient about the risks and benefits associated with the selected treatment.

This Special Issue highlights many recent innovations in the integrated management of breast cancer, their potential advantages and the many open issues that still wait to be properly defined and addressed. The authors' interests span every aspect of breast cancer care: from early breast cancer to metastatic patients, and from surgical assessment to artificial intelligence application in data collection.

Cancer biology is addressed in two pre-clinical studies analyzing breast tissue samples. Santandrea et al. focus on hormone receptor expression in normal breast tissue, in search of a pattern that could favor the development of a breast tumor [2], while a study by Fuso et al. examines breast cancer patients treated with neoadjuvant chemotherapy in search of a miRNA expression associated with survival, and therefore acting as a predictive biomarker in women affected by early breast cancer [3].

An accurate and comprehensive preoperative assessment is crucial in order to prepare the patients for surgery, and breast cancer care still holds many issues waiting to be finetuned. Nonpalpable lesions can compromise and delay an otherwise smooth operation, and the surgeon should be well-prepared with potential solutions to this common problem. This Special Issue offers a review of current image-guided techniques, highlighting the benefits and controversies of each method [4]. Radiology is also tackled in a study focusing on the best imaging technique to assess patients scheduled to receive breast reconstruction via a DIEP flap, and the researchers advocate conventional CT as an alternative to the traditional but costly CT angiography [5].

During the last decade, the goal in surgery has been to make procedures less and less invasive. Much like breast surgery, which has witnessed a gradual diffusion of breast

**Citation:** Franceschini, G.; Sanchez, A.M.; Mason, E.J.; Masetti, R. Innovations in the Integrated Management of Breast Cancer. *J. Pers. Med.* **2022**, *12*, 531. https://doi.org/10.3390/ jpm12040531

Received: 22 February 2022 Accepted: 23 March 2022 Published: 28 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

conserving techniques, axillary surgery has also evolved in an increasingly conservative manner. Where previous surgical approaches tended to favor axillary dissection at all costs, the introduction of sentinel lymph node biopsy (SLNB) has led to the preservation of non-pathological axillary lymph node tissue, and once frequent complications such as post-operative lymphedema have greatly diminished in recent years [6]. In this Special Issue we explore the possibilities of a further evolution in axillary surgery, where treatment with sole SLNB could be extended to include patients downstaged to ycN0 by neoadjuvant chemotherapy [7].

When a breast-conserving approach cannot guarantee both adequate local control and a good aesthetic result, the surgeon has to perform a mastectomy. Innovative surgical procedures called "conservative mastectomies" with immediate prepectoral implant reconstruction have been introduced in order to obtain more favorable aesthetic outcomes and avoid problems caused by manipulation of the pectoralis major muscle, such as breast animation deformity, postoperative pain and injury-induced muscular deficit [8].

The primary goal of management in metastatic disease is the alleviation of symptoms, maintenance or improvement in quality of life and prolongation of survival despite possible treatment toxicity. Patients with metastatic disease receive systemic medical treatments including endocrine therapy, chemotherapy, biologic therapies, targeted and immunotherapy and supportive care measures. However, a subset of patients may benefit from a specific loco-regional treatment [9]: oligometastatic disease has been the object of particular interest because of the possibility to aim for a long-term remission in these patients, and once-discarded options such as liver metastasectomy have been shown to be a possible therapeutic option in selected patients [10,11].

The benefits of a multimodal prehabilitation model are emerging in recent studies, as in this framework patients may be more receptive to health behavior changes in a structured support network. Di Leone et al. shed light on a possible personalized prehabilitation model to enhance patient care in the neoadjuvant setting, which allows each patient to receive the attention of every required specialist in a set frame of time [12,13]. For example, elderly patients can greatly benefit from a preoperative geriatric assessment in order to avoid negative outcomes deriving from otherwise unknown syndromes such as severe sarcopenia [14]. On the other hand, younger women with a new, unexpected diagnosis of breast cancer may face issues related to sexuality and fertility, and studies addressing the impact of treatment on ovarian reserve are paramount to better understand the mechanisms leading to early menopause and subsequent infertility. The clinician's primary objective is to offer a timely oncofertility service, in order to preserve the opportunity for family planning without delaying chemotherapy [15]. Similar strategies must be adopted when confronting pregnancy-associated breast cancer, a rare occurrence that nonetheless threatens the wellbeing of both mother and fetus [16].

Finally, the last few years have seen the creation of new artificial intelligence technologies with the potential to radically change the modern management of breast cancer. Research itself is a viable candidate for the coming high-tech revolution: today, protocol development can be promoted, patient enrollment can be enhanced by a patient-trial matching made possible by the growing diffusion of electronic health records, and patient parameters and adherence to trials can be monitored in real-time by a variety of wearable devices. This Issue witnesses the transformation, thanks to the contribution of authors active in the field of real-world data: Cesario et al. describe the development of a digital research assistant that manages patient enrollment in trials with the employment of an artificial intelligence algorithm [17], while Marazzi et al. exploit text mining to successfully extract data from heterogeneous sources and to generate clinical evidence [18,19].

This Special Issue finds its place in the modern panorama of breast care by promoting a modern, holistic approach to breast disease and encouraging clinicians to tailor patient treatment. The development of appropriate clinical pathways, with a multidisciplinary and standardized approach, is essential for successful, well-rounded treatment in the era of personalized medicine.

**Author Contributions:** Conceptualization/original draft preparation A.M.S. and E.J.M.; Review and editing/supervision G.F.; final draft conceptualization and approval R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Vitamin D and Histological Features of Breast Cancer: Preliminary Data from an Observational Retrospective Italian Study**

**Stefano Lello <sup>1</sup> , Anna Capozzi 1 , Lorenzo Scardina 2, \* , Lucia Ionta 2 , Roberto Sorge 3 , Giovanni Scambia 1 and Gianluca Franceschini 2**


**Abstract:** Background: Vitamin D (vitD) may be involved in different extraskeletal conditions as well as skeletal muscle diseases. It has been hypothesized that, at least in part, a low level of vitD could contribute to facilitating cancer development. Breast cancer (BC) seems to be associated with low levels of vitD. Materials and methods: This was an observational retrospective evaluation of 87 women (mean age: 54 ± 12 years old) who underwent surgery for the treatment of BC. Our main purpose was to correlate the types of BC and the levels of vitD. Results: A positive significant correlation (R > 0.7) was found between non-invasive carcinoma in situ and 25(OH)D levels and age (R = 0.82, *p* < 0.05). A positive, but nonsignificant, correlation was reported between invasive ductal carcinoma and 25(OH)D and age (R = 0.45, *p* > 0.05). A negative but nonsignificant correlation was found between invasive lobular carcinoma and 25(OH)D and age (R = 0.24, *p* > 0.05). Discussion and Conclusions: We did not find a significant relationship between vitD and BC subtypes. Considering the positive significant correlation between vitD levels and age for in situ BC, although preliminary, our results seem to suggest a possible role of vitD in in situ BC. However, these findings need to be confirmed in larger studies.

**Keywords:** vitamin D; breast cancer; ductal breast cancer; in situ breast cancer; lobular breast cancer; histology

#### **1. Introduction**

Breast cancer (BC) is the most common form of female cancer and the second leading cause of death among women worldwide [1]. Many data suggest that several lifestyle and environmental factors—such as a high-fat diet, a lack of physical activity, and chronic alcohol consumption—might play a critical role in the development and risk of recurrence of this cancer [2]. The maintenance of a healthy lifestyle together with regular breast checks, through breast self-examinations, mammography, and/or ultrasonography, represent a cornerstone for primary prevention [3]. Vitamin D (vitD) homeostasis is fundamental for the achievement of bone strength and the prevention of bone and muscle loss [4].

According to the principal international guidelines [5,6], its supplementation is advisable for frail subjects affected by a loss of bone strength and/or hypovitaminosis D. Furthermore, emerging data demonstrate that vitD may produce other important benefits at the extraskeletal level [7]. Hypovitaminosis D is associated with a higher incidence of

**Citation:** Lello, S.; Capozzi, A.; Scardina, L.; Ionta, L.; Sorge, R.; Scambia, G.; Franceschini, G. Vitamin D and Histological Features of Breast Cancer: Preliminary Data from an Observational Retrospective Italian Study. *J. Pers. Med.* **2022**, *12*, 465. https://doi.org/10.3390/jpm12030465

Academic Editors: Raghu Sinha and Christian Singer

Received: 26 December 2021 Accepted: 11 March 2022 Published: 14 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

several cardiovascular, metabolic, autoimmune, endocrine, and neoplastic pathologies [7]. Although large intervention studies about the positive effects of vitD supplementation on these pathologies are limited [8], there is growing interest in the potential involvement of vitD status in the appearance of some of these diseases [7].

An interesting meta-analysis by Hossain et al. [9] showed a direct correlation between vitD deficiency and BC, with a relative risk (RR) of 1.91 (95% confidence interval (CI): 1.51–2.41, *p* < 0.001). At the same time, total vitD intake and supplemental vitD intakes had inverse relationships with BC (RR = 0.99, 95% CI: 0.97–1.00, *p* = 0.022, per 100 IU/day; RR = 0.97, 95% CI: 0.95–1.00, *p* = 0.026, respectively).

Another cohort study [10], evaluating 50,884 women (aged between 35 and 74 years) who had never had BC themselves but had a sister affected by BC, found that high serum 25(OH)D levels and self-reported regular vitD supplementation (≥four times/week) were associated with lower rates of incident BC after menopause over 5 years of follow-up (HR = 0.72 (CI: 0.57–0.93) for high serum 25(OH)D levels, and HR = 0.83 (CI: 0.74–0.93) for regular supplementation).

Our study was an observational retrospective evaluation enrolling 87 women (mean age: 54 ± 12 years old) who underwent surgery for the treatment of BC between December 2019 and March 2020. The objective of this evaluation was to correlate the subtypes of BC with the level of vitD.

#### **2. Materials and Methods**

This was an observational retrospective analysis of 87 patients (mean age: 54 ± 12 years old) who had not been supplemented with vitD in the previous 3 months, selected among patients who underwent surgery for the treatment of BC in the Breast Unit Surgery of Fondazione Policlinico Agostino Gemelli IRCCS of Rome. This retrospective analysis included patients without differences in race and/or ethnicity. The exclusion criteria were being a foreign woman and/or being regularly supplemented with vitD analogues in the previous three months before breast surgery and/or being unwilling to be tested for vitD status.

All the enrolled women were screened for serum 25(OH)D levels during the prehospitalization stage. The total serum 25(OH)D levels were measured using an automated Abbott Architect 25(OH)D immunoassay (bias ng/mL% = + 0.4/1.7–4.7 between 20 and 40 ng/mL, according to the Vitamin D External Quality Assurance Scheme).

According to the US Endocrine Society Clinical Practice Guidelines, vitamin D sufficiency was defined as serum levels of 25(OH)D ranging between 30 and 100 ng/mL [5].

Twenty-four patients underwent a mastectomy combined with immediate reconstruction, whereas 53 patients were treated through breast-conserving surgery (oncoplastic surgery was performed in nine subjects). In particular, after breast surgery, we collected data about the size, type, and histological features of BC.

All the data were analysed by using SPSS 15.0 version for Windows (SPSS, Chicago, IL, USA). For descriptive statistics, the means ± standard deviations (SDs) for parameters with Gaussian distributions (after confirmation with histograms and Kolmogorov– Smirnov tests), and medians and intervals (minimum–maximum) for non-Gaussian variables, were used.

The comparison among normal variables was performed by using one-way ANOVA or Bonferroni tests. We used chi-square (χ2) and Fisher tests for comparisons among the variables of frequency. Pearson linear correlation analysis was used for the calculation of R coefficients. A value of *p* < 0.05 was considered statistically significant.

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board and Ethics Committee of Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy.

#### **3. Results**

The principal characteristics of the study participants are summarized in Table 1.


**Table 1.** Patients' characteristics and ANOVA correlations between vitamin D levels and tumour type, the type of receptors expressed, and grading.

The patients were divided into three main subgroups according to the type of cancer: subgroup one (70 women (mean age: 54.2 ± 12 SD) with ductal carcinoma)); subgroup two (six women (mean age: 54.7 ± 15 SD) with carcinoma in situ); and subgroup three (11 women (mean age: 55.6 ± 14 SD) with lobular carcinoma).

We did not find significant differences among groups according to the major analysed variables (i.e., the tumour size, expression of oestrogen receptors (ERs), expression of progesterone receptors (PRs), Ki67, human epidermal growth factor receptor 2 (HER2), and median level of 25(OH)D). In particular, the age did not significantly differ among the subgroups of cancers (*p* > 0.05).

A positive significant correlation (R > 0.7) was found between non-invasive carcinoma in situ and 25(OH)D levels and age (R = 0.82) (Figure 1).

A positive but nonsignificant correlation was reported between invasive ductal carcinoma and 25(OH)D and age (R = 0.45).

A negative but nonsignificant correlation was found between invasive lobular carcinoma and 25(OH)D and age (R = 0.24).

**Figure 1.** Relationship between age and vitamin D levels in major BC types (tum 1 = ductal BC; tum 2 = in situ BC; and tum 3 = lobular BC).

#### **4. Discussion and Conclusions**

Hypovitaminosis D is notably associated with a loss of bone and muscle strength, and major international guidelines recommend maintaining normal 25(OH)D levels (>20 ng/mL) in vitD-deficient elderly institutionalized patients and postmenopausal women at higher risk of fragility fractures through appropriate supplementation [5,11].

Conversely, the benefits of vitD supplementation for skeletal muscle health in communitydwelling, premenopausal women and younger healthy subjects remain questionable [12].

According to some evidence [13], hypovitaminosis D could favour the development of several extraskeletal conditions (i.e., autoimmune, inflammatory, cardiovascular, and metabolic diseases), although the effective role of vitD in the aetiology of these pathologies needs to be further elucidated. In particular, the risks of colon, prostate, ovarian, and breast cancer have been associated with vitD deficiency by many authors [14,15].

Abbas et al. [16] found, in a population-based case–control study including 289 premenopausal women and 595 matched controls (aged between 30 and 50 years), a significant inverse association between BC risk and plasma 25(OH)D concentrations (*p* < 0.05). Compared with the lowest category (<30 nmol/L) (OR = 1 (95% CI)), the ORs for higher plasma concentrations of 30–45, 45–60, and ≥ 60 nmol/L were 0.68 (0.43–1.07), 0.59 (0.37–0.94), and 0.45 (0.29–0.70), respectively (*p* for trend = 0.0006). Interestingly, this association was stronger for progesterone receptor-negative BC (PR-), with evidence suggestive of effect heterogeneity (*p* for heterogeneity = 0.05, case-only model). Additionally, the authors found a significantly reduced risk of BC, with an OR of 0.90 (0.84–0.96) per 10 nmol/L

increment when considering 25(OH)D as a continuous variable. Meanwhile, no significant interactions between plasma 25(OH)D and the first-degree family history of BC, age at menarche, duration of breastfeeding, parity, alcohol intake, or BMI were reported.

It has been hypothesized that vitD may exert direct protective effects against cancer by promoting cellular apoptosis and differentiation and by inhibiting angiogenesis and tissue inflammation. At the same time, different general risk factors that can favour cancer development by themselves could negatively affect vitD metabolism, such as smoking, obesity, low physical activity, and sun exposure [17,18].

Another study by Alipour et al. [19] compared a control group (364 women; mean age: 44.2 years) with a case group (308 women; mean age: 43.2 years; 172 subjects with a benign mass; and 136 subjects with a malignant mass) regarding vitD status. The results of this study show that the median serum 25(OH)D assessed in the case group was lower than that in the control group (7.7 vs. 8.7 ng/mL), and that the median serum 25(OH)D was higher in benign (7.9 ng/mL) than in malignant cases (7 ng/mL). In the comparison between each of these two case groups with controls, the median 25(OH)D was higher in the control group, lower in the group of patients with benign lesions, and the lowest in the group with cancer. However, the differences between the median 25(OH)D in the benign cases and controls, as well as benign cases and cancers, were not statistically significant (*p* = 0.3 and *p* = 0.1, respectively). The histology of four of the 136 BC was in situ ductal carcinoma; the others were invasive ductal carcinomas. In comparison with subjects with euvitaminosis D (25(OH)D > 35 ng/mL), the ORs for BC were 3 (95% CI: 1.11–8.1) in subjects with severe vitD deficiency (25(OH)D < 12.5 ng/mL), 0.96 (95% CI: 0.3- 2.8) in patients with moderate vitD deficiency (25(OH)D between 12.5 and 25 ng/mL), and 1.79 (95% CI: 0.9–3.5) in subjects affected by mild hypovitaminosis D (25(OH)D between 25 and 35 ng/mL), after adjustment of different variables (i.e., age, menarche, parity, menopausal status, breastfeeding, and family history of BC). Thus, for less-severe hypovitaminosis D, the relationship between vitD status and BC risk appeared to be nonsignificant [19].

The main purpose of our retrospective analysis was to find a possible relationship between the biological finding of BC, according to its histological features, and the level of vitD assessed in all the patients before undergoing breast surgery that would have confirmed the suspicious diagnosis of BC. Firstly, we did not find a significant difference among the subgroups with different types of cancer regarding all the analysed variables (age; tumour dimension; expression of ERs, PRs, Ki67, and HER2; and median level of 25(OH)D) (*p* > 0.05). As mentioned above, the available data in the literature highlight the tendency of both benign and malignant breast masses to be associated with lower median levels of 25(OH)D in comparison with those in healthy subjects. However, the difference in terms of vitD status between benign and malignant lesions did not appear to be clearly significant, as also confirmed by our analysis [19]. Additionally, although our study was small, the results seem to be in line with data reporting a decreased frequency of invasive lobular cancers in the last two decades compared with non-invasive in situ and invasive ductal BC [20]. This phenomenon may be explained by the supposed influence of hormonal exposure, which may contribute to facilitating the appearance of invasive lobular BC and may render this type of tumour more susceptible to incidence variation within population studies [21]. Secondly, a positive significant correlation (R > 0.7) was found between non-invasive carcinoma in situ and 25(OH)D and age (R = 0.82), whereas a nonsignificant but negative correlation was found between invasive lobular carcinoma and 25(OH)D and age (R = 0.45). At the same time, a positive but nonsignificant correlation was reported between invasive ductal carcinoma and 25(OH)D and age (R = 0.45).

A previous retrospective case–control study by Peppone et al. [22] on 224 women diagnosed with Stage 0–III BC showed that suboptimal vitD levels (<32 ng/mL) were more common in women with later-stage disease, non-Caucasians, and those who received radiation therapy (*p* < 0.05). More specifically, the ORs for suboptimal vitD levels were 3.15 (95 CI%: 1.05–9.49) for triple-negative vs. non-triple-negative, 2.59 (95 CI%: 1.08–6.23) for ER- vs. ER+, 2.35 (95 CI%: 1.14–4.84) for premenopausal vs. postmenopausal status

at diagnosis, and 2.29 (95 CI%: 2.05–4.98) for negative family history vs. positive. On the other hand, the OR for suboptimal vitD levels was 2.22 (95 CI%: 0.86–5.71) for invasive vs. non-invasive BC. Our results seem to be consistent with those of the latter study since our evaluation did not show a clear impact of vitD status on the invasiveness of BC.

However, our findings show that vitD status and age could be positively correlated for in situ BC, a type of cancer that is generally associated with lower biological aggressiveness and/or invasiveness than the others. Although the evidence regarding the close correlation between vitD deficiency and in situ BC remains limited, these data show a very interesting picture.

Regarding invasive lobular BC, we observed an inverse, although nonsignificant, correlation between vitD and age, probably due to the small number of patients. These findings may be related, on one hand, to the most frequent biological features of invasive lobular BC, which seem to be influenced more by hormones than by other exogenous and/or endogenous factors, and, on the other hand, to a potentially less-protective role of vitD at the cellular level because of its insufficiency in this type of BC.

According to recent systematic reviews, vitamin D insufficiency is observed in patients with newly diagnosed breast cancer, and supplemental vitamin D intake showed an inverse relationship with this outcome [8,23].

A recent secondary analysis of data from the Women's Health Initiative CaD trial (n = 36,282 cancer-free postmenopausal women aged between 50 and 79 years, randomly assigned to a daily 1000 mg dose of calcium plus 400 IU of vitamin D or to a placebo) found a lower risk of ductal carcinoma in situ (DCIS) throughout approximately 20 years of follow-up (HR = 0.82; 95% CI: 0.70 to 0.96). These results seem to suggest that, since DCIS could be considered a precursor of invasive BC, supplementation with calcium and vitD might reduce BC risk by acting at an early stage in the natural history of the tumour [24]. However, that evaluation has some limitations since it was a post hoc analysis that did not consider calcium and/or vitamin D intake from dietary sources and/or the effects of each supplement separately.

Interestingly, a multicentre randomized, double-blind, placebo-controlled study conducted in the United States in men ≥ 50 years and women ≥ 55 years without cancer and cardiovascular disease at baseline showed that supplementation with vitamin D3 (cholecalciferol, 2000 IU/d) and marine omega-3 fatty acids (1 g/d) could produce a significant reduction in advanced cancers (metastatic or fatal) compared with placebo (226 of 12,927 assigned to vitamin D (1.7%) and 274 of 12,944 assigned to placebo (2.1%); HR = 0.83 (95% CI: 0.69–0.99); *p* = 0.04), particularly in subjects with normal BMIs (HR = 0.89; 95% CI: 0.68–1.17) [9].

However, the cancer incidence was similar in the treatment and placebo groups. Thus, a clear conclusion about the favourable impact of vitD supplementation on the cancer risk for the general population cannot be drawn [25].

Moreover, there are many known, and still-unknown, endogenous and exogenous mechanisms involved in tumorigenesis; thus, these results need to be critically evaluated [26].

