Hormonal-Receptors-Positive and HER2-Negative Patients with Metastatic Breast Cancer Treated with First-Line Palbociclib and Hormonal Therapy: Impact of First-Cycle Neutropenia and Dose Reduction on Therapeutic Outcome

Background CDK 4/6 inhibitors with hormonal therapy are the standard first-line therapy in metastatic hormonal receptors (HR)-positive and HER2-negative breast cancer. This study aims to assess the impact of neutropenia with 1st cycle, dose reduction, HER2-low status, and other clinicopathological factors on survival outcomes with the first-line palbociclib and hormonal therapy. Patients and Methods. In this retrospective study, we recruited patients with metastatic HR-positive and HER2-negative breast cancer. Neutropenia with 1st cycle, palbociclib dose reduction in addition to different clinicopathological and survival data were checked in patients' medical records. Survival outcomes were compared according to the abovementioned factors. Results We recruited 150 patients who received first-line palbociclib with hormonal therapy. 86% of patients developed 1st cycle neutropenia which was more common in patients with high Ki67. Dose reduction was recorded in 46.7% of patients and it was more common in patients with higher Allred scores (scores 7–8). The median progression-free survival (PFS) of the study group was 22 months. No significant difference was observed in PFS according to the 1st cycle of neutropenia or grade of neutropenia. Similarly, no difference in PFS according to palbociclib dose reduction and HER2 low status was observed. Only the Allred score and having a single site of metastasis had an independent significant relation with PFS. The median overall survival (OS) of the study group was 39 months. No significant difference was observed in OS according to the 1st cycle neutropenia, grade of neutropenia, palbociclib dose reduction, and HER2-low status. Only the Allred score and having a single site of metastasis had an independent significant relation with OS. In addition, no difference was observed in PFS and OS according to ECOG PS (2 vs. 0-1) or menopausal status. Conclusion No significant impact of the 1st cycle neutropenia, dose reduction, having ECOG PS2, menopausal status, or HER2 low status on survival outcome was observed. Survival outcome was significantly better in patients with single metastatic sites and higher ER-Allred scores.

Background.CDK 4/6 inhibitors with hormonal therapy are the standard frst-line therapy in metastatic hormonal receptors (HR)-positive and HER2-negative breast cancer.Tis study aims to assess the impact of neutropenia with 1st cycle, dose reduction, HER2-low status, and other clinicopathological factors on survival outcomes with the frst-line palbociclib and hormonal therapy.Patients and Methods.In this retrospective study, we recruited patients with metastatic HR-positive and HER2-negative breast cancer.Neutropenia with 1st cycle, palbociclib dose reduction in addition to diferent clinicopathological and survival data were checked in patients' medical records.Survival outcomes were compared according to the abovementioned factors.Results.We recruited 150 patients who received frst-line palbociclib with hormonal therapy.86% of patients developed 1st cycle neutropenia which was more common in patients with high Ki67.Dose reduction was recorded in 46.7% of patients and it was more common in patients with higher Allred scores (scores 7-8).Te median progression-free survival (PFS) of the study group was 22 months.No signifcant diference was observed in PFS according to the 1st cycle of neutropenia or grade of neutropenia.Similarly, no diference in PFS according to palbociclib dose reduction and HER2 low status was observed.Only the Allred score and having a single site of metastasis had an independent signifcant relation with PFS.Te median overall survival (OS) of the study group was 39 months.No signifcant diference was observed in OS according to the 1st cycle neutropenia, grade of neutropenia, palbociclib dose reduction, and HER2-low status.Only the Allred score and having a single site of metastasis had an independent signifcant relation with OS.In addition, no diference was observed in PFS and OS according to ECOG PS (2 vs. 0-1) or menopausal status.Conclusion.No signifcant impact of the 1st cycle neutropenia, dose reduction, having ECOG PS2, menopausal status, or HER2 low status on survival outcome was observed.Survival outcome was signifcantly better in patients with single metastatic sites and higher ER-Allred scores.

