Prognostic Value of Lymphocyte Subset Levels in Hepatocellular Carcinoma Following Conventionally Fractionated vs. Stereotactic Body Radiotherapy

doi:10.21203/rs.3.rs-4696525/v1

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Research Article

Prognostic Value of Lymphocyte Subset Levels in Hepatocellular Carcinoma Following Conventionally Fractionated vs. Stereotactic Body Radiotherapy

https://doi.org/10.21203/rs.3.rs-4696525/v1

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Background and aim:

Radiotherapy (RT)-induced lymphopenia is associated with poor prognosis. This study aimed to compare the effects of conventionally fractionated radiotherapy (CFRT) and stereotactic body radiotherapy (SBRT) on lymphocyte subpopulations in hepatocellular carcinoma (HCC) patients and to evaluate the prognostic value of lymphocyte subset levels.

Materials and Methods

A retrospective analysis was conducted on 137 HCC patients who underwent CFRT or SBRT between July 2011 and January 2018. Variables were obtained within 1 week before RT, and at 1 day and 2 months post-RT, respectively. Univariate and multivariate Cox regression analyses were conducted to investigate independent prognostic factors for overall survival (OS).

Results

The one-year and two-year OS rates were 80.0% and 55.0%, respectively. Multivariate analysis identified tumor size > 4.5cm, multiple tumors, and post-RT CD4 + T cell count < 231/µL and CD8 + T cell count < 179/µL as independent factors associated with inferior OS in HCC patients. Severe RT-induced lymphopenia (< 0.5*10^⁹/L) occurred in 70.0% of patients following CFRT compared to 23.0% in SBRT patients. Patients receiving SBRT exhibited higher total lymophocyte counts and subset levels at 1 day and 2 months post-treatment compared to those receiving CFRT (P < 0.05). Further analysis revealed that CD19 + B cells were predominantly depleted and recovered more slowly than other populations while CD8 + T cells demonstrated rapid recovery. In the SBRT group, patients with OS > 2 years exhibited higher levels of CD4 + T cell and CD8 + T cell counts compared to those with OS < 2 years (P < 0.05).

Conclusion

SBRT induced less severe lymphopenia than CFRT. Peripheral lymphopenia of CD4 + and CD8 + T cells post-SBRT may independently predict survival for HCC patients.

radiotherapy

hepatocellular carcinoma

lymphopenia

overall survival

Hepatocellular carcinoma (HCC) ranks as a prevalent malignant tumor and the third leading cause of cancer-related mortality worldwide [1]. China contributes to half of the total HCC cases and deaths globally. Currently, surgical interventions remain the primary treatment approach for HCC patients [2]. However, certain patients are deemed ineligible for surgery due to donor shortages, limited liver function, extensive tumor burden, or comorbidities.

Advances in conformal photon-based techniques, such as intensity modulated radiotherapy (RT) and volumetric modulated arc therapy, have enhanced the role of RT in the management of HCC [3]. Irradiation exerts dual effects on HCC treatment. It directly kills tumor cells by inducing DNA damage and breakage while simultaneously eliciting systemic immune-modulating effects [4, 5]. However, it can also decrease the total peripheral lymphocyte count (TPLC) in peripheral blood, thereby weakening anti-tumor immune responses, which is correlated with poor prognosis in HCC patients [6, 7]. Hence, mitigating the incidence of RT-induced lymphopenia (RIL) is of paramount importance.

Conventionally fractionated radiotherapy (CFRT), delivered in 5 to 6 weeks has been used to treat advanced HCC. More recently, stereotactic body radiotherapy (SBRT), capable of delivering a more ablative dose in a short period, has emerged as an effective treatment approach for HCC. Compared to CFRT, SBRT offers substantial local control, improved overall survival (OS), and reduced toxicity for patients with HCC [8]. Additionally, SBRT may present a lower risk of inducing lymphopenia [9].

Increasing evidence has shown that RIL portends poor prognosis in patients with HCC, as well as in pancreatic, cervical, esophageal, lung, and central nervous system malignancies [7, 10–14]. However, the effects of RT-induced decreases in the absolute counts of circulating lymphocyte populations (CLPs), including CD4 + T cells, CD8 + T cells, CD19 B + cells and CD56 + NK cells on HCC patients remain unclear. This study aimed to elucidate the differential impact of SBRT and CFRT on subgroups of circulating lymphocytes and to characterize the influence of RIL on survival outcomes in HCC patients.