Our study presents several limitations, as it was a retrospective analysis and may have included some selection biases: the relatively small number of participants, the absence of a control group, and the exclusion of other potential confounding factors, such as body mass index (BMI), dietary and lifestyle habits (smoking, alcohol consumption, and sport activities), family history of BC, comorbidities, and medications.

Taken together, our analysis did not show significant differences among types of BC regarding vitD status. Additionally, we did not find significant differences among subgroups of BC with respect to tumour size and age (*p* > 0.05).

Despite the small number of cases, since we found a positive significant correlation between vitD levels and age for in situ BC, our results seem to suggest a protective role of baseline endogenous vitD levels in in situ BC, different from that in more invasive types of BC. In other words, vitD, through its putative antiproliferative activity at the cellular level [27], could contribute to reducing the invasiveness of cancer cells.

Therefore, this work provides encouraging data since, even if preliminarily, we can hypothesize that vitD status may affect the occurrence of less-invasive types of BC, rather than others.

Further prospective multicentre trials with larger numbers of patients and longer follow-up are necessary to draw more validated conclusions. Even if clinical studies investigating the synergistic role of vitD in BC treatment are still inconclusive [28], our results could suggest that ensuring an appropriate level of 25(OH)D could become a promising choice in the field of BC cancer prevention. However, these findings need to be confirmed in larger and well-designed intervention studies.

**Author Contributions:** Conceptualization, S.L. and A.C.; methodology, S.L.; software, R.S.; validation, R.S., G.S. and G.F.; formal analysis, R.S.; investigation, S.L.; resources, L.I.; data curation, R.S.; writing—original draft preparation, A.C. and S.L.; writing—review and editing, G.S. and G.F.; visualization, G.F.; supervision, G.S., L.S.; project administration, A.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board and Ethics Committee of Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy.

**Informed Consent Statement:** Informed consent was obtained from all the subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### **Adjuvant Radiotherapy Is Associated with an Increase in the Survival of Old (Aged over 80 Years) and Very Old (Aged over 90 Years) Women with Breast Cancer Receiving Breast-Conserving Surgery**

**Chung-Chien Huang 1,2 , Chia-Lun Chang 3,4 , Mingyang Sun 5 , Ming-Feng Chiang 6 , Shao-Yin Sum <sup>7</sup> , Jiaqiang Zhang <sup>5</sup> and Szu-Yuan Wu 7,8,9,10,11,12,13,14, \***


**Abstract:** This study is the first to examine the effect of adjuvant whole-breast radiotherapy (WBRT) on oncologic outcomes such as all-cause death, locoregional recurrence (LRR), and distant metastasis (DM) in old (aged ≥80 years) and very old (aged ≥90 years) women with breast invasive ductal carcinoma (IDC) receiving breast-conserving surgery. After propensity score matching, adjuvant WBRT was associated with decreases in all-cause death, LRR, and DM in old and very old women with IDC compared with no use of adjuvant WBRT. **Background**: To date, no data on the effect of adjuvant whole-breast radiotherapy (WBRT) on oncologic outcomes, such as all-cause death, locoregional recurrence (LRR), and distant metastasis (DM), are available for old (aged ≥80 years) and very old (≥90 years) women with breast invasive ductal carcinoma (IDC) receiving breastconserving conservative surgery (BCS). **Patients and Methods**: We enrolled old (≥80 years old) and very old (≥90 years old) women with breast IDC who had received BCS followed by adjuvant WBRT or no adjuvant WBRT. We grouped them based on adjuvant WBRT status and compared their overall survival (OS), LRR, and DM outcomes. To reduce the effects of potential confounders when comparing all-cause mortality between the groups, propensity score matching was performed. **Results**: Overall, 752 older women with IDC received BCS followed by adjuvant WBRT, and 752 with IDC received BCS with no adjuvant WBRT. In multivariable Cox regression analysis, the adjusted hazard ratio (aHR) and 95% confidence interval (95% CI) of all-cause death for adjuvant WBRT

**Citation:** Huang, C.-C.; Chang, C.-L.; Sun, M.; Chiang, M.-F.; Sum, S.-Y.; Zhang, J.; Wu, S.-Y. Adjuvant Radiotherapy Is Associated with an Increase in the Survival of Old (Aged over 80 Years) and Very Old (Aged over 90 Years) Women with Breast Cancer Receiving Breast-Conserving Surgery. *J. Pers. Med.* **2022**, *12*, 287. https://doi.org/10.3390/ jpm12020287

Academic Editors: Gianluca Franceschini, Alejandro Martin Sanchez and Riccardo Masetti

Received: 27 January 2022 Accepted: 11 February 2022 Published: 16 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

compared with no adjuvant WBRT in older women with IDC receiving BCS was 0.56 (0.44–0.70). The aHRs (95% CIs) of LRR and DM for adjuvant WBRT were 0.29 (0.19–0.45) and 0.45 (0.32–0.62), respectively, compared with no adjuvant WBRT. **Conclusions**: Adjuvant WBRT was associated with decreases in all-cause death, LRR, and DM in old (aged ≥80 years) and very old (aged ≥90 years) women with IDC compared with no adjuvant WBRT.

**Keywords:** breast cancer; old age; breast-conserving surgery; radiotherapy; survival

#### **1. Introduction**

Standard treatments based on cancer treatment guidelines such as the National Comprehensive Cancer Network (NCCN) guidelines are not suitable for every older patient, because many randomized controlled trials (RCTs) for breast cancer therapy do not enroll patients ≥65 years old [1]. Determining optimal treatments for older cancer patients is challenging, especially for those aged 80 years or more. Although trials have enrolled patients ≥70 years old, the sample size of those ≥80 years old is small, and trials including those ≥90 years old are scant [2,3]. However, cancer is commonly a disease of the old, and the median age at diagnosis for all sites is 65 years [4]. Older patients (≥80 years) constitute a substantial percentage of those with breast cancer [5]. Approximately one in four patients with breast cancer are aged more than 65 years, and approximately 10% of the total breast cancer population is 80 years or older [5]. This age group often presents challenges in terms of treatment because of comorbidities and frailty [6].

It is difficult to evaluate long-time overall survival and disease-free survival for elderly breast cancer patients in RCTs, due to their short life-expectancy. Additionally, there is also the cost of treatment to consider in elderly patients with short life-expectancies. Therefore, all comorbidities should be considered in these kinds of elderly patient studies, and be wellmatched through propensity score matching (PSM). Most patients should have Charlson comorbidity index (CCI) score of 0–1 with relative health, which might be an association with longer life-expectancy. The selection of relatively healthy, suitable elderly breast cancer patients for the consideration of further adjuvant radiotherapy (RT) would be worthwhile.

Adjuvant RT is applied to eradicate any tumor deposits remaining following surgery [7]. This reduces the risk of locoregional recurrence (LRR) and improves breast cancer-specific survival and overall survival (OS) [7]. For most women treated with breast-conserving surgery (BCS), adjuvant whole-breast RT (WBRT), rather than surgery alone, is recommended according to the NCCN guidelines and the results of RCTs [1,7]. Studies with grade 2B evidence (weak recommendation) have suggested that the omission of RT might be considered in women ≥65 years old with node-negative, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative primary tumors up to 3 cm, for whom endocrine therapy is planned [8–12]; alternatively, administering RT to these women is also reasonable depending on their values and preferences, and the biologic features of the tumor. For example, women in this subset who wish to minimize their risk of LRR and accept the toxicities associated with RT may reasonably opt for RT. To date, no study with a sufficient sample size and long-term follow-up for older (≥80 years old) women with breast cancer has been conducted; this is especially true for 90-year-old women and above. A head-to-head PSM study mimicking an RCT might be necessary, especially for old (≥80 years) and very old (≥90 years) women.

The radiation oncologist should discuss the advantages and disadvantages of RT with older women with breast cancer receiving BCS prior to making a decision on its omission. For example, in the real world, compliance with endocrine therapy is a critical aspect of treatment, particularly for those with RT omission. A head-to-head study with a sufficiently large sample size and long follow-up is required to estimate the oncologic outcomes of adjuvant WBRT for older women with breast cancer undergoing BCS. We conducted this PSM study to examine the effects of adjuvant WBRT on oncologic outcomes such as OS,

LRR, and distant metastasis (DM) in old (aged ≥80 years) and very old (aged ≥90 years) women, who have scarcely been enrolled in RCTs; these findings would help determine the value of adjuvant WBRT in these patients.

#### **2. Patients and Methods**

#### *2.1. Study Population*

In this cohort study, data were retrieved from the Taiwan Cancer Registry Database (TCRD). We enrolled old (age ≥80 years) and very old (≥90 years) women with breast invasive ductal carcinoma (IDC) who had received BCS between 1 January 2008 and 31 December 2018. The index date was the date of BCS, and the follow-up duration was from the index date to 31 December 2019. The TCRD of the Collaboration Center of Health Information Application contains detailed cancer-related information of patients, including clinical stage, pathologic stage, chemotherapy regimen, chemotherapy dose, molecular status, drug use, hormone receptor status, HER2 status, radiation modality and dose, and surgical procedure [13–16]. The study protocols were reviewed and approved by the Institutional Review Board of Tzu-Chi Medical Foundation (IRB109-015-B).

#### *2.2. Inclusion and Exclusion Criteria*

The diagnoses of the enrolled women with breast IDC were confirmed after their pathological data were reviewed, and women with newly diagnosed IDC were confirmed to have no other cancers or DMs. Women with IDC were included if they were 80 years or older and had clinical stage IA-IIIC (American Joint Committee on Cancer [AJCC], 8th edition) without metastasis. Women with IDC were excluded if they had a history of cancer before the IDC diagnosis date, unknown pathologic types, missing sex data, unclear staging, or non-IDC histology. In addition, women having unclear differentiation of the tumor grade, missing data on hormone receptor status, or unknown HER2 status were excluded. Other adjuvant treatments such as chemotherapy, hormone therapy, or HER2 inhibitors did not constitute exclusion criteria based on the NCCN guidelines [17]. We also excluded women with unclear data on surgical procedures such as BCS or TM, ill-defined nodal surgery, or unclear Charlson comorbidity index (CCI) scores. Hormone receptor-positivity was defined as ≥1% of tumor cells demonstrating positive nuclear staining through immunohistochemistry [18].

After applying the inclusion and exclusion criteria, we divided the population into two groups based on their adjuvant WBRT status to compare all-cause mortality: Group 1 (older women with IDC who received BCS followed by adjuvant WBRT) and Group 2 (older women with IDC who received BCS and no adjuvant WBRT). We also excluded women in Group 1 receiving nonstandard adjuvant WBRT (contrast with standard adjuvant radiotherapy consisting of irradiation to the whole breast with a minimum of 50 Gy). Contemporary RT techniques (i.e., three-dimensional RT and intensity-modulated RT) were included, and the conventional two-dimensional RT technique was excluded. The incidence of comorbidities was scored using the CCI [19,20]. Only comorbidities observed within 6 months before the index date were included; they were classified according to International Classification of Diseases, 10th Revision, Clinical Modification codes (ICD-10- CM codes) at the first admission or based on more than two repetitions of a code recorded at outpatient department visits.

#### *2.3. Study Covariates and Propensity Score Matching*

To reduce the effects of potential confounders when comparing all-cause mortality between the adjuvant WBRT and nonadjuvant WBRT groups, PSM was performed. A greedy method was used to match the cohorts at a 1:1 ratio by age, tumor differentiation, AJCC clinical stage, AJCC pathologic stage, pT, pN, neoadjuvant chemotherapy, adjuvant chemotherapy, hormone receptor status, HER2 status, nodal surgical type, CCI score, hypertension, ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), diabetes, hospital level (medical center or not), hospital region, and income

with a propensity score within a caliper of 0.2 [21]. Moreover, we separated covariates such as hypertension, ischemic heart disease, cerebrovascular disease, COPD, and diabetes [22] from CCI scores and considered these covariates independently in PSM for more precise matching to control for confounders of all-cause death.

#### *2.4. Statistics*

Multivariable Cox regression analysis was performed to calculate hazard ratios (HRs) to determine the potential independent predictors of all-cause death, LRR, and DM. PSM was applied to control for potential predictors in the analysis (Table 1), and all-cause death was the primary endpoint in the two groups. LRR and DM were secondary endpoints and were estimated using proportional subdistribution hazard regression to overcome the competing risk of death in the analysis of time-to-event data [23,24].

**Table 1.** Demographic information of patients aged ≥80 years undergoing breast conservative surgery.



SD—standard deviation; IQR—interquartile range; WBRT—whole breast radiotherapy; AJCC—American Joint Committee on Cancer; HER2—human epidermal growth factor receptor 2; SLNB—sentinel lymph node biopsy; ALND—axillary lymph node dissection; CCI—Charlson comorbidity index; RT—radiotherapy; T—tumor; N nodal; pT—pathologic tumor stage; pN—pathologic nodal stage; COPD—chronic obstructive pulmonary disease.

The cumulative incidence of death was estimated using the Kaplan–Meier method, and differences in OS, LRR-free survival, and DM-free survival between older women receiving BCS followed by adjuvant WBRT and those without adjuvant WBRT were determined using a log-rank test. We performed all analyses using SAS version 9.3 (SAS Institute, Cary, NC, USA). *p* values < 0.05 were considered statistically significant in the two-tailed Wald test. Risk of all-cause death was calculated, and subgroup analyses by age and cancer were conducted using a log-rank test.

#### **3. Results**

#### *3.1. Study Cohort*

After PSM, 1504 older women with balanced covariates were included (Table 1). Among them, 752 received BCS followed by adjuvant WBRT (Group 1) and 752 with IDC received BCS without adjuvant WBRT (Group 2). After PSM, the results revealed that the covariates between the groups were homogenous. The median follow-up durations after the index date were 70.3 and 64.4 months for Group 1 and Group 2, respectively.

#### *3.2. Impact of Adjuvant WBRT on Oncologic Outcomes of Old and Very Old Women*

In multivariable Cox regression analysis, the adjusted HR (aHR) and 95% confidence interval (95% CI) of all-cause death for adjuvant WBRT compared with no adjuvant WBRT was 0.56 (0.44–0.70). The aHRs (95% CIs) of LRR and DM for adjuvant WBRT were 0.29 (0.19–0.45) and 0.45 (0.32–0.62), respectively, compared with no adjuvant WBRT. The aHRs (95% CIs) of all-cause death for old age (85–89 years) and very old age (≥90 years) were 1.85 (1.28–2.69) and 1.67 (1.47–3.46), respectively, compared with the age of 80–84 years. Other confounders were not significantly different for all-cause death, LRR, and DM between the two groups because of the well-matched PSM design without residual imbalance [25,26].

#### *3.3. Age Stratification in Multivariable Cox Regression Analysis*

Because age remained an independent prognostic factor of all-cause death even after PSM, residual imbalance existed in the confounder of age for all-cause death (Table 2). We performed multivariable analysis of OS that was stratified by the ages of 80–89 years and ≥90 years (Table 3). The aHRs (95% CIs) of all-cause mortality for adjuvant WBRT compared with no adjuvant WBRT in old (80–89 years) and very old (≥90 years) women receiving BCS were 0.60 (0.40–0.91) and 0.64 (0.48–0.87), respectively (Table 3). In addition, the aHR (95% CI) of all-cause death for the age of 85–89 was 1.48 (1.07–2.27), compared with the age of 80–84 years, and that for the age of ≥95 years was 1.50 (1.10–2.04) compared with the age of 90–94 years.

**Table 2.** Multivariate analysis for overall survival, local recurrence, and distant metastasis after propensity score-matching patients aged ≥80 years undergoing breast conservative surgery.


\* All of the covariates listed in Table 2 were adjusted. WBRT—whole breast radiotherapy; aHR—adjusted hazard ratio; CI—confidence interval; AJCC—American Joint Committee on Cancer; HER2—human epidermal growth factor receptor 2; SLNB—sentinel lymph node biopsy; ALND—axillary lymph node dissection; CCI—Charlson comorbidity index; pT—pathologic tumor stage; pN—pathologic nodal stage.


**Table 3.** Multivariate analysis of overall survival of propensity score-matched patients undergoing breast conservative surgery, stratified by old (80 years or over) and very old (90 years or over).

\* All of the covariates listed in Table 2 were adjusted. WBRT—whole breast radiotherapy; aHR—adjusted hazard ratio; CI—confidence interval; AJCC—American Joint Committee on Cancer; HER2—human epidermal growth factor receptor 2; SLNB—sentinel lymph node biopsy; ALND—axillary lymph node dissection; CCI—Charlson comorbidity index; RT—radiotherapy; pT—pathologic tumor stage; pN—pathologic nodal stage.

#### *3.4. Survival Curves with or without Adjuvant WBRT*

Figures 1–3 present Kaplan–Meier curves that illustrate the overall, LRR-free, and DM-free survival curves of the groups. The 5-year OS probability was 90.11% and 83.92% in the adjuvant WBRT and nonadjuvant WBRT groups, respectively (Figure 1A) (logrank test, *p* < 0.0001). Additionally, 5-year LRR-free survival was 97.81% and 87.32% in the adjuvant WBRT group and nonadjuvant WBRT group, respectively (Figure 2A; logrank test, *p* < 0.0001). Moreover, 5-year DM-free survival was 95.74% and 85.61% in the adjuvant WBRT group and nonadjuvant WBRT group, respectively (Figure 3A; log-rank test, *p* < 0.0001).

KM curves for overall survival after propensity score matching in patients aged ≥80 years — — – — – **Figure 1.** KM curves for overall survival after propensity score matching in patients aged ≥80 years undergoing breast conservative surgery. (**A**)—All stages; (**B**)—Stage 0–1; (**C**)—Stage 2–4.

**Figure 2.** *Cont*.

≥

≥ **Figure 2.** KM survival curves for local recurrence after propensity score matching in patients aged ≥80 years undergoing breast conservative surgery. (**A**)—All stages; (**B**)—Stage 0–1; (**C**)—Stage 2–4. ≥80 years undergoing breast conservative surgery. — — – — –

#### *3.5. Survival Curves of Cancer Stages and Age Stratification*

Analysis of the impact of stage (early stage (stage 0-I) or advanced stage (stage II-III)) on oncologic outcomes (OS, LRR, and DM) was conducted with stratification by pathologic stages. The OS, LRR-free, and DM-free survival curves of the adjuvant WBRT group remained significantly superior to those of the nonadjuvant WBRT group regardless of stage (Figures 1B,C, 2B,C and 3B,C). Age stratification by 80–89 and ≥90 years was also performed. The OS, LRR-free, and DM-free survival curves of the adjuvant WBRT group were significantly superior to those of the non-adjuvant WBRT group in both stratifications (Supplementary Figures S1 and S2).

#### **4. Discussion**

#### *4.1. No Solution Regarding Adjuvant WBRT for Older Women with Breast Cancer*

Breast cancer is the most common cancer in women, and one in ten patients affected are aged ≥80 years [5]. However, this age group is generally excluded from clinical trials, and data to inform their care are sparse [7]. Additionally, no RCT with women aged ≥90 years with breast cancer has been conducted. In practice, treatment for older patients with breast cancer involves shared decision-making between physicians and patients based on expected survival lifespan, comorbidities, or prognostic factors of tumor recurrence [8–12]. Nevertheless, few patients in the ≥80 years age group receive RT as part of their treatment, especially those aged ≥90 years [5,8–12,27]. Studies on the omission of RT in older women with a low recurrence of hormone receptor-positive or HER2-negative breast cancer (as a better prognosis) have been conducted, but studies including women aged ≥80 years are scant [8–12]. Breast cancer biologic subtypes of women aged ≥80 years exhibit similarities with those of younger postmenopausal women; thus, treatments should be consistent [6]. Possible problems are the expected survival and comorbidities contributing to the incidence of LRR- and DM-related mortality [22–24]. Nonetheless, if older patients with IDC receiving BCS have consistent comorbidities, molecular types (similar hormone receptor status and HER2), the same cancer stages, and similar treatment protocols relative to younger patients, whether adjuvant WBRT should be omitted is unclear.

#### *4.2. Value of PSM in This Population*

As shown in Table 1, all potential cofounders of all-cause death for women with breast cancer were matched and controlled through PSM. The cofounders—age, differentiation, AJCC clinical stage, AJCC pathologic stage, pT, pN, neoadjuvant chemotherapy, adjuvant chemotherapy, hormone receptor status, HER2 status, nodal surgical type, CCI score, hospital level (medical center or not), hospital region, and income, all mentioned in previous studies—were matched to balance covariates between the two groups [13–15,28–31]. Because the most common causes of death in older patients are hypertension, ischemic heart disease, cerebrovascular disease, COPD, and diabetes [22], we separated the covariates from the CCI scores and included these covariates in PSM independently for more precise matching to control the confounders of all-cause mortality. PSM allows the design of an observational (non-randomized) study that mimics some of the characteristics of an RCT [32]. After PSM design, we believe the balanced covariates mimic an RCT [32] in our study without selection bias for adjuvant WBRT and no adjuvant WBRT in older women receiving BCS. Before PSM, the trends of selection of no adjuvant WBRT (raw population in Table 1) were compatible with those in previous studies, in which women with node-negative, hormone receptor-positive, HER2-negative cancer or small tumor sizes preferred no adjuvant RT [8–12]. Our findings indicate that women with favorable prognostic factors of OS would not receive adjuvant WBRT (Table 1). Conducting an RCT with patients ≥80 years old is difficult. Therefore, a PSM study with balanced conditions is appropriate for evaluating the value of adjuvant WBRT for older women.

