Combined immunotherapy and targeted treatment for primary alveolar soft part sarcoma of the lung: case report and literature review

Primary acinar soft part sarcoma of the lung (ASPS) is a rare malignancy with unique cellular structure and clinical and genetic characteristics. Most patients do not exhibit clear clinical symptoms, with only a few developing respiratory symptoms. The typical histological characteristics are acinoid or organ-like structures. Immunofluorescence in situ hybridization suggests a rearrangement of the transcription factor E3 gene. Patients respond poorly to chemotherapy and are, thus, primarily treated with surgery and targeted therapy. We report herein a unique case of primary alveolar soft part sarcoma of the lung. The patient was a 24-year-old man with metastases to multiple organs, such as the brain, lungs, pancreas, and liver. The craniocerebral lesions attained partial remission after whole-brain radiotherapy and targeted combined immunotherapy, and other distant metastases completely disappeared after targeted combined immunotherapy (anlotinib and camrelizumab), indicating significant treatment efficacy. Anlotinib is an oral multi-target tyrosine kinase inhibitor (TKI) that exerts its anti-tumor effects by acting on various kinases. Camrelizumab is a humanized immunoglobulin G4 monoclonal antibody that can target PD-1 to block the interaction between PD-L1 and programmed death ligand 2, ultimately causing an anti-tumor effect. This is the first report of successful use of anlotinib combined with camrelizumab in the treatment of advanced primary ASPS. The treatment benefit provides preliminary evidence that targeted therapy, combined with immunotherapy, may be a safe and effective approach to treat primary pulmonary ASPS patients, thus warranting further investigation.

tuberculosis antibody, T-SPOT-TB, and other examination results confirmed tuberculosis. We prescribed antituberculosis and hemostatic treatments. The patient also underwent bronchoscopy, but the family did not follow-up on the results. In June 2018, he developed a headache, nausea, vomiting, anorexia, and fatigue. Cranial magnetic resonance imaging (MRI) showed multiple lesions. A retrospective review of the bronchoscopy image showed tumors in the right lower lung, and positron emission tomography/computed tomography (PET/CT) showed malignant lung tumors and widespread metastasis to the lungs, brain, pleura, and right femur. Abdominal B-ultrasound also showed multiple pancreatic metastases. The pathological diagnosis was pulmonary alveolar soft tissue sarcoma. Immunohistochemistry of the tumor performed on September 10, 2018 showed positive staining for TFE (3+) and negative staining for CK8/18, EMA, HMB45, Melam-A, S-100, S/n, CgA, MYOD, and Myogenin. We obtained informed consent from the patient to publish the case details and any accompanying images.

Therapeutic process
We started whole-brain radiotherapy (DT: 39 Gy/13 F) on September 14, 2019, followed by two cycles of systemic chemotherapy (ifosfamide (2 g, d1-5) and doxorubicin (60 mg, d1-3)) combined with apatinib (0.5 g, qd). However, chemotherapy was suspended because of grade IV thrombocytopenia, while apatinib was continued for 11 months. The treatment response was evaluated as stable disease (SD) based on CT findings. However, chest and abdominal CT on August 23, 2019, showed multiple liver metastases, and the patient was determined to have progressive disease (PD). We therefore switched the treatment from apatinib to anlotinib (12 mg, d1-14, q3w) for 2 months. On October 23, 2019, chest and abdominal CT showed a progressive lung lesion (indicating progressive disease (PD)). As such, immunotherapy (carrelizumab 200 mg d1 q2w and anlotinib 12 mg d1-14 q3w) was added to the treatment until December 12, 2020. Multiple evaluations performed during combination therapy showed the disappearance of pancreatic and liver lesions (indicating complete response (CR)) and significant reduction of lung and brain lesions (indicating partial response (PR)). We evaluated blood and immune indicators (including routine blood tests; biochemical, coagulation, myocardial, and thyroid function markers; brain natriuretic peptide; and troponin I) before every treatment cycle. Blood indicators were also evaluated every two cycles. Additionally, we performed electrocardiography and cardiac color doppler ultrasound. Reactive cutaneous capillary endothelial proliferation (RCCEP) occurred after 1 cycle of carrelizumab, following which the patient was prescribed some drugs after consulting a dermatologist. No further significant increase in the rash was observed during treatment. Immune hepatitis toxic reaction occurred after 6 cycles of carrelizumab; the drug was, therefore, stopped for 1 month. Liver function returned to normal after the administration of liver-protecting and enzyme-reducing drugs. However, chest and abdominal CT and brain MRI revealed new lesions in the liver, pancreas, and brain (PD) on December 12, 2020.