Background
Endocrine therapy is a cornerstone in the management of hormonal receptors (HR)-positive and HER2-negative breast cancer [1].However, de novo and acquired endocrine resistance remains a substantial problem as up to 30% of HR-positive breast tumors do not respond to endocrine therapy.Several mechanisms of resistance to endocrine treatment have been elucidated and recent therapeutic strategies to overcome resistance were approved for clinical use [2].
CDK 4/6 generates a complex with cyclin D, which in turn allows for the phosphorylation and inactivation of the retinoblastoma protein (Rb), a tumor suppressor protein, enabling gene transcription [3].Ultimately, this results in the progression of a cell from the G1 to the S phase of the cell cycle and eventually leads to cell division [3].CDK 4/6 inhibitors are a novel class of therapeutics that specifcally target CDK 4/6 and block the phosphorylation of Rb [4,5].
Note that the outcome of patients who receive frst-line CDK4-6 inhibitors with hormonal therapy is variable and there are no established biomarkers to predict the beneft of this therapy apart from ER-positivity [6].HER2-low status recently emerged as a distinct entity which is the target of a new class of antibody-drug conjugate (ADC) such as trastuzumab deruxtecan.It is still unclear whether there is a diferential outcome with CDK4-6 inhibitors in HER2-low vs. HER2-negative patients [10].
Te present study aims to assess the impact of the development of neutropenia (with 1st cycle) and dose reduction in addition to diferent clinicopathological factors such as ECOG PS and HER2 low status, on the outcome of the frstline therapy of palbociclib with hormonal therapy in HRpositive/HER2-negative metastatic breast cancer patients.

Study Design.
In this retrospective study, patients with HR-positive and HER2-negative metastatic breast cancer, who received palbociclib with hormonal therapy in the frst line, were recruited.Neutropenia with 1st cycle, palbociclib dose reduction in addition to diferent clinicopathological and survival data were checked in patients' medical records.Neutropenia is described as an abnormally low number of neutrophils in blood graded as grade 1 (1500-2000), grade 2 (>1500−1000), grade 3 (<1000−500), and grade 4 (<500).Survival outcomes were compared according to the abovementioned factors.Progression-free survival (PFS) is defned as the time from the date of starting palbociclib with hormonal therapy till the date of the frst disease progression or death (any cause).Overall survival (OS) is defned as the time from the date of starting palbociclib with hormonal therapy to the date of patient death, due to any cause, or to the last date at which the patient was known to be alive.

Study Population.
We recruited patients with histologically confrmed invasive breast carcinoma with HRpositive and HER2-negative phenotype and clinical or radiological evidence of metastasis.Patients should have ECOG PS 0-2 and received the frst-line therapy with palbociclib in addition to hormonal therapy.Tis treatment should have started between 1/1/2018 and 31/12/2020.Only patients with complete medical records were included.

Assessment Methods and
Procedures.Eligible patients were checked through hospital medical records.For each patient the following data were collected: age and menopausal status at diagnosis, date of relapse after an early breast cancer diagnosis, if applicable, date of diagnosis of metastatic disease, site(s) of metastasis, ER status, percentage and staining intensity, Allred's score, PR-status, percentage and staining intensity, Ki67% (by manual method), and HER2 status by immunohistochemistry (IHC) with FISH confrmation in cases with (++) by IHC.HER2 low status was defned as HER-2 +1 or HER-2 +2 with a negative FISH.In addition, treatment-related data were collected including the type of hormonal therapy used with palbociclib, neutropenia after the frst treatment cycle of palbociclib with hormonal therapy, and grade of neutropenia in addition to palbociclib dose reduction (if any).Te date of progression after the frst-line systemic therapy with palbociclib and hormonal therapy in addition to the date of death (if applicable) was checked.

Statistical Analysis. SPSS version 17
was used for statistical analysis.Categorical data were presented as percentages and diferences in the distribution of categorical variables according to factors of interest which were assessed using the chi-square test.Survival data were presented by the Kaplan-Meier method and diferences in survival were evaluated using the log-rank test.Factors with signifcant survival benefts in univariate analysis were assessed in multivariate analysis using the Cox regression method.As a general rule, an alpha value of 0.05 will be set as a priori for all two-tailed comparisons.

