The Human Genome Project29 and its efforts to sequence the entire human genome were a milestone in clinical research and marked the end of the so-called pre-genomic era and the beginning of the post-genomic one. Analysis of DNA, RNA and protein sequence data is now an essential part of biomedical research30.

In breast cancer, these achievements have translated into a new way of thinking about neoadjuvant clinical trials. The key questions explored by neoadjuvant trials in the pre-genomic era mainly focused on the possibility of identifying active agents and predicting their success or on fine-tuning their administration schedules. Seminal research in this setting has led to milestones in today’s clinical practice such as the use of docetaxel in sequence with an anthracycline-based regimen instead of anthracycline-based chemotherapy alone31, the administration of weekly instead of 3-weekly paclitaxel32, and the superiority of adjuvant aromatase inhibitors compared to tamoxifen33,34.

All of these studies, conducted in the neoadjuvant setting, shared the peculiarity of being deemed ‘hypothesis generating’, with confirmatory results obtained subsequently in large adjuvant trials (Table 2). Neoadjuvant trials from the pre-genomic era had merit in that they addressed important clinical questions; however, many of these trials were characterized by disappointing results in the search for predictive biomarkers and, ultimately, a failure to identify effective biomarkers of response with the potential to enable the tailoring of treatment with taxanes35,36 and/or aromatase inhibitors33,37.

Table 2 Examples of hypothesis-generating trials and their related hypothesis-confirming trials

Neoadjuvant trials from the post-genomic era have maintained an important role in identifying new active agents worthy of further investigation in the adjuvant setting. An example of this role is provided by dual inhibition of HER2 with pertuzumab plus trastuzumab, which was the first treatment to receive FDA accelerated approval based on pCR results alone from a relatively small (n = 417) neoadjuvant study (NeoSphere)38. The effectiveness of this approach was later confirmed in a larger cohort (n = 4,805) in the adjuvant setting (APHINITY)21, resulting in full approval for use in both settings in December 2017. We believe that the KEYNOTE-522 trial marks the beginning of a new era in the history of neoadjuvant trials: this large (n = 1,174), phase III study led to the approval of neoadjuvant pembrolizumab in combination with chemotherapy for patients with high-risk, early-stage triple-negative breast cancer (TNBC) in July 2021, based on significant improvements in pCR and EFS, its two co-primary end points39,40. An important lesson had been understood: a pCR improvement on its own is not a reliable surrogate of survival and should be supported by a survival end point for regulatory approval. Beyond the important role of identifying new active agents, the neoadjuvant trials of the post-genomic era have prioritized another relevant objective: the deep dissection of disease biology and the search for clinically useful molecular markers of response.

Biomarkers and disease biologyHR-positive, HER2-negative breast cancer

Since the late 1980s, a strong preclinical rationale has supported the hypothesis that a short, preoperative course of endocrine therapy could improve outcomes in HR-positive early-stage breast cancer41. However, the choice of the best end point to predict patient outcomes after such therapy has been a topic of intense academic debate. Data from two small neoadjuvant studies, IMPACT42 and Z1031 (ref.43), showed that levels of the nuclear protein Ki67, assessed 2–4 weeks after starting neoadjuvant endocrine therapy, might better predict patient outcomes than Ki67 levels at baseline. In the POETIC trial44, 4,480 postmenopausal women with HR-positive, HER2-negative early-stage breast cancer were randomly assigned to receive either aromatase inhibitors or placebo for 14 days prior to surgery. Adjuvant treatment was permitted as per standard local practice. The POETIC trial failed to show any significant differences in treatment outcomes; importantly, however, patients with a low Ki67 level at baseline (<10%) and/or after 2 weeks of treatment had a significantly lower risk of disease recurrence compared with those with a high level at 2 weeks44, suggesting that ‘dynamic’ measurements of Ki67 levels could help select patients who might not need further treatment escalation (those showing substantial Ki67 suppression). For patients without a Ki67 response, current escalated treatments (such as chemotherapy) do not provide strong evidence of benefit; for example, the Z1031 trial showed no improvements in pCR for patients without a Ki67 response to short-term neoadjuvant endocrine therapy who subsequently received chemotherapy43. Interestingly, the WSG-ADAPT run-in trial for patients with HR-positive, HER2-negative disease45 confirmed that combining assessments of static (genomic recurrence score) and dynamic (endocrine proliferation response defined as a Ki67 level of ≤10% following induction) biomarkers is feasible and could guide individualized therapy decisions in patients with early-stage breast cancer. Unfortunately, these results were not available at the time of the initiation of several very large adjuvant trials investigating the addition of CDK4/6 inhibitors to endocrine therapy, namely PALLAS46 and MonarchE47, of which only the latter had positive results albeit in a patient population defined by a high disease burden rather than by insufficient sensitivity to endocrine therapy.