Patient selection

We conducted a retrospective examination of medical records from patients diagnosed with unresectable HCC. Inclusion criteria for patients undergoing RT were as follows: 1) HCC diagnosis confirmed by histologic or imaging criteria based on the National Comprehensive Cancer Network guidelines for hepatobiliary cancers, 2) age over 18 years, 3) Child-Pugh class A or B, 4) Eastern Co-operative Oncology Group performance status 0 or 1, 5) laboratory tests conducted within one week before, one month and two months after RT, and 6) one or more radiological assessments performed prior to and following RT. Patients meeting any of the following criteria were excluded from the study: 1) presence of distant metastasis, 2) double primary malignancy, or 3) follow-up duration less than six months post-completion of RT, and 4) receipt of other local treatments within one month before RT. This research received approval from the Institutional Review Board of the Ethics Committee at our institution and adhered to the principles outlined in the Declaration of Helsinki.

Radiation Treatment

All enrolled patients underwent breathing training and respiratory exercises prior to the initiation of RT. The Body Pro-Lok system was utilized for abdominal compression to minimize liver motion. Patients underwent computed tomography (CT) simulation while immobilized in a custom vacuum body mold in the supine position. The gross tumor volume (GTV) encompassed all tumors identified via dynamic CT imaging. For tumors not well visualized on CT scans, pre-treatment magnetic resonance imaging (MRI) studies were co-registered with the planning CT. Four-dimensional CT simulations were employed to generate an internal target volume (ITV), and the planning target volume (PTV) was defined as the ITV plus a radial margin of 3 mm. Patients receiving SBRT were administered 5–10 fractions delivered five times per week, with a median dose of 48 Gray (Gy) and a range of 45–70 Gy. CFRT was characterized by a radiation dose of 1.8Gy or 2Gy per fraction, prescribed to the isodose surface covering 95% of the PTV.

Follow-up and patient outcomes

The follow-up duration was defined as the period from the initiation of RT to determine the median follow-up and time-to-event estimates. Baseline peripheral blood cell counts, including lymphocyte subsets, were obtained before RT, while post-treatment values were assessed at 1 day and two months after RT. In cases where peripheral cell records were unavailable for a specific month, the closest available values were utilized. Follow-up CT or MRI studies were conducted 6–8 weeks following RT and subsequently every three months. OS was calculated at the patient level as the duration from the first RT session until death from any cause or the last follow-up.

Statistics and analysis

Descriptive statistics were presented as means ± standard deviation or as medians and interquartile range, depending on the normality of data distribution assessed using Kolmogorov–Smirnov tests. Quantitative variable comparisons were assessed using two-sided t-tests or Mann–Whitney tests, as appropriate. The effects of RT on lymphocyte subsets at different times were compared using repeated measures analysis of variance. The primary endpoint was OS. Cumulative survival was calculated using the Kaplan–Meier method, and cutoff values for continuous variables affecting patient prognosis were determined using receiver operating characteristic curve. Univariate and multivariate analyses were conducted using Cox regression models, presenting hazard ratios (HRs) and 95% confidence intervals (95% CIs). IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA), was used for data analysis.

1. Patient characteristics and clinical outcomes

A cohort of 137 patients with HCC treated with either SBRT (n=84) or CFRT (n=53) were enrolled in the study. Treatment and tumor characteristics of patients are summarized in Table 1 and stratified by radiation fractionated regimen for comparison. Among the total cohort, 101 patients (73.7%) had received previous treatments including surgery, transarterial chemoembolization, radio frequency ablation and percutaneous ethanol injection. The median tumor size was 2.5 cm for patients treated with SBRT and 5.1 cm (range, 1.2-14.0) for patients receiving CFRT. Median GTV was 16 cc and 285 cc for patients receiving SBRT and CFRT, respectively. The median number of fractions was 6 (range 5-10) for patients receiving SBRT and 25 (range 20-30) for those receiving CFRT. The median biologically equivalent dose (BED) was 77 Gy (range, 65-140 Gy) and 72 Gy (range, 55-91 Gy) for patients treated with SBRT and CFRT, respectively.

