Immune Thrombocytopenia Induced by Immune Checkpoint Inhibitrs in Lung Cancer: Case Report and Literature Review

Immune checkpoint inhibitors (ICIs), including antibodies targeting programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1), are being extensively used on advanced human malignancies therapy. The treatment with ICIs have acquired durable tumor inhibition and changed the treatment landscape in lung cancer. Immune-related adverse events including pneumonitis and thyroiditis have been well described, but less frequent events, such as ICIs-induced thrombocytopenia, are now emerging and may sometimes be severe or fatal. Since early detection and prompt intervention are crucial to prevent fatal consequences, it is of outmost importance that medical staff is aware of these potential toxicities and learn to recognize and treat them adequately. This review focuses on the epidemiology, clinical presentation, mechanisms, and clinical management of ICIs-induced thrombocytopenia in patients with lung cancer. We also present a patient with advanced lung adenocarcinoma who received the PD-L1 inhibitor atezolizumab and eventually developed severe thrombocytopenia. The case indirectly suggests that cytokine changes might contribute to immune dysregulation in ICIs-induced thrombocytopenia.


INTRODUCTION
Immune checkpoint inhibitors (ICIs) lead to a significant improvement of overall survival (OS) in advanced non-small-cell lung cancer (NSCLC), and 8% of responded patients have achieved long-term survival (1). Atezolizumab, a humanized monoclonal antibody binding to programmed cell deathligand 1, is currently approved for use against extensive small-cell lung cancer (SCLC) and has few treatment discontinuation rates due to adverse events (AEs) (2). Similar to other ICIs, atezolizumab can cause immune-related adverse events (irAEs), including endocrine, pneumonitis, hepatitis, thyroiditis, nephritis, and colitis. With the growing application of ICIs, the rate of hematologic toxicity has been observed in many cases (3,4). In particular, ICIs-induced thrombocytopenia has been reported in a few cases with lung cancer (5). Herein, we reported a case of atezolizumab-induced immune thrombocytopenia and discussed the clinical management. We also review the epidemiology, clinical presentation, and prognosis of immune thrombocytopenia caused by ICIs in patients with advanced lung cancer.

CASE PRESENTATIONS
In a case report, a 76-year-old male with stage IV adenocarcinoma (cT2bN2M1b) without targetable genomic alterations, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), rearranged c-ros oncogene 1 (ROS1), was diagnosed by right abdominal muscle surgical resection. Chest computed tomography (CT) revealed the tumor shadow in the right lower lobe of the lung, and it also demonstrated multiple swollen lymph nodes in the mediastinum (Figure 1). 18 F-fluorodeoxyglucose positron emission tomography-computed tomography (PET/ CT) showed multiple 18F-fluorodeoxyglucose (18F-FDG) uptake in the right abdominal muscle, L4, and right iliac bone. Baseline data showed that blood tests were normal, and Eastern Cooperative Oncology Group (ECOG) score was 1. The patient was recruited into a clinical study (IMpower 132) and received 6 cycles of carboplatin, pemetrexed, and atezolizumab (every 3 weeks) treatment. Then the patient showed a partial response indicated by a CT scan, and no severe toxicities were observed in February 2019, followed by 36 cycles of maintenance therapy with pemetrexed and atezolizumab. During the treatment, blood tests were performed every 3 weeks and no hematological abnormality was found. In November 2020, the patient developed thrombocytopenia (platelet level: 91×10 3 /ul) with normal hemoglobin and normal white cell counts and received the interleukin-11(IL-11) therapy to enhance the proliferation of megalokaryocytes for 2 weeks. Unfortunately, his platelet count slightly declined, and no autoimmune or coagulation disorders were displayed. As a result, the diagnosis was presumed as ICIsinduced thrombocytopenia, and he was treated with prednisone for 2 weeks (0.5mg/kg). However, his thrombocytopenia became worse with a sudden decrease in platelet level to 35×10 3 /ul. Thus, the pemetrexed and atezolizumab were discontinued. A bone marrow biopsy examination demonstrated no obvious morphological abnormalities, phagocytosis, or malignant invasion happened to this patient. Furthermore, antinuclear antibodies and other laboratory tests were negative, but antiphospholipid and antiplatelet antibodies were abnormal. After excluding chemotherapy, infection, pseudothrombocytopenia, or other druginduced thrombocytopenia, atezolizumab-induced immune thrombocytopenia was finally diagnosed. Therefore, we gave him a high-dose steroid for 6 consecutive days and recombinant human thrombopoietin (TPO). However, his platelet count showed no improvement and stayed at the level of 23×10 3 /ul. He then received four times of platelet transfusions, mycophenolate mofetil, and infusion of intravenous immunoglobulin (IVIG), but his platelets did not recover. The lowest platelet level was recorded on February 20, 2021, with a level of 20×10 3 /ul. Interestingly, the level of serum interleukin-6 (IL-6) was significantly increased compared to the normal range, and we prescribed him an IL-6 receptor antagonist, tocilizumab, at 400 mg in addition to mycophenolate mofetil. Seven days later, his platelet counts started to increase and reach the normal range (100 to 300×10 3 /ul) by 1 week (Figure 2). The patient stayed a partial response to the treatment during atezolizumab therapy interruptions.

