Racial disparities in neutrophil counts among patients with metastatic breast cancer during treatment with CDK4/6 inhibitors

The three CDK4/6 inhibitors (CDK4/6i) approved for use in HR-positive/HER2-negative metastatic breast cancer (MBC), palbociclib, ribociclib, and abemaciclib, are generally well tolerated; however, neutropenia is a common toxicity. Within the general population, neutropenia has been shown to be more common in individuals of African descent. The landmark CDK4/6i trials in MBC lacked racial diversity in their patient populations. We aimed to assess the toxicity profiles of CDK4/6is in a racially diverse population. We conducted a retrospective study at Montefiore Medical Center in patients with HR-positive/HER2-negative MBC prescribed CDK4/6i as first or subsequent line therapy between January 2015 and April 2020. Baseline characteristics and laboratory data at various treatment timepoints were collected. The final analysis included 182 patients, of whom 46% were Black. Baseline absolute neutrophil count (ANC) was lower in the Black vs. Non-Black cohort (p = 0.001) but the change in ANC from baseline (delta-ANC) was smaller in the Black cohort, and the ANC at different treatment timepoints was similar between groups. There was no difference in the rate of infection or number of dose delays/reductions between racial groups. We did not find any difference in PFS between Black and Non-Black groups, regardless of the presence of CDK4/6i-induced neutropenia. We analyzed toxicity profiles of 182 patients with HR-positive/HER2-negative MBC treated with CDK4/6i. Despite the lower baseline ANC seen in our Black cohort, treatment toxicities were similar between racial groups. Long-term outcomes with CDK4/6i therapy, measured by PFS, were similar between Black vs. Non-Black patients.


Introduction
Cyclin-dependent kinases (CDKs) play an important role in cell cycle physiology. In vitro studies of CDK4/6 inhibitors (CDK4/6i) in human breast cancer cell lines showed hormone positive cell lines to be the most sensitive; moreover, there was synergy when combined with tamoxifen [1,2]. This work led to the phase 2 PALOMA-1 trial, where the CDK4/6i palbociclib improved progression-free survival (PFS, 20.2 vs. 10.2 months) when added to antiestrogen therapy in patients with untreated hormone receptor (HR)positive, human epidermal growth factor receptor 2 (HER2)negative metastatic breast cancer (MBC). As a result, the FDA-approved palbociclib in 2015 for use in combination with antiestrogen therapy as first line treatment in HR-positive, HER2-negative MBC [3]. The three currently available CDK4/6is, palbociclib, ribociclib, and abemaciclib, are all FDA-approved in combination with aromatase inhibitors and with the selective estrogen receptor degrader, fulvestrant.
CDK4/6is are well tolerated and adverse events are usually managed with dose modification and supportive care measures. Neutropenia, the dose-limiting toxicity for palbociclib and ribociclib, was the most common adverse event reported in landmark trials [3][4][5][6][7][8][9][10][11][12]. In PALOMA-2, 80% of patients in the palbociclib group developed neutropenia, with 66% of events being grade 3/4 [4]. Similar results were seen in the MONALEESA trials which studied the use of ribociclib, while the MONARCH trials generally reported lower rates of neutropenia with abemaciclib [6,[8][9][10][11]. CDK4/6i-induced neutropenia is rapidly reversible, reflecting a cytostatic effect on bone marrow precursors, and is not associated with an increased risk for infection. Abemaciclib commonly exhibits gastrointestinal toxicities, whereas neutropenia is less evident due to its stronger selectivity for CDK4 compared to CDK6 [13,14]. CDK6 regulates cytokine expression in hematopoiesis [15], contributes to the "control" of myeloid progenitor expansion, and plays many important roles in myeloid differentiation [16]. Most data about CDK4 and CDK6 are derived from total knockout mouse models. Maurer et al. generated transgenic mice that lack either CDK4 or CDK6 in adult hematopoiesis. Deletion of CDK6 affected all stem cell fractions and led to neutropenia, while deletion of CDK4 resulted in elevated numbers of myeloid progenitors without translating into numeric changes of differentiated myeloid cells [17]. Neutrophils comprise most circulating leukocytes and serve a critical antimicrobial role [18]. Asymptomatic reductions in peripheral blood neutrophil counts are observed in up to 10-30% of individuals of African descent [19]. In the USA, Blacks have lower neutrophil counts than Whites (mean difference 0.83 × 10 9 cells/L) and higher rates of neutropenia (4.5% vs. 0.79%) [20,21].

