放疗与免疫治疗联合治疗的疗效及机制 IV期非小细胞肺癌王明月11第一作者，滨州医科大学 医院， 电子邮件： [email protected]， 隋静， 李硕， 李淑静， 王明霞， 宁方玲22通讯作者， Email:[email protected]*， 田立军33对应 作者， Email:[email protected]*抽象肺癌是全球癌症相关死亡的主要原因。 约85%的肺癌患者患有NSCLC;大多数被诊断为 初次就诊时为 IV 期疾病。随着连续 肿瘤学，免疫疗法或免疫化疗的发展已成为 IV期NSCLC患者的一线治疗。然而，一个 患者比例仍对治疗方案产生耐药性 并经历局部进展，原发性肺部病变进展 IV期NSCLC的主要进展模式。临床前和临床 研究表明，放疗可诱发全身性 抗肿瘤免疫反应和之前加局部放疗 癌症进展可以延长生存期。因此，我们考虑了 是否在肺部进展前进行局部放疗 接受免疫治疗或免疫治疗的IV期NSCLC患者的病变 化疗将是有益的。本次审查的重点是： 放疗和免疫治疗的抗肿瘤疗效，强调 加放疗时间和联合治疗的安全性 用于 IV 期 NSCLC 并了解潜在机制。关键词：IV期NSCLC，联合治疗，免疫治疗， 放射线疗法介绍肺癌是目前最多的 流行的恶性肿瘤和癌症相关死亡的主要原因 全世界估计有3万人死于 1[1].在中国，肺癌的发病率 近年来显著增加。 NSCLC占肺的85% 癌症;IV期非小细胞肺癌（NSCLC）是最常见的 5年生存率低于5%的病理型， 特别是在初步诊断中[1-4].正在进行的免疫疗法或免疫疗法结合的研究 化疗现在是IV.期NSCLC的一线治疗。几个 临床试验，如IMpower132试验[5]和主题演讲-189[6]，已证明其益处 免疫治疗或 IV 期 NSCLC 患者的免疫化疗，尤其是 程序性死亡配体 1 的肿瘤比例评分患者 （PD-L1） 的 50% 或更高。虽然大多数IV期患者 NSCLC——一线治疗 免疫疗法或免疫化疗——取得良好疗效，一部分 的患者出现疾病进展。值得注意的是有多少 疾病进展的方式。其中， 进展模式 已知肺癌是原发性肿瘤扩大、寡转移、 和广泛转移[7]. Xu等人研究了 接受免疫治疗至少 3 次的 IV 期 NSCLC 月份[8]. 寡头进展（55.3%）是 免疫治疗耐药的主要模式。然而，寡核苷酸进展 比较广泛，包括 在少于两个病灶部位进展。 此外，研究发现， 原发性肿瘤病变是 肺癌进展的最重要部位[9, 10].此外，浦内斯等 发现 70% 的 IV 期 NSCLC 患者接受 维持化疗在原发肺上进展 病变[11].对于原发性病变进展，这些 患者可以从局部治疗中受益[12]. 放疗是 晚期肺癌。先前的研究表明， 放疗不仅可以缓解患者的临床症状 有限的转移性NSCLC也延长了生存时间 病人[11, 13].同样，丹尼尔· 调查了IV期NSCLC疾病进展前的治疗情况 接受一线治疗[14].作者 发现添加局部PFS（无进展生存期）更高 治疗与维持治疗（14.2个月 vs 4.4个月）。因此 在免疫治疗或免疫化疗过程中的治疗 IV期NSCLC患者，加法能不能有更好的效果 肺进展前的局部放疗？因此，我们综述了免疫治疗联合 放疗和临床试验证据，以讨论对以下情况下的反应 接受过 IV 期 NSCLC 患者的放疗 免疫疗法或免疫化疗。放疗联合免疫治疗的协同机制临床前研究已经证明了协同效应和 放疗和免疫之间的相互作用。放射线疗法 可以通过调节肿瘤发挥抗肿瘤免疫作用 微[15].同样，免疫疗法可以 也减少了放射治疗的缺点，从而增强了 抗肿瘤作用。放疗的免疫作用放疗可导致肿瘤细胞免疫原性细胞死亡（ICD） 通过活性氧（ROS）介导的DNA 损伤[16].ICD之后是释放 肿瘤相关抗原 （TAA） 和内源性危险 信号损伤相关分子模式 （DAMP），例如高 移动性组框 1 （HMGB1）、三磷酸腺苷 （ATP） 和 钙网蛋白 （CRT）[17-19].这些物质可以 与树突状细胞（DC）的表面受体结合或被吞咽 通过发展中国家，导致 色温[17, 20].然后，激活的 DC 迁移到 附近的淋巴结，将抗原信息呈现给T细胞，从而 促进T细胞的活化和增殖，发挥 免疫调节作用。这个过程离不开 主要组织相容性复合体 1 （MHC-I） 分子的上调 和共刺激分子，如CD80[21]. 最后，活化的T细胞实现对肿瘤细胞的归巢，并发挥 抗肿瘤免疫作用。此外，树突状细胞（DC）也可以吸收 受损的细胞质DNA并诱导DC分泌干扰素β（IFN-β） 可以通过GMP-AMP合成酶-干扰素基因杀死肿瘤细胞 刺激器（cGAS-STING）通路[22, 23].II. IMMUNOTHERAPY ENHANCES THE EFFICACY OF RADIOTHERAPYStudies have reported that not only radiotherapy has immune effects, but immunotherapy also sensitizes radiotherapy, thus playing a synergistic role. Previous research has suggested that radiotherapy does not produce a durable anti-tumor effect, and it can induce an increase in regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). These cells can inhibit anti-tumor immune effects and reduce radiosensitivity through transforming growth factor-β (TGF-β) and interleukin-10 (IL-10)[24, 25]. Immunotherapy, such as the use of PD-1/PD-L1 inhibitors, activates large numbers of CD8+T cells to induce the expression of tumor necrosis factor-α (TNF-α), which in turn suppresses the effect of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby weakening the resistance to radiotherapy and producing a durable anti-tumor effect[26].Some preclinical studies have demonstrated that radiotherapy can up-regulate the expression of PD-L1, mainly expressed on the surface of tumor cells and antigen-presenting cells (APCs) via the JAK–STAT–IRF and cGAS-STING pathways[27-29]. PD-L1 can bind to PD-1 on the surface of T cells, B cells, NK cells, and other lymphocytes, thereby inhibiting the anti-tumor effect of CD8+T lymphocytes[16, 30]. Therefore, anti-PD-L1 inhibitors can consume part of PD-L1, thus weakening the anti-tumor inhibition effect. Researchers have applied radiotherapy and immunotherapy to mice and found that the expression levels of PD-L1 on DC, tumor cells, and macrophages after radiotherapy were increased compared with that of the same cell population in control tumor cells[31]. This inhibitory effect can be broken by adding PD-1/PD-L1 inhibitors. Therefore, radiotherapy combined with immunotherapy can play a