Association of pre-chemotherapy peripheral blood pro-inflammatory and coagulation factors with reduced relative dose intensity in women with breast cancer

Background Chemotherapy decreases the risk of relapse and mortality in early-stage breast cancer (BC), but it comes with the risk of toxicity. Chemotherapy efficacy depends on relative dose intensity (RDI), and an RDI < 85% is associated with worse overall survival. The pro-inflammatory (interleukin (IL)-6, C-reactive protein (CRP)) and coagulation factors (D-dimer) serve as biomarkers of aging. The purpose of this study is to determine if these biomarkers are associated with reduced RDI in women with stage I–III BC. Methods This study enrolled women with stage I–III BC. Prior to adjuvant or neoadjuvant chemotherapy, peripheral blood was collected for biomarker measurement. Dose reductions and delays were captured and utilized to calculate the RDI delivered. Univariate and multivariate analyses were performed to describe the association between pre-chemotherapy IL-6, CRP, and D-dimer levels and an RDI < 85%, controlling for relevant tumor and patient factors (age, stage, receptor status, chemotherapy regimen, and pre-chemotherapy physical function and comorbidity). Results A total of 159 patients (mean age 58 years, range 30–81, SD 11.3) with stage I–III BC were enrolled. An RDI < 85% occurred in 22.6% (N = 36) of patients and was associated with higher pre-chemotherapy IL-6 (OR 1.14, 95% CI 1.04–1.25; p = 0.006) and D-dimer (OR 2.32, 95% CI 1.27–4.24; p = 0.006) levels, increased age (p = 0.001), increased number of comorbidities (p = 0.01), and decreased physical function by the Medical Outcomes Survey Activities of Daily Living (ADL) Scale (p = 0.009) in univariate analysis. A multivariate model, including two biomarkers (IL-6 and D-dimer), age, ADL, BC stage, and chemotherapy regimen, demonstrated a significant association between the increased biomarkers and reduced RDI < 85% (OR 2.54; p = 0.04). Conclusions Increased pre-chemotherapy biomarkers of aging (IL-6 and D-dimer) are associated with reduced RDI (<85%). Future studies are underway to validate these findings. Trial registration ClinicalTrials.gov, NCT01030250. Registered on 3 November 2016. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0895-5) contains supplementary material, which is available to authorized users.


Background
The efficacy of chemotherapy is dependent on relative dose intensity, which is defined as the ratio of standard or planned dose intensity (dosage of drug delivered per unit of time) to actual dose intensity received. A reduction in the relative dose intensity (RDI) of adjuvant chemotherapy is associated with a loss of clinical benefit in women with breast cancer [1][2][3][4][5][6][7]. In particular, an RDI < 85% is associated with worse overall survival and increased age is noted to be a risk factor [1].
Although older adults are at increased risk for reduced RDI, chronological age alone is a poor predictor of biological age [8][9][10][11]. Biomarkers of aging could potentially be utilized to further understand the heterogeneity of the aging process. Growing evidence has demonstrated that pro-inflammatory and coagulation factors such as interleukin-6 (IL-6) [12][13][14], C-reactive protein (CRP) [12], and D-dimer may serve as potential biomarkers of aging. Elevated levels of these factors have been associated with reduced physical performance [15], frailty [16], and an increased risk of death [17] in communitydwelling older adults. The utility of these markers as predictors of chemotherapy tolerance, and the ability to deliver chemotherapy (i.e. RDI) in patients with breast cancer is unknown.
The goal of this study was to explore the association between biomarkers of aging (IL-6, CRP, and D-dimer) and the ability to deliver adjuvant or neoadjuvant chemotherapy. We hypothesized that elevated biomarkers of aging (and/or combinations of these biomarkers) may be associated with a reduced RDI among women with stage I-III breast cancer receiving adjuvant or neoadjuvant chemotherapy.

