Successful Treatment of Aplastic Anemia Induced by Immune Checkpoint Inhibitors for Lung Cancer.

Introduction
Immune checkpoint inhibitors (ICIs) have shown remarkable efficacy for the treatment of various cancers. However, their use is associated with immune-related adverse events (irAEs), which can be life-threatening and pose significant challenges to therapy.
Activated T cells stimulated by ICIs may induce autoreactive inflammatory responses, resulting in damage to normal tissues. Although irAEs can affect many organs, their involvement in the hematopoietic system remains unclear (1).
We herein report a case of ICI-induced aplastic anemia (AA) in a patient with non-small-cell lung cancer. The patient experienced prolonged cytopenia that was refractory to immunosuppressants. Treatment with a thrombopoietin receptor agonist successfully reversed pancytopenia, enabling patients to achieve independence from blood transfusions.

Case Report
A 72-year-old man with abnormal chest findings was referred to the Department of Respiratory Medicine of our hospital. He was undergoing treatment for chronic obstructive pulmonary disease, hypertension, and hypertrophic cardiomyopathy, and had a history of rectal cancer surgery. Computed tomography (CT) and positron emission tomography revealed a tumor in the right lower lobe of the lung with metastases to the left supraclavicular fossa, left axilla, right hilum, mediastinum, abdominal para-aortic lymph nodes, and left adrenal gland. The patient was diagnosed with lung adenocarcinoma, classified as clinical stage IVB (cT4N3M1c), according to the Tumor, Node, Metastasis (TNM) classification. Programmed death ligand-1 (PD-L1) protein expression in the biopsy specimen had a tumor proportion score of 20%, and only the BRAF K601E mutation was detected using next-generation sequencing-based companion diagnostics. No abnormalities were observed in blood counts at the time of the diagnosis.
The patient received a combination therapy of ipilimumab (1 mg/kg) and nivolumab (360 mg/body). After one cycle of treatment, six weeks after the initial treatment, the patient achieved a partial response (PR). However, grade 3 pneumonitis, considered an irAE, developed according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The patient was treated with methylprednisolone (1 g/day for 3 days), followed by oral corticosteroids (1 mg/kg/day). As the pneumonitis improved, the corticosteroid dose was gradually tapered. Subsequently, the patient was monitored for lung cancer and ICIs without further treatment.
The corticosteroid dose was tapered to 10 mg/day over two months without recurrence of pneumonitis. Two weeks later, the patient underwent a routine medical examination. Petechiae were observed in both upper and lower limbs. Blood tests revealed severe thrombocytopenia with a platelet count of 10,000 /μL but no anemia or leukopenia. CT confirmed that his lung cancer remained in PR without pneumonitis exacerbation (Fig. 1). The patient was diagnosed with immune thrombocytopenia (ITP) as an irAE.
He was admitted to the respiratory medicine ward and treated with methylprednisolone 1 g/day for 3 days. However, the patient's platelet count did not improve. A platelet transfusion was performed to prevent life-threatening bleeding. His platelet count was 70,000 /μL at 24 h after transfusion, which is atypical for ITP. Two weeks after admission, he developed severe pancytopenia, with a neutrophil count of 20 /μL, hemoglobin level of 7.6 g/dL, and platelet count of 10,000 /μL. His pancytopenia persisted, leading to febrile neutropenia, which required treatment with antibiotics, antifungal drugs, and granulocyte colony-stimulating factors. Although his infections improved with antibiotic therapy, the pancytopenia remained unresolved (Table 1). The patient was referred to the hematology department for a further evaluation and management.
No morphological abnormalities or paroxysmal nocturnal hemoglobinuria clones were observed in peripheral blood. A bone marrow examination revealed a markedly hypocellular marrow without dysplasia, increased blasts, or bone marrow carcinomatosis (Fig. 2A, B). No chromosomal abnormalities were detected in the bone marrow. Immunohistochemistry showed that the majority of nuclear cells were CD3-positive, with a predominance of CD8+ T cells (Fig. 2C-E). Magnetic resonance imaging (MRI) of the entire lumbar spine indicated diffuse high signals in the vertebral bodies on T2-weighted and in-phase T1-weighted images, along with decreased signals on opposed-phase T1-weighted images, suggesting hypoplastic fatty marrow (Fig. 3). Based on these findings, the patient was diagnosed with severe AA induced by ICIs.
Given that his lung cancer remained in PR, AA treatment was prioritized over lung cancer treatment. While the standard therapy for severe AA includes immunosuppressive treatment consisting of anti-thymocyte globulin (ATG) with cyclosporine and a thrombopoietin receptor agonist (2), we treated the patient with cyclosporine alone to minimize the risk of severe infection and rapid progression of lung cancer. However, the cyclosporine treatment was discontinued because of renal toxicity. The patient was administered eltrombopag (EPAG) at an initial dose of 50 mg/day, followed by dose escalation. His blood count gradually improved, and he eventually achieved independence from blood transfusions with 100 mg/day EPAG. His blood count remained stable, so we tapered off the EPAG and eventually discontinued it, with no recurrence of cytopenia observed. The patient survived without severe complications or cancer progression for 12 months after AA onset. After relapse, the ICIs were not rechallenged because of the patient's history of severe irAEs. He was treated with tegafur/gimeracil/oteracil but developed carcinomatous meningitis and died 20 months after his diagnosis. His blood count did not decrease until his death (Fig. 4).