synergistic and complementary anti-tumor role. One experimental study observed improved survival in mice with combined anti-PD-1 therapy and radiation compared with monotherapy, and only a small percentage (15%–40%) of the animals in the combination group survived more than 180 days after treatment. Immunological data revealed that compared with monotherapy, cytotoxic T cells (CD8+/ IFN-γ +/ TNF-α +) were increased, and regulatory T cells (CD4+/ FOXP3) were decreased in the combined treatment group[21].III. RELATIONSHIP BETWEEN TAMs AND IMMUNOLOGICAL COMBINED RADIOTHERAPYStudies have demonstrated that radiotherapy can activate tumor-associated macrophages (TAMs). TAMs include M1 macrophages and M2 macrophages. Notably, their effects on cancer are opposed. There is evidence that M1 macrophages can play an anti-tumor role by up-regulating the expression of TNF-α and interleukin-6 (IL-6)[32]. Nevertheless, M2 macrophages can promote the expression of TGF-β and epidermal growth factor (EGF) to produce an immune suppressive effect, thereby promoting the growth and metastasis of tumor cells[33].Researchers believe that radiotherapy can increase the aggregation of TAMs, especially M2 macrophages. Radiotherapy activates the STAT pathway through the expression of colony-stimulating factor-1 (CFS-1), activating M2 macrophages. The activated M2 macrophages recruit regulatory T cells (Tregs) that inhibit anti-tumor activity, thereby promoting tumor growth[34, 35]. However, the relationship between radiotherapy and TAMs is still being explored. Some studies have suggested that the radiotherapy dose is related to the type of TAMs. For example, low-dose radiotherapy can promote the activation of M2 macrophages, whereas radiotherapy greater than 1 Gy promotes the polarization of M1 macrophages[36].By contrast, Cao et al. reported that PD-1/PD-L1 inhibitors can induce the transformation of M2 TAMs into M1 TAMs[37]. There is evidence that PD-1/PD-L1 is associated with the polarization of TAMs[38]. Liu et al. found that PD-L1 was significantly elevated in tumor and stromal compartment macrophages compared to other immune cells[39]. PD-1/PD-L1 is highly expressed in TAMs and can induce polarization toward M2 TAMs by transmitting negative regulatory signals and other mechanisms, which are still being explored. In this case, the addition of PD-1/PD-L1 inhibitor can reverse this effect; thereby, it can reduce the amount of M2 TAMs, increase the amount of M1 TAMs, and induce the up-regulation of TNF-α, interleukin-12 (IL-12), and reactive oxygen species, which play an essential role in T cell activation, thus making up for the deficiency of radiotherapy and playing an anti-tumor role[40-42]. Furthermore, other specific mechanisms of immunotherapy-induced polarization of M1 TAMs must be investigated.IV. ABSCOPAL EFFECTS OF RADIOTHERAPYThe abscopal effect, that is, radiotherapy, through its immunomodulatory effect, can play an anti-tumor role at the site of the tumor radiation field and induce adaptive immune response at the site of distant metastasis without radiotherapy[43, 44]. Although the specific mechanism of the abscopal effect is not completely clear, studies have proved that the abscopal effect exists. In the process of radiation therapy, radiation can cause cancer cells to die and increase blood flow, change the tumor microenvironment tumor antigen so that activation of CD8 + T cells throughout the body through the blood circulation, including unexposed sites, is beneficial to the immune system and recognizes tumor cells, leading to reduction or disappearance of distant metastatic sites [45, 46]. In a mouse model of anti-PD-1-resistant 344SQNSCLC adenocarcinoma, radiotherapy was used to treat primary