Association of molecular biomarkers heterogeneity and treatment pattern, disease outcomes in multifocal or multicentric breast cancer patients


 Purpose

To evaluate the rates of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 heterogeneity in multifocal or multicentric breast cancer (MMBC) and its association with treatment pattern and disease outcomes.
Methods

MMBC patients with ER, PR, HER2, and Ki67 results for each tumor focus were retrospectively analyzed and categorized into the Homo group and the Hetero group. Chi-square test were performed to compare the treatment options between the groups. Disease-free survival (DFS) and overall survival (OS) rates were estimated from Kaplan-Meier curves and compared between two groups.
Results

A total of 330 patients were included and 53 (16.1%) were classified into the Hetero group. Adjuvant endocrine therapy was more frequently assigned for patients in the Hetero group than in the Homo group (84.9% vs. 71.7%, P = 0.046). There was no difference in terms of adjuvant anti-HER2 therapy (28.3% vs. 19.6%, P = 0.196) and chemotherapy (69.9% vs. 69.8%, P = 0.987) usage between two groups. At a median follow-up of 36 months, DFS rates were 81.2% for the Hetero group and 96.5% for the Homo group (HR = 2.81, 95% CI: 1.00-7.88, P = 0.041). The estimated 3-year OS rates for the groups were 95.8% and 99.5%, respectively (HR = 4.31, 95% CI: 0.83–22.46, P = 0.059).
Conclusion

Heterogeneity of ER, PR, HER2, or Ki67 was present in 16.1% patients with MMBC. Biomarkers heterogeneity influenced adjuvant endocrine therapy usage and was associated with worse disease outcomes, indicating further clinical evaluation.


Introduction
Breast cancer is a heterogeneous group of diseases in which individual patient differs in morphological features, molecular pro les, therapeutic responses, and prognosis [1]. Morphological variability such as pathological type and histological grade has been well documented for decades and forms the basis for histological classi cation of breast cancer. More recently, different molecular phenotypes of breast cancer have been de ned by genetic or immunohistochemistry testing. For example, the well-de ned 2013 St Gallen subtypes of breast cancer were based on the expressions of estrogen (ER) and progesterone (PR) receptors, human epidermal growth factor receptor 2 (HER2), and Ki67 proliferative index, which provide prognostic information and can be used to tailor systemic adjuvant therapy [2].
The molecular heterogeneity can occur either between different patients (intertumor heterogeneity), or within the same patient (intratumor heterogeneity) [1]. Heterogenous expressions of ER, PR, HER2, and Ki67 have been widely reported between core needle biopsy and surgical samples, between different regions of a primary tumor, between a primary tumor and a matched metastatic lesion, or between metastatic lesions [3][4][5][6][7][8][9]. Beyond spatial heterogeneity, heterogeneity can be observed as the natural evolution of a tumor or as consequences of anticancer treatments [10][11][12].
In a previous study that evaluated the heterogeneity of ER, PR, HER2, and Ki67 between different foci in multifocal/multicentric breast cancer (MMBC), the heterogeneity of these molecular markers was present in 4.4%, 15.9%, 9.7%, and 15.0% cases [13]. However, there are few studies investigating the therapeutic and prognostic impact of such heterogeneity [13][14]. Herein, we performed this retrospective study to evaluate the rates of ER, PR, HER2, and Ki67 heterogeneity in patients with MMBC as well as its impacts on systemic adjuvant therapy decision-making and disease outcomes.

Study population
Patients who received surgery and were diagnosed with multifocal or multicentric breast cancer at Clinicopathological characteristics, adjuvant treatment, and follow-up data were retrieved from Shanghai Jiao Tong University Breast Cancer Database (SJTU-BCDB). The eligibility criteria were as follows: (1) invasive carcinoma for all tumor foci; (2) no distant metastasis at diagnosis; (3) ER, PR, HER2, and Ki67 both tested between different invasive tumor foci. Those who received neo-adjuvant therapy and those with extensive DCIS were excluded from the present study.