Baseline Characteristics.
We recruited 150 patients who were eligible for the study within the specifed timelines.All patients were ER-positive, 84.7% were PR-positive, 44.6% were HER2-low, and 40% had Ki67 >20%.In addition, 52% were premenopausal, 26% had ECOG PS2, and 64% had stage IV disease at the time of diagnosis.
Note that 86% of patients developed neutropenia after the frst cycle.We assessed diferent clinicopathological factors in those who developed neutropenia after the 1st cycle compared to those without neutropenia.First-cycle neutropenia was more common in patients with high Ki67 than in patients with low Ki67 (93.3% vs. 82.6%,p � 0.β02).However, other factors were balanced between the two groups (Table 1 Te Breast Journal 3 (0-1 vs. 2) or menopausal status was found.Similarly, no signifcant diference was observed in dose reduction according to other factors (Table 2).
Te multivariate analysis shows that among variables such as Allred score, PR-status, Ki67 level, bone-only metastasis, and number of metastatic sites (single vs. multiple), only Allred score (p � 0.015) and having a single site of metastasis (p � 0.006) had an independent signifcant relation with PFS (Table 4).
Te median OS of the study group was 39 months.No signifcant diference in OS in those without neutropenia compared to patients who developed neutropenia with the frst cycle (no: NR vs. yes: 39.0 months, HR 0.93, 95% CI: 0.33-2.61,p � 0.89) (Figure 4) was observed.
Te multivariate analysis shows that among variables such as Allred score, PR-status, bone-only metastasis, number of metastatic sites (single vs. multiple), and palbociclib-dose reduction, only having a single site of metastasis (0.04) and Allred score (p � 0.026) had an independent signifcant relation with OS (Table 5).