Efficacy evaluation
Lung lesions (Fig. 1) Before treatment, we observed multiple metastases in both lungs, with a maximum tumor diameter of 3.5 cm. After chemotherapy and apatinib treatment, the maximum tumor diameter was reduced by 28% to 2.5 cm, indicating SD. Before monoclonal antibody treatment with anlotinib and carrelizumab, the maximum tumor diameter was 2.7 cm, which was reduced by 70% to 0.8 cm post-treatment, indicating PR.
Brain lesions (Fig. 2) Before treatment, we observed multiple metastatic lesions in the left cerebellar hemisphere, bilateral occipital lobe, left frontal lobe, right lateral ventricle next to the body, and left parietal lobe, with the largest lesion located in the left frontal lobe and cerebellar hemisphere (maximum tumor diameter: 2.7*2.6*2.8 cm). After treatment with whole-brain radiotherapy and chemotherapy and apatinib, the maximum tumor diameter was reduced by 42% to 1.4*1.5*1.6 cm, indicating PR. Before anlotinib and carrelizumab treatment, the maximum tumor diameter was 1.5*1.0*1.2, with punctate enhancement, indicating PR, posttreatment.
Pancreatic lesions (Fig. 3) We noted multiple metastases before treatment, with a maximum tumor diameter of 2.6 cm. Treatment with chemotherapy and apatinib reduced the maximum diameter to 2.4 cm, indicating SD. Before anlotinib and carrelizumab treatment, the maximum diameter was 3.8 cm. The pancreatic lesions disappeared post-treatment, indicating CR.
Liver lesions (Fig. 4) Before treatment with anlotinib and carrelizumab, multiple metastases, with a maximum tumor diameter of 1.0 cm, were noted in the liver. These lesions disappeared post-treatment, indicating CR.

Discussion
Acinar soft part sarcoma (ASPS) is a soft tissue malignant tumor of unknown origin with the cells and tissues presenting with alveolar or organ-like arrangement. ASPS was first reported by Christopherson et al. [1] in 1952. It occurs in children and adolescents, accounting for about 1% of all soft tissue sarcomas [2]. ASPS develops gradually, and it has a predilection for the head and neck, particularly the orbits and tongue among children. In adults, the tumors mostly develop in the deep soft tissues of the limbs, especially in the thighs and buttocks. Rarely, some tumors can also arise in the female reproductive tract, breast, and meninges. Given its insidious onset, some patients present with extensive metastases in the lungs, brain, and bones at diagnosis. The origin of ASPS remains unclear to date; however, many scholars suggest myogenic involvement. The diagnosis of ASPS mainly relies on typical pathological features and immunophenotypes. Molecular genetic studies of ASPS have confirmed ectopic expression of t(X;17)(P11;25), causing the ASPSA key region gene 1 (ASPSACR-1) and transcription enhancer TFE3 on the 17q25 chromosome to produce a functional fusion gene, ASPSACR1-TFE3 [3]. As such, immunofluorescence in situ hybridization and real-time polymerase chain reaction to detect E3 gene rearrangement and E3 gene fusion, respectively, are useful diagnostic modalities for ASPS [4]. Pulmonary primary ASPS needs to be differentiated from acinar rhabdomyosarcoma, metastatic hepatocellular carcinoma, paraganglioma, large-cell neuroendocrine tumors, and other similar diseases. The pathological and immunohistochemical characteristics found in the presented case are consistent with those reported in the literature. The patient was a young male with no previous history of ASPS or other soft tissue primary lesions. The diagnosis of primary pulmonary ASPS was confirmed by combining imaging findings and typical histopathological features.
Clinically, pulmonary metastatic ASPS is more common than pulmonary primary ASPS. Only four cases of primary pulmonary ASPS have been reported in PubMed to date [5][6][7][8]. To the best of our knowledge, the current case is only the fifth of its kind to be reported, and only this case discussed treatment, while other cases discussed only the specificity of the pathology. Regarding prognosis, the 5-year survival rate of ASPS patients is about 56%. Advanced age, distant metastases at the time of diagnosis, and tumor diameters >10 cm influence prognosis adversely [9]. ASPS primarily involves extensive tumor resection. The 5-year survival rate of patients with localized ASPS who undergo surgical resection is approximately 80%, whereas that of patients who do not undergo resection is only 10% [9]. ASPS is not sensitive to traditional radiotherapy and chemotherapy [10][11][12][13]; chemotherapy only has <10% efficacy [13]. Meanwhile, the benefit of radiotherapy for ASPS is controversial. Some studies reported that radiotherapy could control local recurrence and delay distant metastasis in ASPS, while some studies reported no benefit from radiotherapy [14][15][16]. Our patient had multiple brain and lung metastases at the time of diagnosis; thus, there was no opportunity for surgery. However, the brain and lung lesions were reduced by 42% and 28%, respectively, after total brain radiotherapy and two cycles of chemotherapy combined. This indicates the benefit of radiotherapy and chemotherapy for primary lung ASPS.