Methods
This prospective longitudinal study was open at two participating institutions (City of Hope and Long Beach Memorial Medical Center). Eligible patients had a diagnosis of stage I-III breast cancer, were scheduled to receive adjuvant or neoadjuvant chemotherapy, and were fluent in English (since all measures in the patient questionnaires were not validated in other languages). Patients with stage IV disease were excluded. The study was approved by the institutional review boards (IRB) of the participating institutions (City of Hope IRB and Long Beach Memorial Medical Center IRB). Participating patients completed the informed consent process.

Patient and tumor characteristics
Patient age, race/ethnicity, and socio-demographics were captured. Breast cancer stage, type of treatment, and receptor status (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)) were collected.

Biomarkers of aging
Prior to initiation of adjuvant or neoadjuvant chemotherapy, peripheral blood (7.5 ml) was collected for measurement of pro-inflammatory (IL-6 and CRP) and coagulation (D-dimer) factors. Plasma was stored at − 80°C until assays were run. Quantitative IL-6 and CRP levels were measured using NOVEX® immunoassay (Invitrogen) and D-dimer levels were measured with Nanopia® D-dimer (Sekisui). Information regarding the time from surgery to peripheral blood collection was recorded for each of the patients who received chemotherapy in the adjuvant setting.

Relative dose intensity and chemotherapy toxicities
The chemotherapy regimen was prescribed at the treating physician's discretion. The chemotherapy dose (planned and received) and the reasons for dose reductions and dose delays were captured. RDI was calculated as the ratio of actual dose intensity received to the standard or planned dose intensity (dosage of drug delivered per unit of time). Trastuzumab dosing was not included in RDI calculations. Grade ≥ 3 toxicities defined by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 4.0 were captured. Emergency visits and hospitalizations were collected.

Measures of physical function and comorbidity
All patients completed a questionnaire, which included measures of physical function and comorbidity. Measures of physical function included Activities of Daily Living (ADL) [18], Instrumental Activities of Daily Living (IADL) [19], Karnofsky Performance Status (KPS) [20], and Timed Up and Go (TUG) [21]. The ADL measured by the Medical Outcome Study (MOS) Physical Heath scale evaluated a range of activities including the ability to bathe independently and the ability to run, rated on a scale of 0-100, with a higher score indicating better physical function. The Instrumental Activities of Daily Living (IADL) scale measured the ability to complete activities that are required for independence in the community such as shopping and taking transportation, resulting in a score of 0-14, with a higher score signifying greater independence. The physician-rated KPS is a global measure of performance status measured on a scale of 0-100 with a higher score reflecting better function. Patient self-rated KPS is a global measure of performance status, which the patient self-rates from a scale of 40-100. The TUG is a performance-based measure of functional status that measures the time that it takes (in seconds) to rise from a chair, walk three feet, turn around, walk back to the chair and sit down.
Comorbidity was evaluated using the Older Americans Resources and Services Program (OARS) questionnaire [22] in which patients report if they have comorbidities such as hypertension, arthritis, diabetes mellitus, heart disease, and stroke. Each of these comorbidities has been associated with increased levels of IL-6, D-dimer, and/or CRP [12,[23][24][25].

Statistical analyses
Descriptive analyses were performed to summarize patient, tumor, and treatment characteristics. The mean (SD) and median (inter-quartile) values were calculated for peripheral blood biomarkers (IL-6, CRP, and D-dimer) and physical function measures (ADL, IADL, TUG, patient self-rated KPS, and physician-rated KPS). The main outcome, RDI, was dichotomized using 85% as the cutoff point, with reduced RDI defined as RDI < 85%.
The Pearson product-moment correlation coefficient was computed to assess the relationship among the peripheral blood biomarkers. Logistic regression was used to examine the relationships between reduced RDI (< 85%) and peripheral blood biomarker (IL-6, CRP, and D-dimer) measurements. In the analyses, values of CRP, IL-6, and D-dimer were categorized using the 3 rd quartile as cutoff points (4 th quartile vs. quartiles 1-3) [17]. An IL-6 and D-dimer combination biomarker variable was created indicating 0 (reference group) if neither of the biomarker values were in the 4 th quartile, vs. 1 or 2 if one or two of the biomarkers were in the highest quartiles. In addition, we also examined the relationship between RDI < 85% and the following variables: age, number of comorbidities, body surface area (BSA), breast cancer stage, receptor status, chemotherapy regimen, adjuvant vs. neoadjuvant chemotherapy, time from surgery to peripheral blood collection (in patients treated in the adjuvant setting), and physical function measures (ADL, IADL, TUG, patient self-rated KPS and physician-rated KPS). Age and cancer stage were kept in the multivariate model regardless of their p values. For other variables, stepwise selection was used to select the final variables to be included in the model. All statistical tests were two-sided, and p values <0.05 were considered statistically significant. Data were analyzed using SAS 9.3 (SAS Institute, Cary, NC, USA).

