Treatment-duration is related to changes in peripheral lymphocyte counts during definitive radiotherapy for unresectable stage III NSCLC

Definitive radiotherapy (RT) with platinum-based doublet chemotherapy has been widely recognized over the past few decades as the standard of care for unresectable stage III non-small cell lung cancer (NSCLC). However, the survival outcomes with this approach remain disappointing, with a 5-year overall survival (OS) rate of approximately 15% using conventionally-fractionated radiotherapy (CFRT) with doses of 60–66 Gy in 30–33 fractions [1, 2]. As promising results were observed in the PACIFIC trial, consolidation treatments with immune checkpoint inhibitors (ICIs) are now recommended for patients with stage III, unresectable NSCLC after receiving definitive CRT [3]. However, on the basis of CFRT, increasing the number of fractions and radiation duration to increase the overall radiation dose did not improve outcomes for patients [4]. With higher biological effective dose (BED) over a shorter period, hypofractionated radiotherapy (HFRT) could overcome the accelerated repopulation of tumor cells during treatment and theoretically obtain better efficacies [5, 6]. However, the clinical application of HFRT has been limited due to the side effects of irradiation, particularly grade III or IV toxicities [6]. In recent years, advances in RT techniques have facilitated the safe and effective delivery of high doses of radiation to tumors while preserved the surrounding disease. Several studies reported that HFRT could achieve good local control with well-tolerated toxicity for locally-advanced NSCLC when modern RT techniques were utilized [7‐9]. HFRT is experiencing increasing importance in the treatment of locally-advanced NSCLC.

Numerous studies demonstrated that the host immune system and peripheral lymphocyte populations play crucial roles in oncologic outcomes, and the reduction in total peripheral lymphocyte counts (TLCs) results in impairing antitumor immunity [10, 11]. Lymphopenia is a common consequence of RT in cancer patients, regardless of whether other lymphotoxic agents, such as corticosteroids or cytotoxic chemotherapeutics are administered[12‐15]. The preclinical model suggested that decreasing the target radiation volume and fraction size significantly reduced the circulating blood exposure radiation dose [16]. It is therefore possible that protracted radiation courses with higher fractions will increase exposures to radiation and increase lymphocyte destruction. Such assumptions have led some researchers to conclude that HFRT with shorter treatment periods would reduce radiation exposure dose to circulating blood and spare peripheral lymphocytes, compared to CFRT with treatment period extending.

Despite theoretical superiority, the impact of different fractionation regimes and overall treatment times (OTT) on radiation-induced lymphopenia (RIL) in unresectable stage III NSCLC was largely unexplored until recently. In our institution, apart from CFRT, other fractionation regimes with increased doses per fraction and shorter OTT have been increasingly adopted since 2011 for locally advanced NSCLC. Therefore, we sought to evaluate the potential impact of fraction regime and OTT on TLCs by retrospectively analyzing stage III, locally advanced, unresectable NSCLC patients receiving definitive chemoradiation therapy (CRT) at our institution.

Many studies have shown that RT can dramatically reduce TLCs, and that low TLC nadirs were correlated with poor survival in many solid tumors. Our results further suggested that the level of TLCs was related to the RT treatment duration for unresectable stage III NSCLC. TLCs declined steeply each week for the first 5 weeks, after which, TLCs nadir occurred at approximately the 5th week. Increasing the dose of radiation per fraction to deliver the entire RT regimen in 4 weeks could significantly lower the risk of developing SL. This is in line with a recent study suggesting that SBRT could decrease the severity of RIL compared to CFRT in locally advanced pancreatic cancer [18]. Finally, our results revealed a significantly reduced risk of disease progression and death in patients who did not experience SL during RT.

Radiation can suppress or stimulate the immune system. The contribution of lymphocytes to radiation-induced tumor control was shown in mouse models over 30 years ago [19], and more recently, the availability of T cell receptor (TCR)-transgenic mice made it possible to unequivocally demonstrate that radiation can induce priming of T cells to exogenous model antigens expressed by tumors [20, 21]. Those studies together with the demonstration that radiation induces immunogenic cell death [22], have provided proof that radiation can induce tumor-specific T cells. This study revealed an association between higher lymphocyte levels during treatment and better clinical outcomes. Maintaining an intact adaptive immune system during cancer therapy may be important for enhancing the effectiveness of cytotoxic therapies and improving cancer control. This may impact cancer recurrence by affecting the numbers of tumor-infiltrating lymphocytes, which correlates with the prognosis of multiple cancers [23, 24]. This is consistent with our observation that TLC nadir was associated with worse PFS and OS.

The clinical implications of our findings may also be more pronounced in the setting of the new therapeutic strategy of combining RT and immunotherapy in NSCLC patients. As promising results were observed in the PACIFIC trial, consolidation treatments with immune checkpoint inhibitors (ICIs) are now recommended for patients with stage III, unresectable NSCLC after receiving definitive CRT [16]. Previous work has shown that RIL would further compromise the therapeutic efficacies of ICIs through the loss of effector cells, which identify and destroy tumor cells [25, 26]. The efficacy of ICIs relies on the modulation of lymphocyte activity and are dependent on circulating lymphocyte counts [27]. SL at the onset of ICI was independently associated with poor survival [25]. This further emphasized the importance of preserving and maintaining circulating lymphocytes in the emerging era of clinical immunotherapies. Considering the potential therapeutic abilities of circulating lymphocyte populations, a hypofractionated regimen with fewer fractionations and shorter treatment course (within 4 weeks) may be a superior approach for combination with immunotherapies.

