Consecutive female patients with early stage clinicaly node-negative ER- positive breast cancer tested for the 21-gene RS assay and underwent a surgery for breast cancer at the Acibadem Maslak Hospital with a follow-up more than 5-year after surgery were included into the study. This prospectively maintained data was retrospectively analyzed. Demographic, clinicopathological, treatment and outcome characteristics were analyzed. The study was approved by local institutional ethics committee at the Acibadem University, Faculty of Medicine.
Selected representative paraffine sections of the index tumor patients were studied by Genomic Health (Redwood City, CA, USA) to assess the Oncotype DX recurrence score determined by the 21-gene reverse transcriptase polymerase chain reaction (RT-PCR) using the RNA isolated from formalin-fixed paraffin-embedded tissue as decribed before (19). RT-PCR expression analysis (16 cancer related genes and 5 reference genes) evaluates the expression of 16 cancer related genes normalized to the expression of five reference genes and yields a numeric variable of RS on a scale of 0 to 100. Risk categories were determined according to the Oncotype DX® Recurrence Score® as: a) low risk group for scores < 18, b) intermediate risk group for scores between 18–30 c) high risk group for scores ≥ 31 as previously reported (2). Alternatively, patients were also stratified into three risk categories based on different RS cut-off scores: low risk (RS ≤ 11), intermediate risk (RS 11–25), and high risk (RS ≥ 25), as suggested before (8).
The results of the 21-gene RS assay were prospectively incorporated in the treatment plan as recommended (2). Most low risk patients were treated with adjuvant endocrine therapy, whereas most high risk patients received a combination of endocrine therapy and chemotherapy. Treatment of patients with intermediate RS was variable depending on various clinicopathologic features and individual choices. RS was not routinely considered in the selection of locoregional therapy. Results were correlated with histopathologic factors including: tumor size (≤ 2 cm vs > 2 cm), nuclear grade (NG), histologic grade (HG), lymphovascular invasion (LVI) and different cut-off values of Ki67 (10%≤, 15%≤, 20%≤, or 25%<).
ER or PR positivity were considered for any nuclear immunohistochemistry (IHC) staining > 1%. HER2-positivity was determined by IHC and FISH findings, whereas Ki-67 scores were determined as suggested before (13). The tumor subtypes according to the IHC staining were defined as follows (20): luminal A; ER(+) or PR(+), HER2-neu (-), Ki67 < 20%; luminal B, ER(+) or PR(+), HER2-neu (+) and/or Ki67 ≥ 20%; non-luminal HER2-neu(+), ER (-) PR(-) HER2-neu (+); triple-negative, ER(-) PR (-) HER2-neu (-).
Correlations were assessed between Oncotype DX expressions of ER, or PR, or, HER2, and IHC expressions of ER, PR, or HER2. Clinicopathologic variables included patient age at breast cancer diagnosis, tumor size, histologic type of tumor, LVI, 21-gene RS result, local and systemic treatment, and clinical outcome. For multifocal/multicentric carcinomas, the size of the largest tumor and the highest RS result were recorded. For one patient with metachronous bilateral ER+/HER2 − breast carcinomas with low RS, only the data pertaining to the first tumor were included. The institutional database and electronic medical records were reviewed to record date of last follow-up, date of death, date and type of LRR and distant recurrence.
The statistical software program SPSS 25 (Statistical Package for Social Sciences; SPSS, IBM Corp., Armonk, NY, USA) was used for the statistical analyses. To assess the differences between the groups, categorical variables were evaluated by Pearson Chi-Square, and Fisher's Exact Tests in two-tailed univariate analyses.
Overall survival was calculated from date of pathologic diagnosis of breast cancer to date of last follow-up, estimated using Kaplan-Meier methods and compared using the log-rank test. Kaplan-Meier analyses were used for the survival curves test, and log rank test was used to compare different prognostic including different RS values affecting outcome. Disease-free survival (DFS) was analysed based local and systemic metastases, and disease-specific survival (DSS) rates was analysed based on breast cancer-related mortality. LRR was defined as invasive breast cancer involving the ipsilateral breast parenchyma, axilla, regional lymph nodes, chest wall, identified more than six months from the initial diagnosis of breast cancer. Univariate association of RS score on LRR was also examined among the subset of women treated with endocrine therapy and chemotherapy using the methods described above. All nodal recurrences of the axillary and supraclavicular and mammaria interna region, along with breast and chest wall recurrences were accepted as local recurrence (LR), and local recurrence free survival was analysed by considering local metastases. P-values were two-sided, and a p-value equal or less than 0.05 was considered as statistically significant.