Discussion
ICIs are widely used to treat various types of cancer. However, because ICIs induce hyperactivation of immune cells, patients who respond to ICIs are more likely to develop irAEs, which poses significant challenges to the success of ICI treatment. Generally, irAEs occur within one month of ICIs administration; however, some patients develop irAEs after long-term administration and discontinuation of ICIs (1,3). Notably, the onset of irAEs varies depending on the affected organ and type of ICIs (4). The combination of ipilimumab and nivolumab increased the incidence, severity, and early onset of irAEs. In the present case, pneumonitis developed first, which occurred earlier than typically observed following anti-PD-1/PD-L1 antibody treatment (4), followed by cytopenia. Sequential multi-organ irAEs have been reported, such as a patient who initially had colitis and subsequently developed hepatitis, followed by pneumonitis, nephritis, and pancytopenia (5). This case highlights the importance of monitoring multiple organs that can be involved in irAEs at the time of the diagnosis and during the treatment of the initial irAEs.
Hematological irAEs (hem-irAEs) are rare, occurring in approximately 0.5% of cases (6,7). The most common hem-irAEs are ITP and autoimmune hemolytic anemia; however, other conditions have also been reported, including neutropenia, hemophagocytic lymphohistiocytosis, erythrocyte aplasia, and AA (8,9). Although hem-irAEs are rare, they are often severe (7). The present patient demonstrated severe thrombocytopenia (grade 4) during the tapering of corticosteroids for pneumonitis, suggesting that the cytopenia was potentially refractory to corticosteroids. In addition, he was initially diagnosed with ITP as a hem-irAE; however, his treatment history and response to platelet transfusion suggested that his thrombocytopenia might have been an early indication for AA. The literature showed that cytopenia improved in only 25% of patients with ICI-induced AA (10), and the prognosis was poor, with a mortality rate of 69.2% (11). Generally, ICI-induced AA is diagnosed based on the conventional AA criteria, which include bi- or pan-cytopenia of the blood, exclusion of other diseases, and confirmation of severe hypoplastic marrow using a bone marrow biopsy and MRI. CD8+ T cells are predominant in the bone marrow of patients with pancytopenia after ICIs (12-14). Histological findings in other organs affected by irAEs also showed dominant infiltration of CD8+ T cells (15,16), suggesting that CD8-dominant T-cell infiltration might be a common feature of irAEs.
The discontinuation of ICIs and administration of corticosteroids are generally used to treat irAEs (17). However, the response of AA as a hem-irAE to corticosteroids is poor (11). According to the American Society of Clinical Oncology clinical practice guidelines (18), corticosteroids are not recommended for the treatment of AA as a hem-irAE. They recommended discontinuing ICIs and providing supportive care, such as blood transfusions and granulocyte colony-stimulating factors, as needed for all patients. Furthermore, ATG and cyclosporine should be administered to patients with grade ≥2 disease, along with the consideration of allogeneic hematopoietic stem cells if they are candidates.
For refractory cases, repeated administration of immunosuppressants and EPAG may be considered. EPAG are widely used for the treatment of AA. The addition of EPAG to standard immunosuppressive therapy improves the hematological response in previously untreated patients with conventional severe AA (19). Notably, 40-50% of patients with conventional severe AA who were refractory to immunosuppressive therapy responded to the EPAG monotherapy at 24 weeks after treatment (20,21). In some patients, blood counts were maintained after the discontinuation of EPAG (21). However, there are no comprehensive reports on the efficacy of EPAG as an irAE of AA, and previous case reports have not demonstrated its treatment benefits (Table 2) (12-14,22-28).
In the present case, corticosteroids did not improve the cytopenia. Despite the severity of AA, we were compelled to avoid standard immunosuppressive treatments, including cyclosporine and ATG, owing to the patient's age, comorbidities, and the risk of cancer relapse. We initially treated the patient with cyclosporine alone but could not continue it due to renal dysfunction before we confirmed the response to the treatment. Therefore, we chose EPAG monotherapy, and his blood count gradually recovered. He became independent of blood transfusion without early relapse of lung cancer. Furthermore, cytopenia did not recur even after the discontinuation of EPAG.
Immunosuppressive therapy for steroid-refractory irAEs is often considered, although its efficacy is yet to be established (18). However, in the case of AA as a hem-irAE, all patients were in a tumor-bearing state and likely experienced organ damage and complications from the prior treatment. Thus, many patients are susceptible to adverse effects of immunosuppressive treatment, which may be associated with poor treatment outcomes. We hypothesized that some cases of AA as a hem-irAE might improve with discontinuation of ICIs and transient administration of immunosuppressants and that EPAG might promote recovery from cytopenia. According to the literature, clonal hematopoiesis was found in 47% of AA patients. Mutations in PIG-A, BCOR, BCOR-L1 are associated with a good response to immunosuppressants and a favorable prognosis, whereas mutations in DNMT3A and ASXL1 are correlated with poor outcomes (29). Although we could not evaluate somatic mutations in this case, examining mutation profiles could potentially help predict the treatment response and prognosis for AA as a hem-irAE as well. Further research is required to establish the optimal management of this complication.
In summary, we report a case of multiorgan irAEs after the combination of ipilimumab and nivolumab for the treatment of lung cancer. Although ITP is common as thrombocytopenia after ICI treatment, AA should be considered as an irAE when cytopenia is observed after ICI treatment. To our knowledge, this is the first case demonstrating successful treatment with EPAG monotherapy for severe AA as an irAE, without severe infections or promotion of cancer progression. Although immunosuppressive treatment is recommended as a hem-irAE for severe AA, EPAG may be a promising treatment for vulnerable patients after cancer treatment.

The authors state that they have no Conflict of Interest (COI).