Breast cancer is the most common type of cancer in women and is an important public health problem worldwide (1). Although significant improvements in survival have been achieved in recent years thanks to the advances in systemic treatments, metastatic disease still remains a serious problem affecting prognosis. The main treatment options for metastatic disease have historically been chemotherapy, targeted therapies, and hormone therapy, but in recent years cyclin-dependent kinase 4/6 inhibitor (CDK 4/6i) and immunotherapy have been added to these (2, 3). Breast cancer is classified into three main subgroups according to hormone receptor (HR) (estrogen receptor (ER) and progesterone receptor (PR)) status and human epidermal growth factor 2 (HER2) status: HR+ group, HER2+ group and triple negative group (4). The HR+/HER2- group constitutes approximately two-thirds of patients with metastatic breast cancer (MBC) (5). In this group, endocrine therapy (ET) constitutes the main framework of treatment (6). Aromatase inhibitors (AI) (Anastrozole, Letrozole, Exemestan, etc.), selective estrogen modulators (Tamoxifen), and selective estrogen degraders (Fulvestrant) are widely used as ET (7–9). However, intrinsic or acquired resistance is also encountered in endocrine treatments (10). In order to overcome this problem, combinations with targeted agents, especially CDK 4/6i, have been sought in recent years.

By inactivating the CDK-D-cyclins (CCND) complex, CDK 4/6i increase retinoblastoma protein (pRb), which negatively affects the E2F transcriptional factor, and ultimately induces tumor cell apoptosis by inhibiting cell cycle progression (11). In the vast majority of phase III trials combining CDK 4/6i and ET (AI (Anastrozole, Letrozole) and selective estrogen degraders (Fulvestrant)), progression free survival (PFS) and overall survival (OS) have been significantly improved in the front line as well as in subsequent lines of therapy. Thus, CDK 4/6i have become the main treatment model in the HR+ HER2- patient group with their unique mechanism of action, consistent survival advantages in phase III studies, and different toxicity characteristics (12–14).

It is known that the estrogen signal, which has a fundamental role in breast cancer, has a significant effect on the Cyclin D1-CDK4/6-RB1 complex. This constitutes the rationale for combination therapy based on inhibition of this interaction by combined with ET and CDK 4/6i (15, 16). In this direction, the first studies were carried out in patients with HR+/HER2- MBC (12–14). In general, those with ER positive staining percentage >1% in the pathology material are defined as endocrine sensitive, but those with 1-9% ER+ are called ERlow positive according to the American Society of Clinical Oncology/College of American Pathologists Guideline (ASCO/CAP). However it is well known that these tumors often gain little benefit from ET (17–20). Therefore, in studies evaluating the combination of ET and CDK 4/6i, patients with an ER+ of 10% and above were included (12–14). In these studies, patients were grouped as ER+/PR+ and ER+/PR- according to their HR+, but the percentage of HR positivity was not further categorized in terms of endocrine sensitivity.

On the other hand, when the meta-analysis of the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) is evaulated it is observed that ER positivity is categorized and the benefit from ET is highest especially in the group with ER+>50% (18). In different studies, it has been reported that the therapeutic effect and survival are correlated with the rate of ER positivity in breast cancer patients who is receiving ET (19–22). Similarly, there is information regarding the predictive and prognostic role of a high percentage of predictive biomarker positivity in other tumor groups. For example, in the TOGA study, in which the addition of trastuzumab to systemic chemotherapy in metastatic gastric cancer was investigated the most benefit was observed in the group with immunohistochemically (IHC) HER2-positive 3+/in-situ hybridization (ISH)+ with proportionately less benefit in the IHC 2+/ISH+ and IHC1+/ISH+ subgroups (23). Similarly, it has been reported that the percentage of ALK-positivity in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) is correlated with response and survival (24).

Therefore, when all these results were evaluated together, we suggested that the percentage of hormone positivity in patients receiving CDK 4/6i together with ET may affect the response and overall outcome.

In this study, it was determined that the percentage of baseline HR positivity affected the prognosis in patients with HR+ HER2- MBC treated with CDK 4/6i in combination with an ET. It was observed that the group with ER strong positive/PR moderate or strong positive before treatment was the best prognostic group, followed by the ER moderate positive/PR moderate or strong positive group, followed by the ER moderate positive/PR negative group with a decreasing rate.

