Based on the principles of various classification systems, pCR has been defined unanimously as the complete disappearance of invasive cancer cells in primary breast tumor [67]. However, whether the ductal carcinoma in situ (DCIS) and residual cancer cells present in axillary lymph nodes should be considered when defining pCR is still debated (Table 1) [68910]. Mazouni and colleges [11] from the MD Anderson Cancer Center conducted a retrospective analysis including 2,302 patients treated with NACT and observed similar 5- and 10-year overall survival (OS) and disease-free survival (DFS) between the pCR group (patients achieved pCR both in breast and lymph nodes) and DCIS group (patients without residual disease in primary tumor and lymph nodes, but contained DCIS) after 250 months of follow-up [11]. Concordantly, a meta-analysis of 12 clinical trials conducted by Collaborative Trials in Neoadjuvant Breast Cancer (CTNeoBC) demonstrated similar event-free survival (EFS) and OS rates irrespective of DCIS [5]. Although several studies have suggested that DCIS is not important for defining pCR, other researchers argue that DCIS might lead to higher local recurrence risk for patients after NACT [12]. Indeed, the individual characteristics of patients must be considered when analyzing the effect of DCIS on survival; thus, patients with DCIS should be evaluated and treated individually. In contrast, CTNeoBC conducted a pooled analysis and demonstrated that it was inappropriate to define patients with isolated cells in axillary lymph nodes as pCR [5]. Therefore, researchers of the Breast International Group-North American Breast Cancer Group (BIG-NABCG) defined pCR as the absence of invasive cancer cells in primary tumor and metastatic lymph nodes, while the presence of DCIS has not been explicitly concluded [13]. Concordantly, Morrow [14] indicated that the presence of residual tumor in axillary lymph nodes definitely increased the risk of relapse and death. Thus, the medical community has accepted that the assessment of axillary lymph node status following NACT is indispensable, as it improves the prognostic efficacy of pCR. A comprehensive review of the results of previous clinical studies showed that pCR can be defined as no residual invasive cancer in the breast and lymph nodes but allows for DCIS (ypT0/isypN0). Patients with residual invasive cancer cells in lymph nodes could not be supposed as achieving pCR, while those who remaining DCIS should be analyzed separately.

pCR represents a satisfying treatment response to NACT. Furthermore, ever since studies of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-18 and B-27 showed significant prolongation of DFS and OS in patients who achieved pCR compared to those who retained residual disease after NACT [15], an increasing number of studies have investigated the prognostic value of pCR. Although BC of young women tend to be more aggressive with a relatively unfavorable prognosis [16], reports show that patients aged ≤ 40 years can also obtain significant survival benefit by achieving pCR after NACT [17]. As a prognostic indicator, pCR has the advantage of reflecting chemo-sensitivity in short time after NACT, which underscores the necessity of subsequent adjuvant treatment following surgery.

However, evidence showing that the increase in pCR rate may translate into corresponding improvement in EFS and OS is lacking, and not all patients who achieved pCR had better survival and lower recurrence rates. The limitation of pCR might be because of the following: 1) some other risk factors of patients might weaken the prognostic benefit of pCR. Although several studies have suggested correlation between pCR and better prognosis, a small group of people show disease relapse and metastasis in the short term, even after achieving pCR after NACT. According to previous studies, factors such as HER2-positivity, axillary lymph nodes metastases, identified lymph nodes < 10, premenopausal status, and clinical stage IIIB–C might enhance the recurrence or metastasis rates for patients who have achieved pCR [181920]; 2) the rates of pCR are discordant among different subtypes and the prognostic effects of pCR do not apply to all molecular subtypes of BC. Studies have shown that pCR rate is higher in patients with triple-negative breast cancer (TNBC) and HER2-positivity than in HR-positive/HER2-negative patients [2122]. According to the results of CTNeoBC meta-analysis, patients with highly aggressive subtype (such as TNBC or HER2-positive) who achieved pCR showed more prominent improved outcomes than patients with luminal A subtype [5]. von Minckwitz et al. [12] observed a significant association between improved DFS and pCR in luminal B/HER2⁻, HER2⁺, and TNBC tumors, but not in the luminal A and luminal B/HER2⁺ subset [12]. Concordantly, studies have suggested that failure to achieve pCR is related to unfavorable prognosis in TNBC and HER2⁺ tumors, but not in most of HR-positive patients with BC [12]. Indeed, studies have indicated that HR-positive patients tend to show favorable prognosis although they were less responsive to chemotherapy with relatively lower chances of achieving pCR [5], thereby reflecting the uncertain correlation between pCR and long-term outcomes in patients with HR-positive tumors.