Anlotinib is an oral multi-target TKI that acts on VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, FGFR, c-Kit, Ret, and many other targets to play a role in anti-tumor angiogenesis and inhibit tumor growth and metastasis. The indole group of anlotinib binds to the adenosine triphosphate (ATP) of VEGFR-2 tyrosine kinase to effectively inhibit VEGFR-2, thereby inhibiting angiogenesis. In addition, anlotinib also effectively acts on VEGFR-3, which plays an important role in the process of lymphangiogenesis and inhibits tumor cell metastasis to a certain extent. In June 2019, anlotinib became the first soft tissue sarcoma (STS)-targeted drug to be approved in China as a first-line treatment for ASPS and as a second-line treatment for other types of advanced STS. A m u l t i c e n t e r , s i n g l e -a r m p h a s e I I c l i n i c a l t r i a l (NCT01878448) [17] studied the efficacy of anlotinib in 166 patients with advanced STS who failed conventional treatment. Anlotinib showed good clinical efficacy. The 12-week progression-free survival (PFS) rate was 68%, the mPFS was 5.63 months, and the overall response rate (ORR) was 11.45%. The ASPS group (n = 13, 12 mg qd) had the highest ORR at 46% (6 cases achieved PR); the 12-week PFS rate was 77% and mPFS was 21 months. None of the patients reached the median overall survival (mOS). In 2018, ASCO reported the results of their phase IIb clinical study of anlotinib as a second-line treatment of STS (ALTER 0203) [18]. The mPFS of the anlotinib group was significantly higher than that of the placebo group (18.23 vs. 3 months, P < 0.0001).
Other targeted drugs can also be used for ASPS treatment. treatment for ASPS [19]. Paulina et al. [10] reported that among their 15 ASPS patients with lung metastases treated with sunitinib, 6 and 8 of the patients achieved PR and SD, respectively, and 1 patient developed PD. The clinical benefit rate was 93%, and the mPFS was 19 months. Li et al. [20] retrospectively evaluated 14 ASPS patients who were ineligible for resection or had metastases. The treatment response to sunitinib was PR in 4 patients and SD in 10 patients.
The mPFS was 41 months, and the mOS was not reached. In a follow-up study of 15 patients with advanced ASPS [10], after sunitinib treatment, the mOS was 56 months and the 5-year OS could reach 49%. The mPFS was 19 months, and the 5-  year PFS rate was 30%. Another targeted drug is pazopanib, which was approved by the US FDA and the European Union in 2012 to treat advanced STS in adults [21]. However, since pazopanib cannot cross the blood-brain barrier, its application in ASPS patients with brain metastases is limited [22]. In a study of 6 patients with metastatic ASPS [23], 1 and 5 patients achieved PR and SD, respectively. Within a median follow-up time of 33 months, the mPFS was 5.5 months, the mOS was not reached, and the trial reached the primary endpoint. Another retrospective analysis [24] of 29 evaluable patients reported CR, PR, SD, and PD in 1, 7, 17, and 4 patients, respectively. The median follow-up was 19 months, the mPFS was 13.6 months, the 1-year PFS was 59%, and the mOS was not reached. Based on these findings, pazopanib was recommended as the primary drug for systemic treatment of ASPS (Class 2A evidence) in the 2019 NCCN guidelines. Cediranib is a multi-target small-molecule TKI. In a doubleblind, randomized, phase II clinical trial by Judson et al. [25], the median percentage change in the sum of the target marker lesions for the evaluable population was significantly higher in the cediranib arm than the placebo arm (−8·3% (IQR: −26·5 to 5·9) vs. 13·4% (IQR: 1·1 to 21·3), one-sided p = 0·0010). Meanwhile, a follow-up exploratory phase II clinical trial of cediranib for treating children with metastatic ASPS did not meet the expected ORR [26]. Two retrospective analyses [27,28] reported satisfactory results in advanced ASPS patients treated with apatinib. Additionally, in a clinical trial of 40 MET-positive ASPS patients, the disease control rate of crizotinib treatment reached 87.5% [28]. Immune checkpoint inhibitors, such as the CTLA-4 monoclonal antibodies and PD-1 monoclonal antibodies, were first suggested as effective treatments for drug-resistant ASPS by Conley in 2017 [29]. Carrelizumab is a selective, humanized, high-affinity IgG4 monoclonal antibody that can target and bind to CD4+ and CD8+ T cells, B lymphocytes, and natural killer (NK) cells and dendritic cells (DCs) and other PD-1 on the surface. Carrelizumab blocks the interaction with PD-L1 expressed on the surface of malignant tumor cells, tumorinfiltrating dendritic cells (TIDCs), tumor-infiltrating lymphocytes (TILs), antigen-presenting cells (APC), and PD-L2 on the surface of activated macrophages and DC. It relieves the immunosuppressive effect of T cells mediated by the PD-1 pathway, further induces the activation of T lymphocytes, rebuilds the body's immune system to monitor and kill tumor cells, and has an anti-tumor effect [30,31]. In general, the lower the IC50 and EC50 values of PD-1 