#### *4.3. Conditions Different from Previous Studies*

Adjuvant WBRT can be omitted in older (≥65 years) women with hormone receptorpositive breast cancer, especially for clinically node-negative, small, or HER2-negative breast cancer [8–12]. Moreover, omission of RT in patients with hormone receptor-positive, node-negative, small breast cancer is supported by a meta-analysis that included postmenopausal women, all of whom received systemic therapy (the majority received tamoxifen) [3]. However, most women had T1, node-negative tumors and were aged ≥65 years, with 39% aged ≥70 years [3]. Only approximately 10% of patients were ≥80 years old in the aforementioned studies [3,8–12]. Comorbidities were not considered in the previous studies with unexpected survival lifespans [3,8–12], and the survival benefit of adjuvant WBRT could not be determined in the aforementioned reports. In the current study, all the enrolled women were ≥80 years old, and approximately 40% were ≥90 years old (Table 1). All comorbidities were considered in our study and were well-matched through PSM. In addition, molecular type, cancer stage, and treatment protocols were controlled for through PSM. Therefore, our study is the first head-to-head PSM study mimicking an RCT with consistent conditions to estimate the oncologic outcomes after adjuvant WBRT in old (aged 80–89 years) and very old (aged ≥90 years) women with IDC receiving BCS.

#### *4.4. Cancer Stage and Age Stratification*

Because some reports have indicated that adjuvant RT can be omitted in older women with early-stage breast cancer receiving mastectomy [2,10], we estimated the effects of adjuvant WBRT by using the log-rank test for the PSM population stratified by early or advanced pathologic stage. The results indicated receiving that adjuvant WBRT was significantly superior to not receiving adjuvant WBRT for OS, LRR-free survival, and DMfree survival, even in the earliest stages (stage 0-I) (Figures 1B, 2B and 3B). Previous studies reporting no significant survival difference between adjuvant RT and no adjuvant RT for breast cancer in older women might be attributed to small sample size, short follow-up time, or unknown comorbidities [2,10]. The most common cause of death in these older women is comorbidities [22], but no data on comorbidities have been included in reports [2,10]. Another key concern is that those aged ≥80 years were not the main population, and that those aged ≥90 years were few in the aforementioned studies [2,10]. We used the log-rank test for investigating the effect of adjuvant WBRT or no adjuvant WBRT on oncologic outcomes for different age groups (80–89 years and ≥90 years) in the PSM population (Supplementary Figures S1 and S2). No study of patients aged ≥90 years with breast cancer has been conducted. Our study is the first to demonstrate the benefits of adjuvant RT for women 90 years or older with IDC receiving BCS.

#### *4.5. Limitations*

This study has limitations. First, because all the women with IDC were enrolled from an Asian population, the corresponding ethnic susceptibility compared with non-Asian populations remains unclear; hence, our results should be cautiously extrapolated to non-Asian populations. However, no evidence suggests differences in oncologic outcomes between Asian and non-Asian women with breast IDC receiving BCS. Second, the diagnoses of all comorbid conditions were based on ICD-10-CM codes. However, the combination of the TCRD and the National Health Insurance Research Database (NHIRD) in Taiwan appears to be a valid resource for population research on cardiovascular diseases, stroke, or chronic comorbidities [33–35]. The Taiwan Cancer Registry Administration randomly reviews charts and interviews patients to verify the accuracy of diagnoses, and hospitals with outlier chargers or practices may be audited and heavily penalized if malpractice or discrepancies are identified. Accordingly, to obtain crucial information on population specificity and disease occurrence, a large-scale RCT comparing carefully selected patients undergoing suitable treatments is essential. However, as mentioned, enrolling patients ≥80 or even ≥90 years of age in an RCT is difficult. Despite its limitations, a major strength of this study is the use of a nationwide population-based registry with detailed baseline and

treatment information. Lifelong follow-up was possible through the linkage of the registry with the national Cause of Death database. Considering the magnitude and statistical significance of the observed effects in the current study, the limitations are unlikely to affect our conclusions.

#### **5. Conclusions**

Compared with no adjuvant WBRT, adjuvant WBRT may be associated with decreased all-cause of death, LRR, and DM for older women with breast IDC receiving BCS regardless of stage (early vs. advanced) and age (80–89 vs. ≥90 years). We suggest adjuvant WBRT for old or very old women with IDC receiving BCS, even if the cancer stage is early or the patient is 90 years or older.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jpm12020287/s1, Figure S1: Overall survival, LRR-free survival, and DM-free survival curves for propensity score matched patients aged 80–89 years receiving breast conservative surgery, Figure S2: Overall survival, LRR-free survival, DM-free survival curves for propensity score matched patients aged 90 years or over receiving breast conservative surgery.

**Author Contributions:** Conception and design, C.-C.H. and S.-Y.W.; collection and assembly of data, C.-C.H., M.S., S.-Y.S., S.-Y.W., C.-L.C. and J.Z.; data analysis and interpretation, C.-C.H., M.S., M.-F.C., C.-L.C. and J.Z.; administrative support, S.-Y.W.; manuscript writing: C.-C.H., C.-L.C., J.Z., S.-Y.S., M.-F.C. and S.-Y.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** Lo-Hsu Medical Foundation, LotungPoh-Ai Hospital, supports Szu-Yuan Wu's work (Funding Number: 10908, 10909, 11001, 11002, 11003, 11006, and 11013).

**Institutional Review Board Statement:** The study protocols were reviewed and approved by the Institutional Review Board of Tzu-Chi Medical Foundation (IRB109-015-B).

**Informed Consent Statement:** Informed consent was waived because the data sets are covered under the Personal Information Protection Act. We used data from the National Health Insurance Research Database and Taiwan Cancer Registry database. The authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. The data used in this study cannot be made available in the article, the supplemental files, or in a public repository due to the Personal Information Protection Act executed by Taiwan's government, starting from 2012. Requests for data can be sent as a formal proposal to obtain approval from the ethics review committee of the appropriate governmental department in Taiwan. Specifically, contact information for requesting data appear at http://nhird.nhri.org.tw/en/Data\_Subsets.html#S3 and http://nhis.nhri.org.tw/ point.html.

**Data Availability Statement:** The data sets supporting the study conclusions are included in this manuscript and its supplementary files.

**Acknowledgments:** Lo-Hsu Medical Foundation, LotungPoh-Ai Hospital, supports Szu-Yuan Wu's work (Funding Number: 10908, 10909, 11001, 11002, 11003, 11006, and 11013).

**Conflicts of Interest:** The authors have no potential conflicts of interest to declare. The data sets supporting the study conclusions are included in the manuscript.

#### **Abbreviations**

WBRT, whole-breast radiotherapy; LRR, locoregional recurrence; DM, distant metastasis; IDC, invasive ductal carcinoma; BCS, breast-conserving surgery; OS, overall survival; aHR, adjusted hazard ratio; HR, hazard ratio; CI, confidence interval; AJCC, American Joint Committee on Cancer; TCRD, Taiwan Cancer Registry Database; SD, standard deviation; HER2, human epidermal growth factor receptor 2; SLNB, sentinel lymph node biopsy; ALND, axillary lymph node dissection; CCI, Charlson comorbidity index; ICD-10-CM, International Classification of Diseases, 10th Revision, Clinical Modification; NCCN, National Comprehensive Cancer Network; RT, radiotherapy; T, tumor; N, nodal; pT, pathologic tumor stage; pN, pathologic nodal stage; NHIRD, National Health Insurance Research Database; RCT, randomized controlled trial; PSM, propensity scores matching.

#### **References**


### *Article* **Let-7a-5p, miR-100-5p, miR-101-3p, and miR-199a-3p Hyperexpression as Potential Predictive Biomarkers in Early Breast Cancer Patients**

**Paola Fuso 1,2,† , Mariantonietta Di Salvatore 2,3, \* ,† , Concetta Santonocito 2,4 , Donatella Guarino 2,4 , Chiara Autilio 5 , Antonino Mulè 1,2,6 , Damiano Arciuolo 1,2,6 , Antonina Rinninella 7 , Flavio Mignone 7 , Matteo Ramundo 2,3 , Brunella Di Stefano 2,3 , Armando Orlandi 2,3 , Ettore Capoluongo 2,8 , Nicola Nicolotti 2,9 , Gianluca Franceschini 2,10 , Alejandro Martin Sanchez 2,10 , Giampaolo Tortora 2,3 , Giovanni Scambia 1,2 , Carlo Barone <sup>2</sup> and Alessandra Cassano 2,3**

	- Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy

**Abstract:** Background: The aim of this study is to identify miRNAs able to predict the outcomes in breast cancer patients after neoadjuvant chemotherapy (NAC). Patients and methods: We retrospectively analyzed 24 patients receiving NAC and not reaching pathologic complete response (pCR). miRNAs were analyzed using an Illumina Next-Generation-Sequencing (NGS) system. Results: Event-free survival (EFS) and overall survival (OS) were significantly higher in patients with up-regulation of let-7a-5p (EFS *p* = 0.006; OS *p* = 0.0001), mirR-100-5p (EFS s *p* = 0.01; OS *p* = 0.03), miR-101-3p (EFS *p* = 0.05; OS *p* = 0.01), and miR-199a-3p (EFS *p* = 0.02; OS *p* = 0.01) in post-NAC samples, independently from breast cancer subtypes. At multivariate analysis, only let-7a-5p was significantly associated with EFS (*p* = 0.009) and OS (*p* = 0.0008). Conclusion: Up-regulation of the above miRNAs could represent biomarkers in breast cancer.

**Keywords:** subtypes breast cancer; miRNAs; breast cancer treatment; chemotherapy; integrated therapies; next-generation-sequencing; target therapy; precision medicine; personalized medicine

**Citation:** Fuso, P.; Di Salvatore, M.; Santonocito, C.; Guarino, D.; Autilio, C.; Mulè, A.; Arciuolo, D.; Rinninella, A.; Mignone, F.; Ramundo, M.; et al. Let-7a-5p, miR-100-5p, miR-101-3p, and miR-199a-3p Hyperexpression as Potential Predictive Biomarkers in Early Breast Cancer Patients. *J. Pers. Med.* **2021**, *11*, 816. https://doi.org/ 10.3390/jpm11080816

Academic Editor: Raghu Sinha

Received: 16 May 2021 Accepted: 14 August 2021 Published: 20 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Breast cancer is a heterogeneous disease and many molecular changes occur during the course of the disease; this is the main cause of treatment failure. This characteristic of breast cancer is reflected on the basis of gene expression pattern classification. It falls under five distinct molecular subtypes including luminal A, luminal B, receptor tyrosineprotein kinase erbB-2 (HER2)-enriched, basal-like, and normal-like subtype. Luminal A breast cancer is hormone-receptor positive (estrogen-receptor (ER) and/or progesteronereceptor (PR) positive), HER2-negative, has low levels of the protein Ki-67, and is low-grade. Luminal B breast cancer is hormone-receptor positive (ER and/or PR positive) and either HER2-positive or HER2-negative with high levels of Ki-67. HER2-enriched breast cancer is hormone-receptor negative (ER and PR negative) and HER2-positive. Triple negative breast cancer (TN) is defined as the absence of estrogen receptor, progesterone receptor, and HER2 expression accounting for approximately 15–20% of all breast cancer patients. The majority of TN patients (up to 70%) overlap with the basal-like gene expression subtype.

Tumor evolution is a unique process for each patient and is influenced by intrinsic genetic variability and external factors such as cancer therapy. Neoadjuvant setting is an ideal scenario to understand tumor evolution at a single patient level, because make it possible to identify molecular changes occurring in tumors due to treatment by comparing pre and post-chemotherapy samples [1–3].

Finding the patients most likely to benefit from NAC is a crucial need and increasing experimental and clinical studies are centered on identifying the predictors of long-term benefit. Several surrogate endpoints have been examined in the neoadjuvant setting such as the pCR, which has been identified as a primary endpoint in numerous clinical trials despite the controversies on its power of predicting the outcome [3,4].

It is noteworthy that not all patients with residual disease after NAC relapse, and the prognostic impact of pCR varies among breast cancer-intrinsic subtypes, whereas patients with luminal A-like breast cancer show a low pCR rate, their overall prognosis is favorable, and patients with TN breast cancer show a high pCR rate but may have a poorer outcome; moreover, if all intrinsic subtypes are considered, the prognostic information of pCR is reduced [5–9].

Several studies have been performed to discover molecular breast cancer biomarkers in order to predict response to neoadjuvant therapy.

miRNAs are involved in pathway regulation (one miRNA can target many genes and a single gene can be modulated by several miRNAs), and finally, miRNAs show tissue and cell-specific expression profiles, and their role in the pathophysiology of the disease is supported extensively in the literature [10].

Each miRNA can regulate the expression of several genes; thus, each one can simultaneously modulate multiple cellular signaling pathways. Depending on their modulation (amplification/deletion) and on target gene function (tumor suppressor/oncogene), miRNA can play alternatively an oncosuppressor or oncogene function. MiRNAs expression in tumors can be altered due to epigenetic, genetic, and transcriptional alterations [11,12].

Several studies have demonstrated that many miRNAs are aberrantly expressed in breast cancer, according to breast cancer molecular subtypes and thus potentially play a role of biomarkers for cancer diagnosis and for response to therapy [13].

We hypothesized that miRNA are differently expressed at different steps of the disease, and it could be possible to identify a set of miRNA associated with disease progression or response to therapy and to attribute to them a predictive and prognostic value.

The aim of the present exploratory study was to identify a set of miRNAs able to predict the prognosis of patients who underwent NAC not achieving pCR.

#### **2. Materials and Methods**

#### *2.1. Patients' Characteristics and Tumor Specimen Collection*

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study has the approval of the Ethics Committee of Fondazione Poli-clinico A. Gemelli IRCCS of Rome (Italy) (N protocol 27736/16), and all patients gave written informed consent. We analyzed our database that contains clinical and pathological data on ≈200 cases that underwent neoadjuvant treatment from July 1997 to April 2014 at Fondazione Policlinico A. Gemelli. Patients had measurable breast tumors. Patients were staged according to the American Joint Committee on Cancer (AJCC) Eighth edition [14]. A TRU-CUT biopsy was obtained from each patient. Classification of intrinsic subtypes was defined according to 16th St. Gallen and ESMO guidelines. Histological type, tumor grade, Ki67, ER, PR, and HER2 status were evaluated in the pre-NAC biopsy and in post-surgical neoplastic specimens. Treatment of HER2-negative breast cancer patients consisted of a combination of anthracyclines, taxanes, and cyclophosphamide, while patients with HER2-positive tumors received taxanes and carboplatin combined with trastuzumab, the latter continued after surgery to complete one year of treatment. Patients with ER and/or PR positive tumors received adjuvant endocrine treatment for at least 5 years. Adjuvant radiotherapy was offered according to the national guidelines [15]. The pCR was defined as the absence of any residual invasive cancer on resected breast specimen and on all sampled ipsilateral lymph nodes (ypT0/is ypN0) [16,17] (Table 1).


**Table 1.** Baseline patients' characteristics (N 200).

**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Abbreviations:** TAC, taxanes, anthracyclines, and cyclophosphamide-based regimen; TCH, taxanes, carboplatin, and trastuzumab-based regimen; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor; pCR, pathologic complete response; SD standard deviation.

From the entire database, we selected twenty-four patients homogeneously distributed according to clinical and pathological characteristics not achieving pCR to which the maximum amount of paraffin-embedding samples of both pre- and post-treatment specimen were available (Tables 2 and 3). In particular, we analyzed pre- and post-NAC samples of the three main molecular subtypes, respectively HER2-positive luminal, HER2-positive non-luminal, and TN subtypes, respectively. For each subtype, we selected four patients with good prognosis and four with poor prognosis.


**Table 2.** Clinicopathological characteristics of breast cancer selected patients (N 1–12).

**Table 3.** Clinicopathological characteristics of breast cancer selected patients (N 13–24).


#### *2.2. Purification of miRNA from Paraffin-Embedding Tissue Sections*

Standard formalin-fixation and paraffin-embedding (FFPE) procedures always resulted in significant fragmentation and crosslinking of nucleic acid. For each of the two samples (pre- and post-NAC) for each patient, the starting material for RNA purification was made by up to 4 sections of paraffin-embedding tissue with a thickness of 5 µm combined in one preparation. After microdissection, the total RNA was extracted using miRNeasy FFPE Kit (Qiagen) following the protocol of the manufacturer. The concentration and purity of the total RNA was isolated from tissues and was determined by measuring the absorbance in a spectrophotometer (Nanodrop). The QIAseq miRNA Library Kit (Qiagen) was used for miRNA libraries. In an unbiased rapid reaction, adapters were ligated sequentially to the 3′ and 5′ ends of the miRNAs. Subsequently, universal cDNA synthesis with UMI (Unique Molecular Index) assignment, cDNA cleanup, library amplification, and library cleanup were performed following the manufacturer's recommendation. The integrity and size distribution of the total RNA from the tissue was confirmed using an automated analysis system (Agilent 2100 Bioan-alyzer). Successively, the miRNA sequencing libraries was sequenced using MiSeq® Il-lumina NGS system: the molarity of each sample (in nM) was calculated using the following equation: (X ng/µL)(106)/(112450) = Y nM. Individual libraries were diluted to 4 nM using nuclease-free water and then combined in equimolar amounts.

#### *2.3. MiRNA Discovery*

#### 2.3.1. Analysis Procedure

The QIAseq miRNA-NGS data analysis software (Qiagen) was used. The results were confirmed manually by aligning the fastqs with the sequences corresponding to all human miRNAs. The miRNA sequences were extracted from the miRBase database [18].

The miRNAs were selected based on the number of reads, and those that differed between pre-NAC and post-NAC were taken into consideration.

#### 2.3.2. MiRNA Target Prediction

To know the potential target site, a computational approach was applied for their validation [19]. The miRNA targets were predicted by the instrument MiRDB [20]. This is an online database for miRNA target prediction and functional annotations with a focus on mature miRNAs. It provides a web interface for target prediction generated by an SVM machine learning algorithm. All gene targets were converted by the Human Gene ID Converter tool into their corresponding NCBI entrez gene ID. Some NCBI-gene ID were searched manually on the HUGO Gene Nomenclature Committee (HGNC) database. Perl language scripts have been made to list the NCBI entrez gene ID for each of the miRNAs to be analyzed. For the mapping of the genes, the KEGG Mapper—Search & Color Pathway tool was used. Only the pathways related to the disease were selected and where the mapped genes were more numerous. The pathways related to the disease were selected in consultation with the bibliographic articles in Pubmed-NCBI.

#### *2.4. Statistical Analysis*

The primary endpoint was event-free survival (EFS). The secondary endpoint was overall survival (OS). EFS was considered as the time from diagnosis to any relevant event (progression of disease that precludes surgery, local or distant recurrence, or death due to any cause) and was censored at the last follow-up visit. OS was estimated as the interval from diagnosis to death from any cause, and it was censored at the last follow-up visit for the patients still alive. The Kaplan–Meier method was applied for survival probabilities estimation. For univariate analysis, we used the Fisher exact test. Variables (IHC-based molecular subtypes, histological type, tumor grade, Ki67% value, tumor size, clinical lymph node status, cTNM stage, surgery) were included in the multivariate analysis if the univariate *p*-value was <0.05. Multivariate analysis was done using the Cox proportional

**‐**

hazard model. A two-sided *p*-value < 0.05 was considered statistically significant. Analyses were performed using SPSS statistical package version 13.0.

#### **3. Results**

#### *3.1. Patients Charachteristics*

Within the entire database, we selected 24 early breast cancer patients, who had undergone neoadjuvant chemotherapy at the IRCCS Fondazione Policlinico A. Gemelli, homogeneously stratified according to clinical and pathological characteristics, and not achieving pCR. In particular, we analyzed pre- and post-NAC samples of eight patients for the following subtypes: HER2-positive luminal, HER2-positive non-luminal, and TN subtypes. Median age at time of study entry was 50.2 years (range, 35 to 72 years). Median follow-up was 80 months, median EFS was 40.7 months, and median OS was 63.3 months. ‐ ‐ ‐ ‐ ‐ ‐

#### *3.2. Clinicopathological Variables and Outcome*

We analyzed the correlation between IHC-based molecular subtypes (luminal B/HER2 positive, HER2-positive/non-luminal and TN breast cancer), histological type (ductal invasive breast cancer and others), tumor grade, Ki67% value, tumor size, clinical lymph node status, cTNM stage, surgery, and clinical outcome. Variables showing *p*-values < 0.05 in univariate analyses were used for multivariate logistic regression. However, none of the selected variables were statistically significant at univariate analysis. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

#### 3.3. miRNAs and Outcome

Thanks to the computational algorithms and bioinformatics database, we identified 27 miRNAs that were significantly hypo- or hyper-expressed in pre- versus post-NAC samples: hsa-let-7a-5p, hsa-let-7f-5p, hsa-miR-100-5p, hsa-miR-101-3p, hsa-miR-103a-3p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-143-3p, hsa-miR-191-5p, hsa-miR-196a-5p, hsa-miR-199a-3p, hsa-miR-205-5p, hsamiR-26a-5p, hsa-miR-26b-5p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-30a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-510-3p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-99a-5p, hsa-miR-99b-5p. In Scheme 1, we show the modulation of expression of miRNAs for each subtypes. In Table 4 we presented miRNAs predictive target genes. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

‐ ‐ **Scheme 1.** The chart summarizes—for each miRNA and for each subtype—the number of samples that show the same over/under-expression pattern. Bars above the 0 represent overexpression, while bars below represent under-expression.