Although the estimated rates of pCR have increased after the addition of new drugs in the routine chemotherapy, a large number of patients fail to achieve pCR following NACT, and not all the patients with pCR show good prognosis. pCR is far from a rationale prognostic factor for HR-positive BC. Therefore, the use of pCR as a replacement of DFS and OS to reflect the long-term prognosis still lacks credibility. The general characteristics of patients should also be considered to comprehensively determine the correlation of pCR with prognosis, thereby facilitating the practice of precision medication.

pCR is related to improved prognosis; however, different degrees of residual disease after NACT persisted in some patients, which prompted the use of other indicators to assess the residual tumor and provide prognostic information. RCB is a pathological assessing system for evaluating residual disease following NACT, which has appealed to an increasing number of researchers in recent years. Compared to pCR, the RCB system can easily quantify post-treatment pathological response and can accurately predict long-term prognosis [23]. Furthermore, the RCB index is a highly reproducible tool for effectively predicting distant recurrence free survival (DRFS) and OS [24]; thus, RCB has the potential to act as a widely-used prognostic indicator in clinical practice. The RCB evaluation system considers both the primary tumor in breast tissue (size, cellularity, and in situ disease) and metastatic lymph nodes (number and size) [25], which can be calculated using a web-based calculator of the MD Anderson Cancer Center. The RCB index is calculated both as a continuous score and a category based on the extent of the residual tumor. RCB-0 is equal to pathological complete response, while the residual disease is divided into RCB-I (minimal residual tumor), RCB-II (moderate residual tumor), and RCB-III (extensive residual tumor) with the cut-off values of 1.36 and 3.28 [24].

Several researchers have realized the value of RCB index as an indicator of long-term survival for patients with BC after NACT (Table 2). It is noteworthy that the RCB system has been reported to be a primary or secondary endpoint for NACT due to the prognostic correlation of residual tumors in prospective trials of the Austrian Breast and Colorectal Cancer Study Group (ABCSG) [26]. Peintinger et al. [24] divided RCB into good response class (RCB 0/I) and bad response class (RCBII/III) in terms of the corresponding long-term outcomes. In addition, patients with RCB-I were confirmed to have similar 5-year distant relapse rate as RCB-0 (5.4% and 2.4%, respectively), while patients with RCB-III showed poor prognosis with 5-year distant relapse rate of 53.6% [25]. Symmans et al. [27] conducted a study including five cohorts and reported that RCB index was significantly associated with long-term prognosis in all the molecular subtypes and neoadjuvant treatment regimen cohorts of this study. Furthermore, the RCB index can be integrated with other prognostic biomarkers, which might provide more credible prognostic information for patients with BC. Studies have shown that the prognostic efficacy of tumor-infiltrating lymphocytes (TILs) was limited in patients with TNBC and HER2⁺ subtypes of BC, but not in those with luminal–HER2 BC [28]. Asano et al. [29] concluded that the integrated indicator ‘TILs-RCB’ is also capable of predicting recurrence rates in HR-positive patients and the prognostic efficacy of ‘TILs-RCB’ is higher than that of TILs or RCB alone. A concordant conclusion was obtained by Luen et al. [30], who showed that residual disease (RD) TILs level can further divide RCB-II patients into relatively higher and lower recurrence-free survival (RFS) groups, reflecting that addition of RD TILs into RCB class improved prognostic efficacy. In addition, RCB was also combined with Ki-67 index to form a prognostic indicator called ‘residual proliferative cancer burden’ (RPCB) to predict long-term prognosis after NACT, and the prognostic value of RPCB was confirmed to be more than that of Ki-67 or RCB alone. Furthermore, the predictive value may be further improved when post-treatment tumor grade and estrogen receptor (ER) status are considered [31].