One of the most basic features of cancer is the loss of control in cell cycle regulation (28). Normally, the transition from G1 to S phase in the cell cycle is controlled by the Rb gene, through the sequestration of the E2F family transcriptional factor. CDK 4/6, on the other hand, inactivates the Rb gene by forming a complex with D-type cyclins, thus inducing the transition from G1 to S phase (11). Since Cyclin D1 is a direct transcriptional target of estrogen, it has been reported that ER+ tumor cells are particularly dependent on CDK 4/6 activation in cell proliferation (29). In addition, it has been reported that Cyclin D1 amplification is widely observed (29 to 58%) in ER+ breast cancer (30). All these features have paved the way for clinical trials with CDK 4/6i in patients with HR+ HER2- breast cancer. In this sense, studies were conducted in which palbociclib, ribociclib and abemaciclib were combined with ET (12–14). It is observed that these studies were primarily carried out in postmenopausal patients with HR+ HER2- MBC, followed by premenopausal patients, and also studies on its use in the adjuvant setting. In studies in the postmenopausal group, AI or Fulvestrant was used as ET in both metastatic and adjuvant periods; In studies in the premenopausal group, it is seen that tamoxifen or AI is used together with the LHRH analogue. In all these studies, the addition of CDK 4/6i to ET has been shown to contribute significantly to survival and has become one of the main treatment models for MBC.

To date studies of CDK4/6i with ET have been conducted in postmenopausal and premenopausal patients, both in the metastatic and adjuvant settings. In postmenopausal women, CDK4/6i is generally combined with AI or fulvestrant while in premenopausal women, CDK4/6i can be partnered with tamoxifen or AI with LHRH analogue. Across most studies CKD4/6i combined with ET has been shown to improve outcomes. Most Phase III studies with CDK4/6i have included patients with ER+ ≥ 10% and PR+/- (12–14). As a result, the benefit of adding CDK 4/6i to ET was demonstrated in the whole group, and subgroup analysis was also performed according to PR positivity versus negativity. And although there are differences in both analysis and results between studies, the ER+/PR+ group appears to benefit more than the ER+/PR- group. And also in the Paloma-2 study, patients were not stratified by PR positivity however they were in the Monaleesa-2 study (31, 32). In the latter study, a smaller benefit was seen in the PR+ patients when compared to the PR- patients, though the small PR- sample size may have confounded these results. On the other hand, in the Monarch-3 study no significant difference was observed in terms of PR status (33).

We also reviewed the impact of PR positivity or negative in second lines studies of CDK4/6i paired with fulvestrant. In the Paloma-3 study, the role of PR +/- was not found to impart a significant difference.

And neither the efficacy of fulvestrant plus palbociclib nor the likelihood of disease progression more than 6 months after study entry were significantly associated with the level of expression of estrogen or progesterone receptors (HR: 0.32 vs 0.54, respectively) (34). And also in the Paloma-3 study, it was also stated that endocrine sensitivity was a prognostic factor in favor of CDK 4/6i in patients (35). In the Monaleesa-3 and Monarch-2 studies, no significant difference was found between the ER+/PR+ group and the other group (including PR- patients), as well as between the PR+ group and the PR- group, respectively (36, 37). In the Monaleesa-7 study in premenopausal patients receiving CDK4/6i with ET and ovarian function suppression (OFS) no difference was observed between the ER+/PR+ group and the other group (38). In a meta-analysis that included of all phase III studies on CDK 4/6i in metastatic disease, no significant difference was found between the groups in terms of PR status (39). On the other hand, in adjuvant studies of CDK4/6i, such as the MONARCH-E study, benefit was found independent of PR status, although more benefit was obtained in the PR+ group than the PR- group (HR 0.73 vs 0.81) (40).

Many studies in the literature suggest that patients with ER+/PR- tumors have a worse prognosis and higher risk of recurrence than ER+/PR+ tumors (41). In Gharib KE, et al. (42), predictive and prognostic factors were investigated in patients with MBC receiving palbocicilib and letrozole. They reported negative prognostic and predictive features including liver metastases, line of treatment, and absence of PR. The median PFS in PR+ vs PR- groups was 20.05 months vs 12.99 months (p= 0.046) (42).