The median follow-up time was 35 months (range, 7.5-80 months). Median survival time was 29 months (SBRT 69 months vs. CFRT 17 months). The one-year and two-year OS rates were 80.0% and 55.0%, respectively (93.0% and 77.6% for SBRT, 66.0% and 38.0% for CFRT, respectively).

2. Changes in absolute counts of lymphocyte subsets induced by SBRT versus CFRT

No significant differences were observed in pre-treatment TPLC between SBRT and CFRT-treated patients (Fig. 1). However, severe lymphopenia (<0.5*10^⁹/L) occurred in 23.0% of patients receiving SBRT and 70.0% in those receiving CFRT. Further analysis was performed according to different CLPs. As is shown in Fig. 1, peripheral B cells were profoundly depleted after both SBRT and CFRT (decreased by 78.9% and 90.5%, respectively), followed by NK cells (decreased by 47.3% and 71.8%, respectively). The counts of CD4+ T cells and CD8+ T cells decreased by 46.5% and 50.4% for SBRT and 66.3% and 52.6% for CFRT, respectively (Table S1). Mean values of TPLC and CLPs at different time post-RT were listed in Table 2.

Two months post-RT, TPLC and CLP counts significantly increased. Interestingly, CD8+ T cells recovered to baseline levels post-SBRT and exceeded baseline levels post-CFRT, while CD4+ T cells showed slower recovery, resulting in a pronounced decrease in the CD4/CD8 ratio, particularly post-CFRT. Conversely, the effect of SBRT on the CD4/CD8 ratio was milder (Fig. 1 and Table 2).

3. Univariate and multivariate Cox regression analyses for patients with HCC.

Factors including age; presence of hepatitis and portal venous thrombus, previous treatments, tumor size, tumor number, Child-Pugh grade, baseline AFP, baseline and post-treatment TPLC and CLP counts, GTV, BED and fractions were analyzed at the univariate level. The results demonstrated that portal venous thrombus present, tumor size ≥4.5 cm, multiple tumors, a high level of AFP, GTV and fractions, decreased BED, post-treatment TPLC and CLP counts, were significantly associated with poor OS (Table 3, p<0.05 for each). After adjusting for covariates, the Cox regression model revealed that tumor size ≥ 4.5 cm (HR: 1.25; 95% CI: 1.10-1.43; p=0.001), multiple tumors (HR: 3.39; 95% CI: 1.71-6.74; p<0.001), post-treatment CD4 <231cells/μL (HR: 0.53; 95% CI: 0.32-0.90; p=0.018) and post-treatment CD8 <179cells/μL (HR: 0.74; 95% CI: 0.55-0.99; p=0.043) were identified as independent adverse factors for OS (Table 3).

4. The association of CLP post-RT with patients’ OS.

Regarding CLP after RT, cumulative survivals were calculated using the Kaplan-Meier method and exhibited in Fig. 2. Significant differences were observed in the OS curves between higher and lower levels of CD4+ and CD8+T cells (p<0.05 for each), underscoring the association between post-treatment CLP levels and patients’ OS.

To further evaluate the effects of CLP post-RT on patient survival under different RT modalities, patients were stratified based on whether they underwent SBRT or CFRT. As shown in Figure 3, patients with lower CD4+ and CD8+T cell counts after SBRT tended to exhibit inferior OS (p < 0.05 for each). However, this trend is not observed in the CFRT group. In the CFRT group, the influence of post-treatment CD4+ T cells on OS became evident beyond a two-year survival period, while the level of post-treatment CD8+ T cells after CFRT showed no association with OS.

The pivotal role of immune system in cancer control is widely acknowledged, highlighting the importance of functional lymphocytes in identifying and eliminating malignant cells [15, 16]. The impacts of RT on the immune system are intricate and diverse. While irradiation primarily targets tumor cells for destruction, it can also influence adjacent normal tissue and immune cells [17, 18].