. Discussion
CDK4-6 inhibitors with hormonal therapy are currently the standard of care in the frst-line therapy of advanced HRpositive and HER2-negative breast cancer.A coupling of toxicity with treatment efcacy has been reported with several medications.Tis provides the rationale to explore the outcome of frst-line palbociclib with hormonal therapy according to the development of neutropenia, grade of neutropenia, and palbociclib-dose reduction.
Neutropenia is the most common side efect of this class.Neutropenia was reported in 86% of patients in our study which is comparable to palbociclib data reported in PAL-OMA 2 trial.In PALOMA 2 trial, the most common hematological toxicity is neutropenia followed by leukopenia accounting for around 79% and 39%, respectively [9].Note that, in our analysis, neutropenia was more frequent in patients with higher Ki67 levels which may be explained by the increased palbociclib activity in patients with rapidly proliferating tumors, which may be translated to more drugrelated toxic efects.
Dose reduction was reported in about one-third of the cases in clinical trials which is commonly associated with grade 3-4 hematological toxicity and diarrhea [11].Dose  Te Breast Journal reduction in our study occurred in around 46% of patients, which is higher than that reported in other real-world data.In real-world data from the USA, dose reduction was reported in 20.1% of patients after a median of six cycles [12], while in another report from the Netherlands, 33% had a dose reduction [13].However, these studies included a higher number of patients (763 and 598 patients, respectively) compared with 150 patients only in our study.Meanwhile, the higher frequency of palbociclib-dose reduction in our study may be explained by several factors such as including patients with ECOG PS2 and patients receiving previous chemotherapy (47%).However, it is not clear if racial factor plays a role in this context.
Note that, survival outcomes reported in our study are comparable with that reported in clinical trials [9] pointing out that a higher rate of dose reduction in this study did not impact therapeutic outcomes.Tis may be reassuring that dose reduction when indicated does not compromise treatment efcacy.A similar fnding was reported in the abovementioned Dutch study with no impact of dose reduction on clinical outcomes [13].
In our retrospective study, around 26% of patients had ECOG PS2.Tose patients were almost excluded from clinical trials [9].We reported no diference in survival outcome compared to patients with ECOG PS 0-1.Similarly, no diference in the frequency of neutropenia or dose reduction according to PS status was observed.Our fndings are consistent with the results of the systemic review of CDK4/6 inhibitors which showed similar PFS and OS in patients with ECOG PS2 compared with PS 0-1 [14].Tis may point to the feasibility of palbociclib with hormonal therapy in patients with ECOG PS2.
Recently, there is growing interest on the HER2-low category of breast cancer.Tis category derives a clinically meaningful beneft from new ADCs such as trastuzumab deruxtecan [15,16].In our study, no diference in palbociclib/hormonal therapy outcome in HER-2-low compared with HER2-negative patients was observed.However, the outcome of HER2-low breast cancer with CDK4/6 inhibitors is conficting and not consistent in the available reports.In 8 Te Breast Journal a retrospective analysis of 186 patients with Her2-low breast cancer who received CDK4/6 inhibitors in a single institution, there was no diference in survival outcome compared with HER2-negative patients [17].However, in another retrospective study including 106 women, PFS was inferior in HER2-low compared with HER2-negative patients (8.9 vs. 18.8 months, respectively, p � 0.014) [15].Tis may raise the importance to have data reported from a higher number of patients to elucidate the exact outcome of HER2-low breast cancer with CDK4-6 inhibitors.
In concordance with the results of PALOMA 3 and MONARCH 2 [18,19], we reported similar survival outcomes in premenopausal and postmenopausal patients.Tese studies included a subgroup of premenopausal patients who had survival benefts from CDK 4-6 inhibitors with hormonal therapy compared with hormonal therapy alone, which is comparable to the beneft in postmenopausal patients.Tis fnding was confrmed in a meta-analysis including 9 phase 2 and 3 studies which reported similar benefts from CDK4-6 inhibitors in both premenopausal and postmenopausal patients [11].
In the current study, patients with solitary sites of metastasis had signifcantly better PFS and OS compared to those with multiple sites of metastases.Note that, in PAL-OMA 2 study, the beneft of palbociclib was consistent in patients with single and multiple sites of metastasis [9].Similarly, in MONALEESA 3 study, the survival beneft of ribociclib was consistent irrespective of the number of metastatic sites (<3 vs. ≥3 sites) [20].More prominent survival benefts in patients with a single metastatic site in our report (with no comparator arm) may be explained by the better prognostic outcome in those patients.
In the current report patients with higher ER-Allred scores (who are more endocrine sensitive), had better survival outcomes than those with lower scores (Allred score 7-8 vs. ≤6).In the subgroup analysis of the PALOMA 3 study, the OS beneft of adding palbociclib to hormonal therapy was more pronounced in patients with sensitivity to previous endocrine therapy [18].Nevertheless, accumulating data suggested that patients with a low expression of ER receptors (1%-10%) have a low response to hormonal therapy and behave biologically like triple-negative breast cancer [21].
Tis study has some limitations including a relatively small number of patients, lack of a control arm, and being a single institute study.However, the study provides realworld evidence of the activity and safety of frst-line palbociclib which included some populations of patients not included in clinical practice such as patients with ECOG PS2.
In conclusion, the survival outcome with frst-line therapy of palbociclib with hormonal therapy was comparable with previous reports of palbociclib in clinical trials.No signifcant impact of 1st cycle neutropenia, dose reduction, having ECOG PS2, menopausal status, or HER2 low status on survival outcome was observed.Meanwhile, survival outcome was signifcantly better in patients with single metastatic sites and higher ER-Allred scores.

Table 1 :
Baseline clinicopathological factors and relation to neutropenia after 1 st cycle.
). Dose reduction was recorded in 46.7% of patients.Dose reduction was more common in patients with higher Allred scores (scores 7-8) than in patients with lower Allred scores (score ≤ 6) (50.0% vs. 29.2%,respectively, p � 0.048).No diference in dose reduction level according to ECOG PS 2 Te Breast Journal

Table 2 :
Discussing baseline tumor factors and relation to hormonal therapy interruption in regard to palbociclib dose reduction.
the median PFS of the study group was 22 months.No signifcant diference in PFS in those with neutropenia compared to patients who did not develop neutropenia with the frst cycle (yes: 22.0 vs. no: 24 months, HR 1.13, 95% CI: 0.54-2.36,p � 0.75), (Figure

Table 3 :
Discussing baseline tumor factors and relation to hormonal therapy interruption in regard to HER2-low.