EPIDEMIOLOGY OF ICIs-INDUCED THROMBOCYTOPENIA
ICIs-induced thrombocytopenia in patients with lung cancer have been reported in many studies ( Table 1), and thrombocytopenia has been demonstrated to be one of the most important hematological toxicity of hematological irAEs (21). There is usually a favorable prognosis in patients with thrombocytopenia. However, it is difficult to separate from chemotherapy-induced thrombocytopenia; therefore, the absolute incidence of ICIsinduced thrombocytopenia cannot be calculated. In one retrospective study, the incidence of ICIs-induced thrombocytopenia was less than 1% on ICIs (22), but in the observation study, the frequency of immune thrombocytopenia seems to be relatively high, accounting for 25-29% of hematological irAEs (23,24). Besides, combination therapy was also associated with significantly higher risks of immune thrombocytopenia (5). Table 1 summarizes the reported cases of immune thrombocytopenia during ICIs therapy in patients with lung cancer. Most studies revealed that the onset of immune thrombocytopenia usually occurred within the first 12 weeks of ICIs initiation, but it can be at any time, even after cessation of treatment (25). Here, we reported the first case of one patient with lung cancer who developed late-onset immune thrombocytopenia during atezolizumab therapy. Similar to this case, delayed toxicity has been observed in other reports, which happened 1 to 2 years after monotherapy (26,27). Generally, other irAEs have been shown to be associated with the efficacy of PD-1/PD-L1 inhibitors in patients with lung cancer (28,29). Same as other irAEs, grade 1 thrombocytopenia during ICIs therapy is positively associated with Overall survival (OS), but lacks of Progression-free survival (PFS) benefit (30). Immune thrombocytopenia usually is not fatal, but deaths caused by the adverse events had been reported in uncommon cases (25). Thus, ICIs-induced thrombocytopenia is potentially life-threatening and should be paid close attention in clinical practice.

MECHANISM OF ICIs-INDUCED THROMBOCYTOPENIA
Although the mechanism of drug-induced thrombocytopenia has been well demonstrated, the underlying mechanism of ICIs-induced thrombocytopenia remains unclear (31). Reinvigoration of exhausted CD4 + helper T cells and CD8 + cytotoxic T cells activates inflammatory pathways and ultimately results in damage to hematopoietic stem cells. In addition, ICI-induced antiplatelet antibody production can promote platelet destruction, which is supported by high antiplatelet antibody levels in patients who suffered from ICIs-induced thrombocytopenia (7,18). Furthermore, the expression of PD-L1 on platelets in lung cancer patients was significantly increased, which might render susceptible targets of antibody-based anti-PD-L1 therapies. As a result, the amount of PD-L1-expressing platelets dramatically decreased in the blood of patients receiving PD-L1 therapy (32). Another suggested mechanism was activation of T-cells, leading to the secretion of different cytokines from T-helper cells (33). In the present case, although the patient has a high level of plateletassociated immunoglobulin G antibody and other possible causes of thrombocytopenia, particularly a viral infection, were excluded and multiple treatments were used including steroid, platelet transfusion, IVIG, and mycophenolate mofetil, the platelet counts recovered slightly. Importantly, the serum level of IL-6 was significantly higher than that of the normal range, indicating that the potential mechanism of ICIs-induced thrombocytopenia was relying on the abnormal cytokine secretion of activated lymphocytes. Consequently, the IL-6 receptor antagonist tocilizumab has achieved a partial response.

