Chemotherapy is a risk factor of lymphopenia before adjuvant radiotherapy in breast cancer

Abstract Background Lymphopenia can decrease immune function of the host and is a known risk factor for poor prognosis in malignant tumors. Radiation induced lymphopenia was common in patients with breast cancer and was also reported to have a negative effect on long‐term outcome. Aims Lymphopenia may be associated with baseline immune status before radiotherapy (RT). This study aimed to explore the rate and risk factors of lymphopenia before start of the adjuvant RT in patients with breast cancer. Methods Patients with invasive breast cancer treated from March 2015 to February 2020 and with peripheral lymphocyte counts (PLC) available within 7 days from the beginning of RT were eligible for this study. Data were presented as mean and 95% confidence interval unless otherwise specified. The risk factors of low PLC before RT were identified using univariate and multivariable linear regressions. Results A total of 1012 consecutive patients met the study criteria. The mean PLC before RT commencement was 1.58*109/L (95%CI: 1.55–1.62*109/L) with 15.2% (95%CI: 13.1%–17.6%) CTCAE defined lymphopenia, rendering 12.3%, 2.6%, 0.3%, and 0% for grade 1, 2, 3 and 4 respectively. Univariate and multivariable linear regression showed prior chemotherapy was the most significant risk factor (p < .001) for low PLC, while age, menopausal status and lymph node stage were not (all ps > .05). A total of 912 (90.1%, 95%CI: 88.1%–91.9%) patients had chemotherapy before adjuvant RT in this study. In patients with HR+/HER2‐ breast cancer, 69.0% (95%CI: 63.0%–74.5%) N0 and 98.1% (95%CI: 95.1%–99.5%) N1 had also received chemotherapy. Conclusions Patients with breast cancer might have lymphopenia from prior chemotherapy at the start of adjuvant RT which could have negative effect on long‐term outcome. It is also noted that most of the patients with HR+/HER2‐, early‐stage breast cancer were treated with aggressive chemotherapy without knowing the risk of chemotherapy induced lymphopenia. Future study on predictive or prognostic multigene assays is warranted to avoid unnecessary chemotherapy and subsequent lymphopenia in patients with low risk breast cancer.

multigene assays is warranted to avoid unnecessary chemotherapy and subsequent lymphopenia in patients with low risk breast cancer.

K E Y W O R D S
breast cancer, chemotherapy, lymphopenia, radiotherapy, risk factors

| INTRODUCTION
The immune escape of malignant cells and lack of immune biomarkers such as lymphocyte are associated with the development, metastasis and recurrence of malignant tumors. One meta-analysis showed patients with HIV infection and immunodeficiency after transplantation had higher incidence of malignant tumors. 1 Lymphopenia was found in about 20% of advanced breast cancer, pancreatic cancer, lymphoma and other malignant tumors, while only in 3% of early stage cancers. 2,3 Meanwhile, various studies had reported a significant association between lymphopenia and poor prognosis in patients with various types of malignant tumors, 4-7 including breast cancer. 2,[8][9][10] Indeed, lymphocyte is considered as one of major immunoactive elements which inhibit tumor cell growth.
Lymphocyte also plays an important role for the tumor control effect of immunotherapy including immunocheckpoint inhibitors in malignant tumors and patients with high peripheral lymphocyte count (PLC) had higher immune response rate. 6 Immunotherapy as an important strategy of precision therapy, has been widely used in melanoma, lung cancer, head and neck cancers, breast cancer and other malignant tumors in recent years. However, only about 20% patients with malignant tumors can benefit from immunotherapy. 11 Most patients have primary or acquired resistance to immunotherapy. It is believed that lymphopenia and deficiency of functional lymphocyte subsets contribute to the resistance of immunocheckpoint inhibitors. PLC, CD8 + T lymphocyte infiltration, and the expression of immunocheckpoint inhibitors and inhibitor conjugates may be potential markers to predict the efficacy of immunotherapy. 11 Therefore, in spite of the peripheral white blood cells (WBC), platelets and neutrophils which are related to the toxicity of anti-tumor therapy, it is also essential to pay attention to PLC before and during the course of treatment.
Studying the risk factors of lymphopenia or low PLC has a clinical significance.
Radiation induced lymphopenia was also reported to be a potential risk factor for poor survival in breast cancer 12,13 and several other malignant tumors. 14-16 RIL was common in patients with breast cancer with approximately 50% patients had lymphopenia after radiotherapy (RT) in Sun's study. 13 We recently reported that 60.5% patients had lymphopenia after RT, and baseline lymphocyte counts and radiation dosimetric factors to the immune system in radiation field might have contribute to RIL. 17 This study aimed to preliminarily explore the rate and risk factors of lymphopenia before adjuvant RT in patients with breast cancer, so as to provide a basis for subsequent study of RIL.

