Radiation-Induced Lymphopenia Prognosis and Risk Factors in Postmastectomy Radiotherapy Patients

Introduction

Breast cancer, the leading cancer in women and the second most common cancer globally,¹ is typically treated through a multidisciplinary comprehensive methodology. Radiation therapy is an essential component of the treatment of breast cancer. Postmastectomy radiotherapy (RT) can reduce both local regional recurrence rate and breast cancer mortality in women with positive lymph nodes.² Also, meta-analysis demonstrates that postoperative RT after breast-conserving surgery enhances local control rates and decreases breast cancer-specific mortality.^3,4 Therefore, RT is the standard treatment after breast-conserving surgery and high-risk patients received mastectomy.^5,6 However, it is reported that radiation may suppress host immunity, manifesting as lymphopenia.^7,8 It is generally considered that lymphocytes are considered the most radiosensitive cells in humans, the counts of which decrease on the first post-RT day.⁹ Therefore, severe radiation-induced lymphopenia (RIL) often occurs during cancer treatment involving RT.¹⁰

A meta-analysis revealed that severe RIL can reduce survival rates associated with several solid tumors.¹¹ According to recent studies, patients with G4 RIL during radical or postoperative adjuvant chemoradiotherapy of esophageal cancer have a poor OS.^12,13 Another study on definitive concurrent chemoradiotherapy (CCRT) for esophageal cancer demonstrated that the occurrence of G4 RIL during radiotherapy was an independent prognostic factor for impaired OS rates, and this adverse prognosis persists even when lymphocyte counts return to normal or near-normal levels after treatment completion¹⁴. Kobzeva et al reported that the absolute levels of B-, T- and natural killer cells significantly reduced after RT regardless of whether the patients previously underwent chemotherapy courses in breast cancer.¹⁵ However, only a few studies have explored the relationship between RIL and survival in patients with breast cancer. The results of a recent post hoc analysis on 598 patients from a Phase III randomized clinical trial demonstrated significantly inferior 5-year disease-free survival (DFS) in breast cancer patients with a nadir-peripheral lymphocyte count (PLC)/pre-PLC ratio < 0.8 treated with mastectomy followed by adjuvant RT.¹⁶ However, the risk factors for RIL in patients with breast cancer remain unknown. Therefore, here, we investigated the relationship between RIL and survival rate in postmastectomy RT patients, as well as the related risk factors, specifically treatment-related risk factors such as radiation dosimetric factors and RT modalities.

Materials and Methods

Patients

Breast cancer patients who received adjuvant RT after mastectomy at Beijing Shijitan Hospital Affiliated with Capital Medical University over June 2016 to December 2022 were enrolled. Peripheral blood counts were obtained before and during treatment and at the first posttreatment follow-up. We excluded patients who lacked complete blood count data or who paused RT for >3 consecutive days due to personal reasons during the RT period (Figure 1).

Figure 1 Study flowchart.

All patients received postmastectomy RT, with the clinical target volume including the supraclavicular area and chest wall, to which varied RT techniques were applied. The patients received either hybrid RT [involving three-dimensional conformal RT (CRT) or intensity-modulated RT (IMRT) to the supraclavicular area and two-dimensional (2D) electron-beam RT to the chest wall; Figure 2A] or integrated RT [involving IMRT or volume intensity-modulated arc RT (VMAT) to both the supraclavicular area and chest wall; Figure 2B]. Moreover, 50-Gy doses were delivered in 25 fractions.

Figure 2 (A) Hybrid RT. (B) Integrated RT.