In the present study, we observed that CFRT resulted in more severe lymphopenia, consistent with previous findings by Wild et al [9]. Our previous study has also confirmed that smaller GTV and fractions can reduce the risk of RIL in patients with HCC [19]. These findings closely align with the hypothesized mechanisms underlying treatment-induced lymphopenia [10, 20, 21]. The phenomenon of RIL was initially delineated in 1916 [22–24]. Exposure primarily through circulating blood leads to a substantial decrease of 60–80% in circulating lymphocytes, persisting for years following exposure to RT [25]. Previous studies have established a negative correlation between fraction number and TPLC following RT, while keeping the total radiation dose consistent [24]. This proposition was corroborated in research comparing the impacts of conventionally fractionated (50.4 Gy in 28 fractions) and hypofractionated (30 Gy in 10 fractions) neoadjuvant chemoradiotherapy in pancreatic cancer patients. CFRT was associated with pronounced RIL, encompassing both CD4 + and CD8 + subsets, while hypofractionated chemoradiotherapy led to a milder decline in T cell levels with a swifter recuperative trajectory [26].

Among the various subsets of lymphocytes, CD19 + B cells exhibited pronounced depletion, with a notably slower recovery rate compared to other populations, particularly evident in CFRT. Clave et al. assessed the in vivo radiosensitivity of lymphocytes through the administration of whole-body irradiation to patients before undergoing bone marrow transplantation [27]. Similar to our conclusion, the research revealed that B cells exhibited the highest degree of sensitivity, with T cells (CD4+, CD8+) and NK cells following as the subsequent sensitive cell types. CD19 + B cells are recognized for their potential significance in tumor immune surveillance, as they contribute to the identification and elimination of malignant cells. Through antibody production and immune response modulation, CD19 + B cells facilitate the enhancement of the immune system’s recognition and response to tumor cells [28, 29]. Relative to CFRT, SBRT is posited to attenuate the influence of RT on the body’s immune surveillance prowess. Simultaneously, it may mitigate the decline in the body’s capacity to identify and eradicate tumors resulting from the reduction of B cells subsequent to RT, thereby potentially impeding the development and progression of tumors.

Our study also found a rapid resurgence of CD8 + T cells within a two-month period following the conclusion of RT, particularly notable among participants receiving CFRT. In contrast, the recovery trajectory of CD4 + T cells was characterized by a sluggish pace, initially marked by a modest elevation in the CD4+/CD8 + ratio, followed by a notable reduction below the threshold of 1.0. Yang et al. similarly documented a decline in the CD4+/CD8 + T cell ratio among individuals with head and neck cancer [30]. This alteration in the ratio appeared unrelated to minor variations in the radiosensitivity of CD4 + and CD8 + T cells. Instead, it likely stemmed from radiation-triggered activation and recruitment of CD8 + T cells, which counterbalanced the reduction in CD8 + T cells.

Prior investigations have indicated that RT exerts an influence on the prognosis of tumor patients through the depletion of T lymphocytes in the bloodstream [31]. Wild et al. found that for unresectable pancreatic cancer, CFRT was associated with a higher proportion of severe RIL compared to SBRT (71.7% of CFRT vs 13.8% of SBRT patients; P < .001), and higher post-treatment TPLC was associated with improved survival (HR for death: 2.059; 95% confidence interval :1.310–3.237; P = 0.002)[9]. Moreover, studies have demonstrated that employing hypofractionated regimens in the treatment of HCC results in reduced lymphocyte depletion and expedited post-treatment recovery[32]. Our findings indicate that the diminished levels of CD4 + and CD8 + T lymphocytes subsequent to RT serve as independent prognostic indicators for unfavorable outcomes among HCC patients, aligning with prior scholarly research [7, 31]. Furthermore, our study revealed a notable correlation between diminished counts of CD4 + and CD8 + T lymphocytes after RT and diminished OS rates within the SBRT cohort. Hence, for patients undergoing SBRT, the preservation of elevated lymphocyte levels holds paramount importance for enhancing patient survival outcomes.