tumors, and a trend was found to increase M1 TAMs in abdominal tumors[47]. In another study on the model of primary anti-PD-1 resistance, the combination of anti-PD-1 and radiotherapy for primary tumors significantly reduced the growth of primary tumors and significantly controlled tumors without radiotherapy and reduced spontaneous lung metastasis (abscopal effect)[48].CURATIVE EFFECTRecently, preclinical and clinical trials have demonstrated a prolonged survival benefit of immunotherapy combined with radiotherapy in patients with stage IV NSCLC.Michel et al. found that adding NHS-IL2 immune cytokine and local radiotherapy to first-line palliative chemotherapy reduced tumor size and delayed tumor growth in a lung cancer animal model[49]. The NHS-IL2 consists of human NHS76 (antibody specific for necrotic DNA) fused to genetically modified human interleukin-2 (IL-2). Furthermore, mice models of lung cancer treated with low-dose radiotherapy (LDRT) and anti-PD-1 inhibitors achieved better tumor control and prolonged survival[50].The enhanced anti-tumor effects of immunotherapy combined with radiotherapy have been confirmed in preclinical studies and demonstrated in several clinical trials.A study demonstrated a considerable benefit of local control and survival by adding stereotactic radiotherapy (SBRT) in patients with oligoprogression after required resistance to checkpoint inhibitors (ICIs) in stage IV NSCLC [51]. The trial enrolled advanced NSCLC patients receiving at least two cycles of immunotherapy and had minimal progression. The sites of oligometastases in these patients included the lungs, brain, lymph nodes, adrenal glands, liver, and cervical spine. Overall survival after oligoprogression (OS-PO) was 34 months in patients treated with immunotherapy combined with SBRT versus 22 months in patients without SBRT. Many studies have found that immunotherapy combined with radiotherapy has survival benefits, and the radiotherapy site includes not only the primary tumor, such as the lung, but also metastatic sites, such as the liver and brain. As mentioned previously, the most common site of progression of lung cancer after systemic therapy is the primary lung lesion. Therefore, we must explore whether adding lung radiation before the progression of the primary lesion can provide additional benefits or prevention.The PEMBRO-RT Phase 2 randomized trial enrolled patients with metastatic NSCLC who progressed after at least one chemotherapy regimen[52]. Enrolled patients were randomly assigned using a 1:1 ratio to receive treatment with pembrolizumab after SBRT to the site of lung lesions or lymph node metastases (experimental arm) or without SBRT (control arm). The ORR (Overall-Response-Rate) at 12 weeks was 18% in the control arm vs. 36% in the experimental arm (P = 0.07). Median PFS was 1.9 months vs. 6.6 months (P = 0.19), and median OS (Overall-Survival) was 7.6 months vs. 15.9 months (P = 0.16). Although the experimental arm did not meet the study’s pre-defined criteria, it showed an increase in ORR, median PFS, and OS and no increase in toxicity in the SBRT group. Many clinical trials have demonstrated the feasibility of radiotherapy for pulmonary lesions. Li et al. performed a real-world analysis and also found that the median PFS (9 months vs. 5 months) and median OS (30 months vs. 16 months) were both higher with sintilimab plus radiotherapy for stage III or IV NSCLC than with sintilimab alone[53]. Notably, CRT was performed in patients with positive margins or gross residual tumors after surgery. Patients with isolated or local metastases received SBRT or CRT, indicating that lung radiotherapy combined with immunotherapy can improve survival. Regretfully, the study did