**Table 4.** miRNAs predictive target genes.

**miRNAs Gene Target Predicted miR-101-3p** ASCC3 RAB27A BEGAIN ZNF510 RFPL4B CCDC68 TLK2 TAGAP FUCA2 ZNF549 RAB15 OTUD4 CCSER1 ZBED4 RASGRP3 GRIN2A ANXA10 WWC3 HNRNPF KAT6B HAS2 DCUN1D1 CTCF CCDC88A FAM73A MTSS1L BBX FAM60A RNF19A RCN2 PKD2 ATRX POLR3K MAP3K9 N4BP1 DNM1L MRPL42 KHDRBS2 STX6 CSNK1G3 NOTCH1 GABRB2 SPOP GLIPR1L1 KIF5B C9orf72 DENND2C SACM1L LRRC4 MAP3K2 SPG11 DCAF7 ARHGEF10 KLF2 ZCCHC2 KPNB1 KIAA1432 CRLS1 BTLA NSD1 MAPK1 TMEM167A PDS5B OGT KDM6B GCNT1 C11orf70 ANKZF1 RNF38 ROBO2 SGMS2 EPT1 SLMO2 HIVEP3 FAR1 CAPS2 TMEM231 TKTL1 TMEM68 ZNF469 SGPL1 RXRB WDR72 DESI2 NACA2 MTMR4 LGI2 CREBRF XPO5 PTCH1 NACA GABBR2 PRR11 CTDSPL DCLRE1B DDX3X MAB21L3 MLEC FAM103A1 GNB1 SPATS2L PRRC2C UBR7 FYTTD1 CD86 RIPK1 CNIH3 NAIP MON2 ATRNL1 KIAA1462 BCL2L11 RANBP1 FMNL3 PHTF2 TMF1 LANCL3 ZNF33A TIMM17A PLEKHG1 PBX3 MTX3 UNKL TEX2 RANBP6 AGAP1 ZNF235 CCDC126 FAM169A PTBP3 CADM2 KCNE1 FAM216B OTUD3 MAP10 FLRT2 PIK3C2B PCK1 PYGO1 TMEM201 C7orf73 C1orf52 SPRED1 B3GNT3 NDUFB5 TKTL2 ATP11B NEGR1 CADM1 TMED5 SMARCA4 SMN2 IKZF4 ZNF24 XKR6 PLA2R1 CDKN1A NAV1 PYGO2 NAA15 FRYL PCDH20 KIAA1377 PACRG NF1 SUPT7L C2orf88 RRM1 SMN1 FAM53B INPP4B IPO5 SRPK2 BBS7 STAR GDE1 FBXW11 JDP2 CRISPLD1 MAD2L1 SLTM DPY19L2 TBC1D12 ADH5 VSX1 LONRF1 COTL1 RBBP7 JAK2 SOAT1 NEK4 UBE2F MNX1 AGFG1 PTPRJ KTI12 PHACTR2 C16orf72 ARHGAP32 POGK IQGAP3 FAM122C USP38 CCNT2 DTD2 TMEM170B STMN1 PITPNB PCDH7 ZIC1 LRAT PDP1 CISD2 FOXN2 ZNF260 EPB41L5 DENR SLC25A4 ZC3H7A GRSF1 TMEM132D RHOT1 C10orf12 JAKMIP2 AP1S3 CASP3 BAZ2A **miR-199-3a** ETNK1 CELSR2 ADAMTSL3 KLHL3 ACVR2A LRP2 BCAR3 SERPINE2 NOVA1 MAP3K4 FAM110C KIAA0319L RB1 ZHX1 KDM5A PSD2 LIN28B LLGL2 ITGA3 CHMP5 TUBGCP3 FAM60A NLK CD2AP NID2 UTP20 PAK4 C9orf40 KDM6A CDK7 C2orf49 KATNBL1 CDK17 PPP2R2A APLP2 MCFD2 CDNF PRPF40A CXADR PPP2R5E G3BP2 FUBP1 NEDD4 SLC24A2 RASEF SDC2 PDGFRA SCD SUMO3 ITPK1 ARHGEF3 ESRP1 ATAD1 MAP3K5 APLF ASTN1 EMC1 GGNBP2 CYB5R4 PAWR NXPH1 PIP5K1B ATRX NUFIP2 KTN1 RNGTT MDGA2 GORAB PNRC1 VGLL2 FAM199X DEPDC1B GNPTAB NFIA DNHD1 RAPH1 TPPP WDR7 ARL15 ADAM10 NLRP1 CBLB RAPGEF4 SEMA3A COL12A1 TACC2 KLF13 SPIRE1 FAM115C ANKRD44 MS4A7 LRRC1 PTPN3 AEBP2 COL4A5 CBLL1 CISD2 CCDC85C FN1 ATP6V1A NRBP2 PTPRZ1 SP1 ATL1 DNMT3A NET1 FOS PROSER1 RFX3 WFDC8 MFSD6 TAOK1 ZBTB18 PTPRC C20orf194 ITGA6 RPS6KA6 LPAR4 LCOR MAPRE1 CD151 FXR1 PLCB1 MPP7 YWHAE EPG5 SMARCC2 EPB41L5 SLC25A46 C21orf91 SMIM8 GPBP1L1 KIDINS220 GPM6A VPS33A PON2 TMED5 HNF1B WAPAL DCBLD2 CNIH2 C9orf170 RALGPS2 LAMP3 BEND7 FAM129A ITGB8 ANKRD61 CETN3 KCMF1 FAM76B PDE4B HYPK SLC39A10 NAA25 NTRK2 KDM3A GLT8D2 WDR47 MBNL1 MTOR SOWAHC RGS4 FGL2 ALX4 YWHAG STARD9 ENOX2 MAP3K1 GALNT7 YWHAZ CREBRF TENM1 TAB2 EML4 RP1 FMN1 CHKA PVRL2 VAMP3 ZCCHC17 TEAD1 SYNJ1 SLC16A12 PCDH7 ABHD4 DUSP5 KCND2 SECISBP2L DIMT1 PPP1R9A ATP6V1C2 MEIS2 ARG2 CHAD SORL1 RNF216 ELAVL2 CAPRIN1 FCGR3A LONRF3 ADD3 RRM2B CNOT7 SRR IL1RL1 ECM2 MVB12B ADRB1 CLDN8 FCGR3B CCSAP CA5B VLDLR UBQLN1 EFCAB14 TMEM62 PTPRU ABCA1 CABLES1 SH3GLB1 ERO1L ANK2 TMEM218 KIAA0907 ASAP2 ACOX1 SYPL1 BRWD3 DPAGT1 PIK3CB NF1 ZNF614 SLC39A9 SLC5A7 HRNR CYP1B1 ZC3H14 LOC101929844 PCDHB12 HECTD2 PLEKHH1 UCK2 HNMT CDC42BPB RFX7 CCSER1 KCTD7 CITED2 CFL2 RHOT1 UBXN2B HGF KIAA0141 FBXW11 GPR160 KCNH2 TRMT61B GNA12 GRHL1 SLC44A5 PHF6 KLF12 CYP24A1 CDK5R1 MAP3K2 ATP1B4 CCDC88C ADAM22 C10orf2 TXLNG CEP85L KAZN PRKCB BAG4 FAM46D CALCRL PRC1 KIAA1244 SEC16B FKBP14 CDC14A CTNNA2 NAP1L1 UNC45A DDIT4 PAQR3

> Up-regulation of let-7a-5p, mirR-100-5p, miR-101-3p, and miR-199a-3p in post-NAC specimens was significantly correlated with better EFS and OS compared to those with normal or lower expression, independent from breast cancer subtypes.

> At subgroup analysis, the overexpression of mentioned miRNAs in post-NAC samples was linked with an improvement in EFS and OS only in HER2-positive non-luminal subtypes (Table 5). Furthermore, when we stratified patients according to a sort of miRNA signature (let-7a-5p, mirR-100-5p, miR-101-3p, miR-199a-3p), we found that patients who concurrently overexpress all four miRNAs experimented a significantly better prognosis in terms of EFS and OS (Table 5; Figures 1–5). However, at multivariate analysis, EFS (*p* = 0.009) and OS (*p* = 0.0008) showed a statistically association exclusively with upregulation of let-7a-5p.


**Table 5.** Prognostic impact of miRNA expression profile on EFS and OS in all populations and in HER2 non-luminal subtypes.

‐ ‐ **Figure 1.** Prognostic impact of Let7a-5p on EFS (on the left) and on OS (on the right) in all population: blue line refers to patients with overexpression of Let7a; red line refers to patients without overexpression of Let7a-5p. ‐ ‐

‐ ‐ ‐ ‐ ‐ **Figure 2.** Prognostic impact of miR100-5p on EFS (on the left) and on OS (on the right) in all population: blue line refers to patients with overexpression of miR100-5p; red line refers to patients without overexpression of miR100-5p.

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months months

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**Figure 3.** Prognostic impact of miR101‐3p on EFS (on the left) and on OS (on the ‐ ‐ **Figure 3.** Prognostic impact of miR101-3p on EFS (on the left) and on OS (on the right) in all population: blue line refers to patients with overexpression of miR101-5p; red line refers to patients without overexpression of miR101-5p.

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‐ ‐ ‐ **Figure 4.** Prognostic impact of miR199a-3p on EFS (on the left) and on OS (on the right) in all population: blue line refers to patients with overexpression of miR199a-3p; red line refers to patients without overexpression of miR199a-3p. ‐ ‐ ‐

**Figure 5.** Prognostic impact of miRNA signature on EFS (on the left) and OS (on the right) in all population: blue line refers to patients with overexpression of miRNA signature; red line refers to patients without overexpression of miRNA signature.

#### **4. Discussion**

 ‐ ‐ ‐ ‐ Recent suggestions have revealed that the miRNAs can modulate the expression of oncogenes or tumor suppressor genes. Based on this evidence, miRNAs appear as hopeful biomarkers of breast cancer [21].

‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ Bertoli et al. analyzed the role of several miRNAs in breast cancer and showed that some of them could be useful for diagnostic tools (i.e., miR-9, miR-10b, and miR-17-5p); other miRNAs (i.e., miR-148a and miR-335) may have a prognostic role, while still others (i.e., miR-30c, miR-187, and miR-339-5p) may be predictive of treatment response [22].

‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ In our study, we investigated the potential role of miRNAs as predictors of outcome in early breast cancer patients. We found a significantly differential miRNA expression

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among some breast cancer subtypes in pre-NAC and post-NAC paraffin-embedding tissue: in particular, we found that the up-regulation of let-7a-5p, miR-100-5p, miR-101-3p, and miR199a-3p in post-NAC samples was significantly associated with better prognosis in terms of EFS and OS, but at multivariate analysis, only overexpression of let-7 was correlated with survival.

Although miR100, miR101, and miR199 did not maintain a statistically significant correlation with survival outcome in multivariate analysis, there is a strong biological rationale supporting their role in breast cancer prognosis and, in our opinion, they deserve further studies.

Interestingly, all these miRNAs have shown to be normally down-regulated in breast cancer and have a role in cancer pathogenesis affecting cell cycle, proliferation, and metastasis diffusion.

Let-7 employs its antiproliferative activities and its tumor-suppressor role by controlling key checkpoints of several mitogenic pathways and by suppressing different oncogenes, including HMGA2, RAS, and MYC [23,24]. Let-7 expression levels have a role as a prognostic marker in several cancers, and the loss of its expression is a marker for less differentiated cancers [25,26]. It is newsworthy that HMGA2 and H-RAS oncogenes are targeted by an induced expression of let-7 in breast cancer cells, and in a murine model of breast cancer, exogenous let-7 delivery represses mammosphere formation, cell proliferation, and the undifferentiated cell population by downregulating both H-RAS and HMGA2 oncogenes [27]. Barh demonstrated that in silico analysis, apart from repressing HMGA2, RAS, and MYC, let-7 may also target CYP19A1, ESR1, and ESR2, thereby potentially blocking estrogen signaling in ER-positive breast cancers [28]. Moreover, Kim et al. affirmed that let-7a inhibits breast cancer cell migration and invasion through the down-regulation of C-C chemokine receptor type 7 expression (CCR7) [29]. Other authors described a new role of let-7a in regulating energy metabolism in neoplastic cells [30]. To underline the role of Let-7 restoration to prevent tumor progression, our study found that the overexpression of let-7 family members in post-NAC samples is associated with a better prognosis in patients with no pCR. From the therapeutic viewpoint, let-7 is an attractive molecule for preventing tumorigenesis and angiogenesis; thus, it could be a potential therapeutic target in several cancers that lose let-7.

miR-100, miR-99a, and miR-99b belong to the miR-100 family. The miRNA-100 controls several genes playing an important modulatory role. mTOR, PI3K, AKT1, IGF1- R, HS3ST2, HOXA1, RAP1B, and FGFR3 are some of the multiple targets of miR-100. Modulating these important genes, miRNA 100 could block proliferation by promoting cell cycle arrest and apoptosis in tumor cells. Furthermore, recent findings suggest that in breast cancer, the miR-100 may act as a pro-differentiating agent for cancer stem cell modulating Wnt/β-catenin pathway and Polo-like kinase 1 gene. It was found that miR100 overexpression has the capability to inhibit the Wnt pathway. Recent evidence showed that miRNA-100 downregulates Polo-like kinase 1 in basal-like cancer, blocking the maintenance and expansion of breast cancer stem cells (BrCSCs), inducing BrCSC differentiation, thus favoring the transition from undifferentiated tumors into well-differentiated ones [31,32]. Petrelli et al. analyzed 123 early node-negative breast cancer tumor specimens: patients were categorized on the basis of the miR-100 expression status. Patients with low miR-100 levels experienced worst distant metastasis-free survival [32]. According to the literature, the miR-100 family could convert an aggressive tumor into a well differentiated, biologically favorable, phenotype. In support of this potential role, miRNA-100 family members are understudied as targets for differentiation therapy: this therapeutic strategy aims to induce the transformation of aggressive cancer cells into well-differentiated ones, which are more sensitive to therapy [31,32].

miR-101 is known to be involved in many important cancer processes such as inhibition of proliferation, chemoresistance, angiogenesis, invasion, and metastasis [33]. According to this hypothesis, several reports showed that the loss of miR-101 is frequent and is associated to a worse outcome in many types of tumors [34–39]. Several studies

demonstrated that EZH2, a mammalian histone methyltransferase, is emerging as one of the most important targets of miR-101: loss of miR-101 function induces the overexpression of EZH2, which is related to cancer evolution [40,41]. A meta-analysis showed that the down-regulation of miR-101 expression is correlated with a poor prognosis [13]. Liu at al. revealed that a high expression of miR-101 inhibits TNBC progression and increases chemotherapeutic drug-induced apoptosis in TNBC by directly targeting myeloid cell leukemia 1 (MCL-1) [42]. Other authors demonstrated that miR-101 is hypo-expressed in different breast cancer subtypes and stimulates cellular proliferation and invasiveness by targeting Stathmin1 (Stmn1) [43]. According to these findings, our study showed that higher levels of miR-101-3p were correlated with a better EFS and OS, independently from breast cancer subtypes in patients not achieving pCR. Therefore, it is possible to say that miR-101 could be a potential therapeutic target and a novel prognostic factor.

The role in breast cancer progression is unclear regarding miR-199a/b-3p. Some studies showed a loss of miR-199a/b-3p expression in aggressive breast cancer [44]; other evidence demonstrated the ability of miR-199a/b-3p to inhibit proliferation, migration, and multi-drug resistance. miR-199a/b-3p seems to be down-expressed in many types of cancer [45–52]. According to Shou-Qing Li et al., PAK4 could be a possible target of miR-199a/b-3p with an oncosuppresive role: in human breast cell lines, ectopic expression of miR-199a/b-3p blocks the PAK4/MEK/ERK pathway to inhibit breast cancer progression by inducing G1 phase arrest [52]. Xuelong et al. have shown that the hyper-expression of miR-199a-3p inhibits mitochondrial transcription factor A (TFAM) expression, enhancing sensitivity to cisplatin in breast cancer cells. Hence, miR-199a/b-3p could represent a good prognostic and predictive biomarker [53]. It was found that the overexpression of miR-199a-3p regulates the activation of the G protein coupled receptor (GPER), which is involved in tumorigenesis, and suppresses cells' proliferation, invasion, and epithelial–mesenchymal transition in TNBC [54].

Taking into consideration all our findings, our hypothesis is that miRNA patterns of expression could help identify, in the group of patients not achieving pCR, a population with better outcome. Moreover, in our opinion, the present study is interesting because it gives further support to the fundamental role of the miRNAs in cancer biology and their potential application as target cancer therapies. Several studies have been conducted in order to modulate cellular miRNA levels as inhibiting the oncogenic miRNAs and as restoring the tumor-suppressive ones, with encouraging results [55–58].

Although larger case series are needed, our findings provide a basis for broader, prospective, and multicenter trials to support the potential role of miRNAs as predictive and prognostic biomarkers not only in early but also in advanced disease. We hope that the identified miRNAs will help in comprehensively understanding their pathway mechanism in breast cancer and improve the therapeutic strategies [59].

#### **5. Conclusions**

miRNAs have changed our understanding of cell pathway modulation and opened fields not only for the development of novel cancer target therapies but even for new diagnostic tools. At present, important topics in cancer research are discovering the underlying pathways involved in miRNA expression and secretion and understanding miRNA modulation in different phases of cancer progression. Large cohort studies are still required to analyze and confirm the diagnostic, prognostic, and therapeutic application of miRNA.

**Author Contributions:** Conceptualization, P.F. and M.D.S.; methodology, P.F., M.D.S., G.T., G.S., C.B. and A.C.; Writing—review and editing, all authors; supervision, P.F., M.D.S., G.T., G.S., G.F., C.B. and A.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of FONDAZIONE POLICLINICO GEMELLI (protocol code 27736/16 and date of approval (20 October 2016).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

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### *Systematic Review* **Ovarian Reserve after Chemotherapy in Breast Cancer: A Systematic Review and Meta-Analysis**

**Alessia Romito 1 , Sonia Bove 1 , Ilaria Romito 1, \*, Drieda Zace 2 , Ivano Raimondo 1 , Simona Maria Fragomeni 3 , Pierluigi Maria Rinaldi 4,5 , Domenico Pagliara 1 , Antonella Lai 6 , Fabio Marazzi 5 , Claudia Marchetti 2,3 , Ida Paris 3 , Gianluca Franceschini 3,7 , Riccardo Masetti 3,7 , Giovanni Scambia 1,2 , Alessandra Fabi 3 and Giorgia Garganese 1,2**


**Abstract: Background**: Worldwide, breast cancer (BC) is the most common malignancy in the female population. In recent years, its diagnosis in young women has increased, together with a growing desire to become pregnant later in life. Although there is evidence about the detrimental effect of chemotherapy (CT) on the menses cycle, a practical tool to measure ovarian reserve is still missing. Recently, anti-Mullerian hormone (AMH) has been considered a good surrogate for ovarian reserve. The main objective of this paper is to evaluate the effect of CT on AMH value. Methods: A systematic review and meta-analysis were conducted on the PubMed and Scopus electronic databases on articles retrieved from inception until February 2021. Trials evaluating ovarian reserves before and after CT in BC were included. We excluded case reports, case-series with fewer than ten patients, reviews (narrative or systematic), communications and perspectives. Studies in languages other than English or with polycystic ovarian syndrome (PCOS) patients were also excluded. AMH reduction was the main endpoint. Egger's and Begg's tests were used to assess the risk of publication bias. Results: Eighteen trials were included from the 833 examined. A statistically significant decline in serum AMH concentration was found after CT, persisting even after years, with an overall reduction of −1.97 (95% CI: −3.12, −0.82). No significant differences in ovarian reserve loss were found in the BRCA1/2 mutation carriers compared to wild-type patients. Conclusions: Although this study has some limitations, including publication bias, failure to stratify the results by some important factors and low to medium quality of the studies included, this metanalysis demonstrates that the level of AMH markedly falls after CT in BC patients, corresponding to a reduction in ovarian reserve. These findings should be routinely discussed during oncofertility counseling and used to guide fertility preservation choices in young women before starting treatment.

**Keywords:** breast cancer; AMH; ovarian reserve; chemotherapy; pregnancy desire

**Citation:** Romito, A.; Bove, S.; Romito, I.; Zace, D.; Raimondo, I.; Fragomeni, S.M.; Rinaldi, P.M.; Pagliara, D.; Lai, A.; Marazzi, F.; et al. Ovarian Reserve after Chemotherapy in Breast Cancer: A Systematic Review and Meta-Analysis. *J. Pers. Med.* **2021**, *11*, 704. https:// doi.org/10.3390/jpm11080704

Academic Editor: Raghu Sinha

Received: 30 June 2021 Accepted: 21 July 2021 Published: 23 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Breast cancer (BC) is the most common cancer in the female population, with an estimated 2.3 million new cases worldwide in 2020 [1].

While the death rate has dropped by 40% since 1989, an increase in BC among young women has been reported, with around 10% of new cases diagnosed in patients younger than 40 years old [2].

Physicians should carry out counseling on fertility issues and fertility preservation in patients who have not completed childbearing before starting treatment [3].

Recent evidence has shown that age and the use of cyclophosphamide-based chemotherapy are the main factors influencing ovarian failure. In particular, the risk of chemotherapyinduced amenorrhea (CIA) was 55% [95% CI 50–60%], ranging from 26% to 77% in women younger than 35 years old compared to those older than 40 [4]. ]. However, CIA might not be the best indicator of fertility because resumption of menses can resume even one year after chemotherapy treatment [4]. In the last two decades, there has been increased interest in the role of anti-Mullerian hormone (AMH) as a marker of ovarian reserve. Preantral and antral follicles, which remain in the growing phase for many weeks, independently of FSH or menses fluctuation, release AMH [5]. AMH is superior in terms of accuracy compared to other conventional indicators of ovarian function such as menstruation, estradiol or FSH [6,7]. Recently, a growing body of literature has been published on the effect of chemotherapy on the ovarian reserve in women with BC, assessed by AMH [8–25]. The summarization and synthesis of these results could provide BC patients and healthcare professionals with crucial information on the reproductive health consequences of chemotherapy treatment and the possibility of achieving pregnancy after completion of endocrine therapy.