Laboratory Data

The absolute lymphocyte counts (ALCs) of the included patients at different time points were collected. In particular, we included the ALCs at baseline (pre-ALC; within 1 month before RT), during RT (once a week during RT), and within 3 months after treatment (Figure 3). Lymphopenia was graded according to the Common Terminology Criteria for Adverse Events (version 4.03). The nadir ALC during the RT course was used to classify lymphopenia degrees: ALC ≥ 1.0 × 10⁹/L, grade 0 (G0); ALC = (0.8–1.0) × 10⁹/L, grade 1 (G1); ALC = (0.5–0.8) × 10⁹/L, grade 2 (G2); ALC = (0.2–0.5) × 10⁹/L, grade 3 (G3); and ALC < 0.2 × 10⁹/L, grade 4 (G4).

Figure 3 The typical time schedule of the RT and blood sample collection.

Dose–Volume Parameters

The sternum was contoured with other organs at-risk (Figure 1). Moreover, the relative volume of normal tissues at risk of receiving x Gy (V_x) and mean dose (D_mean) were calculated from the dose–volume histogram.

Follow-up

After treatment, all patients were followed up every 3 months for the first 2 years, every 6 months for the next 2 years, and once a year thereafter. Recurrence was confirmed through diagnostic imaging or histopathology. Chest wall recurrence was considered local recurrence. Moreover, recurrence at axillary lymph nodes, internal mammary lymph nodes, and supraclavicular lymph nodes was considered recurrence at regional lymph nodes. The spread of a tumor to distant organs or nonregional lymph nodes was considered to indicate distant metastasis.

Statistical Analysis

DFS was defined as the period from the RT end date to the date of the first recurrence or death due to any cause or censorship. Overall survival (OS) was defined as the interval from the RT end date to death due to any cause or censorship. The Kaplan–Meier method was used to calculate DFS and OS, and the log-rank was used to determine the significance of between-group differences. Logistic regression analysis was used to identify the factors associated with G3 lymphopenia. Cox multivariate regression analysis was used to identify risk factors affecting DFS. Receiver operating characteristic (ROC) curves were used to determine the thresholds for G3 lymphopenia prevention.

All statistical analyses were performed on SPSS (version 23.0; IBM, Armonk, NY, USA). A two-tailed p value of <0.05 was considered to denote statistical significance.

Results

Patient Characteristics

We recruited a total of 156 breast cancer patients who received a mastectomy; of them, 59 patients received neoadjuvant chemotherapy, 107 patients received hybrid RT (15 IMRT vs 92 CRT cases in the supraclavicular area), and 49 patients received integrated RT (11 IMRT vs 38 VMAT cases). A total of 153 patients completed the prescribed dose of 50 Gy, while 3 patients received a reduced dose of 48 Gy (due to personal reasons resulting in incomplete treatment). Table 1 presents the characteristics of patients with varied degrees of lymphopenia. In total, 132 and 24 patients were assigned to the G0-2 and G3 groups, respectively. The ALCs before treatment were comparable between the G0-2 and G3 groups [(1.76 ± 0.56) × 10⁹/L vs (1.62 ± 0.56) ×10⁹/L; p = 0.293]. Moreover, 29 (18.6%), 36 (23.1%), 67 (42.9%), and 24 (15.4%) patients developed G0, G1, G2, and G3 lymphopenia during their RT course, respectively.

Table 1 Characteristics of Patients with Different Degrees of Lymphopenia

ALC Changes

The median RT duration was 5.0 weeks. The ALC decreased gradually during treatment and reached the nadir in week 5 (Figure 4). In all patients, the ALC before RT was (1.74 ± 0.56) ×10⁹/L. It reduced to (1.35 ± 0.41) × 10⁹/L, (1.09 ± 0.33) × 10⁹/L, (0.92 ± 0.29) × 10⁹/L, (0.77 ± 0.27) × 10⁹/L, and (0.72 ± 0.28) × 10⁹/L after 1, 2, 3, 4, and 5 weeks of RT, respectively. Within 3 months after RT, the ALC was (1.20 ± 0.40) × 10⁹/L.

Figure 4 ALC distributions before, during, and after treatment.