| Data collection
Patient factors (e.g., age, menopausal status), tumor factors (e.g., ER/PR/HER2 subtype, N stage, stage), treatment related parameters (e.g., chemotherapy, anti-HER2 target therapy and surgical approach) and PLC within 7 days from the beginning of RT were retrospectively collected. Modified N stage and modified stage were applied in this study for patients received neoadjuvant chemotherapy (NACT). Modified (N) stage used the higher (N) stage between clinic (N) stage and pathological (N) stage for patients who had received NACT, and used pathological (N) stage for patients who did not receive NACT. According to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, lymphopenia was classified using PLC cut-off of lower limits of normal (LLN)-0.8*10 9 , 0.8-0.5*10 9 , 0.5-0.2*10 9 and 0.2*10 9 /L, for grade 1, 2, 3, and 4, respectively. LLN of lymphocyte count was 1.06*10 9 /L in our institution and PLC less than 1.06*10 9 /L was defined as lymphopenia in this study.

| Statistical considerations
The primary endpoint of this study was PLC before RT, which was a surrogate endpoint for lymphopenia before RT. The effects of potential risk factors such as age, menopausal status, stage, ER/PR/HER2 subtype, chemotherapy, anti-HER2 target therapy and surgical approach were estimated using univariate analysis initially and those significant (p < .05) in univariate linear analysis were further evaluated by multivariable regression. To avoid the unstable and imprecise estimates of the coefficients, the potential presence of collinearity was assessed using variance inflation factor (VIF) <10 and some of the highly correlated variables with VIF >10 were excluded in the final multivariable regression analysis. Data were presented as mean (95% CI) unless otherwise specified. p-value (p) less than .05 was considered to be statistically significant. Statistical analysis was performed using R software (version 3.6.2; https://www.R-project.org).  (Table 1).
Time interval between last chemotherapy and the start of RT was a significant risk factor for low PLC in all patients treated with various regimens of chemotherapy in univariate linear regression ( Table 1). As  to have recovered according to traditional assessment of the neutrophils and platelets. This study showed prior chemotherapy was the significant risk factor for low PLC before RT, which was consistent with previous report of Sage EK's study. 18 The benefit of chemotherapy in aggressive type of breast cancer is known, thus the vast majority of these patients had received chemotherapy before adjuvant RT.
As unexpected, most patients with HR+/HER2-, early-stage breast cancer were also treated with aggressive chemotherapy in this study while chemotherapy is considered to have low clinical benefits in this population.
The most common chemotherapy regimens for breast cancer are anthracycline, taxane and cyclophosphamide. Ménétrier-Caux et al had reported cyclophosphamide, cisplatin, methotrexate and taxane were the strongest chemotherapeutic agents that induced lymphocyte deficiency. 11 Kotsakis et al also found taxane can induce lymphocytosis in solid tumors. 19  predictive or prognostic multigene assays should be more applied in clinic practice in patients with HR+/HER2-, early-stage breast cancer.
With the affirmed low risk by quantized genomic results, physicians could be more confident to omit unnecessary chemotherapy in these cases as NCCN recommended 25 and the risk of lymphopenia in this low risk population might be reduced.
As for high risk patients with locally advanced breast cancer or aggressive subtypes such as triple negative breast cancer (TNBC), it is difficult to omit chemotherapy with the irreplaceable cytotoxic effect and survival benefits in these cases in spite of the possibility of chemotherapy induced lymphopenia. 28,29 TNBC is considered to be immunogenic with higher PD-L1 mRNA expression 30 and CD8+ T cell

| CONCLUSIONS
This study of 1012 patients with breast cancer demonstrated that patients might have lymphopenia before RT which was associated with prior chemotherapy. Since lymphopenia has a negative effect on long-term survival, further study on predictive or prognostic multigene assays for risk stratification and personalized decision of chemotherapy may help avoid unnecessary chemotherapy and reduce chemotherapy induced lymphopenia in patients with low risk breast cancer.
ACKNOWLEDGMENT Lingyu Ma, Pingfu Fu and Hao Yu are responsible for statistical analysis.

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

ETHICS STATEMENT
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of the University of Hong Kong-Shenzhen Hospital # 2019 098 and individual consent for this retrospective analysis was waived.

DATA AVAILABILITY STATEMENT
The datasets analyzed during the current study are not publicly available due to privacy but are available from the corresponding author on reasonable request.