Patient, tumor, and treatment characteristics
Between July 2009 and December 2014, 206 patients with stage I-III breast cancer were accrued to this study. The current analysis included 159 patients who consented for the pre-chemotherapy blood biomarker measurements. There were no significant differences between the 159 patients who contributed and the 46 patients who declined blood sample, in terms of age, race/ethnicity, disease stage, hormone receptor status, and functional status. However, patients who declined participation had increased comorbidities (p < 0.01).

Peripheral blood biomarkers of aging
The median time from surgery to peripheral blood sample collection in patients treated with adjuvant chemotherapy was 52 days (range 18-193 days). The mean and median values of the three biomarkers in peripheral blood are shown in Table 4. There was modest positive correlation between IL-6 and CRP levels (r = 0.37; p < 0.001). No correlation was found between D-dimer levels and either IL-6 (r = 0.12; p = 0.12) or CRP levels (r = 0.07; p = 0. 35). No significant changes in the mean biomarker levels were found when patients who received neoadjuvant chemotherapy were excluded from the analysis (Additional file 1: Table S1).

RDI
The median RDI was 90% (range 20-100%), with 23% of patients (N = 36) receiving an RDI < 85%. Patients who experienced grade 3-5 toxicities were more likely to receive an RDI < 85% (40% vs. 9%; p < 0.001). Compared to patients who never experienced grade 3-5 toxicities, those experiencing grade 3-5 non-hematological   Patients who were hospitalized during chemotherapy were more likely to receive an RDI < 85% than those who were not hospitalized (55 vs. 15%; p < 0.001). Patients with an emergency department visit during chemotherapy were as likely to receive an RDI < 85% as those without emergency department visits (24% vs. 22%; p = 0.81). In univariate analysis, older age (p = 0.001), increased number of comorbidities (p = 0.01), and reduced physical function as measured by the ADL scale (p = 0.009) were associated with an RDI < 85% (Table 5). In patients treated with adjuvant chemotherapy, the time from surgery to peripheral blood draw was not significantly associated with either biomarker levels or with an RDI < 85%. The various chemotherapy regimens used, and their association with an RDI < 85%, are also shown in Table 5. Increased IL-6 (OR 1.14, 95% CI 1.04-1.25; p = 0.006) and D-dimer levels (OR 2.32, 95% CI 1.27-4.24; p = 0.006) were associated with an RDI < 85%. No association was found between increased CRP levels and an RDI < 85% (p = 0.47) analyzed as a continuous variable (Table 6). When examined as categorical variables, patients with IL-6 (OR 2.49, 95% CI 1.12-5.56; p = 0.02) and/or D-dimer (OR 3.52, 95% CI 1.6-7.78; p = 0.002) levels in the highest (4 th ) quartile were more likely to have an RDI < 85% compared to those with IL-6 and D-dimer levels in quartiles 1-3. This association was not found for CRP levels (OR 1.25, 95% CI 0.53-2.89; p = 0.61). When combining IL-6 and D-Dimer, those patients with one or more biomarkers in the 4 th quartile had an increased risk of an RDI < 85% (OR 3.59, 95% CI 1.64-8.87; p = 0.001). No significant changes in the association between biomarkers of aging and reduced RDI were found when patients who received neoadjuvant chemotherapy were excluded from the analysis (Additional file 2: Table S2).
In multivariate analysis, the association between an IL-6 and D-dimer level in the highest (4 th ) quartile and an RDI < 85% remained significant (OR 2.54, 95% CI 1.03-6.23; p = 0.04) after adjusting for patient age, physical function (ADL score), breast cancer stage, and receipt of an anthracycline regimen (Table 7). Although the number of comorbidities was associated with an RDI < 85% in univariate analysis, it was no longer significant after stepwise selection, and thus was not selected to be included in the final model.