In this context, this systematic review aims to evaluate the impact of chemotherapy treatment on the ovarian reserve in fertile women with BC through the quantification of AMH.

#### **2. Materials and Methods**

The present work has been reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [26].

#### *2.1. Research Question*

To address our objective, we structured a specific research question based on the PI/ECOS framework (Population, Intervention/Exposure, Comparison, Outcome, Setting/Time) as follows:


#### *2.2. Literature Search*

The research was conducted on the PubMed and Scopus electronic databases. A search string was first built for PubMed, using MeSH terms, Boolean operators, and free text words. The string was subsequently adapted for use in the other database. The search was restricted to articles published in English, without any further restrictions and was last performed on 17 February 2021 for all databases. Supplementary Material (Table S1) shows the complete search strategy.

The reference lists of included studies were hand-searched for additional articles. Reference lists with trials, previous reviews or meta-analyses were also reviewed.

#### *2.3. Study Selection and Inclusion/Exclusion Criteria*

All studies that compared AMH levels at baseline and after chemotherapy in BC patients younger than 50 years old were considered pertinent. Only studies that reported a minimum of one-month follow-up were included in the systematic review. Only peerreviewed articles reporting primary data, with no time limits, were included. We excluded case reports, case-series (reporting data for fewer than ten patients) reviews (narrative or systematic), communications and perspectives. We also excluded studies in languages other than English or with polycystic ovarian syndrome (PCOS) patients.

All articles retrieved from the search strategy were imported to Rayyan QCRI [27] and duplicates were removed. Two independent reviewers (A.R. and I.R.) selected the identified studies based on the title and abstract. We evaluated the full-text version of the studies if the title or abstract did not clarify the topic.

Discussion by a multidisciplinary team, including breast surgeons (S.B., S.M.F. and D.P.), gynecologists (Iv.R. and C.M.), a radiologist (P.M.R.) and medical oncologists (A.L., F.M. and I.P.) resolved uncertainties about the eligibility of the papers. A statistician experienced in meta-analysis (D.Z.), along with the two reviewers, analyzed data. We discussed the results in a multidisciplinary setting.

Finally, an expert committee (G.F., R.M., G.S., A.F. and G.G.) performed an independent review and gave the final approval.

#### *2.4. Data Extraction*

Two researchers (A.R. and I.R.) performed the data extraction process. We used a standardized Excel spreadsheet to extract following data: first author; year of publication; study design; sample size; mean age of patients; type of chemotherapy; follow-up duration; AMH assay; basal AMH; AMH immediately after chemotherapy; and AMH at 6 months, 1 year, 2 years and 3 years of follow-up.

#### *2.5. Data Synthesis and Statistical Analyses*

Treatment effect, defined as the difference between basal AMH and the value of AMH immediately, 6 months, 1 year, 2 years and 3 years after treatment, was calculated for each included study. If the AMH was undetectable after chemotherapy, we used the lower detection limit for the specific assay. The variance in the treatment effect, derived from standard deviations or standard errors of paired differences between baseline and the end of follow-up, was calculated. If these statistics were not given, they were calculated, where appropriate data were available [28]. The mean effect size was calculated using the inverse variance method in random-effect models. Forest plots were used for the graphical representation of each study. Heterogeneity was examined using the I<sup>2</sup> test, with I<sup>2</sup> > 50% considered important [29]. Publication bias was examined using the analyses described by Egger and Begg, where *p* < 0.05 indicated significant publication bias [30,31]. Galbraith's test and sensitivity analysis were conducted to investigate the impact each study had on the overall estimate and its contribution to Q-statistics [32]

An overall analysis of data from all studies was performed (at 1 month, 6 months, 1 year, 2 years and 3 years of follow-up). Subsequently, to account for confounding factors, subgroup analyses were performed based on the patient's age, AMH kits used, BRCA status, chemotherapy regimen and hormone therapy. Statistical analysis was performed using Cochrane Collaboration RevMan 5.1 software (http://www.cochrane.org, accessed on 16 May 2021) and STATA software (StataCorp, 2015, Stata Statistical Software: Release 15; StataCorp LP 4905 Lakeway Drive, College Station, TX, USA).

#### *2.6. Quality Assessment*

The methodological quality of the included studies was assessed based on the study design. The Newcastle–Ottawa scale was applied for cohort studies to evaluate the following quality parameters: selection of study groups, comparability of study groups and ascertainment of outcome, giving scores that range from 0 to 9. The Jadad tool was used

to assess the methodological quality of randomized controlled trials (RCTs) included in the systematic review. It evaluates the randomization process, blinding, dropouts and withdrawal and assigns up to 5 points. To summarize the overall evidence quality, we grouped the articles into three categories: good methodological quality (studies that met at least 75% of the quality criteria), moderate methodological quality (studies that met between 50% and 74% of the quality criteria) and poor methodological quality (studies that met less than 50% of the quality criteria).

#### **3. Results**

–

Figure 1 shows the number of studies assessed and excluded through the stages of the meta-analysis.

#### *3.1. Bibliographical Search*

The defined search strategy retrieved 833 studies from PubMed and Scopus. After the first screening process, 96 articles were deemed pertinent, and their full texts were thoroughly read. Based on this step, 79 articles were excluded for the following reasons: outcome other than AMH, incomplete data and full text in languages other than English. One study was excluded because it reported the same sample of patients as another [33]

and three studies were excluded because they included fewer than ten patients [34–37]. One article was additionally added after hand-searching the references of the included studies [8]. A total of 18 studies were deemed eligible for this systematic review and meta-analysis [8–25]. Anderson et al. carried out two studies on the same population but with different follow-up periods; thus, both studies were included [10,11].

#### *3.2. Description of the Included Studies*

The main characteristics of the included studies are shown in Supplementary Material (Tables S2 and S3). Nine studies (50%) were carried out in Europe [10,11,15,16,18,21–24], three (16%) were carried out in the USA [17,20,25], four (22%) were carried out in Asia [8,9,12,19], one (6%) was carried out in Africa [14] and one (6%) was carried out in South America [13].

All included studies had a cohort design [8–11,13–15,17–23], except for three RCT [16,24,25] and one case–control study [12]. The study by Trapp et al. was a subanalysis of the SUCCESS-A study [37]. It compared the disease-free survival (DFS) of patients receiving three cycles of fluorouracil, epirubicin and cyclophosphamide (FEC) followed by three cycles of docetaxel (D) versus three cycles of FEC chemotherapy followed by three cycles of gemcitabine and docetaxel (DG). This study included only premenopausal patients, younger than 40 years old, treated with FEC-D. The RCT of Hadji et al. and Yu et al. randomized patients to receive zoledronic acid versus a placebo after the standard treatment. For both studies, the two aims were combined in the overall analysis [16,25].

The sample size varied from 23 [13] to 250 patients [15], for a total of 1219 women. The mean age varied from 26 [13] to 41 [10,11]. The chemotherapy regimen was variable (cyclophosphamide, FEC, FEC-D, FEC-D plus methotrexate plus fluorouracil (CFM), doxorubicin plus cyclophosphamide (AC) in association with taxanes (AC-T), treatment with aGnRH during or after chemotherapy, and hormone therapy often not reported (in 52%, 32% and 37% of the studies, respectively). Six different AMH kits were used. Elecsys AMH assay was used in four studies [13,18,21,22], AMH Gen II assay was used in six studies [8,12,16,17,19,24], four articles used the Immunotech (IOT) [10,11,14,15], one article used the Diagnostic Systems Laboratories (DSL) [25], one used the ultrasensitive AMH ELISA kit [23] and one used the picoAMH ELISA kit [20]. One study did not specify the AMH kits used [9].

Seven studies reported AMH value immediately after chemotherapy [8,9,14,17,21,24,25], four studies reported it six months after chemotherapy [10,14,16,25], ten studies reported it one year after chemotherapy [10,13,16–20,22,23,25], four studies reported it two years after chemotherapy [11,15,20,24] and three studies reported it three years after chemotherapy [11,18,22].

#### *3.3. Quality Assessment*

Five out of fifteen cohort and case–control studies were considered of high quality (satisfied 75% or more of the quality criteria), while two out of three RCT were of medium quality (satisfied between 50% and 74% of the quality criteria) (Supplementary Material: Tables S4 and S5).

#### *3.4. Meta-Analysis*

All studies were analyzed, followed by a subgroup analysis for age, AMH kits and BRCA status. The insufficient data did not permit the subgroup analysis for a chemotherapy regimen and endocrine therapy.

#### 3.4.1. Ovarian Reserve after Chemotherapy

An analysis of seven studies including 410 patients revealed a statistically significant decline in serum AMH concentration immediately after chemotherapy, with an overall reduction of −1.97 (95% CI: −3.12, −0.82). There was significant heterogeneity in the pooled analysis (*p* for heterogeneity < 0.00001, I2 = 99%). After six months, there was a slight recovery (Mean Difference (MD): −1.61 (95% CI: −2.38, −0.84)) with a pooled population of 190 patients (*p* for heterogeneity < 0.00001, I2 = 89%). One year after chemotherapy, there

was a further reduction (MD: −2.21 (95% CI: −2.95, −1.48)) (*p* for heterogeneity < 0.00001, I2 = 98%) to achieve a steady state after two and three years of follow-up (MD after 2 and 3 years: −2.59 (95% CI: −3.95, −1.24) (*p* for heterogeneity < 0.00001, I2 = 99%) and −2.57 (95% CI: −3.99, −1.15) (*p* for heterogeneity < 0.00001, I2 = 97%), respectively (See Figure 2)). Galbraith's test was conducted, but heterogeneity remained high (more than 80%). Egger's and Begg's tests showed no significant publication bias for studies assessing AMH values immediately after chemotherapy (*p* = 0.5 and *p* = 0.3, respectively) and for those assessing AMH values 1 year after chemotherapy (*p* = 0.4 and *p* = 0.1, respectively).

#### 3.4.2. Subgroup Analysis

Considering the high heterogeneity in the overall analysis, subgroup analysis was performed when data were available based on factors that could contribute to heterogeneity between the included studies. − − −

#### Subgroup Analysis: Age

The first subgroup analysis was conducted among patients with a median age of more than 40 years old, from ages 35 to 40 years old and from ages 30 to 35 years old, one year after chemotherapy. We included ten studies. Only one study reported results on women younger than 30 years old [13]; thus, a meta-analysis was not performed. ff − − − − − −

An analysis of three studies including 140 patients older than 40 years revealed a statistically significant decline in serum AMH concentration of −1.01 (95% CI: −1.37, −0.65). Two studies (n◦ of pts = 134) included patients from 35 to 40 years old, reporting a higher impairment (MD −2.69 (95% CI: −2.87, −2.50). Higher toxicity was shown in younger patients (MD −2.73 (95% CI: −3.77, −1.70) aged between 30 and 35 years) assessed in four studies (N = 307). Heterogeneity was acceptable (I<sup>2</sup> = 53%, I<sup>2</sup> = 0% and I<sup>2</sup> = 85%) (See Figure 3). − − − − − −


(**A**)

### (**B**)

**Figure 2.** *Cont.*




(**D**)

(**E**)

**Figure 2.** Forest plot for post-operative AMH levels in all BC women. AMH level were assessed (**A**) immediately after chemotherapy, (**B**) 6 months later, (**C**) 1 year later, (**D**) 2 years later and (**E**) 3 years later.

#### Subgroup Analysis: AMH Assay

We performed a subgroup analysis accordingly with different AMH kits one year after chemotherapy (more data available). A pooled analysis of three studies (N = 225) using Elecsys AMH kits showed a statistically significant decline in serum AMH concentration one year after chemotherapy with an overall reduction of −2.75 (95% CI: −4.28, −1.22) (*p* for heterogeneity < 0.0001, I<sup>2</sup> = 89%). The level of AMH was higher than that in the analysis of the three studies (*n* = 165) using AMH Gen II kits, which revealed an overall reduction of −1.79 (95% CI: −3–17, −0.41), also with high heterogeneity (*p* for heterogeneity < 0.00001, I<sup>2</sup> = 92%). Only one study used each of the other four AMH assays (DSL, IOT, pico-AMH and ultrasensitive AMH); therefore, meta-analysis was not performed (See Figure 4).

− − −

(**C**)


− − −

− − −



#### **Figure 3.** Forest plot for post-operative AMH levels accordingly with age. AMH levels were assessed in women (**A**) older than 40 years, (**B**) between 35 to 40 years, and (**C**) between 30 to 35 years.

(**C**)


− − −


(**A**)

(**B**)

**Figure 4.** Forest plot for post-operative AMH levels accordingly with AMH assay. AMH levels were assessed with (**A**) ELECSYS kit (**B**) and AMH Gen II Elisa.

− −

−

−

− −

### (**B**)

### (**A**)

Subgroup Analysis: BRCA Subgroup Analysis

A subgroup analysis was performed accordingly with BRCA status one year after chemotherapy (more data available). An analysis of two studies including 49 patients revealed a decline in serum AMH concentration one year after chemotherapy of −2.50 (95% CI: −2.97, −2.04) for BRCA mutated (m-BRCA) patients (p for heterogeneity < 0.00001, I <sup>2</sup> = 0), similar to the reduction for wild-type BRCA (wt-BRCA) (MD: −2.47 (95% CI: −2.68, −2.25) on 172 patients (*p* for heterogeneity < 0.00001, I<sup>2</sup> = 0)) (See Figure 5). − − − − − −


(**A**)

(**B**)

**Figure 5.** Forest plot for post-operative AMH levels accordingly with BRCA status. AMH levels were assessed at (**A**) m-BRCA (**B**) and wt-BRCA.

#### **4. Discussion**

This paper is the first systematic review and meta-analysis to evaluate the effect of chemotherapy on AMH in BC patients. The overall analysis revealed a marked decline of AMH value after chemotherapy treatment. AMH levels drop significantly soon after chemotherapy, with some recovery 6 months later. However, combining the detrimental effect of older age and the damage caused by chemotherapy, the ovarian reserve was almost completely depleted for women over 35 years old.

Fertile women represent almost 10% of cases with BC diagnosis [2]. In 2018, to improve the quality of life in cancer survivors [38,39], the American Society of Clinical Oncology suggested a discussion of this reproductive issue and offered fertility preservation strategies before starting cancer treatment [40]. Especially for BC, generally undergoing a polychemotherapy regimen, the risk of ovarian reserve loss is very high [41].

Different authors proposed chemotherapy-induced amenorrhea (CIA) as markers of ovarian reserve to assess the effect of chemotherapy. Women older than 40 years of age had between 77% to 100% risk of developing CIA (77–100%) compared with younger people (0–40%) [42–44].

However, amenorrhea is a poor surrogate for fertility because it serves as an instant measure of ovarian function. Above all, resumption of menses could occur after a long time. In addition, pregnancy can also arise during amenorrhea due to sporadic ovulation [45].

In our study, we evaluate chemotherapy-induced gonadotoxicity by AMH levels. In the last 15 years, AMH showed a strong correlation with ovarian reserve, with higher accuracy compared with other markers such as FSH [7].

This metanalysis demonstrated a marked fall in the ovarian reserve after chemotherapy. Although a few months later there was a slight recovery, AMH values remained in the

poor responder's threshold. Therefore, BC women, after ovarian stimulation, will probably obtain a low number of oocytes, resulting in poor chances of becoming pregnant [46].

In our paper, AMH recovers following the first chemotherapy decline, dropping again after 6 months, probably due to physiological aging decline [47]. Previously, Andersen et al. have shown that recovery slope and peak depend on age and type of treatment; in fact, while BC patient's recovery was slight [10], in other cancers such as lymphoma, for which median age is younger, recovery was greater and longer [48].

The high statistical heterogenicity found in the overall meta-analysis could be due to the high clinical variability, including different ages, chemotherapy regimens, hormone therapies, AMH assay kits and BRCA mutations [49]. In order to reduce heterogeneity, we could perform a sub-group analysis for some of these items, with an overall decrease in heterogeneity that was significant for age and BRCA status and slighter for AMH assay kit.

Instead, given the lack of data, a subgroup analysis for other items, such as chemotherapy regimens, aGnRH treatments and endocrine therapies, remained unaddressed in this meta-analysis. Furthermore, the differences in the methodological quality of the included studies could contribute to the high statistical heterogeneity.

Regarding age, in our study, we showed a different chemotherapy effect accordingly for each age category. Indeed, a lower reduction was shown for older compared with younger women (M D was −1.01, −2.69 and −2.73 for the >40 years, 35 to 40 years and 30 to 35 years subgroups, respectively). The lower baseline level of AMH in older patients probably explains this effect. In fact, through the years, serum AMH levels gradually decline even in healthy women (5.6% per year) [47].

However, the most important finding from this analysis is the AMH value after chemotherapy. It appears to be very low for women older than 35 years of age (mean AMH values one year after CHT for the age category were 0.24 ± 0.69, 0.15 ± 0.77 and 1.14 ± 1.65 ng/mL for the >40 years, 35 to 40 years and 30 to 35 years subgroups, respectively). For these patients, pre-treatment counseling should be mandatory to inform them about the expected fertility drop. Physicians should inform women with pregnancy desire about fertility preservation strategies that could be implemented before starting BC treatment.

Among the 18 included studies, six different AMH assays were used. To reduce bias, standardizing the methodology might be useful in future research, especially since each test reported different sensitivities, detection limits and inter-variability [50]. Indeed, heterogeneity was slightly reduced in the subgroup that used the best performing kit [51].

We also analyzed the impact of CT on AMH levels among BRCA mutation carriers (m-BRCA). Indeed, several studies focused on their fertility potential, since the baseline ovarian reserve is expected to be reduced because of the lack of DNA double-stranded break repair [52]. However, studies on the reduction of AMH levels in the m-BRCA women compared to wild type before cancer treatments are controversial, with opposite results [53].

Moreover, data on the effects of chemotherapy depending on BRCA status are poor. In our sub-analysis, we identified only two studies. Our results suggest that BRCA mutation do not seem to change the effect of chemotherapy on ovarian reserve (MD: −2.50 (95% CI: −2.97, −2.04) versus −2.47 (95% CI: −2.68, −2.25) in mBRCA and wtBRCA, respectively). Further studies are needed to clarify this issue, as the total population in the meta-analysis was small (mBRCA *n* = 172; wtBRCA *n* = 49).

This systematic review and meta-analysis highlighted several gaps in current knowledge and could potentially help guide future research. Indeed, many sub-analyses that would be clinically very helpful and informative cannot be performed with the available data. Many relevant questions remain unsolved: First, what is the possible role of different chemotherapy regimens? Chemotherapy drugs are already known to impact menses in different ways. Anthracycline has a higher risk of amenorrhea than other drugs, but it is not known whether it directly affects ovarian reserve [4].

Second, what are the effects of GnRHa administered during and after chemotherapy on ovarian reserve? No studies in the literature investigate this topic in patients with BC, although several authors have demonstrated that GnRHa could influence AMH levels in healthy women. This question is particularly relevant for BC women since 2015. Based on evidence from the SOFT and TEXT trials, all high-risk patients younger than 35 years of age undergoing chemotherapy are currently receiving ovarian suppression with GnRHa as a standard [54,55].

Third, how do AMH levels vary during adjuvant endocrine therapy? We already know that tamoxifen could induce amenorrhea. Despite this, the effect on AMH remains unknown. There are no data in the literature, but as 75% of women with BC are suitable for endocrine therapy [56], we should clarify the effect of tamoxifen or aromatase inhibitors on ovarian reserve.

The results of this meta-analysis should be interpreted in the light of some limitations, such as publication bias, having included only peer-reviewed, English-language articles; the failure to stratify the results by factors such as chemotherapy regimen, ovarian suppression administration and endocrine therapy because data were unavailable; in general, the lowto-medium methodological quality of the studies were unrepresentative and carried out on small sample sizes, for which no stratified analysis had been performed to contain possible confounding factors.

However, despite the above limitations, this meta-analysis provides a practical tool for predicting ovarian reserve in BC patients undergoing chemotherapy. These findings should be routinely discussed during oncofertility counseling and used to guide fertility preservation choices or even simply to reduce the emotional stress associated with unexpected reproductive health impairment. Future efforts should be made to improve knowledge with more systematically collected data, precisely oriented toward clinical stratification based on the key risk factors.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jpm11080704/s1, Supplementary Material Table S1: Search strategy; Supplementary Material Table S2: Data extraction 1; Supplementary Material Table S3: Data extraction 2; Supplementary Material Table S4: Quality assessment 1; Supplementary Material Table S5: Quality assessment 2.