The addition of CDK4/6 inhibitors to endocrine therapy has also been explored in the neoadjuvant setting for patients with HR-positive, HER2-negative early-stage breast cancer. These trials revealed a low pCR rate (0–5%) but also demonstrated that CDK4/6 inhibitors induced more profound reductions in Ki67 levels from baseline to 2 weeks and to surgery48,49,50,51,52,53.

Next to Ki67 levels, the preoperative endocrine prognostic index (PEPI) score is another tool that allows patients to be selected as candidates for treatment escalation. The PEPI score is determined by summing the partial scores corresponding to pathological tumour and nodal stages, Ki67 levels, and ER status following neoadjuvant endocrine therapy and has been shown to predict recurrence-free survival in the context of the IMPACT trial54. In the ALTERNATE study55,56, women with locally advanced luminal breast cancer were randomly assigned to receive either anastrozole, fulvestrant or a combination of the two; the primary end point was endocrine-sensitive disease rate, defined as the proportion of patients with a PEPI score of 0 among all eligible patients who started neoadjuvant endocrine therapy. Of note, patients were removed from randomization if their 2-week Ki67 levels did not decline from baseline in response to endocrine therapy. Neither fulvestrant nor fulvestrant plus anastrozole significantly improved endocrine-sensitive disease rate compared with anastrozole alone56. Relapse-free survival data remain immature.

The search for gene expression signatures that might enable patients receiving endocrine therapy to safely avoid chemotherapy has been another area of intense research interest. Owing to the more widespread use of screening, which has modified the epidemiology of breast cancers, the question of de-escalation has become crucial. The risk of disease relapse can now be characterized by transcriptomic signatures, two of which (Oncotype-DX and MammaPrint) have proven clinical utility in the selection of patients who will have excellent outcomes on adjuvant endocrine therapy alone57,58,59.

The intrinsic tumour subtype is also being investigated as a potential predictive biomarker. In a retrospective, exploratory analysis of data from patients with luminal metastatic breast cancer from the MONALEESA phase III studies, all PAM50 intrinsic subtypes were associated with a consistent OS benefit with the addition of ribociclib to endocrine therapy, except for the basal-like subtype60. Similar data from patients with early-stage disease are not yet available.

Further complexity exists when questioning the optimal definition of ER positivity. Although 1% of cells staining ER positive on immunohistochemistry is considered the official cut-off for ER-positive breast cancer, a cut-off of 10% is often used to guide clinical decision-making, which is supported by literature reports indicating that tumours with 1–9% ER expression (so-called ER-low tumours) have similar clinical characteristics to those classed as ER-negative (<1%)61.