Study design and data collection
We conducted a single-center retrospective study at Montefiore Medical Center. Using the institutional clinical software, "Clinical Looking Glass", we identified patients with HR-positive, HER2-negative MBC who were prescribed a CDK4/6i as first or subsequent line therapy between January 1st, 2015 and April 28th, 2020. We excluded patients with inaccessible medical records, patients whose race was not available, patients who were lost to follow up, patients who did not take the CDK4/6i, those treated at outside institutions, and those who took the CDK4/6i for less than 14 days. Baseline characteristics including age, race, ethnicity, prior treatment lines for metastatic disease, tumor grade, visceral involvement, and menopausal status were collected through chart review. The Standard Charlson Comorbidity Index was calculated for each patient. Medication compliance and adherence were carefully tracked through diligent review of clinical notes. Patients who were not adherent to CDK4/6is as documented in clinicians' notes were excluded from the study.

Statistical analysis
Baseline characteristics were summarized using descriptive statistics. To assess associations between two categorical variables, we used the Pearson's Chi-square test or Fisher's exact test. The Wilcoxon rank sum test was used to compare neutrophil counts at each time point between Black vs. Non-Black patients. Change in neutrophil count over time was compared using the Wilcoxon signed rank sum test.
Toxicities were recorded and graded using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The number of dose reductions/delays were also recorded. The Pearson's Chi-square test was used to compare adverse event rates between Black vs. Non-Black patients.
PFS was calculated as the time in days between the CKD4/6i initiation date and the date of disease progression or death. Data was censored at the date each patient was last seen at our institution. The Kaplan-Meier method was used to estimate PFS, and the log-rank test was used to compare PFS between groups. To assess the effect of neutropenia and dose reduction on PFS, a landmark analysis was conducted excluding patients who died or had disease progression within 28 days of CDK4/6i initiation. Multivariable Cox Proportional-Hazards model, adjusting for age, visceral disease, menopausal status, prior endocrine therapy (ET) lines, prior chemotherapy lines, and neutropenia, was used to compare the PFS between Black and Non-Black patients.
Statistical analysis was conducted using STATA version 14.0 and R statistical software. This study was approved by the Albert Einstein Institutional Board Review.
When accounting for missing data, we started by excluding patients who had missing data for race, since this was our main variable of interest. We conducted detailed chart review to obtain most values for collected variables. For the evaluation of ANC over time in Black vs. Non-Black patients, we encountered 10-15% missing data for some ANC values. It was deemed that values were missing completely at random and there was no indication that any missing data were systematic. Therefore, we removed records with missing data to conduct statistical analysis of ANC at certain time points. To assess disease progression, we obtained information from imaging and clinical notes; only 1 patient had missing data for PFS analysis.

Baseline characteristics
A total of 233 patients were identified. Of those, 45 were excluded for the following reasons: 11 were treated at outside institutions, 4 were lost to follow up, 13 never took the prescribed CDK4/6i, 2 had HER2 + disease, 6 had inaccessible medical records, 8 were treated with CDK4/6i for less than 14 days, and 1 patient did not have stage IV disease.