**Author Contributions:** Conceptualization, A.R., I.R.(Ilaria Romito) and G.G.; methodology, A.R. and D.Z.; software, A.R. and D.Z.; validation, S.B., S.M.F., D.P., I.R.(Ilaria Romito), C.M., P.M.R., A.L, F.M., I.P. and G.G.; formal analysis, A.R. and D.Z.; investigation, A.R. and I.R (Ilaria Romito).; resources and data curation, A.R., I.R. (Ilaria Romito) and I.R. (Ivano Raimondo); writing—review and editing, A.R., I.R.(Ivano Raimondo) and G.G.; visualization, S.B., S.M.F., D.P., I.R. (Ivano Raimondo), C.M., P.M.R., A.L., F.M. and I.P.; supervision, G.F., R.M., G.S., A.F. and G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Oligometastatic Breast Cancer: How to Manage It?**

**Vittoria Barberi 1 , Antonella Pietragalla 2 , Gianluca Franceschini 3 , Fabio Marazzi 4 , Ida Paris 5 , Francesco Cognetti 1 , Riccardo Masetti 3 , Giovanni Scambia <sup>2</sup> and Alessandra Fabi 6, \***


**Abstract:** Breast cancer (BC) is the most frequent cancer among women and represents the second leading cause of cancer-specific death. A subset of patients with metastatic breast cancer (MBC) presents limited disease, termed 'oligometastatic' breast cancer (OMBC). The oligometastatic disease can be managed with different treatment strategies to achieve long-term remission and eventually cure. Several approaches are possible to cure the oligometastatic disease: locoregional treatments of the primary tumor and of all the metastatic sites, such as surgery and radiotherapy; systemic treatment, including target-therapy or immunotherapy, according to the biological status of the primary tumor and/or of the metastases; or the combination of these approaches. Encouraging results involve local ablative options, but these trials are limited by being retrospective and affected by selection bias. Systemic therapy, e.g., the use of CDK4/6 inhibitors for hormone receptor-positive (HR+)/HER-2 negative BC, leads to an increase of progression-free survival (PFS) and overall survival (OS) in all the subgroups, with favorable toxicity. Regardless of the lack of substantial data, this subset of patients could be treated with curative intent; the appropriate candidates could be mostly young women, for whom a multidisciplinary aggressive approach appears suitable. We provide a global perspective on the current treatment paradigms of OMBC.

**Keywords:** oligometastatic breast cancer; locoregional therapy; CDK4/6 inhibitors; multidisciplinary

#### **1. Introduction**

Breast cancer is the most frequent cancer among women and represents the second leading cause of cancer-specific death [1]. Metastatic breast cancer (MBC) includes about 6% of cases of de novo disease, and about 20–30% of early-stage cancers recurred at distant sites [2]. The behavior of stage IV breast cancer may differ, depending on the biology of the tumor, the likelihood of spreading to certain sites (e.g., bone in hormone receptor-positive disease), and the disease burden. A subset of patients with MBC presents limited disease, termed 'oligometastatic' breast cancer (OMBC) [3].

The concept of oligometastases represents a condition midway between locoregionally confined cancer and disseminated disease, in which tumor burden is low and the number of affected organs is limited, typically with 1 to 5 secundarisms [4–8].

**Citation:** Barberi, V.; Pietragalla, A.; Franceschini, G.; Marazzi, F.; Paris, I.; Cognetti, F.; Masetti, R.; Scambia, G.; Fabi, A. Oligometastatic Breast Cancer: How to Manage It? *J. Pers. Med.* **2021**, *11*, 532. https://doi.org/ 10.3390/jpm11060532

Academic Editor: Valeria Bertagnolo

Received: 21 March 2021 Accepted: 2 June 2021 Published: 9 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Even though the incidence of OMBC is not clearly defined (1–10%), it seems that a considerable amount of all new MBC presents as oligometastatic. A tri-institutional retrospective analysis of 2249 patients with stage I–III disease who had first treatment failure showed that 21.9% were characterized by oligometastatic disease [9]. This boundary between oligo- and polymetastatic disease is increasingly recognized because of treatment and survival implications [3].

Given the likelihood of limited spread, it is possible to achieve longer survival, and, in 2–3% of cases, cure, with aggressive metastasis-directed therapy [5,8].

Moreover, OMBC is characterized by its chronicity and evolvement: primary cancer may present synchronous limited metastases, or the primitive tumor over time can develop a few metachronous metastases. We define oligorecurrence as the development of metachronous oligometastases with a controlled primary site [10], whereas oligoprogression represents a condition where a limited number of metastases progress, while all other sites of the disease remain stable, commonly during systemic treatment [11,12]. This distinction is representative of different scenarios and related prognosis, and it has a clinical implication in terms of survival [4].

For example, the patients with oligometastatic disease included in the previously cited study present a significantly longer overall survival (OS) as compared to polymetastatic patients with a follow-up of more than three years.

Prior reviews on oligometastatic disease investigated the effect of local techniques, namely surgical and radiotherapy. Recently, new techniques directed to disease biology provide information about next-generation treatment strategies, leading to a deeper biological understanding of OMBC and related treatment options. We provide a global perspective on the current treatment paradigms of OMBC [3].

#### **2. Options for Treatment of Oligometastatic Breast Cancer**

The oligometastatic disease can be managed with different treatment strategies to achieve long-term remission and eventually cure. In Figure 1 a flow chart of treatment options is presented.

Several approaches are possible to cure the metastatic disease: locoregional treatment of the primary tumor and the metastases; systemic treatment, including target-therapy or immunotherapy, according to the biological status of the primary tumor and/or of the metastases; or the combination of these approaches [13].

Locoregional options both of the primitive tumor and of the metastases lead to longlasting remissions reported in several case series; however, unlike other tumor entities, prospective data are lacking [13].

#### *2.1. Surgery*

In oligometastatic cancer, several trials involve surgery [14] (Table 1). The role of surgery in metastatic disease is unknown in terms of prognosis. Retrospective analyses demonstrate that patients who underwent surgery on the primitive tumor show a better prognosis compared to those who received only systemic therapy [15,16].

To corroborate a possible role of local treatments for the prognosis at the beginning of the metastatic disease, there is evidence that a multidisciplinary approach (surgery + radiotherapy, axillary dissection) is better for locoregional control of the disease, despite it being only surgery of the mammary node/mastectomy [17]. However, the findings of these studies are weakened due to selection bias: for example, patients with a less extended metastatic disease and/or who are responsive to medical treatments have more opportunities to undergo surgery on the primitive tumor than those who present a more advanced disease and/or who are less responsive to medical treatments.

**Figure 1.** Diagram flow of therapeutic options in oligometastatic breast cancer.

In the literature, three randomized trials evaluated the efficacy of surgery in MBC at the beginning of the disease.

In Tata Memorial Trial [18], among 350 women who enrolled, 173 underwent surgery and medical treatment and 177 received only medical treatment. This trial demonstrates that there are no differences in OS between the two groups. Surgical treatment is related to a better locoregional PFS, but also a worse DPFS (distant progression-free survival).

In the MF0701 [19] study, of 274 women who were enrolled, 138 underwent surgery and systemic treatment, while 136 were administered only systemic treatment. Patients with HR+ could receive hormone therapy. The protocol permitted upfront randomization (before the beginning of medical treatment) and the option of surgery on the primitive tumor during the local progression in the systemic treatment group. This trial showed a

significant increase in median survival in those patients who underwent surgery upfront (46 vs. 37 months, HR 0.66 *p* < 0.005). An analysis of the subgroups showed that the survival was superior to locoregional treatment in women with luminal tumors, age < 55 years, and solitary bone metastases.

ECOG-ACRIN E 2108 [20] studied 258 patients with de novo MBC with no progression after 4–8 months of systemic treatment that were randomized to continue systemic treatment or to receive radical locoregional treatment (surgery with free margins and subsequent radiotherapy, if indicated). About 60% presented an HR+/HER-2 negative tumor, 26% HER-2+, 15% were triple negative. In addition, 37% of these patients presented only bone metastases. The survival analysis showed no difference in OS and PSF in the two cohorts in the general population. The subgroup analysis suggests a possible detrimental effect of locoregional treatment in the subgroup of patients with triple-negative BC. Thus, even though we observed an increase of 2.5 times in the risk of locoregional progression in patients who received systemic therapy without locoregional treatment, there is no benefit in terms of quality of life from locoregional treatment.

Moreover, a prospective cohort trial [21] shows that in patients who have responded to first-line treatment, surgery on the primitive tumor does not improve PFS and OS, so that the predominant prognostic role is given by medical treatments, histopathologic features, and tumor burden. Conclusively, in patients with de novo MBC, the surgery approach has a palliative role (e.g., ulcerative lesions). In the absence of results of the effectiveness in OS, this procedure is considered in selected cases and after discussion with the patient.

There are three other randomized trials, one of which has finished the accrual, and it could furnish other elements to the argument.

In clinical practice, surgery is reserved for vertebral metastases with medullary compression, pathological fractures, pleural or pericardial effusion, and single visceral metastasis (e.g., liver, lung, brain).

In this regard, the resection of liver metastases in MBC is little explored, although in other tumors such as colorectal cancer it is widely recognized [6].

Different case series [6,22–36] show different survival rates (22–61 months) for liver metastases resection. A monocentric experience with 51 patients reported a 16% increase of 10-year OS rate [26]; 8.9% of these patients never presented any recurrence after surgery. However, this result is affected by a selection bias of the sample: the resection, but also the indolent course of the disease, the specific genetic profile of the tumor, and the ability of subclones to metastasize to a certain organ likely play a crucial prognostic role. Therefore, these reports need confirmation with prospective randomized trials [37].

A prospective data collection of 41 patients, who underwent liver metastases resection, revealed that positive resection margins and a short disease-free interval until the detection of liver metastases may lead to poor long-term survival [38]. Comparable results can be assumed for pulmonary lesions metastasectomy [39,40]: a short disease-free interval, the presence of several metastases, incomplete resection of them, and a non-luminal subtype are considered negative prognostic factors [41].

In summary: in OMBC, surgery on the metastases is still experimental because there are no data from prospective randomized trials with large samples. In addition, OMBC, even the indolent behavior, is a widespread disease, where local treatments alone could not be sufficient. However, these preliminary results may identify subgroups of patients with more favorable outcomes and for whom the surgery could lead to long-term survival [13].


**Table 1.** Randomized trials that evaluate the efficacy of surgery in MBC.

#### *2.2. Radiotherapy*

Patients with oligometastatic disease or with oligorecurrence in a single area could be treated with local therapy such as stereotactic body radiotherapy (SBRT), even associated with chemotherapy. Possible target lesions include brain, lung, liver, and lymph nodes.

Oligorecurrent metastases in the brain, lung, and liver can be definitively treated with SBRT. Instead, there are some controversies regarding lymph node oligometastases, thus further phase III trials are needed [42].

The use of stereotactic ablative radiotherapy (SABR) produces favorable outcomes, since it presents high accuracy to the target lesion, very conformal dose distributions, and delivers a highly ablative dose over a treatment duration of 1–5 treatments maximum.

Several works strengthen the use of SABR in OM disease, mostly randomized controlled trials (RCTs) [4].

Patients with a limited number of brain metastases and controlled extracranial disease may benefit from locoregional treatment combined with systemic therapy, which crosses the blood–brain barrier. Currently, stereotactic radiosurgery (SRS) is the recommended option for resected cavity and non-resected brain metastases [43] and achieves longer overall survival (OS) compared to whole-brain palliative irradiation [44,45].

Concerning lung OM disease, stereotactic techniques demonstrate a 2-year local control rate of 77.9% and a 2-year OS of 53.7%, according to a systematic review [46].

At the same time, a regional nodal recurrence after conservative breast treatment affects about 1% to 5.4% of patients with early-stage breast cancer [47–49]. A phase II study with SBRT or intensity-modulated radiation therapy for OMBC showed encouraging results [50]. Even though the principal site of metastases was the bone, several cases of lymph node metastases were treated with SBRT or intensity-modulated radiation therapy, without reporting severe toxicity. Furthermore, 90% of patients with oligorecurrence had

an objective response to salvaging radiotherapy and the 3-year treated tumor control rate was 93% [51]. However, despite the lack of reports about SBRT for oligorecurrent lymph node metastases of breast cancer, this subgroup of patients seems to be well suited for SBRT, especially those who did not receive previous irradiation, because of the indolent behavior of the disease. Nonetheless, patients should be carefully monitored over time, because of the risk of late toxicities.

The research is moving forward, with an ongoing randomized phase II/III trial (NRG-BR002), which evaluates the role of these techniques in OMBC [42,52].

#### *2.3. Systemic Treatments*

Systemic treatment remains a milestone in the management of metastatic breast cancer. Considering hormone receptor (HR) positive, HER2-negative metastatic breast cancers, certainly CDK4/6 inhibitors in combination with endocrine therapy have changed the paradigm of the treatment [53].

Concerns about the difference among the three CDK4/6 inhibitors involve the significant OS improvement, demonstrated from MONALEESA-3, MONALEESA-7, and MONARCH-2 trials, but not reported in PALOMA-1, PALOMA-3, and MONALEESA-2 trials [54–59].

As a result, a meta-analysis of all these randomized controlled trials evaluated the OS improvement among Palbociclib, Ribociclib and Abemaciclib, and focused on the efficacy of these compounds in some relevant subgroups of patients.

Of 5862 patients from MONALEESA-2, MONALEESA-3, and MONALEESA-7 trials, 2429 presented visceral (lung or liver) disease, 929 had bone-only disease, and 2504 had visceral and bone disease. Of 2845 patients, grouped by the number of metastases, 782 had only one metastatic site, 635 two, and 1428 three or more. The pooled results of the metaanalysis showed no heterogeneity for all these subgroups, with a statistically significant improvement in PFS with a similar hazard ratio [60].

Therefore, this meta-analysis demonstrates that CDK4/6 inhibitors plus endocrine therapy are beneficial in terms of PFS, regardless of the presence of visceral metastases, the number of metastatic sites, and the length of the treatment-free interval. Consequently, the pooled estimate for the overall population is also feasible for OMBC patients [60].

However, in luminal breast cancer, even after a first-line systemic treatment, OM disease could be persistent; therefore, due to the introduction and approval from FDA and EMA of Alpelisib, it is advisable to test the presence of PIK3CA mutation. Patients with PIK3CA mutation may benefit from Alpelisib plus Fulvestrant association, both with bone metastases and visceral metastases, as shown by the subgroup analysis of the SOLAR-1 study [61]. Instead, patients without the expression of PIK3CA mutation should receive a further line of hormonal treatment; this can be Everolimus plus Exemestane or Fulvestrant alone or, in selected patients, chemotherapy; confirmed data about the use of CDK4-6 inhibitors beyond progression are still unknown, and to date there are ongoing phase III studies comparing Alpelisib plus Fulvestrant versus Fulvestrant alone (CBYL719C2303 study-EPIK-B5).

In summary, a key role in the OMBC treatment is maintaining hormonal target therapy, reserving chemotherapy in cases of visceral crisis or widespread disease.

Unlike the luminal BC, often HER-2-like and triple-negative tumors have a different presentation since they have more aggressive behavior. Therefore, in these subtypes the strategy overlaps with a polymetastatic disease: in case of an HER-2 like OMBC, the use of anti-HER-2 molecules remains the first goal; instead, the current targets for triple-negative tumors are PD-L1 and BRCA mutations, and the use of Atezolizumab plus Nab-paclitaxel and Olaparib, respectively, showed better outcomes in terms of PFS and quality of life [62,63].

#### *2.4. Combination of Radiotherapy and Systemic Treatment*

Although CDK4/6 inhibitors are largely involved in the treatment of MBC, preliminary findings suggest a possible synergic effect of these compounds when combined with radiotherapy, especially in OM disease [64].

CDK4/6 inhibitors can act as a DNA double-strand break repair inhibitor, thus amplifying the anticancer effect of RT [65].

Therefore, the simultaneous administration of a radio-sensitizing drug could significantly improve symptoms and disease control. Despite the potential benefit of this combination, there is little literature on this topic, and clinicians could be frightened, since the radio-sensitizing effect may also increase the toxicity, involving healthy tissues as well [66,67]. The consequence might lead to, on one hand, improperly interrupting the systemic treatment or the radiotherapy.

Table 2 shows the preliminary results from small patient samples with the combination of CDK4/6 inhibitors with RT.

Hans et al. described five patients treated with Palbociclib and concurrent palliative RT without severe toxicity [68]: all patients experienced pain relief, but follow-up time and local control were not reported.

Meattini et al. described five patients treated with Ribociclib and concurrent palliative RT for bone metastases [69]: two patients developed grade 3–4 toxicity (one neutropenia and one vomit and diarrhea) and two needed temporary suspension of Ribociclib; radiotherapy was never suspended. At a 3-month assessment, three stable diseases and two partial responses were observed.

Chowdary et al. evaluated 16 patients treated with Palbociclib and RT for symptomatic metastases [64]. No side effect differences were found compared to the use of Palbociclib alone; all patients experienced prolonged pain control, and no local failures were described. However, only 31.3% of patients did not interrupt Palbociclib during the RT, while the other patients suspended the CDK4/6 inhibitor 14 days before or after RT, with a median interval of 5 days.

Ippolito et al. analyzed 16 patients treated with Palbociclib or Ribociclib concomitant to RT [70]. First, 68.7% of patients received palliative RT for bone metastases with a median dose of 30 Gy, while the remaining with OM disease were treated with higher doses (median 50 Gy). At 6.3 months follow-up, the only toxicity reported was neutropenia, apparently not worsened by radiotherapy, because it had already existed during the previous cycles of systemic treatment. Patients with bone metastases experienced all pain relief; the other subgroup developed complete responses (two patients with visceral and/or soft tissue), partial responses (two patients with bone disease), and stable disease (one patient with bone involvement) [71–76].

Two other retrospective analyses evaluated risks and benefits from the concomitant therapy with CDK4/6 inhibitors and RT. In one experience 16 patients under treatment with Palbociclib and radiotherapy were studied. At a follow-up of 14.7 months, none reported relevant acute or late toxicities: all reported that side effects were mild. All the patients achieved pain relief, and no local failures were developed [64]. The second study analyzed 18 patients treated with radiotherapy and concomitant CDK4/6 inhibitors for bone involvement. The hematologic toxicity was mild during the end of RT and the subsequent cycles of systemic treatment (grade 3–4 neutropenia) [72]; the other relevant side effect was grade 1 gastrointestinal toxicity. Three months after the end of RT, 88.9% of patients experienced pain relief, with no pain recurrence. With a median follow-up of 13.7 months, only one patient developed local recurrence. This study involves the largest cohort with concomitant CDK 4/6 inhibitors and RT published, but numbers are still limited.

These preliminary works suggest that the combination of CDK4/6 inhibitors and RT, particularly on bone metastases, is safe, with limited toxicities in terms of time and grade. The hematologic toxicity is comparable between the combination of these approaches and

the medical treatment alone, while the gastrointestinal side effects could be more relevant; therefore, clinicians should be careful in case of RT of the abdominal or pelvic area.

Although the results of these trials are limited by the small number of the sample, the clinical and radiological outcomes are promising. Future studies with a larger population and a longer follow-up will validate these results [64,77].


**Table 2.** Trials that evaluate the efficacy of concomitant RT and CDK4/6-i in MBC.

#### **3. Conclusions**

Even though metastatic breast cancer is considered incurable, OMBC presents a better prognosis [78].

Regardless of the lack of substantial data, this subset of patients could be treated with curative intent, mostly young women for whom a multidisciplinary aggressive approach appears suitable [3,78].

For these patients with a favorable nature for their disease, a multidisciplinary aggressive approach might improve survival [78].

Specifically, a combination of local and systemic treatment can achieve such long-term effects [13].

Local ablative options (radiotherapy/surgery) play a key role in this setting, as can be assumed from retrospective trials, but these encouraging results need confirmation by prospective randomized studies [78].

Moreover, preliminary data suggest an increase of disease-free survival after surgery on distant metastases; however, the selection of the appropriate candidates concerns the biology of the disease, and unfortunately, valuable comparative data are still missing. For this reason, surgery on breast cancer metastases remains an experimental approach.

Systemic therapy, e.g., the use of CDK4/6 inhibitors for HR+/HER2 negative BC, leads to an increase of PFS and OS in all the subgroups, with favorable toxicity.

Therefore, combined strategies increase the probability of producing results such as tumor-size reduction, long-lasting responses, and, eventually, cure [79].

All of these treatment strategies present a higher rate of success when the metastatic disease is detected early, so it is crucial to involve modern imaging equipment and liquid biopsies to model a personalized and multidisciplinary treatment [13].

#### **4. Future Directions**

The lack of strong data concerning the management of OMBC clearly emerges, due to the quality and heterogeneity of the systematic reviews and meta-analyses.

However, the increasing interest in the OM phenotype is emerging, and several prospective phase II/III randomized controlled trials involving new strategies for OMBC are ongoing (Table 3). A phase III study in the Netherlands (NCT01646034) is evaluating the role of high-dose chemotherapy with carboplatin, thiotepa, and cyclophosphamide in homologous recombination-deficient OMBC, since it seems that these tumors are particularly sensitive to alkylating agents which disrupt double-stranded DNA. Several trials are assessing the use of SABR and/or traditional surgery associated with systemic therapy in the first-line setting for newly diagnosed OMBC (e.g., CLEAR, NCT03750396; STEREO-SEIN, NCT02089100; NCT02364557). For instance, a pilot phase I study in Australia is evaluating the role of SABR followed by 6 months of anti-PD1 therapy with pembrolizumab, intending to show both safety and enhanced immune activation (BOSTON-II, NCT02303366).

The comparison of these trial results is weakened by the different definition of 'oligometastatic disease', which could include from two to five distant lesions. For further future studies, it would be reasonable to employ a universal definition of 'oligometastatic' within the breast cancer investigative community [3].


**Table 3.** Ongoing trials in oligometastatic BC.

**Author Contributions:** Conceptualization, A.F.; methodology, A.P., G.F., F.M., I.P.; resources, A.F. and V.B.; writing—review and editing V.B., A.F.; supervision, R.M., F.C. and G.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Breast Cancer-Related Lymphedema: Recent Updates on Diagnosis, Severity and Available Treatments**

**Marco Pappalardo 1 , Marta Starnoni 1,2, \*, Gianluca Franceschini 3 , Alessio Baccarani <sup>1</sup> and Giorgio De Santis 1**


**Abstract:** Breast cancer-related lymphedema (BCRL) represents a global healthcare issue affecting the emotional and life quality of breast cancer survivors significantly. The clinical presentation is characterized by swelling of the affected upper limb, that may be accompanied by atrophic skin findings, pain and recurrent cellulitis. Cardinal principles of lymphedema management are the use of complex decongestive therapy and patient education. Recently, new microsurgery procedures have been reported with interesting results, bringing in a new opportunity to care postmastectomy lymphedema. However, many aspects of the disease are still debated in the medical community, including clinical examination, imaging techniques, patient selection and proper treatment. Here we will review these aspects and the current literature.