Although several studies have proved the prognostic effect of RCB in neoadjuvant chemotherapy, its ability to accurately identify high-risk patients is still conflicting [2732]. The reliability of the RCB evaluation system might be challenged, as it places importance on the presence of residual tumor in post-treatment pathological specimens without considering the baseline data. The crucial problem in the clinical decision-making for patients with RCB-I is the similarity in their prognosis with those who have achieved RCB-0. Furthermore, the reliability of RCB as a surrogate endpoint following NACT should be investigated.

Ki-67 is a nuclear antigen present in all phase of the cell cycle except in the G0 phase. It is a tumor proliferation marker and is mainly assessed using immunohistochemistry (IHC). Based on the theory that highly proliferative tumors are highly sensitive to chemotherapy even in HR-positive patients, the pre-treatment Ki-67 level may be a potentially positive predictive factor of NACT response. Indeed, several studies have investigated the function of pre-treatment Ki-67 expression in predicting NACT response of patients across various BC subtypes (Table 3). Results obtained from various patient groups have confirmed that higher baseline Ki-67 expression was significantly associated with better treatment response and improved pCR rate [333435]. Compared to patients with HER2⁺ and TNBC BC, ER-positive patients with higher pretreatment Ki-67 expression are more likely to show clinical response to NACT [36]. Chen et al. [35] concluded that higher baseline Ki-67 level can be considered a predictive indicator for pCR following NACT only in patients with the luminal subtype, and showed that 25.5% was ideal as the cut-off value for Ki-67 level using receptor operator characteristics (ROC) curve analysis. On the contrary, Alba et al. [37] analyzed the data from four clinical trials and concluded that baseline Ki-67 level > 50% was associated with higher pCR rate, especially in patients with ER⁻/HER2⁻ and ER⁻/HER2⁺ subtypes [37]. In contrast, Kim et al. [38] showed that pretreatment Ki-67 index > 25% was as an independent predictor of pCR, especially in HER2⁺/ER⁻ patients.

In general, the predictive significance of Ki-67 index varies with studies, which might be because of lack of statistical verification in the cut-off values used in each study. Furthermore, as the ER⁺ patient groups usually have lower pCR rate irrespective of the baseline level of Ki-67, significant discrepancy in the pCR rate between high and low Ki-67 level patient groups is not easily observed. Nevertheless, it is indubitable that the combination of baseline Ki-67 level based on core needle biopsy with other pre-NACT evaluating biomarkers can provide additional predictive information for treatment response, which can assist in identifying patients who may benefit from NACT.

Evidently, both changes in Ki-67 level during NACT and post-treatment Ki-67 level in residual disease are effective predictors of long-term survival [3139]. Previous studies (Table 3) showed that the latter generally tends to have a stronger prognostic impact than the former [4041]. Keam et al. [34] investigated post-NACT Ki-67 level in 105 patients with TNBC and residual disease, and indicated that > 10% Ki-67 expression in residual disease was significantly associated with poor RFS (p = 0.0013) [34]. Guarneri et al. [42] built a prognostic model based on post-NACT node status, as well as Ki-67 expression level, and concluded that compared to low-risk (nodes negative and Ki-67 < 15%) and intermediate-risk (nodes positivity or Ki-67 ≥ 15%) patients, high-risk patients (nodes positive and Ki-67 ≥ 15%) show significantly higher probability of recurrence and death. It is noteworthy that the prognostic efficacy of the Ki-67 index might differ in various BC subtypes. von Minckwitz et al. [12] concluded that the evaluation of post-treatment Ki-67 provided more prognostic information than evaluation of pCR alone for ER⁺ patients. The DFS of ER⁺ patients who had low Ki-67 level (0%–15%) in residual disease was comparable to that of patients who achieved pCR, while post-NACT Ki-67 level > 35% was usually accompanied by higher risk of relapse and required subsequent adjuvant chemotherapy [40]. Furthermore, post-NACT Ki-67 expression has also been confirmed to be an independent prognostic indicator for DFS in HR-negative BC [43]. Similarly, Diaz-Botero et al. [44] and Montagna et al. [45] concluded that Ki-67 expression decreased during NACT correlated statistically with more favorable prognosis than high Ki-67 level retained before and after treatment, although the cut-off values for Ki-67 expression level were different (15% and 20%, respectively).