Lymphopenia–Survival Correlation

The date of the final follow-up was July 8, 2024, and the median follow-up duration was 45.5 months. Two G0-2 group patients died of cancer progression. The median OS was not achieved in the G0-2 and G3 groups. The 1-, 3-, and 5-year OS rates in the G0-2 group were 100%, 99.2%, and 98.3%, respectively. Moreover, the 1-, 3-, and 5-year OS rates in the G3 group were all 100% (p = 0.531). The median DFS in the G3 group was 54.0 months; however, this value was not obtained for the G0-2 group. The 1-, 3-, and 5-year DFS rates in the G3 group were 83.3%, 69.2%, and 39.5%, respectively; these values were lower than those in the G0-2 group [97.0%, 90.3%, and 87.4%, respectively; hazard ratio (HR) = 0.206, 95% confidence interval (CI) = 0.062–0.686, p < 0.001; Figure 5]. The Cox univariate and multivariate analysis results demonstrated that the pathological stage and lymphopenia degree were independent prognostic factors for DFS in patients with breast cancer (Table 2).

Table 2 Multivariate Analysis of Prognostic Factors for DFS

Figure 5 DFS in patients with RIL.

Lymphopenia Predictors

Table 3 presents the relationship between lymphopenia during treatment and different clinical characteristics. The age, tumor location, and chemotherapy modality of patients were nonsignificantly associated with G3 lymphopenia risk. In contrast, BMI and RT modality were significantly associated with G3 lymphopenia risk (both p < 0.05). Regarding the RT dosimetric predictors, the radiation doses at the sternum and heart were both associated with higher rates of G3 lymphopenia (all p < 0.05). Finally, our ROC curve analysis revealed the optimal cutoff points of the dosimetric variables significantly associated with G3 lymphopenia (Table 4).

Table 3 Logistic Regression Analysis of Factors Associated with G3 Lymphopenia

Table 4 ROC Curve Cutoff Points for G3 Lymphopenia Prevention

Discussion

This study revealed that lymphocytes exhibit exquisite radiosensitivity during postoperative radiotherapy for breast cancer, and G3 lymphopenia was significantly associated with impaired DFS. As we all know, lymphocytes are extremely sensitive to low doses of ionizing radiation. In the 1990s, in vitro studies reported that the entry of even a relatively low radiation dose into the bloodstream can significantly reduce lymphocyte counts; in particular, a lethal dose of only 2-Gy can reduce lymphocyte survival by 50%.¹⁷ Yovino et al¹⁸ designed a typical malignant glioma planning (8-cm tumor and 60-Gy radiation in 30 fractions) to monitor the radiation dose received by circulating blood cells. The results demonstrated that after a single dose of 2-Gy radiation, 5% of circulating blood cells received a 0.5-Gy dose, whereas 62%, 92%, and 99% of circulating blood cells received ≥0.5-Gy doses after 10, 20, and 30 fractions of 2-Gy radiation, respectively. In the present study, lymphocytes demonstrated extreme sensitivity to irradiation, and their counts gradually decreased as the RT course progressed, reaching their lowest value in RT week 5 and gradually recovering to baseline levels after the end of RT. This result is consistent with that of studies reporting lymphocyte decline after RT for esophageal cancer.^13,14