**Keywords:** breast cancer; lymphedema; lymphaticovenous anastomosis; vascularized lymph node transfer; lymphatic microsurgery; radiotherapy

#### **1. Introduction**

Breast cancer-related lymphoedema (BCRL) remains a significant clinical issue for breast cancer survivors in that it causes severe physical and psychological discomfort. With the ever-increasing incidence of breast cancer, more patients are undergoing breast surgery that may include sentinel lymph node biopsy (SLNB) and/or axillary lymph node dissection (ALND) [1,2]. Chest wall radiotherapy is also commonly performed in patients with previous ALND, whereas axillary radiotherapy is sometimes indicated as an alternative to ALND in selected patients [3,4]. Both axillary surgery or radiotherapy can cause lymphedema with significant impairment of the normal lymphatic drainage producing an abnormal collection of protein-rich fluid within the upper limb. Despite improved early detection and evolving approaches to minimize surgical intervention increasing conservative surgery procedures with fewer ALND [5]; BCRL remains however a significant healthcare burden [6].

According to reports the incidence of BCRL varies and is approximately 20% at one year and increases to 40% at ten years after breast cancer treatment with a cumulative incidence of 28% [4,7]. Indeed, lymphedema is significantly more likely to occur following ALND than after SLNB alone [8,9]. Lymphedema can to develop within days postoperatively and can continue to present until 11 years after breast cancer treatment [10].

The impact of a lower quality-of-life on patients with lymphedema is unquestionable and there is a higher likelihood of poorer general health [11]. Besides, complications of

**Citation:** Pappalardo, M.; Starnoni, M.; Franceschini, G.; Baccarani, A.; De Santis, G. Breast Cancer-Related Lymphedema: Recent Updates on Diagnosis, Severity and Available Treatments. *J. Pers. Med.* **2021**, *11*, 402. https://doi.org/10.3390/jpm 11050402

Academic Editor: Stephen Opat

Received: 5 April 2021 Accepted: 7 May 2021 Published: 12 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

lymphedema including repeated episodes of cellulitis and ulceration, may require antibiotic therapy and hospitalization.

Cardinal principles of lymphedema treatment are patient education and control of concomitant diseases that may worsen swelling. Upper limb swelling is primarily controlled through the use of complex decongestive therapy (CDT) such as manual lymphatic drainage, bandages, compression garments and individualized exercises to reduce limb swelling [12]. Historical surgical treatments for lymphedema such as Homans' operation and Charles' procedure are palliative and nowadays largely abandoned [13]. Instead, a more recent volume reduction approach is circumferential liposuction [14,15]. In recent years, microsurgical and supermicrosurgical techniques, such as lymphaticovenous anastomosis (LVA) [16,17] and vascularized lymph node transfer (VLNT) [18] have gained popularity as they can potentially reconstitute lymphatic flow and, ideally, reduce the use of compression garments.

The recent introduction of severity staging using lymphoscintigraphy [19,20], and indocyanine green (ICG) [16,21] has helped the patient selection and improved the reported outcomes as it allows preoperatively to evaluate the lymphatic obstruction and the lymphatic flow patterns. This review article will focus on the current issues and debates in BCRL including diagnosis, severity, patient selection criteria and type of treatment available.

#### **2. Diagnosis of BCRL and Clinical Symptoms**

In order to properly manage upper limb lymphedema, the physician should first have a detailed knowledge of the diagnosis and severity of the disease. Traditionally health-care professionals have clinically diagnosed BCRL with subjective interpretations of swelling [22]. Diagnosis of upper limb lymphedema depends on a combination of comprehensive history, physical examination with subjective/objective symptoms and physiologic measures [6]. The patient's medical history including risk evaluation, medical conditions and medications that may cause edema should be meticulously reviewed. The differential diagnosis of BCRL is wide and can include: infection, congestive heart failure, primary/recurrent malignancy, vascular anomalies, electrolyte imbalances, hypoproteinemia, renal or hepatic failure, and peripheral neuropathies [23]. The common subjective clinical symptoms of patients with lymphedema in the upper limb are swelling, numbness, heaviness, tightness, stiffness, decreased coordination and mobility, limb fatigue or weakness. However, symptom presentation is broad and not all patients experience these symptoms. Next, during the physical examination, evaluation of the swollen limb should provide information regarding size, presence of scars, comparison with the healthy limb, skin condition and sensation. Objective clinical signs can include skin changes such as reddening, hyperkeratosis, thickening/firmness of tissues. Pitting edema is commonly seen at the end of the latent phase, with a depression formed in the skin after a fingertip pressure as the lymph is pushed into the surroundings. Later, non-pitting edema is characterized by hypertrophied adipose tissue with fibrosis. Stemmer's test is commonly performed and it is considered positive when it is difficult or impossible to pinch the skin at the base of the toes or at proximal phalanx of the fingers due to severe fibrosis. Patients with BCRL are susceptible to recurrent episodes of cellulitis that may increase adipose tissue deposition [24].

Limb volumetric measurements are considered the mainstay of the diagnosis and to track the progression of the disease. Many non-invasive tools such as tape circumferential measurements, water displacement, perometry, bioimpedence spectroscopy and threedimensional laser scanning are available to measure lymphedema (Table 1). However, there is not a universally accepted method.


**Table 1.** Comparison between Different Diagnostic Tools for the Diagnosis of Breast Cancer-related Lymphedema.

LNs: lymph nodes; CT: computed tomography; MR: magnetic resonance.

#### *2.1. Tape Circumferential Measurements*

Circumferential limb measurements at designated anatomic distances are the most common and easy method for quantification of lymphedema by measuring limb size or girth. Generally, a circumferential difference of greater than 2 cm or a volumetric differential of more than 200 mL is considered significant [25]. Sequential circumference measurements measured at standardized anatomical locations are widely used. The distance of each designated point is measured and total upper limb volume calculated based on the truncated cone formula [26].

Cheng et al. have described a sequence of measurements at 10 cm proximal and distal to the elbow [27,28]. These data are compared to the healthy limb, producing a quantitative limb measurement of lymphedema as well as a tool to check the progress during the follow-up.

Tape limb circumferential measurements are considered an easy and practical method for monitoring the progress of lymphedema. However, several critiques have been moved against this tool for not allowing a precise assessment of limb volume. Conversely, a study showed that circumferential and CT measurements are highly complementary in the assessment of volume in the lymphedematous limb [29].

#### *2.2. Water Displacement*

Water displacement offers perhaps the most precise tool for the assessment of the limb volume; however, this method is impractical in clinical setting and thus seldom used. In this procedure, the patients immerse the lymphedematous limb in a container full of water. The water overflow is transferred in another box, then it is weighed and measured. Disadvantage of this method include: (1) hygienic concerns, (2) it does not provide information about swelling location, (3) is contraindicated in patients with open wounds. It is thus rarely used in clinical practice.

#### *2.3. Perometry*

Perometry uses an infrared optoelectronic device that can measure the volume of the swollen limb and then compared to the healthy limb. The perometer works using infrared scanning to calculate the circumference of multiple areas of the limb [30] creating a 3-D image of the limb, with the limb volume calculated in ml. A great advantage of the perometer is its capacity: (1) to measure bilateral lymphedema, (2) to localize swelling, and (3) to detect a 3% limb volume change [31].

#### *2.4. Bioimpedence Spectroscopy*

Bioimpedence spectroscopy (BIS) calculates the rate of electrical current transmission through the tissues by comparing impedance and resistance in the extracellular fluid between the lymphedematous limb and the healthy limb using a low-level current (<30 kHz) [32]. Advantages of this method are: (1) it is safe, painless and rapid, (2) provides objective data even for the early detection of lymphedema and (3) it is repeatable. BIS uses the impedance ratio values between the lymphedema and the healthy limb, with the latter acting as a control, to calculate the Lymphedema Index (L-Dex) ratio. L-Dex outside the range (−10 to +10) reveals early signs of lymphedema. L-Dex value increases of +10 units from baseline also support the diagnosis of lymphedema. A disadvantage is that BIS is not useful for assessing bilateral limb lymphedema.

#### *2.5. Three-Dimensional Laser Scanning*

Recently, three-dimensional laser scanning has been used as a promising method for the measurement of upper limb volume [33,34]. This tool allows real-time reconstruction of 3D upper limb images. Three-dimensional laser scanners showed similar accuracy and reproducibility compared to water displacement for the measurement of arm volume [33,34]. Indeed the technique shows higher intra-rater reliability compared to water displacement. Furthermore, three-dimensional laser scanners are able to identify very small differences of limb volume, including increases or reductions of swelling as a consequence of CDT [35]. Conversely, the high costs of the devices, difficulties in the detection of upper limb reference points and time-consuming nature for the elaboration of data are the main issues of this tool. A recent study showed the reproducibility and reliability of three-dimensional laser scanner compared to tape circumferential measurements to assess arm volume in BCRL patients before and after CDT pointing out the easy learning curve of this method [36].

#### *2.6. Lymphoscintigraphy*

Lymphoscintigraphy is currently the 'gold standard' imaging technique for the diagnosis of extremity lymphedema when the clinical diagnosis is uncertain and, indeed, provides a clear image of the lymphatic drainage status of the upper limb [37,38]. Lymphoscintigraphy involves injection of a radiotracer in the hand and analysis of proximal lymph node uptake. It is, generally, performed as a qualitative analysis to evaluate the following features: (1) presence or absence of axillary/elbow lymph node uptake; (2) presence of linear, dilated or absent lymphatic ducts; (3) presence and location of dermal backflow. Some centers have reported also quantitative analysis based on decay-adjusted uptake and lymphatic transport index; however these are not commonly performed [39,40]. Recently, single photon emission computed tomography-computed tomography (SPECT-CT) lymphoscintigraphy has been used for the diagnosis of lymphedema providing 3-D live images of lymph flow [38,41,42]. A recent study reported significant association between the type of dermal backflow, the lymph flow pathways, and the visualization of lymph nodes around the clavicle [42].

#### *2.7. Computed Tomography (CT)*

This imaging study is able to differentiate between lymphedema, cellulitis, and generalized edema [43]. CT can detect lymphedema features including skin thickening, honeycombing or presence of fat lobules. It provides a standardized and reproducible method to measure the limb volume providing a 3-D representation of the lymphedematous limb [29].

#### *2.8. Indocyanine Green (ICG) Lymphography*

Nowadays, indocyanine green (ICG) lymphography is the most used imaging modality for the assessment of the severity and treatment in extremity lymphedema. This imaging technique involves the intradermal injection in the distal limb of the fluorescent dye ICG. Using a near-infrared camera, a laser light source is able to show the fluorescence in the dye when functioning lymphatics are present. Instead, non-functioning lymphatics will not be visualized. Several advantages have been described for ICG lymphography such as: (1) less invasiveness without radiation and (2) the capacity to clearly observe superficial lymphatic channels in real time bedside or even intraoperatively [44]. However, the main drawback of this imaging technique is its inability to visualize deep lymphatic at more than 1 cm in depth.

#### *2.9. Magnetic Resonance Lymphangiography*

Magnetic resonance (MR) lymphangiography is a safe imaging technique, with high spatial resolution with the possibility to provide visualization of the function of the lymphatics. Additional MR lymphangiography features include: (1) the amount of fat deposition, (2) the muscle compartments and (3) precise limb volume [45].

#### **3. Severity of BCRL and Patient Selection**

Since the severity of lymphedema starts from a soft pitting edema to an irreversible non-pitting edema with fatty and fibrotic deposition, it is imperative to understand the different lymphedema stages. A number of classifications and staging systems, based on clinical and imaging findings have been proposed in the medical literature. These classification systems are further explained in Table 2.


**Table 2.** Staging and Classification for the Severity of Breast Cancer-related Lymphedema.

LE: Lymphedema; ICG: Indocyanine Green (ICG) Lymphography; LVA: Lymphovenous anastomosis.

#### *3.1. International Society of Lymphology (ISL) Classification*

The International Society of Lymphology (ISL) classification is the most widely used one and divides the severity of lymphedema into three stages [46]. Briefly, patients are classified as Stage 0 (latent or sub-clinical lymphedema) when lymphatic channels have been injured with impaired lymph transport, but swelling or edema is not measurable. Stage I (spontaneously reversible lymphedema) is considered with measurable swelling and pitting of the skin due to accumulation of lymph, which decreases with limb elevation or compression garments. Stage II (spontaneously irreversible lymphedema) occurs when significant adipose tissue deposition and protein-rich fluid accumulation prevent limb elevation alone or compression garments from being an effective method to reduce symptoms. In late Stage II, the limb may present increase of fat and fibrosis. Finally, Stage III (lymphostatic elephantiasis) is the most severe stage of lymphedema. It is characterized

by severe swelling, excess deposition of fat and fibrosis and significant skin thickening in the form of acanthosis or hyperkeratosis.

Campisi et al. have published a similar classification with Stage I described as initial or irregular edema, Stage II defined as persistent lymphedema, Stage III as persistent lymphedema with lymphangitis, Stage IV as fibrolymphedema, and Stage V when elephantiasis is manifest [47].

#### *3.2. NECST Classification*

Mihara et al. have advocated a four-stage classification based on the pathological progression of post-mastectomy lymphedema. These stages are based on the histochemical changes of the lymphatic channels after axillary dissection. The changes in lymphatic channels were classified as normal, ectasis, contraction and sclerosis (NECST) [48].

#### *3.3. Arm Dermal Backflow and MD Anderson Classifications*

The Arm Dermal Backflow classification (ADB) [21,49], and the MD Anderson staging (MDA) [16] methods are widely used to define the severity of BCRL and both use ICG lymphangiography. The first was based on the examination of 20 patients, and the latter on 30 patients. Both staging systems include 6-stages of lymphedema severity, with stage 0 as normal linear lymphatics with no dermal backflow and stage 1–5 showing abnormal lymphatic patterns with various degrees of dermal backflow. Recently, Jørgensen et al., validated the two staging systems based on ICG lymphography, MDA Scale and ADB scale, in 237 unilateral BCRL [50]. They found near-perfect inter-rater and intra-rater agreement for both ICG lymphography staging and substantial agreement between the MDA and the ADB scales. Indeed, they found a slight correlation between the two ICG lymphography staging systems' results to conventional circumferential measurements. They concluded that the two ICG lymphography staging were reliable, safe tools with the MDA scale providing better disease stratification than the ADB scale.

#### *3.4. Cheng's Lymphedema Grading and Taiwan Lymphoscintigraphy Staging*

Cheng's Lymphedema Grading is a 5-grade classification that includes objective symptoms, limb volume measurements, and functional evaluation of lymphatic system using lymphoscintigraphy [51]. The five grades are divided based on the limb circumferential difference between the two limbs, the affected and non-affected as follows: grade 0 (<9%), grade I (10–19%), grade II (20–29%), grade III (30–39%) and grade IV (>40%).

Recently, the Taiwan Lymphoscintigraphy Staging has been validated and incorporated into the Cheng's Lymphedema Grading being it more objective and with the aim to offer a reliable and useful lymphedema staging system for diagnosis, severity and treatment of extremity lymphedema [19,20,37]. Patients selection for surgical treatment using the Cheng's Lymphedema Grading is as follow: Patients with Cheng's Grading 0 showing a range of circumferential difference between 0 and 10% and Taiwan Lymphoscintigraphy Stages L-0, P-1 or P-2 are suggested to be treated with compression garment treatment. Patients with Cheng's Grade I and early Grade II presenting respectively a circumferential difference range of 11–20% and 20–30% are commonly treated with LVA when presenting Taiwan Lymphoscintigraphy Stages P1–P3 and linear lymphatic ducts at ICG lymphography. Instead, when they show Taiwan Lymphoscintigraphy Stages P-3/T-4/T-5 with dermal backflow at ICG lymphography, they are suggested to be treated with VLN transfer. Patients with Cheng's Grade III and IV showing respectively a range of circumferential difference 30–40% and >40% with Lymphoscintigraphy Stages T4-T6, a single or double VLNT transfer is performed [52].

#### **4. Treatments for BCRL**

Current treatment options for BCRL include conservative and surgical treatments; however, determining the best treatment method for each patient remains challenging.

#### *4.1. Conservative Treatments*

CDT is widely accepted the universal first-line therapy for extremity lymphedema. It includes manual lymph drainage (MLD), skin care, specialized exercises, compression garments and self-education [6]. CDT is divided into Phase I Decongestion, and Phase II Maintenance and should be individualized to improve its effectiveness and contain costs.

Several advantages can be obtained by a CDT including: (1) reduction of lymphedema volume, pain and arm heaviness, (2) improvement of lymphatic drainage, (3) acceptable quality of life and (4) reduction of episodes of cellulitis [53,54]. Although conservative therapy alone may provide enough symptomatic relief, it depends essentially on patient compliance and their capacity to wear life-long compression garments.

#### 4.1.1. Manual Lymphatic Drainage

Manual lymphatic drainage (MLD) is a massage method increasing the transport capacity of the lymph collectors and moving lymph fluid and protein absorption when the lymphatic ducts are still functioning. A meta-analysis showed that, compared with other CDT modalities, additional MLD is unlikely to produce a proper reduction in the lymphedematus limb circumference [55]. In the other hand, another systematic review found that when MLD was used in combination with compression garments, provide increased swelling reduction in BCRL patients compared to the compression bandages alone, especially for moderate lymphedema stages [56].

#### 4.1.2. Compression Bandages and Compression Garments

Compression bandages are an important part of CDT maintaining the therapeutic effects of MLD. Compression bandages apply: (1) a resting pressure during the limb relaxed and (2) a working pressure when muscles contraction push the skin against resisting bandages. Low-stretch bandages produce the highest working pressure with multi-layered compression bandaging.

Compression garments are an essential part of CDT and with the aim to keep the volume reduction achieved with MLD and bandaging. Compression garments produce a two-way stretch in both longitudinal and transverse direction with the greatest pressure above the wrist and less pressure in the arm. The longitudinal pressure facilitates the joint movements. Generally, patients with BCRL wear a full arm sleeve and, frequently, a glove to prevent dermal backflow. There is no consensus regarding suitable compression values. Class 2 compression garments with 30–40 seamless are often recommended to be wear at least 12 h per day [19]. Of note, compression garments should be custom-made by a certified and experienced therapist in fitting garments for lymphedema patients.

#### 4.1.3. Exercises and Life-Style

Exercises are an integral part of CDT with the aim (1) to promote lymph flow, (2) to mobilize the joints, and (3) to strengthen the muscles. It is widely known that participation in exercises during and after oncological treatment can improve the physical and psychosocial condition, ameliorating the quality-of-life [57]. Recent studies reported that gradual weight-lifting program does not worsen the risk of BCRL compared to patients without exercises [58,59].

#### *4.2. Surgical Treatments*

Many surgical procedures to treat BCRL have been propose as follow: (1) physiologic procedures (lymphaticovenous anastomosis, vascularized lymph node transfer) and (2) excisional procedures (reduction or liposuction) (Table 3).


**Table 3.** Available Treatments for Patients with Breast Cancer-Related Lymphedema.

CLG: Cheng's Lymphedema Grading.

#### 4.2.1. Physiologic Procedures

In recent years, with the advent of microsurgical and supermicrosurgical techniques [60–64], lymphatic microsurgery procedures have gained popularity for the treatment of BCRL. Commonly practiced procedures include lymphovenous anastomosis (LVA) and vascularized lymph node (VLN) transfer. These surgeries try to deal with physiologic impairment resulted from cancer-related lymphedema and have the ability to provide venous shunting of lymphatic fluid bypassing areas of damaged lymphatics creating new lymphatic connections or by replacing the damaged lymph nodes and lymphatic channels [65].

#### Lymphovenous Anastomosis (LVA)

Lymphovenous anastomosis (LVA) is not a new procedure as it was initially described in 1969. It is a delicate supermicrosurgery technique, diverting lymph into the venous system bypassing proximal obstruction [66]. LVA has been shown to be especially beneficial in patients with early-stage upper limb lymphedema (Cheng's Grade I and early II) [16]. In a prospective study of 100 LVAs, symptomatic improvement was described in 96% of BCRL patients. Other advantages of LVA include decreased episodes of cellulitis. Recently, Cheng's group reported more effective lymph drainage in both proximal and distal sites using side-to-end LVA configuration compared with end-to-end LVA, without need of postoperative compression garment [17].

Previous studies have reported that LVA seems more effective in early-stage lymphedema due to the unavailability of functional lymphatic ducts in advanced stage lymphedema [16]. Therefore, advanced stage lymphedema was considered a relative contraindication for LVA [67]. However, recently Hong's group showed promising results using LVA for advanced stage lymphedema [68]. The authors pointed out the crucial role of preoperative magnetic resonance lymphangiography and ultrasound for the success of the procedure.

Prophylactic LVA have been also performed and has successfully prevented upper limb lymphedema in 23 patients who underwent oncologic resection for breast cancer treatment and ALND [69,70].

Disadvantages of these procedure include (1) its technical difficulty for the execution anastomosing lymphatic ducts with a diameter of 0.5–0.8 mm with subdermal venules of 0.6–1.0 mm in diameter. (2) the requirements of supermicrosurgery instruments, high resolution microscope, and ICG lymphography (3) difficulty to monitor the anastomoses patency. Reported complications of LVA include infection (3.9%), lymphorrea (4.1%) and necessity of reintervention (10%) [71].