Similarly, Canino F, et al. (43) evaluated the prognostic role of the intrinsic subtype detected by PAM50 in patients with HR+/HER2- MBC. As a result, it has been reported that the response to ET is low and the prognosis is worse in non-luminal subtypes. In addition, they stated that the response to endocrine therapy was significantly lower in patients whose non-luminal subtype was detected not from the primary tumor but from the metastatic area (43). In a biomarker study conducted in the intrinsic subtypes of the Monaleesa studies, it was stated that the addition of ribociclib to endocrine therapy contributed significantly in all subgroups except the basal-like group. Compared to the luminal A group, the risk of disease progression was found to be 1.44, 2.31 and 3.96 times higher in the Luminal B, HER2-enriched and basal-like groups, respectively (44). Although no separate analysis was performed in the Luminal B group according to PR percentage or Ki-67 index in this analysis, the high risk of progression in this group compared to Luminal A may be an indirect indicator of the importance of hormone receptor positivity.

It has also been postulated that the level of ER expression has a prognostic role in patients with breast cancer undergoing ET (19–22). In Yoon KH et al. patients with ERlow (ER 1-9%) benefitted less fromendocrine therapy and had a significantly higher risk of recurrence compared to the ERhigh group (20). Apart from this, the percentage of hormone receptor positivity as well as its presence or absence have prognostic importance, and in a study conducted by Bae et al., single HR+ tumors without HER2 overexpression (ER + PR-HER2- or ER-PR + HER2-) were found to be of prognostic importance. has been shown to have a poorer survival rate than triple-positive tumors, and this group even has a poorer prognosis comparable to triple-negative breast cancer (45). On the other hand, in the EBCTCG meta-analysis, it was observed that ER positivity was categorized and the benefit from endocrine therapy was highest especially in the group with ER+>50% (18). Similarly, in the P024 study, in which Letrozole and TMX were compared in neoadjuvant therapy in patients with ER+/PR+ breast cancer, patients with ER≥10% positive were included and a linear correlation was reported between ER expression levels and response (46). However, studies investigating the role of hormone receptor levels in the effectiveness of CDK 4/6i are limited in the literature.

Of these, Shikanaj A et al. (47), clinicopathological factors associated with efficacy in patients with MBC were analyzed. As a result, it was reported that tumor grade in the primary lesion and initial neutrophil/lymphocyte ratio (NLR) were associated with efficacy, while expression levels of hormone receptors had no significant effect. In this study, patients were divided into “high” and “low” groups according to the proportion of cells staining positive for ER and PR, and the cut-off value was taken as 66% for this distinction. Patients in which both ER and PR were expressed over 66% were termed the “high” group. However, while separate risk groups were defined for ER and PR, risk groups formed by combining the two were not defined (47). In our study, besides defining separate risk groups for ER and PR according to hormone receptor expression levels, combined risk groups were formed by combining these two. Thus, the prognostic risk groups were better defined and it was determined that the group with ER ^{strong positive}/PR ^{moderate or strong positive} was the best prognostic group. This was followed by the ER^{moderate positive}/PR^{moderate or strongly positive} group, followed by the ER^{moderate positive}/PR^negative group in a decreasing fashion. In our study, the positive effect on the prognosis, especially of being ER-strong positive, was more remarkable. In this sense, it was observed that both the response and survival were better in the ER-positive group, even if it was PR-negative. Therefore, our results, although not statistically significant, pointed out the prognostic importance of a high ER positivity. In this respect, it can be thought that our work has a different originality. In the above-mentioned Monaleesa-2 study, the fact that the strong positivity of the ER in both groups was not fully known may also have a role in the lower benefit observed in the PR+ group compared to the PR- group.

In line with all these studies, cyclin D1 is a direct transcriptional target of estrogen and therefore it is known that patients with high hormone receptor expression may obtain more benefit from CDK4/6i when combined with ET.

Our study has some limitations, such as its relatively small sample size, reflecting a single center experience, and retrospective design.

In conclusion, the results of this study suggest that the percentage of HR positivity may have a predictive and prognostic role in patients with HR+ HER-2- MBC who received CDK 4/6i with ET. As far as we know, our study is one of the few studies in the literature conducted with CDK 4/6i in breast cancer patients. We believe that the percentage of hormone receptor positivity and especially the strong positive ER should be taken into account in defining the patient group who will benefit more from the treatment in patients treated with ET plus CDK 4/6i. Our results are hypothesis generating and more comprehensive studies may be needed to further elucidate our findings.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Non-Invasive Research Ethics Committee of Dokuz Eylul University Faculty of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

MK: Formal analysis, Methodology, Writing – original draft, Writing – review & editing. HSS: Writing – original draft, Writing – review & editing. TY: Supervision, Writing – original draft, Writing – review & editing. IO: Supervision, Writing – original draft, Writing – review & editing.