The beneficial role of Asian-based RecurIndex test in the prognostic prediction in Chinese male breast cancer patients

RecurIndex, a multigene profiling assay, can predict the risk of local recurrence and distant metastasis in female breast cancer (FBC), but its role in male breast cancer (MBC) remains unclear. In this study, the clinicopathological data of 43 consecutive MBC patients undergoing surgeries between 2009 and 2018 were retrospectively analysed. Their paraffin-embedded tissue sections were examined by RecurIndex test which comprised 2 models: recurrence index for local recurrence (RI-LR) and recurrence index for distant recurrence (RI-DR). Of 43 patients, there were 26 low-risk and 17 high-risk patients assessed by RI-LR, while 17 low-risk and 26 high-risk patients by RI-DR. For RI-LR, tumor N stage showed statistically significant (P < 0.001) between low- and high-risk patients; for RI-DR, differences were pronounced in tumor grade (P = 0.033), T stage (P = 0.043) and N stage (P = 0.003). In terms of clinical outcomes, the overall survival (OS) of low- and high-risk patients stratified by RI-LR showed no statistically significant differences (P = 0.460), while high-risk patients identified by RI-DR had a significantly worse distant recurrence-free survival (DRFS) (P = 0.035), progression-free survival (PFS) (P = 0.019) and OS (P = 0.044) than low-risk patients. Overall, RI-DR can effectively predict the DRFS, PFS and OS of MBC patients and identify those at low risk of recurrence, which may serve as a potential prognostic tool for MBC.


Association of RI-LR and RI-DR with clinicopathological features. The clinicopathological features
of low-and high-risk patients stratified by RI-LR and RI-DR were compared in Tables 2 and 3, respectively. For RI-LR, tumour N stage showed statistically significant between low-and high-risk patients (P < 0.001; Table 2). For RI-DR, significant differences were presented in tumour grade (P = 0.033), T stage (P = 0.043) and N stage (P = 0.003) between low-and high-risk patients ( Table 3).

Association of RI-LR and RI-DR with clinical outcomes.
For RI-LR, analysis of overall survival (OS) of low-and high-risk patients showed no statistically significant differences (P = 0.460; Fig. 1A). For RI-DR, however, high-risk patients had a significantly worse OS (69.2% vs. 100.0%, P = 0.044; Fig. 1B), distant recurrence-free survival (DRFS) (73.1% vs. 100.0%, P = 0.035; Fig. 1C), and progression-free survival (PFS) (65.4% vs. 100.0%, P = 0.019; Fig. 1D) than low-risk patients. During the follow up, 17 (100.0%, 17/17) low-risk patients identified by RI-DR did not suffer from any recurrences or deaths, while 9 (34.6%, 9/26) high-risk patients were subjected to recurrences or deaths. According Table 2. Correlation between RI-LR and clinicopathological features, n (%). LVI lymphovascular invasion, ER/PR estrogen receptor/progesterone receptor, HER2 human epidermal growth factor receptor 2, RI-LR recurrence index for local recurrence. www.nature.com/scientificreports/ to the subtypes, 22 patients not receiving adjuvant chemotherapy were screened out. Despite no statistically significant difference, the low-risk patients without adjuvant chemotherapy seemed to have a longer OS than the high-risk patients without adjuvant chemotherapy (P = 0.140; Fig. 2). Among the patients without adjuvant chemotherapy, 12 cases were predicted at low risk of recurrence. The final follow-up showed that these 12 patients did not experience any recurrences or deaths. All these findings suggested that RI-DR was conductive to identifying the patients at low risk of recurrence for MBC, and these patients might be exempt from postoperative adjuvant chemotherapy.