inhibitors, the higher the binding affinity to PD-1 and the stronger the anti-tumor effect. Studies have shown that the IC50 value of carrelizumab combined with PD-1 is 0.7 nmol/L, and the EC50 value is 0.38 nnmol/L, which are IC50 and EC50 values are similar to those of pembrolizumab. The NCT02961101, NCT03250962, and NCT03155425 trials [32,33] all showed the efficacy of carrelizumab for classic Hodgkin's lymphoma (CHL). Accordingly, carrelizumab was officially approved by the National Medical Products Administration (NMPA) in May 2019 for treating relapsed or refractory CHL after at least second-line systemic therapy [34]. After reviewing the literature, we found only one case report of successful treatment of ASPS using carrelizumab combined with apatinib [35]. After 6 months of treatment, the tumor regression continued with a 69% decrease from the baseline, and the mPFS was 10 months. Although there are not many clinical trials involving the use of a c d b carrelizumab for treating ASPS, the drug is used in many other solid tumors, such as osteosarcoma [36], esophageal cancer [37,38], gastric cancer [39], liver cancer [40,41], lung cancer [42], and nasopharyngeal cancer [43]. Its safety and effectiveness will be validated further in a series of large-scale clinical trials. The common adverse reactions of carrelizumab include RCCEP [32,33], elevated blood bilirubin, altered liver function [39], fever, fatigue, hypothyroidism, proteinuria, and lung infections. Most of the adverse reactions of carrelizumab are reversible, with prompt diagnosis being instrumental [44]. Patients commonly choose carrelizumab over the more expensive pembrolizumab or nivolumab. In the current case, RCCEP and liver function abnormalities developed during the therapy but were successfully controlled through active drug treatment. Accordingly, carrelizumab was continued, and good results were achieved. Other immunological drugs for treating ASPS have also been reported. Wilky et al. [45] conducted a randomized, single-arm, phase II clinical trial of axitinib and pembrolizumab in patients with sarcoma. The 3-month PFS rate was 72.7, and the mPFS in ASPS patients was higher than that in patients with other sarcomas (12.4 months vs. 3.0 months). In the current case, anlotinib combined with carrelizumab showed high clinical effectiveness. The pancreatic and liver lesions disappeared, and the brain and lung lesions were significantly reduced. The PFS was 14 months, which was similar to that reported in the literature. Notably, pembrolizumab was added in the 2019 edition of the NCCN guidelines (2B recommendation). Lewin et al. [46] compared durvalumab monotherapy with combination treatment using durvalumab and tremelimumab to treat 2 patients with advanced ASPS. PD occurred after 12 months in the single-agent arm, while PR was achieved in the combination treatment arm, reducing the tumor volume by 73%. Further, the duration exceeded 18 months. Moreover, they conducted genome analysis of ASPS and found that immune checkpoint inhibitors may be an effective treatment strategy for ASPS patients. Toripalimab is a recombinant humanized anti-PD-1 monoclonal antibody, independently developed in China. In a phase I clinical trial (JS001) of toripalimab for advanced or refractory ASPS, 1 and 2 of the 12 patients achieved CR and PR, respectively. The mPFS is expected to be 12.4 months [47].
In the current study, the patient was a young male treated for ASPS using the standard treatment of anlotinib with carrelizumab. After the combined immunotherapy and targeted treatment, the patient's tumor burden significantly reduced from baseline (PR, CR). Moreover, the combination treatment (anlotinib and carrelizumab) resulted in significant regression and disappearance of malignant lesions. Current clinical studies have confirmed that the combination treatment for unresectable ASPS achieves a higher response rate, disease control rate, and a longer PFS than before. However, the current research is mostly comprised of case reports that are incapable of screening out those patient characteristics that benefit from the combination therapy of anlotinib with carrelizumab. Therefore, large randomized controlled trials should be conducted to examine the potential benefits of PD-1/PD-L1 blockade, either alone or in combination with other therapies, to treat patients with ASPS.

Conclusion
This case study reported an extremely rare primary pulmonary ASPS with metastases in multiple distant organs. Primary pulmonary ASPS can recur and metastasize; thus, imaging and follow-up are necessary. Although surgery remains the optimal treatment strategy, targeted therapy, combined with immunotherapy, may be a more effective approach for treating primary ASPS patients with advanced distant metastases. The treatment benefit observed in the current case provides preliminary evidence that targeted therapy, combined with immunotherapy, may be a safe and effective modality for treating primary pulmonary ASPS patients, thus warranting further investigation.