Discussion
This study demonstrates an association between elevated pre-chemotherapy peripheral blood biomarkers of aging (IL-6 and D-dimer) and reduced RDI in women with stage I-III breast cancer receiving neoadjuvant or adjuvant chemotherapy. These biomarkers could potentially be of utility in oncology practice to understand "biological age" and the ability to deliver chemotherapy.
RDI represents the proportion of the dose intensity actually delivered compared with the standard dose intensity for a chemotherapy regimen [26]. Dose reductions or delays in the administration of chemotherapy due to toxicity, or with the intention to avoid toxicity, lead to a reduction in the dose intensity of treatment, and may decrease its therapeutic effect. Maintenance of chemotherapy dose intensity is necessary to maintain chemotherapy efficacy. A reduction in RDI to < 85% has been shown to correlate with worse survival in women with both early and advanced-stage breast cancer [27,28], and optimizing RDI may be a valuable strategy to improve outcomes. Nevertheless, RDI reductions are common in clinical practice, and a large nationwide study of community practices identified RDI reductions of 15% or more in over half of patients with early-stage breast cancer [29]. Across several studies, advanced age has consistently been found to be a risk factor for a decreased RDI, and older women with breast cancer are more likely to have a reduced RDI compared with younger women [2,[30][31][32][33][34].
Although advanced age is a known risk factor for reduced RDI, chronological age is often a poor reflection of the physiological and functional status of older adults, and new tools are needed to determine which older patients are at greater risk of toxicity and a reduced RDI. At a molecular level, aging involves complex processes such as oxidative stress, inflammation, DNA damage, shortening telomeres, genotoxicity and genomic changes [35,36]. In an attempt to measure the aging process,   AC-T doxorubicin plus cyclophosphamide followed by paclitaxel, TCH docetaxel, carboplatin and trastuzumab, AC-TH doxorubicin plus cyclophosphamide followed by paclitaxel plus trastuzumab, A-T-C sequential doxorubicin, paclitaxel and cyclophosphamide several potential biomarkers have been evaluated both in community dwelling older adults and in older adults with cancer. Among these, some of the most widely studied biomarkers of aging are inflammatory mediators and coagulation factors, such as IL-6, TNF-α, CRP and D-dimer. Inflammatory and coagulation markers are easily measured, increase with age, and are associated with functional decline and increased mortality in community-dwelling older adults [8,12,13,25,[37][38][39][40][41].
The aforementioned biomarkers have also been studied in the context of oncology patients; however, the data focus more on the association between these biomarkers at the time of diagnosis and disease characteristics and cancer outcomes rather than their utility as biomarkers of aging. For example, elevated pretreatment serum IL-6 correlates with a more advanced cancer stage and poorer response to chemotherapy [40]. Elevated CRP levels at the time of diagnosis of breast cancer are associated with reduced overall and diseasefree survival and an increased risk of death from breast cancer [24]. In addition, among untreated patients with operable breast cancer, elevated plasma D-dimer levels are associated with lymphovascular invasion, higher clinical stage, and lymph node involvement [41].
In contrast with our study, most previous publications have focused on changes in biomarkers of aging before and after adjuvant chemotherapy. One study demonstrated that expression of p16INK4a, an inhibitor of cyclin-dependent kinase 4 associated with cellular senescence, increased after adjuvant chemotherapy in 33 patients with breast cancer, and remained elevated 12 months after treatment [42]. In another study of 57 patients age 70 years and older with breast cancer undergoing chemotherapy, there were modest decreases in IL-10 and insulin-like growth factor-1 (IGF-1), and increases in TNF-α and monocyte chemotactic protein 1 (MCP-1) from baseline to 1 year, which were significantly more pronounced in the chemotherapy group   compared to the control group, suggesting accelerated biological aging [43]. A recent study by Extermann et al. found that there were no differences in peripheral biomarkers of aging (IL-6, D-dimer, IGF-1, and TNF-α) between patients (N = 27) with breast cancer at 1-2 years after completion of adjuvant chemotherapy and nonchemotherapy controls (N = 29) [44]. While previous publications show chemotherapy may lead to accelerated aging, information on the relationship between biomarkers of aging and chemotherapy tolerance is lacking, and this study is the first to our knowledge to show an association between pre-chemotherapy biomarkers of aging and the ability to deliver chemotherapy successfully.
Limitations of this study include the modest sample size and the fact that the biomarker component was optional; however, more than 75% of patients participated in the biomarker component. Furthermore, while the majority of the studies of biomarkers of aging were conducted among older adults, in this study we included patients across the aging spectrum. This could be considered a limitation or a strength, as our results could potentially be applied to patients across the aging spectrum. Patients receiving both neoadjuvant and adjuvant chemotherapy were included in this study. However, only a small proportion of the patients were treated with neoadjuvant chemotherapy, and the results of our analyses were not modified by the exclusion of this group of patients.
This study is hypothesis generating, and our findings need to be validated in an independent cohort of patients before biomarkers of aging can be integrated into the clinical care of women with breast cancer. However, if these findings are confirmed, biomarkers of aging could potentially be utilized before starting chemotherapy, since they could help identify those patients who are less likely to successfully complete planned treatments. This, in turn, could lead to the selection of less toxic chemotherapy regimens for those patients, or more intensive follow up aimed at the earlier detection of dose-limiting toxicities. An ongoing multicenter study of older adults with breast cancer (N = 500) undergoing adjuvant chemotherapy is close to completing accrual, and will be able to verify our findings and provide information on which specific chemotherapy regimens are more likely to be successfully completed (ClinicalTrials.gov identifier NCT01472094).