Pulmonary circulation is the main portion of the cardiovascular system in which oxygen-depleted blood is pumped away from the heart via the pulmonary artery. A separate system known as bronchial circulation also supplies blood to the tissues of the larger airways. Arteries are further divided into very fine capillaries, which are extremely thin-walled and cover the lung. Some tumors may also be adjacent to heart, thoracic aorta, and vein which are filled with blood. Thus, the distribution of low-dose irradiation could include part of the heart or large vessels. One proposed mechanism for RIL is via radiation exposure of the circulating blood pool, since lymphopenia occurs after irradiation of tissues, including breast and brain, which contain little bone marrow or lymphatic tissues, respectively [28, 29]. Peripheral blood lymphocytes are known to be extremely sensitive to radiation despite mitotic inactivity [30]. Nevertheless, an unexpected finding of our study was that the TLC nadirs occurred at approximately the fifth week, not at the completion of RT or upon delivery of maximal doses.

Multiple studies demonstrated that pretreatment with low doses of irradiation can induce resistance to damage from subsequent high doses of irradiation [31, 32]. Yovino et al. analyzed a modeled “typical” radiation plan for glioblastoma (60 Gy/30 fractions) and found that a single fraction caused 0.5 Gy exposure to 5% of all circulating blood cells. L. Basler et al. also regarded that the proportion of circulating lymphocytes exposed to at least 0.5 Gy was a surrogate parameter for radiation-induced immunosuppression [33]. The circulating blood dose appeared to depend on the target volume and fraction number in early treatments. However, as treatment progressed to the 5th week of RT, nearly all the circulating blood cells were projected to receive at least 0.5 Gy (mean dose 2.2 Gy) [16]. This simulation model might explain our finding that by the 5th week of RT, nearly all circulating lymphocytes had received a low-dose (at least 0.5 Gy) of irradiation and developed resistance. The underlying mechanisms of this phenomenon are not well understood and our findings provide an important basis for further investigations.

In addition to the treatment duration, pre-RT TLCs, GTV, and MLD were also found to strong impact RIL in our study. A higher GTV and MLD tended to trigger greater depletion of peripheral lymphocytes due to greater exposure of those lymphocytes to radiation. We had excluded patients who had received any other anticancer treatments besides CRT before disease progression, had used any systemic corticosteroids during RT except for pretreatment before chemotherapy, had any ongoing infection, rheumatoid disease, or immune system disease to eliminate potential clinical factors influencing the lymphopenia. As for underlying respiratory system disease, our data show that it has no impact on lymphopenia. The contribution of chemotherapy to lymphopenia has been disputed [14, 34]. Our study showed that concurrent chemotherapy was not associated with SL, which was consistent with previous study [14]. Additionally, there was no difference in the median baseline TLCs or pre-RT TLCs in our study population, whether stratified by the chemotherapy regimen or cycles (all P > 0.05). This was consistent with the notion that the choice of chemotherapy regimens and the cycles had no correlation with lymphopenia in patients with stage III NSCLC [35]. Thus, the chemotherapy regimen used in our study population did not play a major role in causing lymphopenia. Bone marrow suppression caused by chemotherapy primarily resulted in the reduction of peripheral neutrophil cell counts in our patients (data not shown). Given the potential effect of corticosteroids on circulating lymphocytes, patients who had received systemic steroids during RT were excluded from our analyses, unless steroids were used as pretreatments before chemotherapy [36, 37]. However, logistic regression analyses showed that the dose of corticosteroids (equivalent to approximately 60–120 mg of prednisone) was not associated with an increased risk of developing SL. Clinical trials for ICIs typically excluded patients who received systemic steroids exceeding 10 mg of prednisone daily (or the equivalent) [38]. Collectively, our findings indicated that the doses of transient corticosteroid administration and chemotherapy had no obvious cytotoxicity to circulating lymphocytes.

Several limitations should be noted in interpreting our results. First, the frequencies of blood tests during RT were subject to variations, although the general practice at our institution was to perform blood testing prior to, and weekly during, RT. Second, considering the TLC nadir was at the 5th week of RT, we divided patients into two groups based on whether the RT course was completed within 4 weeks. However, the small number of patients in the STRT group decreased the analysis power. For example, despite a large absolute difference in 3-year OS rates favoring the STRT group after adjustment, the data did not reach statistical significance. Third, the selection of fractionation and dosing schemes was generally based on the treating physician’s preference. Fourth, unscheduled treatment interruptions due to malfunctions prolonged the OTT. Therefore, we cannot exclude the possibility that such occurrences biased the results.

In summary, our observations suggest that changes in TLCs during definitive RT was related to treatment-durations in unresectable stage III NSCLC. TLC nadirs occurred at approximately the fifth week of RT, not upon completion of RT or upon administration of the maximal dose. Decreasing the total number of RT fractions and increasing the dose of each fraction to complete treatment within 4 weeks while maintaining same overall dose is a potential strategy to protect circulating lymphocytes and improve clinical outcomes. The clinical implications of our findings may be more pronounced in the setting of a new therapeutic strategy combining RT and immunotherapy for unresectable stage III NSCLC, particularly given the promising results observed in the PACIFIC trial. Additional large-scale prospective investigations are needed to test the potential lymphocyte-sparing of hypofractionation, particularly when used in combination with immunotherapeutic approaches.