Histopathology assessments
Histopathology analysis for different tumor foci on surgical specimens were independently performed and reviewed by two pathologists at the Department of Pathology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine [15][16]. In this study, multifocality was de ned as the presence of more than one focus of invasive carcinoma in one breast quadrant; and multicentricity was de ned as the presence of a focus in a different breast quadrant from the main lesion [13]. Immunohistochemistry (IHC) of ER, PR, HER2 and uorescence in situ hybridization (FISH) of CerbB-2, if necessary, were performed on surgical specimens according to the ASCO/CAP guidelines [17][18][19][20]. In brief, ER/PR were considered positive if a minimum of 1% of the invasive tumor cells were positive for ER/PR staining and HER2 positivity was de ned as IHC 3 + or IHC 2 + with FISH CerbB-2 ampli cation. Molecular subtypes were determined based on 2013 St Gallen system: luminal A-like (ER+/PR ≥ 20%/HER2-/Ki67 < 20%), luminal B-like (HER2-) (ER+/HER2-/Ki67 ≥ 20% or ER+/PR < 20%/HER2 or ER-/PR+/HER2-), luminal B-like (HER2+) (ER + or PR+/HER2+), HER2+ (ER-/PR-/HER2+) and triple negative (ER-/PR-/HER2-) [2]. Patients with concordant status of ER, PR, HER2, and Ki67 among all invasive tumor foci were categorized into the Homo group, while the Hetero group was de ned as the existence of at least one discordance for ER, PR, HER2, or Ki67 between different foci. The main focus referred to the largest tumor focus and the other foci were named minor foci.

Treatment and follow-up
Adjuvant treatment decisions were made through multidisciplinary team (MDT) meetings attended by surgical oncologists, medical oncologists, radiation oncologists, and pathologists [21]. The patients were followed up every 3 months during the rst two years after surgery, every 6 months from the third to the fth years, and once per year hereafter till death. DFS was de ned as the period from the date of surgery to rst local-regional relapse, contralateral breast cancer, secondary new malignant tumor, distant relapse, or death. OS was calculated from the date of surgery to the date of death.

Statistics
Kappa test were performed to evaluate the concordance rates of pathological type, histological grade, ER, PR, HER2, Ki67, and molecular subtype between the larger tumor focus and the smaller focus. For tumors with 3 or 4 foci, the results were considered concordant only when the biomarkers status of all tumor foci were concordant. The adjuvant therapy options were compared between the Homo group and the Hetero group using chi-square test or Fisher exact test. DFS and OS rates were estimated from Kaplan-Meier curves and compared between the two groups via log-rank test. Cox proportional hazard model was used to calculate the hazard ratios for relapse and death. Two-side P < 0.05 was considered statistically signi cant. All the statistical procedures were performed on SPSS (version 26.0).

Baseline clinicopathological characteristics
There were 584 women with multifocal or multicentric breast cancers and 330 of them were included in the study (Fig. 1). Physical examination, sonography, mammography, and MRI identi ed 16.5%, 68.9%, 35.7%, and 78.1% of these patients, respectively (Supplementary Figure S1). As shown in the Supplementary Figure S2, the median distance between the main and minor foci was 12.5 (IQR: 7.1-20.0) mm, which showed no signi cant difference between the Homo and Hetero groups (12.4 mm versus 14.0 mm, P = 0.359). The demographic and clinicopathologic characteristics for the cohort are summarized in Table 1. The median age for the patients was 56 (IQR: 47-65) years and 43.8% patients were pre/perimenopausal at diagnosis. Patients with 2 and 3/4 foci accounted for 92.4% and 7.6% of the cohort, respectively. Twenty-six (7.9%) patients received breast-conserving surgery and sentinel lymph node biopsy was performed in 103 (31.5%) patients. A total of 45.7% and 41.2% patients with multifocal and multicentric diseases received radiotherapy, respectively (Supplementary Figure S3). There were 130 Rates of molecular markers heterogeneity ER, PR, HER2, and Ki67 mismatches of the main tumor focus between CNB and surgical samples were present in 15 (6.6%), 32 (14.0%), 9 (5.5%), and 57 (25.1%) patients, respectively (Supplementary Table  S1). As shown in Table 2

Heterogeneity of molecular markers and adjuvant therapy
A total of 45 (84.9%) patients in the Hetero group received adjuvant endocrine therapy, which was signi cantly higher than that of patients in the Homo group (71.7%, P = 0.046, Fig. 2A). There were no signi cant differences in the usage rates of adjuvant anti-HER2 therapy (28.3% versus [vs.] 19.6%, P = 0.196, Fig. 2B) and chemotherapy (69.9% vs. 69.8%, P = 0.987, Fig. 2C) between the two groups.

Heterogeneity of molecular markers and disease outcomes
At a median follow-up time of 35 (IQR: 19-57) months, 21 DFS events were recorded: 16 in the Homo group and 5 in the Hetero group (Table 4). As shown in Table 5, status of molecular markers was signi cantly associated with DFS (P = 0.041). The estimated 3-year DFS rate was 81.2% for the Hetero group, which was signi cantly higher than that of the Homo group (96.5%, HR = 2.81, 95% CI: 1.00-7.88, Fig. 3A). Two patients in the Hetero group and 5 in the Homo group died during the follow-up period ( Table 4). Patients in the hetero group tended to have shorter OS, with the estimated 3-year OS rates for these two groups being 95.8% and 99.5%, respectively (HR = 4.31, 95% CI: 0.83-22.46, P = 0.059, Fig. 3B).