The prevention of RIL represents a significant area of concern. Although complete avoidance of this phenomenon may not always be achievable, there exist strategies aimed at alleviating its impact. Increased focus should be directed towards monitoring the lymphocyte count in patients undergoing RT for tumors, both during and after treatment. Early detection of lymphocyte depletion enables timely intervention, thus mitigating the risk of complications and ultimately enhancing patient prognosis. Moreover, the dosage and regimen of RT can be tailored based on the patient’s condition to minimize lymphocyte damage. For instance, the application of SBRT technology allows for the escalation of single irradiation doses while decreasing the overall number of irradiations. Besides, during target delineation, it is advisable to minimize exposure to major blood vessels, spleen, and other hematopoietic organs. When designing RT plan, reducing radiation through bloodstream may help reduce the occurrence of RIL. Several investigations have indicated that adjunctive therapies, such as cytokine interventions, hold promise in ameliorating the diminishment of lymphocytes induced by radiation therapy. Byun et al. found that injection of exogenous Interleukin 7 after RT not only restored the RT-induced decrease in lymphocyte count, but also enhanced the antitumor effect compared with RT alone [33, 34]. Additional investigations are currently in progress, aiming to provide further insights into RIL.

One limitation of this study is the relatively small sample size and its retrospective nature, which may introduce bias. Additionally, over 73.7% of patients in this study received other treatments prior to RT, including surgery, transarterial chemoembolization, radio frequency ablation and percutaneous ethanol injection. These treatments might impact patients’ immune function, thereby influencing lymphocyte counts. Although multivariate Cox regression analysis accounted for their effects on patient OS, potential data biases may persist. We plan to conduct prospective studies to address these issues.

In summary, our study revealed that SBRT has less effect on lymphocytes in HCC patients than CFRT, and CD4 + and CD8 + T cell counts after SBRT are independent prognostic factors for OS in HCC patients. Therefore, investigating therapeutic approaches aimed at mitigating RIL holds promise for enhancing treatment efficacy and prolonging survival among HCC patients.

Acknowledgements

The authors thank all the participants who took part in the study.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 82102913 and No. 82102823) and the Ministry of Science and Technology of the People’s Republic of China (No. 2022YFC2503704).

Authors' contributions

All named authors contributed to the paper. S-T W drafted the manuscript. Y-X C, X-N G, P Y and Q-Q Z participated in the collection, analysis and interpretation of data. Y-X C, Y Z and Z-C Z conceived the study, and participated in its design and coordination and critically revised the manuscript. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The study was ethically reviewed and approved by the Ethics Committee of Zhongshan Hospital Affiliated to Fudan University. Informed consent was given by accepting the invitation to participate and follow-up for each patient.

Competing interests

The authors declare that they have no competing interests.

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TABLE 1. Patient characteristics.

Characteristic	All patients (n=137)	SBRT-treated patients (n=84)	CFRT-treated patients (n=53)	p-value
Median age (range)	62 (22-87)	59 (22-86)	58 (30-87)	0.259
Sex				0.230
Male	113 (82.5%)	71 (84.5%)	42 (79.2%)
Female	24 (17.5%)	13 (15.5%)	11 (20.8%)
Child-Pugh				0.295
A	118 (86.1%)	71 (84.5%)	47 (88.7%)
B	19 (13.9%)	13 (15.5%)	6 (11.3%)
Median tumor size (cm)	3.0 (0.8-14.0)	2.5 (0.8-6.8)	5.1 (1.2-14.0)	<0.0001
Mean AFP (ng/mL)	3741 ± 12057	977 ± 2344	7524 ± 17449	<0.0001
TPLC before RT (10^9/L)	1.31± 0.59	1.39± 0.58	1.23± 0.60	0.060
Viral etiology Hepatitis				0.152
Yes	95 (69.3%)	57 (67.9%)	38 (71.7%)
No	42 (30.7%)	27 (32.1%)	15 (28.3%)
Previous treatments				0.231
Yes	101 (73.7%)	64 (76.1%)	39 (73.6%)
No	36 (26.3%)	20 (23.9%)	14 (26.4%)
Median of fractions (range)	10 (5-30)	6 (5-10)	25 (20-30)	<0.0001
Median GTV (cc)	38 (1.6-1530)	16 (1.6-465)	285 (17-1530)	<0.0001
Median BED (Gy)	77 (55-140)	77 (65-140)	72 (55-91)	0.087
Abbreviations: AFP, alpha-fetoprotein; BED, biologically equivalent dose; CFRT, conventionally fractionated radiotherapy; GTV, gross tumor volume; RT; radiotherapy; SBRT, stereotactic body radiotherapy; TPLC, total peripheral lymphocyte count.