Discussion
Genomic testing is thought to play an important role in aiding clinical decision-making and better balancing the efficacy and detriments of adjuvant therapies, which is valuable in avoiding overtreatments or harmful treatments 21,22 . In this study, RecurIndex, a multigene prognostic test, was used to assess the risk of LRR and DR in MBC. The results showed no statically significant association of RI-LR with MBC prognosis. However, RI-DR could effectively predict the DRFS, PFS and OS of MBC patients and identify those at low risk of recurrence to make them exempt from adjuvant chemotherapy. These findings suggested that RI-DR could be a potential prognostic tool for MBC. RI-DR, a clinical-genomic model generated by clinical variables and genetic information, has a relatively high sensitivity and a relatively high negative predictive value. It is useful in the identification of low-risk breast cancer patients, particularly in those with Asian genetic backgrounds 23 . A previous study showed that RI-DR contributed to classifying Asian endocrine-responsive breast cancer patients with both negative and positive lymph nodes into the low-and high-risk groups based on 10-year DR 24 . In another study performed by Huang et al., it was also found a significant difference in 10-year DR-free intervals between low-and high-risk groups classified by RI-DR 19 . These studies all confirmed the utility of RI-DR in clinic.
Currently, National Comprehensive Cancer Network (NCCN) guidelines have proposed that multigene assays Oncotype Dx and MammaPrint can be used to evaluate the risk of recurrence in FBC according to certain cancer-gene expression patterns. In addition to providing information on recurrence prognosis, they also have predictive ability and can indicate who may benefit from additional chemotherapy 25 . With a high concordance rate with Oncotype Dx, RecurIndex possesses potential for helping clinicians make more informed decisions on adjuvant chemotherapy in Asian FBC patients 20,26 . Among the MBC patients without adjuvant chemotherapy in our study, the low-risk patients stratified by RI-DR tended to have better OS than the high-risk patients, and Table 3. Correlation between RI-DR and clinicopathological features, n (%). LVI lymphovascular invasion, ER/PR estrogen receptor/progesterone receptor, HER2 human epidermal growth factor receptor 2, RI-DR recurrence index for distant recurrence. 10-year OS rates of patients with lymph node-negative disease and those with lymph node-positive disease were 75% and 43%, respectively, indicating that the death risk of MBC patients with lymph node-positive disease did not decrease significantly after adjuvant chemotherapy 27 . There is another study showing that adjuvant chemotherapy may be skipped for stage I-IIA MBC patients 28 . Additionally, our results also revealed that 4 high-risk patients identified by RI-DR who developed recurrence or deaths might benefit from adjuvant chemotherapy, suggesting that adjuvant chemotherapy may be taken into consideration in MBC patients at high risk of recurrence or metastasis 29 . Hence, RI-DR can not only provide clinical outcome information before treatment, but also contributes to risk-benefit assessment of systematic adjuvant chemotherapy.
The major superiority of our study was that it first employed RecurIndex, a multigene prognostic test, to assess the prognosis of Chinese MBC patients and showed the clinical utility of RI-DR in MBC. Additionally, RI-DR can identify the patients at low risk of recurrence, probably leading to a reduction of adjuvant chemotherapy. It may be a necessary study whether MBC patients should receive postoperative adjuvant chemotherapy or not. However, there were also several limitations in our study. First, our study was a retrospective, single-center study with the small sample size, which may affect the statistical power and reliability of results. Second, we did not find statically significant association of RI-LR with MBC prognosis, which might be associated with the small sample size. In the future, more well-designed, large-scale studies should be implemented to further investigate the correlation between RI-LR and the prognosis in MBC. The clinicopathological data of patients were collected by reviewing the electronic medical record, including age, LVI, tumour grade, tumour stage, Ki-67 expression, ER/PR and HER2 status, as well as presence or absence of adjuvant endocrine therapy, adjuvant chemotherapy and adjuvant radiotherapy.

RecurIndex test.
As an in vitro test, RecurIndex utilized real-time fluorescent quantitative nucleic-acid amplification technology to detect the ribonucleic acids (RNA) extracted from FFPE breast cancer samples, which was used for analysing gene-expression profiling of breast cancer. The primer pairs of genes included in RecurIndex test were complementary to the messenger RNA sequence of each target gene, and PanelStation was used for real-time fluorescent quantitative amplification. Based on gene-expression profiling of breast cancer and clinical factors, the risk scores of LRR and DR were calculated using analysis software.
A previous study has demonstrated that the best cut-off values of RecurIndex test for predicting LRR and DR are 8% and 4%, respectively 19 . According to the RI-LR and RI-DR generated from RecurIndex test, patients were separately divided into the low-and high-risk groups. Postoperatively, all the patients were followed up by further consultations and telephones. The follow-up deadline was February, 2020.
Statistical analysis. The data in this study were managed using R software (version 4.0.1, The R Foundation). Normally distributed measurement data were expressed as mean ± standard deviation ( − x ± s), t-test was used. Non-normal distribution data were presented as the median and interquartile [M (Q1, Q3)], the Mann-Whitney U rank sum test was performed. The ranked data were presented as number of cases and percentiles n (%), χ 2 test or Fisher's exact test was performed. The survival curves were drawn using Kaplan-Meier method and compared by Log-rank test. P < 0.05 was considered statistically significant.
Ethics approval and consent to participate. The study was approved by the Institutional Review Board of The Fourth Hospital of Hebei Medical University (Approval No.: 20211372). All the patients included in this study had given informed consent, and all the procedures performed were in accordance with relevant guidelines/regulations, as well as the 1964 Helsinki declaration and its later amendments.