In conclusion, monitoring of Ki-67 expression level both at pre-treatment and post-treatment stages is necessary to guide subsequent treatment. However, the accuracy of previous conclusions might be affected by errors present in the process of pre-surgical coarse needle biopsy (CNB) and post-surgical pathological specimen slicing [46]; therefore, the pathological evaluation procedure must be further standardized, such that Ki-67 can be used as an effective prognostic indicator more reliably.

The Ki-67 expression level reflects the cell proliferative status, while the mechanism of endocrine therapy involves induction of stasis in the cell cycle; therefore Ki-67 analysis has the potential to predict treatment benefits of NET. In a study containing 158 patients who received pre-surgical antiestrogen treatment, Dowsett et al. [47] demonstrated that patients with high Ki-67 level after two weeks of NET revealed an obviously lower RFS than those with low Ki-67 level (p = 0.004), while pre-treatment Ki-67 did not show any prognostic effect. This suggested that short-term change in Ki-67 expression is a valid prognostic indicator of NET. Preoperative endocrine prognostic index (PEPI) score has been accepted as an effective prognostic model that incorporates post-treatment Ki-67 index, tumor histological stage, ER status, post-treatment residual disease size, and metastatic nodal status [48]. PEPI has been shown to be a prognostic marker that can better predict RFS for patients receiving NET, as a result of which some patients can avoid the post-surgical adjuvant treatment [49]. The relevance of PEPI in the context of RFSs has been investigated in a study of 203 patients enrolled in the IMPACT trial. The results showed that patients with pathological stage 1 or 0 and PEPI score 0 had the lowest recurrence rate of 10%, while patients with pathologic stage > 3 and PEPI score 3 showed the highest recurrence rate of 48% [50]. According to the results of The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial (n = 236, ER-positive), patients with PEPI = 0 showed a significantly lower recurrence rate than the patients in the PEPI > 0 group (3.7% vs. 14.4%, p = 0.014). However, 35 patients (2- to 4-week NET, Ki-67 > 10%) who were changed to receive NACT showed lower pCR rate (5.7%) than expected; therefore, this group of patients should urgently follow a new effective alternative treatment strategy [51].

Currently, the Ki-67 index is not being applied in routine clinical practice, as the consensus of rationale cut-off values for defining high versus low Ki-67 level has not been reached. A meta-analysis including 23 studies showed that the pooled HR for OS was significantly higher in the subgroup in which cut-off value of Ki-67 was > 25% than that in which the cut-off value was < 25%, which suggested that selecting a cut-off value > 25% might improve the prognostic prediction ability of Ki-67 [52]. The other disadvantage of using the Ki-67 index might be the lack of standardized measurement technique and the limited reproducibility of the study results. In conclusion, the usefulness of Ki-67 with discordant cut-off values in predicting the prognosis of patients in various molecular subtypes is still uncertain. To address this, more studies should be conducted to determine a standard method of accurately estimating the Ki-67 level.

It is well known that lymphocytes infiltrating tumors modulate the cancer cell killing effect of chemotherapy, and an increasing body of evidence supports the correlation between pre-treatment TIL level and pathological response to NACT (Table 4). High levels of T cells, especially of the CD8⁺ subtype, have been demonstrated to be predictive of higher pCR rates [53545556]. A meta-analysis involving 3,251 patients reported that higher pre-treatment TILs correlated well with higher pCR rate in TNBC and HER2⁺ BC, but not in ER tumors [57]. Tumors that contain more than 50% TILs are defined as lymphocyte-predominant breast cancer (LPBC). Patients with LPBC were confirmed to have higher chance of achieving pCR (p < 0.001) and usually yielded lower RCB class following NACT than non-LPBC patients (p < 0.001) [58]. Furthermore, the extent of elevation in TIL level in the process of NACT can be an indicator of tumor microenvironment response and may influence post-treatment tumor reduction [59].