HER2-positive breast cancer

The discovery that 15–20% of breast cancers have an aggressive clinical course linked to overexpression of HER2 marked the beginning of an era of steady progress, with the registration of four HER2-targeted therapies in the early-stage disease setting62. Based on a growing understanding of both the HER2 signalling pathway and the mechanism of action of HER2-targeted therapies, translational research efforts have focused on identifying predictive biomarkers of response that are both directly related to HER2 itself (such as the ratio of ERBB2 copy number to CEP17 determined using FISH, polysomy, mRNA and protein expression) and go beyond HER2, namely evaluating other receptors and/or ligands (such as HER3, EGFR, EGF, IGFR), HER2 downstream signalling pathways (such as PIK3CA/PTEN or RhoA), features associated with the tumour stroma (such as tumour-infiltrating lymphocytes (TILs) or immune-related gene signatures), and other patient-specific variables (such as the presence of HER2 and/or Fcγ receptor polymorphisms). Unfortunately, none of these putative biomarkers has thus far reached clinical utility62, although some have generated early promising results, for example, in the WSG-ADAPT trial63,64.

Stratification based on tumour-intrinsic subtypes is another attractive strategy that could be incorporated into patient selection for treatment escalation or de-escalation. The PAM50-based HER2-enriched intrinsic subtype is associated with a higher likelihood of a pCR following neoadjuvant therapy65, and tools combining traditional clinical, pathological and molecular characteristics to better predict clinical outcomes in patients with early-stage HER2-positive breast cancer are currently in development66.

DECRESCENDO (NCT04675827) is an ongoing, single-arm, prospective trial testing de-escalation of chemotherapy for patients with HER2-positive, HR-negative and node-negative early-stage breast cancer. After neoadjuvant taxane-based chemotherapy and dual HER2 blockade, patients with a pCR will receive an additional 14 cycles of adjuvant pertuzumab plus trastuzumab but no further chemotherapy. This approach is based on the hypothesis that the HER2-enriched subtype (roughly 65–70% of the trial population) has ‘HER2-addicted’ tumours that are therefore good candidates for anthracycline omission. Nonetheless, an ambitious 3-year invasive recurrence-free survival of at least 94% will have to be demonstrated in this molecularly defined subpopulation for this trial to meet the primary end point.

Triple-negative breast cancer

TNBC has been traditionally considered a ‘targetless’ breast cancer subtype, for which chemotherapy has long been the only effective and available treatment strategy67. Nevertheless, advances in clinical research in the past decades have been changing the treatment landscape of TNBC, both in terms of chemotherapy options (such as the introduction of dose-dense regimens and platinum-based therapies in the neoadjuvant setting) and in terms of other therapies that are now available for patients with this tumour type, including PARP inhibitors, immune-checkpoint inhibitors and antibody–drug conjugates. Immune-checkpoint inhibitors, in particular, have revolutionized the treatment and clinical trajectory of several cancer types, and relevant efforts are being made to identify predictive biomarkers of response, including in breast cancer. PD-L1 (ref.68), TILs68, a high tumour mutational burden (typically ≥10 mutations per megabase)69, mismatch repair deficiency70, immune gene signatures71 and intrinsic molecular subtypes71 are under investigation, but, so far, no biomarkers have been proven to have a clear role in predicting response to immune-checkpoint inhibitors in early-stage TNBC. A further level of complexity derives from the existence of several different diagnostic assays, scoring algorithms, intersample and intrasample heterogeneity, and differences according to the site of evaluation (metastatic versus primary lesions)72. Furthermore, beyond tumour characteristics, several additional factors could affect the outcomes of patients receiving immune-checkpoint inhibitors73, including the general characteristics of the patient (including immune function74, obesity75 and/or the microbiota76). Thus, the search for biomarkers capable of predicting a response to immune-checkpoint inhibitors in patients with early-stage TNBC remains a huge challenge.

A portion of the tumours that were traditionally considered to be TNBCs match the definition of the so-called HER2-low category (namely those with a HER2 immunohistochemistry score of 1+ or 2+ without detectable amplification on FISH)77. Robust evidence so far supports the use of the HER2-targeted antibody–drug conjugate trastuzumab deruxtecan in these patients78, thus underlining that the traditional distinction between these three breast cancer subtypes might be too simplistic and unhelpful in certain scenarios given that the level of expression of both HER2 and hormone receptors is not binary but rather a continuous spectrum of expression, which intrinsically limits most attempts at classification.