Other toxicities
There were no differences in the rates of any thrombocytopenia (45% vs. 49%) or grade 3/4 thrombocytopenia (4% in each group), between racial groups (p = 0.42;

Dose reductions and delays
At least one dose reduction was required in 53 patients (29%) (

Progression-free survival
There was no difference in median PFS in Black vs. Non-Black (316 vs. 407 days, p = 0.51) (Table 3, Figure 4; see appendix). Among patients who received CDK4/6i as first line treatment, the median PFS for Black vs. Non-Black was 390 and 518 days (p = 0.48), respectively (Table 3, Figure 7; see appendix). We stratified breast cancer outcomes by neutropenia, to evaluate whether the drug's effects on the bone marrow could predict disease response. Among patients who developed neutropenia, the median PFS was 336 vs. 421 days (p = 0.45) for Black and Non-Black, respectively (Table 3, Figure 5; see appendix). In patients who did not develop neutropenia, the median PFS was 141 vs. 259 days (p = 0.41) for Black and Non-Black, respectively (Table 3, Figure 8; see appendix). Next, we assessed the effect of dose reductions on breast cancer outcomes by racial groups. Among patients who required a dose reduction, the median PFS was 312 vs. 627 days (p = 0.26) for Black vs. Non-Black (  Figure 9; see appendix). In a univariate analysis, the use of prior endocrine therapy and prior chemotherapy for metastatic disease (p = 0.01 for both) and presence of visceral disease (p = 0.04) were statistically significantly associated with PFS. Black race (p = 0.51), neutropenia (p = 0.09), and age (p = 0.27) were not associated with PFS (Table 8; see appendix). We then created a multivariable analysis using Cox Proportional-Hazards model including Black race, neutropenia, age, visceral status, prior endocrine therapy for metastatic disease, and prior chemotherapy for metastatic disease. In this model, the use of prior endocrine therapy for metastatic disease was inversely associated with PFS (HR 1.87, 95% CI 1.22-2.87, p < 0.01), and the presence of visceral metastasis was inversely associated with PFS, with a trend towards statistical significance (HR 1.57, 95% CI 0.95-2.60, p = 0.08) ( Table 8; see appendix).