With the exception of baseline TIL level, the presence of post-NACT TILs in residual disease can also reflect the response of the tumor immune microenvironment to chemotherapy, which can provide additional prognostic information. A study investigated the prognostic value of TIL level before, during, and after NAC in HER2⁺ BC; results suggested that stromal TIL level > 25% in residual disease had an adverse prognostic efficacy. However, no association between baseline TIL levels and long-term survival was observed [60]. For HER2-positive patients who received trastuzumab, the prognostic role of TILs has not been definitely ascertained, and further studies should be conducted to determine whether HER2-positive patients with higher TIL level can obtain survival benefit from trastuzumab therapy [5661]. Dieci et al. [62] observed that the higher post-treatment TIL subgroup showed significantly elevated 5-year metastasis-free survival (MFS) and OS rates for patients with TNBC. Pruneri et al. [63] showed that each 10% increase in TILs correlated significantly with better survival. Denkert et al. [28] confirmed that the predictive value of enhanced TIL level for NACT response was potent across all molecular subtypes. However, patients with luminal subtype BC and increased TIL infiltration showed poor prognosis, whereas patients with HER2 enrichment and TNBC tumor gained survival benefit from the high level of TILs. This supported the hypothesis that the cellular composition of immune infiltration in tumors is different among each molecular subtypes, which determines different clinical outcomes and NACT response [64]. On the other hand, the predictive efficacy of TILs on OS for patients with luminal subtype is opposite for TNBC and HER2⁺ BC, which may be explained by the resistance of luminal patients to adjuvant endocrine therapy induced by TILs, while TNBC and HER2⁺ BC patients with higher post-treatment TILs level have more chances of obtaining survival benefit from NACT. Furthermore, infiltrated T cells contain various subpopulations, such as CD8 and Foxp3 cells [65]; the presence of CD8⁺ T cells was associated with improved outcome, while the presence of Foxp3⁺ T cells limited anti-tumor immunity [666768]. Ladoire et al. [69] investigated the prognostic role of intratumoral CD8⁺/FOXP3⁺ ratio and observed significant association between CD8⁺/FOXP3⁺ ratio and RFS/OS, especially in the HER2-positive group. In conclusion, T cells in tumors can modulate the pathological response to NACT and have the potential to act as a surrogate endpoint for long-term outcomes. Therefore, evaluation of detailed tumor immune response will be important for improving prognostic prediction and screening immunotherapy candidates.

In current clinical practice, BC is divided into diverse intrinsic molecular subtypes using immunohistochemical analysis, including HR-positive subtype (luminal A and luminal B), TNBC, and HER2-positive subtype (HER2-enriched and luminal B/HER2⁺) [80]. The intrinsic subtype analysis has been demonstrated to significantly affect the prognosis of patients after NACT. Parker et al. [81] established a risk of relapse model combining intrinsic subtype and tumor size, which provides more prognostic and predictive information than other standard parameters. As has been reported previously, the survival benefit derived from NACT varied among different BC molecular subtypes. HR-positive patients have relatively more favorable outcome, although they are less responsive to NACT than TNBC and HER2⁺ tumors [82]. Indeed, it is widely accepted that patients with TNBC and HER2-enriched tumor showed higher pCR rates than luminal subtype [83848586]. Prat et al. [83] evaluated the recurrence risk based on BC subtype and confirmed the independent predictive and prognostic value of intrinsic subtype at diagnosis for patients after NACT. The correlation between pCR and survival is significant in partial patients based on molecular subtypes. Díaz-Casas et al. [84] reported longer EFS and OS in patients who achieved pCR, which was statistically significant only in patients with TNBC. In a study involving 13,939 patients, pCR was demonstrated to be significantly associated with 5-year OS in patients with luminal B, HER2⁺, and TNBC (93.0%, 94.2%, and 90.6%, respectively, HER2⁺ vs. TNBC, p = 0.016) [87]. Meyers et al. [88] reported high locoregional recurrence (LRR) rate in basal-like subtype patients on NACT, who should be offered stronger local therapy to improve prognosis. Similarly, Yoo et al. [85] and Haque et al. [87] separately concluded that the TNBC subgroup showed poorer survival than other subgroups after NACT, although pCR rate was highest in the group of patients with TNBC. The molecular subtype of breast tumor is an important biomarker in clinical practice for selecting NACT candidates who may benefit more from the treatment.