#### Vascularized Lymph Node (VLN) Transfer

VLN transfer is the latest physiological procedure added to the treatment repertoire and it is commonly indicated in more advanced cases of lymphedema. Several donor sites have been described of VLN transfer including groin, submental, supraclavicular nodes, thoracic, and omental. In 2006 Becker et al. popularized for the first time the procedure with the publication of groin VLN transfer for postmastectomy lymphedema [72]. After that, Cheng and colleagues described anatomic and clinical application of both groin and submental VLN transfer transferred into the distal limb [28,73]. Three recipient sites have been described for upper limb lymphedema such as axilla, elbow and wrist. The decision of recipient site is taken based on the severity of the lymphedema, recipient vessel availability, and surgeon preference.

Recent studies have shown the benefit of VLN flap with significantly improvement of lymphedema limb without patent lymphatic ducts compared to CDT or LVA [74]. Indeed, microsurgical breast reconstruction do not improve the outcome of postmastectomy lymphedema [74,75]. A meta-analysis compared the outcome of VLN transfer and LVA in extremity lymphedema [71]. The result showed that although both procedures were both efficient in a short-term outcome, patients with VLN transfer presented significant better improvement in the long-term with good likelihood of discontinue to wear compression garments.

VLN transfer is suggested for Cheng's Grade II-IV who did not present patent lymphatic channels using ICG lymphography. Additional procedures such as flap debulking and liposuction following VLN transfer are suggested for Cheng's Grade III and IV. In a recent study, patients with different grades of bilateral limb lymphedema underwent LVA in the less severe limb and VLN transfer in the more severe limb. This individualized treatment achieved effective improvement in the reduction of each limb swelling and cellulitis, as well improvements in quality-of-life [76]. Although VLN transfer has shown favorable results, however it could carry the risk of donor site lymphedema [25,77,78]. Other complications include flap loss, lymphocele, infection, and wound healing complications.

#### 4.2.2. Excisional Procedures

The first surgical method used to treat BCRL lymphedema was reported by Sistrunk in 1927 [79]. The excess skin and soft tissue were removed using a spindle-shaped incision in the medial region of the arm with removal of the deep fascia and creating a connection between superficial and deep lymphatics. Later, with Thompson a further step forward in the BCRL treatment was achieved using a lymphatic transposition method. A deepithelialized rectangular hinge skin flap was harvested from all length of the arm with the flap tip embedded near the neurovascular bundle with the aim to bridge the superficial and deep lymphatics [80].

Nowadays, excisional procedures, such radical reduction with preservation of perforators [81], and suction-assisted lipectomy [82] aim to eliminate the affected tissue in severe lymphedema stages. All excisional procedures produce the following advantages: (1) decrease limb size, (2) reduce episodes of cellulitis, and therefore improve the quality of life of the patients. Although these surgical procedures can be immediately effective to reduce the lymphedema volume, however they can carry some risks including wound

complications, swelling recurrence, and the need for the patient to wear compression garments lifelong to prevent recurrence.

#### Liposuction

Fat accumulation is one of the pathologic findings of BCRL. Adipose tissue deposition is probably due because it is an endocrine organ in which complex structures of cytokine-activated cells, and chronic inflammation play a role [82]. However the pathophysiological mechanism of adipose tissue accumulation in lymphedema still remains controversial. Tashiro et al. reported adipose tissue alterations in extremity lymphedema using macroscopic and ultrasound findings [83]. They found in adipose tissue samples larger adipose lobules in lymphedema limb compared to non-lymphedema samples. Indeed, lymphedema samples presented hypertrophic changes of adipocytes and increased collagen fibres. Finally, adipose-derived stem cells and M2 macrophages were less in in lymphedema adipose tissue than in the healthy controls [83].

Liposuction is currently the most accepted excisional procedure. Brorson et al. showed that BCRL with nonpitting edema treated with liposuction presented 68% to 93% of fat, 32% of interstitial fluid, and 7% of lymph [84,85]. This excisional technique is able to remove fat producing significant arm reduction [84,86,87]. Indeed, a reduction in episodes of cellulitis was reported. A possible explanation of reduced cellulitis may be the increased skin blood flow after liposuction that could eliminate bacteria that entered through skin wounds [88]. However, the main drawback is the need to use life-long compression garments [84,89].

#### 4.2.3. Combined Treatments

Due to lack of consensus among the experts regarding the most appropriate protocol for lymphedema treatment, each surgeon applies a surgical procedure based on his personal approach. A combined treatment have been proposed as an alternative to the single strategy [65,90]. Recently, Di Taranto et al., reported that patients with extremity lymphedema treated with combined VLN transfer, LVA and liposuction LVAs showed better improvement in terms of circumference reduction compared to patients treated only with VLN transfer and liposuction [91].

Later Baumeister et al. described a new method for the treatment of 28 BCRL patients in which autologous lymphatic grafting is initially performed to bypass the axilla reestablishing lymphatic flow and later on liposuction is performed as a second step [92] without the need for additional treatments.

#### **5. Conclusions**

BCRL is a debilitating and chronic and condition that can severely affect the patient's quality of life. An improvement in identification, prevention, and management of affected patients is imperative in reducing BCRL. A particular attention should be given to all stages of breast cancer treatment in order to reduce the incidence of BCRL. The use of new technologies for performing mastectomies and sentinel lymph node biopsy or axillary lymph node dissection could be useful [93–96]. Accurate physical examination and assessment of the lymphedema severity are essential to provide more predictable outcomes. A prompt management of the disease in a multidisciplinary team is the key to obtain good results [97–105]. Despite the fact lymphedema is still considered an incurable disease, in the last decade promising results with significant reduction of the limb swelling and improvement of psychosocial well-being have been shown.

**Author Contributions:** M.P.: Conceptualization, Writing. M.S.: Conceptualization, Writing. G.F.: Review, Supervision. A.B.: Review, Supervision. G.D.S.: Review, Supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, because this study is just a review of the literature.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Hormone Receptor Expression Variations in Normal Breast Tissue: Preliminary Results of a Prospective Observational Study**

**Giacomo Santandrea 1,2 , Chiara Bellarosa 3 , Dino Gibertoni 4 , Maria C. Cucchi 5 , Alejandro M. Sanchez 6, \* , Gianluca Franceschini 6 , Riccardo Masetti <sup>6</sup> and Maria P. Foschini 3, \***

	- gianluca.franceschini@policlinicogemelli.it (G.F.); riccardo.masetti@policlinicogemelli.it (R.M.)

**Abstract:** Normal breast tissue undergoes great variations during a woman's life as a consequence of the different hormonal stimulation. The purpose of the present study was to examine the hormonal receptor expression variations according to age, menstrual cycle, menopausal state and body mass index. To this purpose, 49 tissue samples of normal breast tissue, obtained during surgery performed for benign and malignant conditions, were immunostained with Estrogen (ER), Progesterone (PR) and Androgen receptors (AR). In addition, Ki67 and Gross Cystic Disease Fluid Protein were studied. The data obtained revealed a great variability of hormone receptor expression. ER and AR generally increased in older and post-menopausal women, while young women presented a higher proliferative rate, evaluated with Ki67. PR increase was observed in women with BMI higher than 25. The different hormonal receptor expression could favor the development of breast cancer.

**Keywords:** breast cancer; normal breast; breast pathology; hormone receptor; hormone expression

#### **1. Introduction**

Physiological variations in the expression of Estrogen receptor alpha (ER) and Progesterone receptor (PR) play an important role in breast in breast remodeling during physiological changes: from embryological development to puberty [1] as well as during menstrual cycle, pregnancy and even after menopause [2].

As it happens for physiological variations of breast glandular tissue, the expression of hormonal receptors is thought to be an underlying mechanism involved in breast cancer onset, with determinant variations induced by well-known risk factors, such as age [3,4] exogenous hormone use [5] or Body Mass Index (BMI) [6,7]. Indeed, breast cancer is now classified according to a combination of hormone receptor expression, Ki67 labelling index and HER2 [8,9].

Tot [10,11] proposed the theory of the "sick lobe", according to which breast cancer arises in a genetically predisposed breast epithelium. Tot based his theory on cytokeratin expression; nevertheless, hormone receptor expression variations occurring during life could predispose the breast epithelium to malignant transformation.

**Citation:** Santandrea, G.; Bellarosa, C.; Gibertoni, D.; Cucchi, M.C.; Sanchez, A.M.; Franceschini, G.; Masetti, R.; Foschini, M.P. Hormone Receptor Expression Variations in Normal Breast Tissue: Preliminary Results of a Prospective Observational Study. *J. Pers. Med.* **2021**, *11*, 387. https://doi.org/ 10.3390/jpm11050387

Academic Editor: Hisham Fansa

Received: 17 March 2021 Accepted: 4 May 2021 Published: 8 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Furthermore, breast cancer presents differences in young pre-menopausal women and in older post-menopausal women [12], with ER negative cases being more frequently in the young.

Among hormonal receptors that are normally expressed in breast tissue, prior studies confirmed that the expression of ER and PR may be associated with subsequent breast cancer risk [5,13–18]. However, there is still scarce evidence regarding a larger panel of breast tissue receptors, including Androgen Receptor (AR), Gross Cystic Disease Fluid Protein 15 (GCDFP-15) and Ki67.

The aim of this study is to investigate the expression of ER, PR, AR, GCDFP-15 and Ki67 in breast normal tissue according to age, BMI, menstrual cycle and the onset of a breast neoplasm (benign vs. malignant).

#### **2. Materials and Methods**

#### *2.1. Patients Selection*

All patients who underwent surgery for benign or malignant breast lesions between June 2015 and January 2016 at the Breast Surgery Unit of Bellaria Hospital (Bologna, Italy), were asked to participate to the present study. Seventy-nine patients accepted.

Among them, 2 pre-menopausal patients who experienced post-chemotherapy menstrual cycle arrest and 28 patients who had only a little amount of normal glandular tissue, insufficient for the analyses, were excluded.

The 49 remaining patients constituted the study population and were grouped as follows:


#### *2.2. Tissue Selection Process*

Histologic diagnoses and immunohistochemistry were obtained at the Section of Anatomic Pathology, Department of Biomedical and Neuromotor Sciences, University of Bologna, at Bellaria Hospital, Bologna, Italy. All tissues were fixed in 4% buffered formalin and paraffin embedded according to routine protocol. Serial 2µm sections were obtained from each block and stained with Haematoxylin and Eosin (H&E) for histologic evaluation.

Cases were retained for the present study when normal breast tissue was present around the lesion leading to surgery and the block containing the largest amount of normal breast tissue was selected for immunohistochemical studies. When possible, tissue obtained from the upper outer quadrant (UOQ) was chosen. Apocrine cysts, sclerosing adenosis and all the benign changes observed in aging breast were excluded from evaluation.

After histological evaluation on H&E, areas with at least 5 normal terminal ductular lobular units (TDLU) were selected for Tissue Micro-Arrays (TMA) construction. TMA were constructed following the technique described by Zimpfer et al. [19].

#### *2.3. Tissue Immunohistochemical Evaluation*

Immunohistochemical evaluation was made on TMA sections.

Evaluation and quantification of biomarkers was performed by counting the percentage of positive cells at 40x magnification. A minimum of 4 terminal-ductular-lobular units were evaluated for each marker. Immunohistochemical staining was performed on a Ventana Automatic Stainer (Ventana Medical Systems, Inc). The following pre-diluted antibodies were supplied by Ventana: Estrogen Receptor (ER) (clone SP1), Progesterone Receptor (clone 1E2), Androgen Receptor (clone SP107), Ki67 (clone 30-9) and Gross Cystic Disease Fluid Protein 15 (clone EP1582Y).

#### *2.4. Statistical Analysis*

For each patient who participated in the present study, the following data were collected: age, Body Mass Index (BMI), contraceptive therapy, post-menopausal hormonal replacement therapy, date of surgery, type of surgical procedure, and site and size of the lesion leading to surgery.

BMI was evaluated as a three-level categorical variable with cutoffs at 18.5 and 25 kg/m<sup>2</sup> .

The variability of expression of ER, PGR, AR, GCDFP-15 and Ki-67 markers was very limited in the myoepithelial and stromal cells; therefore, it was evaluated only in the epithelial cells. Due to the limited population size and the skewed distribution of most markers (Figure S2), and although the hypothesis of normal distribution was not always rejected by the Shapiro–Wilk test (Table S1), median and interquartile range (IQR) were used as descriptive statistics. For each marker the percentage of patients with positive expression and the percentage of positive cells among positives were calculated. Comparisons of subgroups of patients according to the percentage of patients with positive expression were conducted using Fisher's exact test when the expected frequency of each cell was < 5, or using chi-square otherwise. The differences in the percentage of positive cells across subgroups defined by menstrual cycle and nature of lesions were evaluated by Mann–Whitney U test, and those across phenotypes and BMI subgroups by Kruskal–Wallis test. Post hoc analyses by Dunn's test with Holm adjustment for multiple comparisons were carried out after significant Kruskal–Wallis tests. Stata v.15.1 was used for all analyses, specifically the dunntest procedure [20] was used to perform the post hoc analyses. Statistical significance was set at *p* = 0.05.

#### **3. Results**

Descriptive statistics of the study population are reported in Table 1. The median age of patients was 50 years, with younger patients in group A and older patients in group C. Median BMI was lower in group A (22.81 kg/m<sup>2</sup> ) and higher in group C (27.92 kg/m<sup>2</sup> ). Most patients had a malignant diagnosis (75.0%), with a lower incidence in group A (61.9%). Only 5 patients, all in group A, were receiving contraceptive therapy and 1 patient (of group C) was under hormonal therapy.

**Table 1.** Characteristics of the study population.


The observed expression of epithelial markers is reported in Table 2. AR and GCDFP-15 were expressed in the large majority of patients (84.2% and 72.2%, respectively), while Ki-67 (38.6%) and Estrogen receptor alpha (43.2%) showed lower prevalence.

**Table 2.** Epithelial expression of markers.


GCDFP-15 and ER were the most evidenced markers (median rates of 55% and 36%, respectively). (Figure S1).

Most of the breast cancers here were ER positive, with 16 cases being Luminal A, 13 cases Luminal B cancers (two of which HER2 enriched) and 4 cases triple negative (TNBC) [8,9].

Hormone expression variations according to age:

There were significant differences among groups, specifically the proportion of positive cases for PR and AR was lower in group C. As for the distribution of expression among positive patients (Table 3), women in group A showed significantly lower values of GCDFP-15 with respect to group C (median: 30 vs. 90) and borderline lower values of ER with respect to group B (median: 8.5 vs. 48.5).


**Table 3.** Expression of hormone receptors according to age.

\* χ 2 -test.

Hormone expression variations according to the menstrual cycle:

The proportions of positives for each marker evaluated in the 22 patients of group A were not significantly different for menstrual cycle phase; however, the expression of PR and Ki67 was remarkably higher in women in follicular phase (41.5 vs. 18 and 8.25 vs. 2.5, respectively) and not far from reaching statistical significance (Table 4).

**Table 4.** Expression of hormone receptors according to menstrual cycle.


Hormone expression variations according to BMI:

ER positive cases increased with higher BMI, not significantly. PR positive cases were less frequent in the BMI ≥ 25 patients with respect to underweight and normal weight patients (33.3% vs. 75.0% and 73.7%; Fisher's exact test: *p* = 0.028, Table 5); on the contrary, underweight women showed a higher, but not significant, proportion of Ki-67 positives. The median values of expression did not differ according to BMI.

**Table 5.** Expression of hormone receptors according to BMI.


Hormone expression according to the type of lesion leading to surgery (benign versus malignant):

The proportion of Ki-67 positives was higher among patients who showed benign lesions than malignant lesions (66.7% vs. 25.8%, *p* = 0.032). The amount of hormone receptor expression did not change significantly for any marker (Table 6). Specifically, ER and PR expression was similar in breast tissue adjacent to benign and malignant lesions. Most patients included in the present study were affected by ER positive breast cancers.


**Table 6.** Expression of hormone receptors according to the nature of the treated lesion.

\* χ 2 -test.

Examples of positivity obtained with ER, PR, AR and Ki67 are shown in Figure S1.

#### **4. Discussion**

The present study confirms the great variations in hormone receptor expression occurring in adult women. The data here shown reveal hormone receptors variations mainly according to age and BMI, while little changes were observed during the menstrual cycle. While ER, AR and GCDFP-15 were higher in post-menopausal patients, PR expression decreased as age increased, reaching very low values in patients older than 60 (Group C).

The ER increase in older women shown here is consistent to the data published by Lawson et al. [21] who observed higher ER levels in post-menopausal compared to pre-menopausal women.

The progressive increase in ER expression in older women lead to some considerations about the ER role in breast cancer development. Breast cancer is known to have a peak of incidence in the 6th decade of life. Moreover, hormonal receptor positive breast cancers, classified as Luminal A or Luminal B, according to the St. Gallen definition [8,9], are the most frequent cancer types encountered in elderly women [22].

The association between hormonal expression and cancer has been studied extensively. Khan et al. [14] observed a significant ER expression increase in normal breast tissue of patients who underwent surgery for breast cancer. Steroid hormone receptors play an important role in regulating cell cycle and cell proliferation [23]. In Luminal A and B breast cancer, ER binds to the CCND1 promoter favoring cell proliferative activity. The ER higher expression here observed in post-menopausal women can lead to ER-driven transcription of CCND1, that is a crucial factor in neoplastic transformation [23].

ER expression in our study showed a peak in group C (patients older than 60 y/o) while PR gradually reduces after the 6th decade of life, supporting the concept that estrogen plays a role in developing Luminal A and Luminal B cancers (typically ER+ and PR +/−). Moreover, in the present study, ER mean values observed in normal breast epithelial cells surrounding cancers were slightly higher than those found in normal breast tissue surrounding benign breast lesions.

Similar considerations can be done for AR. AR is expressed in the majority of breast cancers [24–26]. Cancers being ER/PR/HER2 negative but AR positive showed better outcome compared to those AR negative [24,25]. AR expression in our study was higher in older patients and significantly lower in younger patients. AR positive breast cancers, are also of apocrine histotype [27]. This finding is consistent with the present data that AR expression is paralleled by GCDFP-15 expression. GCDFP-15 is strongly expressed in Apocrine carcinomas which are well known to be AR positive [27,28]. This suggests that AR could promote the development of Apocrine carcinomas in post-menopausal women [24,25].

Ki67 showed higher expression in younger patients. Ki67, an antigen expressed in cells G1, S, G2 and M phases, is widely used in daily practice as proliferation marker. Higher levels in Ki67 in normal breast tissue from younger patients may be justified by a higher regenerating tissue levels under the influence of the periodic hormonal variation of the menstrual cycle.

Several studies demonstrated that a BMI greater than 25 kg/m<sup>2</sup> represents a risk factor for the development of breast cancer [29]. The risk of breast cancer raises significantly in obese women (BMI > 35 kg/m<sup>2</sup> ) compared to those having a BMI within normal ranges [6]. Estrogen circulating levels are significantly higher in overweight and obese patients which usually develop ER+ cancers; this, together with the evidence that expression of ER and PR levels is significantly higher in obese patients' breast cancers, led to the conclusion that estrogens could play a role in breast cancerogenesis [6,29,30]. In the present series, ER expression showed an increasing trend according to BMI even if it did not reach statistical significance. It should be underlined that, in our series women with BMI < 18.5 were predominantly in the group A (mean age < 42 y/o) while women in overweight group belonged from groups B (mean age 54 y/o) and C (mean age 66 y/o). Therefore, the increased ER expression could be related to the older age and not only to increasing BMI.

The limited number of young obese or overweight patients, in the pre-menopausal period, does not allow definitive conclusions to be drawn.

In the present study, hormonal expression variation according to menstrual cycle phase was not so evident as expected. The present data demonstrated a tendency for reduced hormonal expression from follicular to luteal phase. Data here shown, even if not reaching statistical significance, are in keeping with those of Battersby et al. [31] who observed a marked reduction in ER expression during the menstrual cycle, while PR did not show a significant variation. The same result was achieved by Khan et al. [32].

Among the limitations of the current study, its limited sample size prevented us from obtaining results with robust statistical significance; therefore, our findings should be carefully interpreted.

#### **5. Conclusions**

Our study highlighted a high variability in the expression of hormonal receptors in healthy breast epithelial cells. The combination of different expressions of ER, PR, AR, GCDP-15 and Ki67 could be a risk factor in the development of breast cancer. Even the molecular subtypes of breast cancer could be influenced by the normal expression of hormones at a certain age: for example, triple negative cancer are more frequent at younger age when we demonstrated that ER expression is at its lowest value. On the contrary, post-menopausal women's breasts, characterized by higher expression of ER, PR and AR in the epithelial component, tend to develop ER+/PR+/AR+ cancers.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jpm11050387/s1, Table S1: Results of the Shapiro-Wilk test of normality conducted on the markers used in the study, Figure S1: Examples of positivities obtained with ER, PR, AR and Ki67, Figure S2: Histograms of markers used in the study.

**Author Contributions:** G.S., M.P.F.—Designed study, analysis and interpretation of data, drafted paper and revised it critically, approved the submitted version; C.B., D.G., M.C.C., A.M.S.—Designed study, analysed and interpretation of data, drafted paper; G.F., R.M.—Designed study, drafted paper and revised it critically, approved the submitted version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding

**Institutional Review Board Statement:** The study was approved by the local ethics committee (number of study: Protocol Number: 15005/CE AUSL Bologna, Bologna, Italy). The study protocol conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki and Ethical Principles for Medical Research Involving Human Subjects.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** Disclosure of financial interests and potential conflicts of interest. MPF received grants from Roche, Devicor Mammotome as support for course organization and participation, and from MSD and Biocartis as speaker fee. All the remaining authors submitting this manuscript confirm and attest that they have no conflict of interest. There are no source of support in any form nor funding for this work. There are no financial relation-ships for this work.

#### **References**