not focus on the timing of adding radiotherapy, such as before or after the onset of disease progression. Similarly, Ratnayake et al. found that patients with stage IV NSCLC treated with nivolumab and radiotherapy as second-line or subsequent therapy had significantly better PFS compared with patients who did not receive radiotherapy (2.8 months vs. 1.3 months, p = 0.02). The study also found that prior or concurrent thoracic radiotherapy was well tolerated and not associated with an increased incidence of pneumonia[54].Based on these trials, we verified that radiotherapy to the lung lesion combined with immunotherapy prolonged survival among patients with stage IV NSCLC. As studies have demonstrated that the site is most likely to progress after systematic treatment is the primary site, we wanted to explore the feasibility of adding local radiation to the lung before disease progression to prevent disease progression. Michael et al. found that patients with stable or regressed stage IV cancer after systemic therapy had a longer 2-year OS rate with SBRT for metastatic disease than with progressive disease (55% vs. 15%)[55]. Although our literature search was limited, it cannot be denied that radiotherapy of the primary tumor before disease progression is acceptable.The most frequently used radiotherapy modality in the literature is SBRT. Nevertheless, conventional radiotherapy is commonly used in our actual clinical practice. Can the addition of conventional radiotherapy bring some benefits? Taugner et al. conducted a real-world study of stage III NSCLC and revealed that concurrent chemoradiotherapy followed by the addition of a PD-L1 inhibitor resulted in higher rates of PFS (60.0% vs. 31.8%) and OS (100.0% vs. 70.5%) at 12 months than chemoradiotherapy alone[56]. This study was applied to conventional fractionated thoracic radiotherapy. Although the patients in this trial were not stage IV patients, it suggests that conventional radiation therapy combined with immunotherapy seems to have unexpected benefits. Similarly, the PORT-C randomized trial demonstrated a higher incidence of 3-year disease-free survival (DFS) with the addition of conventional radiotherapy to postoperative adjuvant chemotherapy than with no radiotherapy in patients with pIIIA-N2 NSCLC (40.5% vs. 32.7%)[57].Thus, we realized that despite their differences in sensitivity and tumor cell death patterns, SBRT and conventional radiotherapy are both essentially radiotherapy[16, 58, 59]. Both SBRT and conventional radiotherapy can induce cell morphology enlargement and cytoskeletal recombination, causing cancer cells to recognize these stimuli and alter gene expression and cell signals and thereby altering the biological process and cell function of cancer cells; cell morphology and cytoskeletal enlargement are found to be dose-independent[60]. Radiotherapy can target the designated dose to the tumor lesion, whereas normal structures outside the tumor receive less dose, minimizing toxic effects on adjacent normal tissues[61]. Radiotherapy can also benefit cancer patients by relieving pain, possibly curing, preserving organ integrity, and cost effectiveness[62]. Radiotherapy, including SBRT and conventional radiotherapy, contributes to local tumor control and enhances anti-tumor immune effects[63]. The antitumor effects of these two kinds of radiotherapy have also been verified in the following clinical practice..The MDACC trial found that pembrolizumab combined with radiotherapy had a longer median PFS compared with pembrolizumab alone (9.1 vs. 5.1 months, p = 0.52) for stage IV NSCLC[64]. In this study, the ORR (10% vs. 38%, p = 0.11) and median PFS (6.8 vs. 20.8 months, p = 0.03) were increased in the SBRT group