Time to re-think trial design?Improving the end points of neoadjuvant trials

The establishment of an accelerated approval pathway for drugs designed to treat serious diseases that are also unmet medical needs dates back to the early 1990s and clearly addressed the need to expedite the entire process in the best interest of patients79,80. Such pathways typically rely on improvements on one or more surrogate end points to provide an early indication of efficacy. However, considerable academic debate exists on how and when a surrogate end point can truly be considered as ‘validated’. According to certain international guidelines81,82, surrogate validation should be based on robust correlation with survival end points, both at the study level and at an individual patient level.

As explained previously, pCR is only weakly correlated with OS at the study level28, thus questioning the role of pCR as a valid surrogate end point. Moreover, pCR appears to lose some of its prognostic power when used to assess the efficacy of drugs that are not chemotherapies. This limitation applies to immune-checkpoint inhibitors. For example, the GeparNUEVO study69,83 was a randomized, phase II study testing the addition of durvalumab to neoadjuvant chemotherapy for patients with early-stage TNBC. No significant improvement in pCR rate was observed69; however, after a median follow-up duration of 43.7 months, durvalumab was associated with a significant improvement in survival outcomes (3-year OS 95.1% vs 83.1%, hazard ratio 0.26, 95% CI 0.09–0.79; P = 0.0076)83. This observation might reflect that immune-checkpoint inhibitors have a different mechanism of action to that of cytotoxic agents, and, by activating an immune response against cancer cells instead of acting directly against them, they are able to produce more durable and, in some cases, delayed responses84,85,86. Furthermore, pCR might also not be the best end point for the evaluation of responses to neoadjuvant endocrine therapy. Compared with other breast cancer subtypes, HR-positive, HER2-negative tumours are characterized by lower pCR rates than neoadjuvant therapies and a simple distinction between pCR versus non-pCR might be not useful because few patients reach a pCR. Compared with other breast cancer subtypes, HR-positive, HER2-negative tumours are traditionally characterized by lower pCR rates than neoadjuvant therapies87. Thus, a dichotomous distinction between pCR and non-pCR could be an overly simplistic and inaccurate indicator of patient outcome, while the extent of residual disease could provide important additional information.

The issues with pCR justify a renewed interest in residual cancer burden (RCB). RCB is a continuous index based on the assessment of pathological measurements of the primary tumour (dimensions and cellularity fraction) and nodal metastases (number and size). Based on these parameters, the RCB index can be divided into one of four categories: RCB 0 (pCR), RCB 1, RCB 2 and RCB 3 (Fig. 1). The association between RCB and long-term outcomes has been robustly demonstrated in all breast cancer subtypes (HR-positive and HER2-negative, HER2-positive, and TNBC) independently of other clinical and/or pathological characteristics88. Evaluations of the RCB index should increasingly be adopted as a clinical end point in neoadjuvant trials involving patients with early-stage breast cancer.

Fig. 1: Residual cancer burden as an end point for neoadjuvant clinical trials.

Replacing pathological complete response (pCR) with a residual cancer burden (RCB) profile and event-free survival (EFS) is likely to improve the accuracy of data from neoadjuvant clinical trials as an early indicator of clinical benefit. RCB is a validated surrogate88,117 of longer-term survival outcomes (such as EFS) that is also more granular than dichotomous comparisons of pCR versus non-pCR, with improved survival durations often seen for patients with more limited residual disease. Thus, post-neoadjuvant trials should increasingly explore treatment strategies directed at addressing the residual disease profile.

Considerable debate currently exists regarding the role of survival end points. In the list of surrogate end points to be used for regulatory approval purposes released by the FDA in 2018, beyond pCR, DFS, EFS, objective response rate and PFS are all permitted89. The initial justification for this approach comes from the desire to expedite the approval process owing to the shorter time required for assessments based on these end points compared with OS, thus allowing patients with unmet medical needs earlier access to drugs that might be effective. However, the strength of the associations of these surrogate end points with OS is not well documented. In 2020, Gyawali et al. explored the underlying evidence for these surrogate end points90, showing that considerable variability exists and that, in some scenarios, the association with OS is either weak or absent.