Discussion
The study was designed to evaluate the rates of molecular markers heterogeneity as well as its associations with systemic adjuvant therapy and disease outcomes in MMBC. Molecular markers showed good concordance among different invasive tumor foci. Heterogeneity of ER, PR, HER2, and Ki67 were present in MMBC, which was associated with more adjuvant endocrine therapy usage (P = 0.046) and shorter DFS (P = 0.041), indicating the necessity of molecular assessments for different tumor foci in patients with MMBC.
With limited knowledge on the consequences of molecular heterogeneity for therapeutic decision-making, it has been accepted that biomarkers can be assessed only in the largest individual tumor focus [26]. This is based on the observations that molecular markers in MMBC are usually homogenous. However, if the tumor foci demonstrate different pathological or histological features, biomarkers evaluation of the smaller focus will be necessary. According to Buggi et al, 14 out of the 113 (12.4%) patients received additional systemic treatments with the biomarkers analysis for the smaller tumor focus [13]. In the present study, heterogeneity of biomarkers was also found to be signi cantly associated with the usage of adjuvant endocrine therapy.
To the best of the authors' knowledge, this was the largest study to evaluate the prognostic signi cance  [14]. Taken together, these results suggested the necessity of evaluating molecular markers for different tumor foci in patients with MMBC [27]. And it would strengthen our point if we can verify that patients with multicentric disease who do not undergo a second biopsy have worse outcome. However, only 3 out of 30 patients with multiple invasive diseases who lacked biomarkers data in our cohort experienced DFS events, indicating a large cohort with more patients are needed to validation this recommendation.
The present study enrolled both multifocal and multicentric diseases, which were heterogenous and could be further differentiated into multifocal breast cancer (MFBC) and multicentric breast cancer (MCBC). In our cohort, ER status, PR status, and breast surgery were signi cantly different between patients with MFBC and MCBC (Supplementary Table S1, S2). While for DFS and OS, no signi cant differences were observed between the two groups (Supplementary Figure S4). To date, relatively few reports have directly compared the clinical-pathological features, treatment patterns and survivals of patients with MFBC and MCBC, which warrant further research [28][29][30].
The incidence of MMBC increases with the advancement of preoperative imaging, and intratumor molecular heterogeneity has attracted the attention of clinicians. For example, MRI can identify 74.6%, 54.2%, and 67.3% of MMBC that were not identi ed by physical examination, sonography, or mammography, respectively (Supplementary Figure S1). We performed the study for the rst time to evaluate the rates of biomarkers heterogeneity in MMBC as well as its impacts on adjuvant therapy and survival. However, there were several limitations in the present study. Firstly, this was a single-institutional retrospective study, so there might be selection bias and limited applicability. Further validation in other cohorts will provide us more insights to the therapeutic and prognostic role of biomarkers heterogeneity. Secondly, the Hetero group consisted of a relatively small number of patients and cases, which warranted longer follow-up. Thirdly, the present study enrolled patients over a nine-year period from Jan 2009 through Dec 2018, which might exert an in uence to disease outcomes. However, no signi cant disease outcome difference was observed between patients who received surgery before or after 2015 in our cohort (Supplementary Figure S5).
In conclusion, heterogeneity of ER, PR, HER2, or Ki67 was present in 16.1% patients with MMBC. Biomarkers heterogeneity was associated with more adjuvant endocrine therapy usage and worse disease outcomes, indicating the necessity of molecular assessments for different tumor foci in patients with MMBC. Ruijin Hospital, Shanghai Jiao Tong University School of Medicine-"Guangci Excellent Youth Training Program" (GCQN-2017-A18). All these nancial sponsors had no role in the study design, data collection, analysis or interpretation.

Con ict of interest
The authors declare no con ict of interest.
Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Code availability Not appliable.
Authors' contributions XC conceived and designed the study. SL analyzed and interpreted the data for presentation and was a main contributor in writing the manuscript. XC revised the manuscript and all authors approved the nal manuscript.

Ethical approval
This article does not contain any studies with human participants performed by any of the authors. 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.

Consent to participate
Not appliable. Figure 1 Folw chart of 330 patients in the study Figure 2 Adjuvant systemic therapy by molecular markers status