DIAGNOSIS AND TREATMENT
ICIs-induced thrombocytopenia mimics virtually any other type of thrombocytopenia, making it a diagnosis of exclusion and is difficult due to the lack of specific testing. Any new platelet count decrease should be considered as immune thrombocytopenia for patients receiving ICIs therapy. Generally, bone marrow examination is necessary to exclude dysplasia or cancer invasion. In addition, other examinations should be administrated to distinguish ICIs-induced thrombocytopenia from other etiologic agents, such as drug- induced thrombocytopenia (heparin/HITT, chemotherapy, etc.), infections, hematological malignancies (myelodysplastic syndrome, etc.), platelet sequestration (spleen, liver diseases), platelet consumption (thrombotic thrombocytopenic purpura, etc.) (9,34,35). Furthermore, the presence of a high plateletassociated IgG titer may be helpful to diagnose ICIs-induced thrombocytopenia (14). Therefore, effective recognition and diagnosis for immune thrombocytopenia are important because of the different prognosis and therapeutic management. In our case, absence of liver, spleen, or rheumatologic disease; malignant infiltration of the bone marrow; and other causes of thrombocytopenia suggest that thrombocytopenia was induced by atezolizumab. Sometimes, the lack of efficacy of transfusions during and after ICI administration is indicative of ICIsinduced thrombocytopenia. The targeted therapies are not well defined for immune thrombocytopenia induced by ICIs. According to the American Society of Clinical Oncology (ASCO) guidelines, grading treatment on severity classification is the current principle for immune thrombocytopenia (36). Generally, the management of grade 1 toxicities (<100×10 3 /ul) should comply with the clinical and laboratory evaluation. Sometimes ICIs need not stop, and platelet changes should be continued with close monitoring. Withholding ICIs therapy is generally recommended for grade 2 toxicities (<75×10 3 /ul) until the platelets recover to grade 1 toxicities, and oral corticosteroids (0.4-1 mg/kg/day of prednisone or equivalent) should be presented with 2-4 weeks and/or conjunctive use of IVIG. But the dose adjustment is not advised if ICIs therapy is readministrated because irAEs are not dose-dependent (37). For grade 3 toxicities (<50×10 3 /ul) or grade 4 toxicities (<25×10 3 /ul), ICIs therapy must be definitively discontinued until return to grade 1 toxicities, with the administration of high-dose corticosteroids and optional IVIG, with permanent ICIs, withdrawal is necessary if platelet do not resolute to normal. Other therapy strategies include recombinant human TPO, romiplostim, platelet transfusion, and immunosuppressive agents, such as azathioprine and rituximab. Steroids are generally essential for treating immune thrombocytopenia by ICIs but are not always effective in severe thrombocytopenia. Many studies have demonstrated that the presence of specific single-nucleotide polymorphisms, such as PD-1 -606 AA genotype and +63379 TT genotype, affects the susceptibility to prednisolone treatment (38). In addition, HLA-DRB1*0410 or HLA-DRB1*0405 allele in the patients' immune thrombocytopenia has been reported to contribute to steroid therapy resistance (20,39). This can explain why the patient had a weak response to steroid therapy.

CONCLUSION
Although ICIs-induced thrombocytopenia is rare in patients receiving ICIs, we still need to pay more attention to this issue because of its life-threatening characteristic. Any new abnormality of platelet counts should be considered as a potential clinical significance for immunotherapy patients; thus, careful recognition and accurate diagnosis are extremely important. Although its response to steroids, IVIG, and platelet transfusion is relatively good, the underlying mechanism of immune thrombocytopenia remains elusive, and further study is awaited.

AUTHOR CONTRIBUTIONS
LC developed the idea for the study. WX and NH did data collection, data analysis, and manuscript preparation. All authors have reviewed and approved the final version of the manuscript and have consented to its publication.