With the exponential increase in cancer drug costs in the past years, agreeing on a ‘prioritization’ list based on the real added value of such drugs is a legitimate response. This much-needed but difficult exercise has been conducted by ASCO, with the development of the ASCO Value Framework91, and by ESMO with the development of the ESMO Magnitude of Clinical Benefit Scale (MCBS)92, which values improvements in OS and/or quality of life far more heavily than improvements in ORR or extension of PFS. Interestingly, DFS has been the subject of an intense debate, leading to a reasonable consensus: living additional months or years without detectable disease has greater value than living somewhat longer with stable, non-progressive disease; thus, improvements in median DFS of sufficient magnitude are credited with high scores on the ESMO-MCBS until OS data become available, at which point a lack of OS benefit induces a one-point downgrade (ESMO-MCBS version 2.0 will soon be published93). Although the scale does not include a dimension of ‘cost’, its application might help health authorities to prioritize drugs that more strongly warrant financial investment.

The post-neoadjuvant setting: a promising scenario gaining in popularity

Among the several advantages provided by the neoadjuvant approach is the possibility of selecting patients for treatment escalation or de-escalation based on their response to neoadjuvant therapy. Despite imperfect surrogacy, patients who do not have a pCR generally have worse long-term survival outcomes than those with a pCR after the completion of neoadjuvant therapy27. Hence, the post-neoadjuvant setting is an attractive scenario for the design of pivotal clinical trials as it involves selecting patients with high-risk residual invasive disease at surgery who might be candidates for treatment escalation. Furthermore, compared to the traditional adjuvant setting, such trials are less likely to enrol patients already cured by standard therapy (Fig. 2). Two systemic post-neoadjuvant treatments for patients with residual invasive disease at surgery are already approved for use in clinical practice: capecitabine for patients with TNBC, based on the results of the CREATE-X trial94, and trastuzumab emtansine (T-DM1) for patients with HER2-positive disease, based on the results of KATHERINE95.

Fig. 2: The post-neoadjuvant setting: an attractive scenario for future clinical trials.

The post-neoadjuvant setting offers the possibility of selecting patients with residual invasive disease at surgery who might benefit from additional adjuvant treatments (white text box) and also the avoidance of enrolling patients who are not likely to benefit from further therapy owing to a complete response to neoadjuvant therapy. Moreover, this approach allows translational analyses to be performed on the residual tumour material, thus enabling potential biomarkers to be identified (such as Ki67, tumour-infiltrating lymphocytes (TILs), circulating tumour DNA (ctDNA), and/or genetic and genomic alterations) (sepia text boxes), which could subsequently be validated prospectively. LVI, lymphovascular invasion; NGS, next-generation sequencing.

In the CREATE-X trial, 910 patients with early-stage, HER2-negative (HR-positive or HR-negative) breast cancer and residual disease after the completion of neoadjuvant chemotherapy were randomly assigned to either 6–8 cycles of capecitabine or to no treatment. In the overall population, post-neoadjuvant capecitabine resulted in significant improvements in invasive DFS (hazard ratio 0.70, 95% CI 0.53–0.92; P = 0.01) and OS (hazard ratio 0.59, 95% CI 0.39–0.90; P = 0.01), respectively, with a greater magnitude of benefit in the TNBC subgroup (n = 286) (hazard ratio 0.58, 95% CI 0.39–0.87 for invasive DFS, and hazard ratio 0.52, 95% CI 0.30–0.90 for OS, respectively)94. In the KATHERINE trial, 1,486 patients with early-stage, HER2-positive breast cancer and residual disease at surgery, after neoadjuvant chemotherapy and HER2-targeted therapy, were randomly assigned to receive either T-DM1 or trastuzumab for 14 cycles. T-DM1 was associated with a significant improvement in invasive DFS (hazard ratio 0.50, 95% CI 0.39–0.64)