Conclusions
This study is novel in connecting the fields of geriatrics and oncology by demonstrating the potential utility of biomarkers of aging in patients with early-stage breast cancer across the aging spectrum who received chemotherapy. This finding expands our current understanding of the association between the biomarkers of aging and delivery of chemotherapy. Future studies are underway to verify these findings.

Additional files
Additional file 1: Table S1. Biomarkers measurement in neo-adjuvanttreated vs. adjuvant-treated patients. (DOC 39 kb) Additional file 2: Table S2 Availability of data and materials All data generated or analyzed during this study are included in this published article.
Authors' contributions YY, NV, and AH made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the manuscript, and final approval for publication. TS, VT, and SM performed biomarker measurements. CS and DS performed data analysis for the study. ES and SY participated in manuscript preparation and revision. DL, JM, TL, GS, JW, and JC contributed to patient enrollment and manuscript revision. VK, AL, AA, JC, and LZ contributed to data collection and database management for the study. YY and AH agree to be accountable for all aspects of the work. YY and CS made substantial contributions to analysis and interpretation of data. All other authors made substantial contributions to acquisition of data, revising the manuscript, and final approval.
Ethics approval and consent to participate This prospective longitudinal study was open at two participating institutions (City of Hope and Long Beach Memorial Medical Center). The study was approved by the institutional review boards of both participating institutions. Participating patients completed the informed consent process.

Consent for publication
Not applicable.
Competing interests I confirm that I have read BioMed Central's guidance on competing interests and have included a statement indicating that none of the authors have any competing interests in the manuscript.