compared with the conventional radiotherapy group, but the increases were not significant. Therefore, we believe that conventional radiotherapy for lung lesions in combination with immunotherapy can benefit stage IV patients. The same holds for lung lesions before their development. However, most studies involved in this aspect are SBRT, and a few studies have related to conventional radiotherapy (added before primary lesions progression combined with immunotherapy), which requires further research and exploration.SAFETY OF COMBINATION THERAPY:As conventional radiotherapy combined with immunotherapy can benefit patients with stage IV NSCLC, can the adverse effects (AEs) of combination therapy be tolerated?Adverse effects can occur with any anti-tumor therapy, so toxicity should be considered in the choice of treatment[43]. The ETOPNICOLAS trial studied the safety of nivolumab when added to chemoradiotherapy in patients with stage III NSCLC[65]. Adding nivolumab to conventional radiotherapy was safe and tolerable, with a 23.5% incidence of ≥ grade 3 pneumonia. Combination therapy is tolerated in patients with stage III NSCLC and those with stage IV disease. A secondary analysis of KEYNOTE-001 found that after a median follow-up of 32.5 months, there was an acceptable safety profile about previous radiotherapy combined with Pembrolizumab for stage IV patients[66]. Although treatment-related pulmonary toxicity occurred in 13% of patients who received chest radiation, compared with 1% of patients who did not, there was no statistical difference in grade 3 or higher toxicity between the two studies. Similarly, William et al. also found that adding thoracic radiotherapy to treating stage IV lung cancer with ICIs can increase toxic effects, especially pulmonary toxicity. However, it is not conspicuous and can be tolerated by patients (3.3% vs. 4.1%)[67]. Notably, a small percentage of patients who received a median dose of 40cGy of chest radiation during or after immunotherapy did not develop symptomatic pneumonitis, compared with a median dose of 60 cGy for most patients. Tian et al. found that it is safe to receive concurrent treatment of lung lesions SBRT and ICIs, defined as ICIs received within 30 days after pulmonary SBRT[68]. In this study, although 10.7% of SBRT+ICIs patients developed grade 3 or higher pneumonia, compared with 0% of SBRT patients (p<0.01), the patients tolerated it. In addition, it was found that radiotherapy for two lobes had a higher risk of pneumonia than that for a single lobe (66.75% vs. 7.55%, P=0.028). Here, there seems to be a relationship between the size of the radiotherapy site and toxicity. A toxicity analysis of thoracic radiotherapy combining with immunotherapy found that 3.8% of patients receiving the combination developed grade 3 pneumonia[69]. Among them, the incidence of grade ≥3 pneumonia was slightly higher in patients treated with concurrent ICIs and chest radiotherapy compared with sequential therapy (7.1% vs. 3.9%), but the difference was not significant (P = 0.014). Unfortunately, the distinction between concurrent and sequential treatment is not described here.When exploring the toxicity of lung radiotherapy combined with immunotherapy, we found that the dose of radiotherapy, the area of the irradiated lung, and the time interval between the two treatments were all related to the toxicity. However, we believed that adding radiotherapy before progression could benefit the aforementioned parameters, but we were unsure about the specific time. Therefore, this present article next focuses on the relationship between the interval time between radiotherapy, immunization, and toxicity.The PACIFIC study found a slight increase in pulmonary toxicity (33.9% vs. 24.8%) with durvalumab administered between 1 and 42 days after the completion of thoracic radiotherapy[70]. However, the study did not compare conditions at other time points. Another systematic analysis of real-world studies found rates of all grade pneumonia of 35% and grade ≥3 pneumonia of 6%[2]. By contrast, the interval between radiotherapy and immunotherapy was at least 42 days in more than half of the patients. The two studies used the same treatment regimen, but the latter was a real-world systematic analysis. The incidence of pulmonary toxicity was similar. Radiotherapy was added before lung disease progression occurred in the two studies. Andrew et al. conducted a multicenter study and found that local radiotherapy combined with immunotherapy was acceptable[71]. There were 66 patients (50%) who had received radiotherapy before the start of immunotherapy, with a time interval of 71 days between the two treatments, and 56 patients (42%) who started radiation within 14 days of immunotherapy. Patients who received radiotherapy after immunotherapy had an increase in grade 3-5 ir-AEs compared to another arm (8% vs. 4%), but the timing of the addition of radiotherapy was not distinctly associated with grade 3 or higher toxicity (P = 0.45). Other studies have also found that combining radiotherapy and immunization has a high safety profile and tolerable toxicity. Similarly, Donata et al. found that only 6% and 8% of patients receiving immunotherapy combined with chest radiotherapy developed grade 2 pneumonia and grade 2 esophagitis, respectively[72]. In combination therapy, there are inevitably overlapping toxicities, especially pneumonia. However, concurrent (immunotherapy and radiotherapy were started within 1 month) and sequential therapy (within > 1 month and ≤6 months) were not significantly associated with lung toxicity in subgroup analyses.With the aforementioned studies, the toxicity of concurrent immunotherapy and radiotherapy is similar to that of sequential therapy, and there is no significant difference. What’s more, we found that the toxicity of radiotherapy combined with immunotherapy before lung disease progression was tolerable. Nevertheless, we did not rule out that the more the time of radiotherapy and immunotherapy coincide, the more detrimental to patients. It makes sense for more studies to target intervals exceeding 1 month. However, studies in this area are not abundant, and we must further explore the specific time of adding radiotherapy for patients with stage IV NSCLC.CONCLUSION:Most preclinical studies have demonstrated that radiotherapy can enhance the anti-tumor immune response, and immunotherapy can also synergistically promote the anti-tumor effect of radiotherapy. More clinical studies have also demonstrated that radiotherapy combined with immunotherapy, compared with other treatments alone, has a higher survival benefit, and patients can tolerate its toxic effects. It is also feasible to add local radiotherapy before lung disease progression. It is believed that the same is true for conventional radiotherapy. Although the published literature has limited data, conventional radiotherapy combined with immunotherapy has great promise in treating stage IV NSCLC before the progression of primary focus. However, there are some limitations in the research, such as the few studies on conventional radiotherapy combined with immunotherapy, the specific time to add conventional radiotherapy, a dose of radiotherapy, and anti-tumor predictors are also major challenges. With the continuous development of medicine, more preclinical and clinical studies are urgently needed to explore these issues and challenges before this protocol can be widely used.