Discussion
Our study included 182 patients with HR-positive/HER2negative MBC treated with CDK4/6i in a racially diverse cohort (Black = 83, Non-Black = 99). We report that Black patients have a lower baseline ANC (3.0 vs. 4.0 × 10 9 /L, p = 0.001), and smaller decreases in ANC over time compared to Non-Black patients (delta-ANC at C2D1: − 1.5 vs. − 2.6 × 10 9 /L, p = 0.001) while on CDK4/6is. A similar trend was observed for the total WBC count. Most patients   Figure 4, Table 3; see appendix). Of note, the PFS results should be interpreted with caution, given the number of patients included in each analysis. Further, our multivariable analysis did not show an association between PFS and Black race or neutropenia. These data support the notion that the lower baseline ANC seen in Black patients does not negatively impact survival or toxicity outcomes during CDK4/6i treatment. Our results also suggest that baseline ANC alone may be a poor indicator of infection risk during CDK4/6i therapy. Benign ethnic neutropenia (BEN), an entity where an ANC < 1.5 × 10 9 /L is seen in certain populations, is most common in people of African descent [22]. Although the etiology has not been elucidated, studies have identified a chromosome 1q22 polymorphism containing the Duffy antigen and receptor for chemokine (DARC) gene which strongly influences leukocyte counts in African Americans and is one potential mechanism [23]. BEN is not associated with an increased risk for infection or febrile neutropenia. Despite this, a proportion of patients with BEN may be inadvertently excluded from oncology clinical trials (which typically have an eligibility criteria of ANC ≥ 1.5 × 10 9 /L for inclusion), or have unnecessary dose interruptions/reductions, due to their lower than normal baseline ANC [22,24]. Our study is the first to evaluate racial disparities in toxicities and breast cancer outcomes for patients treated with CDK4/6i for MBC, and has the largest proportion of Black patients (46%; N = 83) reported in the literature in this clinical scenario.
Prior studies have evaluated racial disparities in neutrophil counts in breast cancer patients receiving chemotherapy. Smith et al. found no racial differences in the frequency of febrile neutropenia in breast cancer patients receiving chemotherapy, despite a lower baseline ANC in Black patients [25]. A retrospective analysis by Hershman et al. found that Black patients had a lower baseline WBC prior to starting adjuvant chemotherapy but a similar mean percentage decline in WBC from baseline to treatment completion compared to White patients [26]. In another study analyzing differences in WBC counts and dose delays/discontinuations between Black and White breast cancer patients receiving adjuvant chemotherapy from two clinical trials, the rates of neutropenic fever were similar between racial groups despite a lower baseline WBC/ANC in Black patients [27]. These studies call into question the clinical relevance of a lower baseline WBC and ANC that may be seen in Black patients, and whether clinical trial inclusion criteria should more frequently be liberalized. Vastola et al. explored the ANC inclusion criteria of prostate cancer clinical trials and found that 41.4% of trials excluded patients with BEN, arguing that Black patients may be disproportionately excluded from cancer clinical trials due to benign racial laboratory variations [28]. Hsieh et al. argues that rigid eligibility criteria for WBC in cancer clinical trials may cause undue exclusion of Black patients from participation [22]. Our work provides additional evidence that clinical trial inclusion criteria for ANC should be reexamined, to provide equitable and evidence-based opportunities for participation.
Inclusion criteria for most CDK4/6i landmark trials required an ANC ≥ 1.5 × 10 9 /L, which may have impacted the underrepresentation of Black patients in these studies [4,7,11,12,29]. The phase 2 single arm PALINA trial analyzed the hematologic safety of palbociclib in combination with letrozole or fulvestrant in self-reported African American, African, or Black women with HR-positive/ HER2-negative MBC [29]. The ANC inclusion cutoff was 1.0 × 10 9 /L. The median baseline ANC was 3.1 × 10 9 /L, and 35 patients were enrolled. No patients experienced febrile neutropenia or required drug discontinuation due to neutropenia. Lower baseline ANC (2.4 vs 4.3 × 10 9 /L, p = 0.006), grade 3 neutropenia (66.7% vs. 23%, p = 0.029), and dose reductions (55.6% vs. 7.7%, p = 0.008) were more common in patients found to have the Duffy null polymorphism [29]. Interestingly, our study reports similar conclusions as the PALINA trial, that CDK4/6is may be safely administered in Black patients even if they have a lower baseline ANC.
Our study has certain limitations. Given its retrospective nature, information on medication compliance was limited to review of clinical notes. We encountered missing data points, which were subsequently excluded from statistical analysis and potentially limited the magnitude of the results. Despite careful attempts to reduce missing data, it is possible that systematic bias was introduced by inclusion of only those without missing data. There is also the possibility of selection bias, as perhaps only the compliant patients were included in this retrospective analysis. Our sample size is indeed smaller than those studied in landmark CDK4/6i clinical trials. However, although small, our study includes the highest proportion of Black patients for analysis of toxicities and PFS of CDK4/6i ever reported in the literature. This is the first study to elucidate that despite lower baseline neutrophil counts seen in Black patients, there are no differences between racial groups in the frequency of infections, dose reductions, or dose delays seen with CDK4/6i use in MBC. Based on these results, the lower baseline neutrophil counts that may be seen in Black patients should not cause concern or hesitancy when initiating CDK4/6i, as they may not be clinically relevant.

Conclusion
In our cohort, Black patients had a lower baseline ANC compared to Non-Black patients, but they experienced less of a decline in ANC from baseline (delta-ANC) during treatment with CDK4/6i. Our results are the first to examine racial disparities in toxicities of patients receiving CDK4/6i. Our data suggest that CDK4/6is can be safely administered to patients who may have BEN, since their baseline ANC does not necessarily translate into increased rates of infections, dose reductions, or dose delays.

Appendix
See Tables 4, 5      Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Conflict of interest All authors declare that they have no relevant financial or non-financial interests to disclose.
Ethical approval This retrospective chart review study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The Human Investigation Committee (IRB) of Montefiore Medical Center/Einstein College of Medicine approved this study.