RT during cancer treatment can influence tumor immunogenicity by increasing the expression of certain tumor-specific antigens. The immune system can process these antigens, stimulating the transformation of naive lymphocytes into tumor-specific lymphocytes. Lymphocytes are key effector cells in tumor immune responses; thus, a decrease in lymphocyte counts may reduce the immune system’s clearance efficiency of malignant tumor cells. Qiu et al¹⁹ identified a correlation of effective doses to immune cells with poor clinical outcomes and severe RIL, indicating that high doses to the immune system are related to cancer progression and death. Moreover, several studies have demonstrated that severe RIL is a negative prognostic factor for numerous cancers, such as brain tumor, pancreatic cancer, lung cancer, stomach cancer, cervical cancer, esophageal cancer, nasopharyngeal cancer, and breast cancer.^{11,13,14,16,20–24} A recent study reported that severe RIL can compromise survival benefits from durvalumab after CCRT for non–small-cell lung cancer.²⁵ A study on CCRT for esophageal cancer demonstrated that the occurrence of G4 lymphopenia is an independent prognostic factor for worsened OS. Although lymphocyte counts may gradually recover to near-normal or normal levels after CCRT, the worsening of the OS cannot be reversed.¹⁴ Tseng et al²⁶ also reported that inadequate lymphocyte recovery was significantly associated with worse OS and local recurrence-free survival in esophageal cancer patients who received RT. Therefore, maintaining a sufficient lymphocyte count during RT is crucial for cancer patient survival.

Only a few studies have assessed the relationship between RIL and breast cancer prognosis. Kobzeva et al¹⁵ reported that RT led to significant reductions in the absolute counts of B, T, and natural killer cells in all breast cancer patients, regardless of whether they had previously undergone chemotherapy. Another study revealed that RIL after breast-conserving surgery can affect prognosis.²⁷ Sun et al¹⁶ assessed the relationship between RIL and survival after mastectomy and noted that the 5-year DFS rate was 71.8% in patients with nadir-PLC/pre-PLC ratio < 0.8, which was significantly lower than that in patients with nadir-PLC/pre-PLC ratio ≥ 0.8 (82.6%; p = 0.01); no such between-group difference was noted in the patients’ OS rate. Similarly, the current results indicated that G3 lymphopenia after postmastectomy RT is associated with poor DFS in patients with breast cancer.

The causal relationship between RIL and poor prognosis in cancer patients remains unclear. Naive T cells can be categorized into helper T (T_h) cells (CD3⁺CD4⁺) and cytotoxic T_cyt cells (CD3⁺CD8⁺).²⁸ T_cyt cells can directly kill abnormal cells.²⁹ Regulatory T (T_reg) cells (CD4⁺CD25⁺Foxp3⁺)—a subset of T_h cells²⁸—are involved in immune suppression.³⁰ Muroyama et al³¹ found that after exposure to 10-Gy radiation, the number of T_reg cells in the tumor microenvironment increased in tumor-bearing mice. Oweida et al^32,33 reported that RT combined with T_reg-cell inhibitors can inhibit tumor growth. Because of the relative resistance of T_reg cells to radiation, surviving T_reg cells may be able to inhibit effector T-cell recovery during lymphocyte recovery.³⁴ A clinical study also indicated that an increased proportion of CD8⁺T/T_reg cells predicts improved cancer prognosis.³⁵ Therefore, the effects of RIL on survival might be indicated by changes in the circulating T-cell numbers and subpopulations during RT. In the current study, Cox multivariate analysis revealed that the pathological stage and lymphopenia degree were independent prognostic factors for DFS in our patients—consistent with the results of Sun et al.¹⁶

In the present study, low BMI, integrated RT use, and increased heart and sternum exposure were associated with G3 lymphopenia. The lymphocyte counts may have decreased mainly because irradiation reduced circulating mature lymphocyte numbers, as well as diminished lymphocyte production in the hematopoietic organs. In the human body, the blood volume accounts for 7%–8% of body weight. Patients with a higher BMI have larger blood volumes, indicating the presence of a richer reserve of mature lymphocytes in peripheral blood; as such, RT has less impact on lymphocytes. Therefore, high BMI may be a protective factor for lymphocyte depletion. Low BMI increases RIL risk in patients with esophageal cancer and breast cancer.^16,36 However, the underlying mechanism warrants further research. Modern radiation techniques in which a large volume of tissue is irradiated with low doses of radiation can increase lymphopenia risk.