TABLE 2. The mean values of TPLC and CLP at different time after RT.
Counts of lymphocytes	All patients (n=137)	SBRT-treated patients (n=84)	CFRT-treated patients (n=53)	p-value
TPLC before RT (×10⁹/L)	1.32±0.60	1.42±0.60	1.19±0.60	0.056
CD4 (cells/μL)	489.13±229.47	513.29±208.78	463.52±249.10	0.212
CD8 (cells/μL)	322.81±184.89	352.35±204.56	290.86±156.82	0.152
NK (cells/μL)	294.76±258.67	322.88±286.19	264.96±224.96	0.295
B (cells/μL)	141.77±103.59	172.52±84.48	109.18±112.50	＜0.001
TPLC at 1 day post-RT (×10⁹/L)	0.58±0.33	0.70±0.30	0.40±0.30	＜0.001
CD4 (cells/μL)	222.74±146.17	274.60±138.02	156.26±129.76	＜0.001
CD8 (cells/μL)	158.60±136.96	174.70±101.97	137.97±171.04	＜0.01
NK (cells/μL)	128.31±123.15	170.13±141.76	74.69±62.92	＜0.001
B (cells/μL)	25.02±27.29	36.41±29.16	10.41±15.43	＜0.001
TPLC at 2 months post-RT (×10⁹/L)	1.08±0.56	1.18±0.61	0.84±0.35	0.050
CD4 (cells/μL)	312.25±240.26	377.47±273.17	199.20±100.41	0.020
CD8 (cells/μL)	368.10±249.01	345.42±233.46	407.40±277.89	0.716
NK (cells/μL)	296.35±230.40	381.55±242.04	148.67±101.03	0.001
B (cells/μL)	82.29±56.34	100.76±57.61	50.27±37.53	0.018
Abbreviations: CFRT, conventionally fractionated radiotherapy; CLP, circulating lymphocyte population; RT; radiotherapy; SBRT, stereotactic body radiotherapy; TPLC, total peripheral lymphocyte count.

TABLE 3. Univariate and multivariate analysis of prognostic factors for OS.
Variables	Univariate analysis		Multivariate analysis
Variables	HR (95% CI)	p-value	HR (95% CI)	p-value
Age (≥60 years)	0.98 (0.96–1.00)	0.09
Presence of hepatitis	1.71 (0.98–2.99)	0.06
Previous treatments present	1.07 (0.61–1.86)	0.82
Portal venous thrombus present	2.59 (1.60-4.20)	<0.001	1.24 (0.60-2.57)	0.562
Tumor size (≥4.5cm)	1.20 (1.11–1.27)	<0.001	1.25 (1.10-1.43)	0.001
Multiple tumors	2.47 (1.51-4.05)	0.04	3.39 (1.71-6.74)	<0.001
Child B vs Child A	1.11 (0.52–2.37)	0.78
AFP	1.11 (0.91–1.32)	0.01	1.01 (1.0-1.02)	0.24
Baseline TPLC	0.70 (0.45–1.08)	0.098
TPLC at 1 day post-RT≤0.5（10^⁹/L）	2.55 (1.47–4.40)	<0.001
CD3 T cells	0.71 (0.60-0.83)	<0.001
CD4 T cells	0.49 (0.35-0.67)	<0.001	0.53 (0.32-0.90)	0.018
CD8 T cells	0.36 (0.53-0.91)	0.007	0.74 (0.55-0.99)	0.043
NK cells	0.69 (0.51-0.95)	0.023	1.20 (0.84-1.69)	0.33
B cells	0.78 (0.61-1.10)	<0.001	1.01 (0.99-1.03)	0.07
GTV	1.002 (1.001-1.003)	0.017
BED	0.97 (0.95-0.99)	0.001	1.00 (0.98-1.03)	0.642
Fractions	1.07 (1.04-1.11)	<0.001
Abbreviations: AFP, alpha-fetoprotein; BED, biological equivalent dose; GTV, gross tumor volume; OS, overall survival; RT; radiotherapy; TPLC, total peripheral lymphocyte count.

No competing interests reported.

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Prognostic Value of Lymphocyte Subset Levels in Hepatocellular Carcinoma Following Conventionally Fractionated vs. Stereotactic Body Radiotherapy

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