Platinum agents (i.e. carboplatin and cisplatin) are cytotoxic DNA damaging agents causing DNA strand breaks and consequent cell apoptosis[12]. This mechanism makes them useful in cancers with DNA HR repair deficiency, especially those harbouring deleterious mutations in the BRCA1/2 genes[6, 7]. BRCA gene abnormality and TNBC are closely related. Up to 20% of TNBC patients are carriers of a BRCA germline mutation[22]. Moreover, several other mechanisms involving in the HR pathways have been indicated to platinum response, defining the concept of BRCAness. This genetic signature is defined by epigenetic inactivation of BRCA, mutations in other genes or post-translational modifications of key proteins involved in HR system[23, 24]. Since the functions of BRCA1/2 gene and the phenomenon of BRCAness in HR repair mechanism was revealed, several studies attempted to investigate the role of the platinum-based chemotherapy in the neoadjuvant TNBC setting. A series of phase II studies, such as the GEICAM/2006-03, the GeparSixto and the CALGB 40603 trials[25–27], suggested a possible activity and survival benefit of the addition of platinum in the NACT, however, available results are mixed and controversial. According to current breast cancer guidelines, the routine use of platinum agents as part of neoadjuvant therapy for TNBC is not recommended for most patients, however, it may be considered in select patients requiring better local control[9]. Therefore, predictive biomarkers for platinum in BC represent an unmet clinical need.
Recently, three independent DNA-based measures of genomic instability resulting from HR repair defects were developed. The test was based on genomic alterations with loss of heterozygosity (LOH)[28], telomeric allelic imbalance (TAI)[29] and large-scale state transitions (LST)[30]. The combination of the three scores, called HRD score,could distinguish homologous recombination deficient tumors from non-deficient tumors[31, 32]. HRD is defined by a threshold of HRD score equal or over 42 [33, 34]. It has been reported that HRD-high tumors were sensitive to platinum-containing regimens, indicating a clinical utility of HRD score for the selection of patients who were more likely to respond to platinum [34]. However, the BrighTNess study showed that higher pCR rates was not related to HRD status in the platinum-containing NACT [35]. Additionally, Tutt et al. also found similar response rate to carboplatin between HRD-high and HRD-low tumors in the metastatic settings (TNT trial)[36]. Therefore, it is necessary to carry out further studies to confirm the clinical utility of HRD scores as a predictor for response to platinum-containing regimens.
Genomic alterations detected by NGS technique for HRD status actually reflect the pre-transcriptional events, while gene expression profiling can provide a current transcriptional state of tumor samples, since the quantity of RNA varies dynamically during cellular process. In 2014, Pitroda et. al. developed a recombination proficiency score (RPS) system with gene expression profiling to evaluate HRD status and tumor sensitivity to chemotherapy[10]. The RPS score from Pitroda et. al. is calculated by the expression levels of four genes (Rif1, PARI, RAD51, and XRCC5) involved in DNA repair pathway. Initially, the RPS system was studied in patients with non–small cell lung carcinomas (NSCLC), which showed low-RPS tumors are especially sensitive to platinum-based chemotherapy[10]. This indicated RPS has the potential to determine sensitivity to platinum-based chemotherapy. And in their later study, such RPS was used to evaluate sensitivity of breast cancers to anthracycline-based NACT [11]. Compared with the work of Pitroda et.al., our study focused on the response to platinum-based NACT, not on anthracycline-based NACT. Additionally, the final genes used in our study was obtained from the correlation analysis with the pCR results, while the gene list from Pitroda’s study were selected from cell line database on topotecan sensitivity.
In our study, eight target genes (RIF1, PARI, RAD51, XRCC5, BRCA1, PARP1, C-Met, and E2F1) were included in primary analysis. Beside the four genes (Rif1, PARI, RAD51, XRCC5) referred to Pitroda’s work, four additional genes (BRCA1, PARP1, C-Met, E2F1) were added as well. As reported previously, these genes played important role in HR repair process. BRCA1 protein was an upstream effector and considered as a permanent factor during the whole process despite of the complex mechanism involved in double-strand break repair pathway[12, 13]. E2F1 was a transcription regulator participating various pathways such as cell cycle, proliferation, apoptosis, development and differentiation[15]. E2F1 accumulation was a response to DNA double strand damage in tumor cells and promotes DNA repair[14]. Compared with RIF1 and PARI, PARP1 not only mediates excision repair in single-strand break but also acted as a sensor of DNA double-strand break involving the control and recruitment of important HR proteins[16]. In addition, TNBC were shown to express PARP1 more frequently than other breast cancer subtypes. And high levels of PARP1 expression in breast cancer correlated with improved response to chemotherapy[2]. Importantly, it has been revealed that active PARP-1 could enhance E2F1 transcription factor activity in HR related DNA repair process[17]. Furthermore, several studies showed that C-MET inhibition reduced RAD51 phosphorylation by impairing its nuclear translocation and decreased the formation of the RAD51/BRCA2 complex in DNA damage response[18, 19]. Additionally, C-Met is not only a clinical prognostic marker but also a predictive marker of response to chemotherapy in patients with breast cancer. Interestingly, previous studies have also indicated that c-Met related with and phosphorylated PARP-1 at Tyr907, and inhibiting both c-Met and PARP-1 could synergize to suppress the growth of breast cancer cells [20]. Considering the evidence showed above, these eight genes were put into primary analysis. As a result, the expression level of BCRA1 and PARP1 presented significant correlation with different MP grading subgroups, while RIF1 and PARI didn’t. Thus, two genes (RIF1 and PARI) referred in Pitroda’s final formula were replaced by BRCA1 and PARP1.
Our study showed that, TNBC with higher score had nearly quadruple likelihood to achieve pCR to platinum-based NACT compared with a lower score. Moreover, a test result below − 2.6440 might be used to rule out the patient with less sensitivity to platinum regimen; and a result above − 1.9692 might be rule in the patient with increased possibility to achieve pCR. However, if the subset acquired the value between − 2.6440 and − 1.9692, the predictive ability of 4-gene score was significant attenuated.
We also showed that high level Ki-67(≥ 40%) correlated with pCR in breast in TNBC. As demonstrated in other studies, the definitions of Ki-67 cutoff values differed widely, ranged from 10–61% in TNBC[37]. In our study, the median Ki-67 index was 70%. It is consistent with that baseline Ki-67 values for TNBC are much higher than those for luminal tumors[37]. Due to interobserver variations there was always misclassification in assessment of Ki67 when the level of expression of Ki67 lay in grey zone. In the PACS01 study, when Ki-67 expression ranged between 10% and 25%, there was a risk of misclassification with 37%; while the risk of misclassification was only 11% with Ki-67 expression either < 10% or ≥ 25% [38]. In our study the median Ki-67 index was significant high with 70%, thus it is almost impossible to misclassified the value of Ki-67. However, using such a high threshold to make further analysis was unwise. Several studies suggested using high Ki-67(≥ 30%) proliferative index could identify those patients with significantly higher breast-related events[39, 40]. Thus, we defined Ki-67 as a classification variable at a 40% threshold. This cutoff value is in line with that reported earlier by another TNBC study from Wei Wang et.al[41]. Additionally, the 4-gene score is positively correlated with Ki-67, indicating that higher 4-gene score represented higher proliferation rate of tumors.