The heart is rich in blood, and the sternum is an adult hematopoietic organ; both are located near the radiation field used for breast cancer. Therefore, compared with hybrid RT, integrated RT demonstrates a larger low-dose area distribution; this results in an increase in the radiation doses delivered to the heart and sternum, accelerating lymphocytopenia development. Studies have reported that radiation doses to the heart, lungs, sternum, thoracic vertebrae, and spleen are predictive factors for RIL in cancer patients receiving RT.^13,37–42 Therefore, low-dose irradiation of structures containing large amounts of blood or demonstrating high-velocity blood flow may be associated with RIL development. Hence, optimizing treatment plans to decrease radiation doses to immune cells is essential for improving relevant clinical outcomes.

Several studies have focused on RIL prevention during RT. Proton or carbon-ion RT, which can protect organs at-risk because of its physical advantages, is gradually being applied in cancer treatment. Compared with photon RT, proton RT can effectively reduce G3 or G4 RIL occurrence.^43–50 Similarly, carbon-ion RT can reduce severe RIL development in patients with locally advanced pancreatic cancer compared with photon RT.⁵¹ However, the suitability of proton or carbon-ion RT as postoperative adjuvant RT for patients with breast cancer remains unclear. The RT segmentation method can also affect the degree of RIL during RT. McLaughlin et al⁵² reported that treatment with stereotactic RT was associated with a low degree of RIL in patients with early non–small-cell lung cancer. Sun et al¹⁶ also reported that RIL risk after hypofractionated RT was lower than that after conventional fractionated RT. McCullum et al⁵³ used dynamic four-dimensional blood flow simulations to predict RIL severity in individual proton RT patients by varying dose rates and fractionation. Their results indicated increasing the dose rate at constant fractionation can reduce ALC depletion more significantly than reducing the number of fractions. Moreover, when shortening the fractionation regimen, higher dose rates are associated with increased lymphocyte sparing, particularly in high-risk patients with radiosensitive lymphocytes. However, relatively few studies have focused on the role of chemotherapeutic drugs in preventing or reversing RIL. Zheng et al⁵⁴ reported that cinnamon effectively reversed T-cell subpopulation imbalance and promoted effective anticancer immunity by increasing T_h1-cell proliferation and inhibiting T_h17- and T_reg-cell expansion in a mouse lung melanoma model after a single low-dose whole-body irradiation. In addition, other animal studies have demonstrated that administering exogenous interleukin (IL) 7 can not only restore lymphocyte counts but also enhance RT’s anticancer effects. Exogenous IL-7 can aid in overcoming lymphocyte depletion, and in combination with RT, it can improve treatment efficacy.⁵⁵ However, these findings require validation through further clinical research.

In the current study, we analyzed the relationship between RIL and survival in patients who received postmastectomy RT and, for the first time, elucidated the relationship between dosimetric parameters of organs at-risk and RIL development. The major limitations of this study are the retrospective design, small sample size (mainly because of few eligible patients opting for routine complete blood analysis), and relatively short follow-up duration. Moreover, we included patients who received adjuvant or neoadjuvant chemotherapy. Therefore, the current results may be validated by additional prospective studies with larger sample sizes and longer follow-up periods.

In summary, RIL, commonly occurring during RT, influences cancer prognosis during and after treatment. However, the causal relationship of RIL with poor survival and the underlying mechanisms remain unclear and warrant further research. Strategies to reduce RIL risk include using hypofractionated RT, increasing dose rate, optimizing RT for new critical organs such as the heart and sternum, using particle RT, and applying other procedures to reduce the integral radiation dose.

Conclusions

G3 RIL was noted to be a crucial prognostic predictor of postmastectomy RT in patients with breast cancer. Significant predictors in the prediction model of G3 lymphopenia during RT were identified to be BMI, RT modality, and heart and sternum exposure dose. The current findings highlight the need for RT dose optimization and planning to minimize RIL risks. Thus, the sternum adjacent to the heart should also be considered a routine organ for RIL risk evaluation during RT planning for patients with breast cancer.