Immune Checkpoint Inhibitors Induced Autoimmune Haemolytic Anemia: Case Series and Literature Review

Immune checkpoint inhibitors (ICIs) have brought a revolution to the anti-cancer treatment, however, they also triger a unique spectrum of immune-related adverse events (irAEs). Among irAEs, haemopoietic AEs are rarely reported and mostly severe or even life-threatening, especially autoimmune haemolytic anemia (AIHA). AIHA is presumed to relate to the abnormal formation of circulating autoantibodies against red cell membrane antigens. It usually cannot be discovered timely because of atypical symptoms. It is diagnosed according to presence of hemolysis evidences such as decrease of haemoglobin, increase of indirect bilirubin and lactate dehydrogenase (LDH), urobilinogen, and positive direct antiglobulin test (DAT). Treatments of AIHA are according to clinical experience and consensus, which have not been veried by prospective trial. Here we investigate previous reported ICIs induced AIHA cases including thirty detailedly documented patients. On the other hand, we report three patients who developed AIHA after three different anti-PD-1 antibodies. Most of them were aged patients with melanoma or NSCLC, developed AIHA by anti-PD-1 antibodies and relived with glucocorticoid. 43.3% of previous cases and all of our observed cases had anemia before ICIs treatment, which reminds us of anemia as a risk factor for ICIs induced AIHA. By screening parameters like complete blood examination, reticulocyte, liver function test or DAT test prior to immunotherapy, doctors could exclude pretreatment haemolytic anemia or be aware of post ICIs AIHA. Thus, it is possible to avoid the potentially life-threatening AIHA, or improve the level of pre-alarm and treatment ability of AIHA. appearance, dizziness, fatigue and dark brown urine. The laboratory tests can show hemolysis related evidences such as a decrease of haemoblobin, an increase of serum indirect bilirubin and lactate dehydrogenase (LDH), urobilinogen, reticulocytosis, low serum haptoglobin and spherocytosis.(Leaf et al., 2019) Diagnosis is based on the direct antiglobulin test (DAT, Coombs test), which is usually positive for anti-immunoglobulin G and/or anti-C3d antisera.(Leaf et al., 2019) Recommendations for treating ICIs induced AIHA come from treatment of primary AIHA,(Jaime-Perez et al., 2013; Zanella and Barcellini, 2014) clinical experiences(Leaf et al., 2019; Tanios et al., 2019) and consensus(Brahmer et al., 2018; Haanen et al., 2018; Thompson et al., 2019) because no prospective trial has been conducted.(Davis et al., 2019) The information shortage of ICIs induced AIHA is because of its uncommon nature or doctors’ poor recognition. As such, reporting more cases and retrospectively summarizing previous cases are helpful for enriching the understanding on ICIs induced AIHA.


Introduction
In recent decades, the anti-tumor immunotherapy has been rapidly discovered and widely developed due to its favourable anti-tumor effect and well tolerance. respectively. With these treatments, 28/30 patients completely recovered from AIHA while 2/30 expired. Although 76.7% patients suffered grade 3 or higher AIHA, one third of patients re-challenged ICIs with well tolerance.  (Table 3) presented to the department of oncology complaining with chest distress for half a year, occasional chest pain for half a month and vomiting after eating for a week. He also felt mental in rmity, loss of appetite and troubled sleep. During this past half year, he lost two kilograms. In the past fty years, he suffered from a skin allergy very often but allergen was unknown. Seven years ago he was diagnosed with silicosis due to ve years work history in coal mine. His smoking and alcohol history were both going back almost fty years. His physical examinations were unremarkable except for a mild anemia appearance, while no hemorrhage was found. Blood routine showed a red blood cell count of 2.87×10 12 /L with hemoglobin level of 87 g/ L. Iron, vitamin B12, and folate de ciency were ruled out. Positron emission tomography-computed tomography (PET-CT) revealed nodules or masses with increased glucose metabolism in both lungs, bilateral hilar and mediastinal lymph nodes, sacrum, left adrenal gland and pleura. After tissue biopsy, pathological diagnosis was made as squamous cell carcinoma of lung with PD-L1 expression over 50%, while molecular diagnosis did not found any valuable mutation.
Thus, pembrolizumab was administered as rst line treatment. However, one day later the patient's complete blood examination showed an astonishing decrease of RBCs to 1.34×10 12 /L and hemoglobin to 50 g/L with normal leukocyte and platelet counts. Supplement of iron and vitamin B12 was not helpful.
Furthermore, hemoglobin level lowered to 45 g/L with RBCs of 1.30×10 12 /L in three days. The absolute reticulocyte count was 0.031×10 12 /L and percentage of reticulocyte was 3.49%. Liver function test revealed an increase in lactate dehydrogenase (LDH, 289.7 U/L), total bilirubin (27.6 umol/L) and indirect bilirubin (13.4 umol/L). Besides, the patient complained with dark brown urine and an increased urobilinogen was found in his urine, while urine and fecal occult blood test were both negative. The DAT test was positive for IgG and C3 antibodies. Additionally, a positive antinuclear antibody was detected (granular pattern) at a titer of 1:80. Anti-U1-nRNP antibody, anti-Ro-52 (52kDa) antibody, anti-histone antibody were also found positive. Echocardiography showed slight pulmonary arterial hypertension with pulmonary arterial systolic pressure of 33mmHg, while bone marrow biopsy was normal and abdominal ultrasonography did not nd hepatomegaly or splenomegaly. In accord with the evidences stated above, the patient was diagnosed as AIHA and immediately received intravenous 80mg/day methylprednisolone together with other supportive treatment like gastric protection. RBC transfusion was not performed because of increased risk of additional hemolysis. Luckily, he responded well with rising level of hemoglobin and RBCs. Subsequently, his intravenous steroid was gradually tapered, and replaced with oral steroid. Finally he weaned off oral steroid in a month when his hemoglobin completely recovered.

Case 2: Intrahepatic cholangiocarcinoma
A 63-year-old woman (Table 3)  After intraperitoneal perfusion chemotherapy of cisplatin and two cycles of intravenous chemotherapy of paclitaxel and carboplatin, the tumor progressed unexpectedly. Anti-angiogenic therapy of anlotinib could not control the disease either. Thus, anti PD-1 antibody camrelizumab (200mg) was applied. Four days after the second cycle, the patient complained with dizziness, fatigue, and soy-colored urine. Immediate blood routine test indicated severe anemia in which RBCs was 1.85×10 12 /L and hemoglobin level was 51 g/L, while previous RBCs was 4.02×10 12 /L and hemoglobin was 106 g/L before camrelizumab therapy. Suspended leukocyte-reduced RBCs transfusion was performed, however, the hemoglobin level decreased to 40 g/L instead. Urobilinogen became positive in the urine. Besides, chemical analysis revealed an increase in total bilirubin (40.7 umol/L) and indirect bilirubin ( (Table 1, Table 2), and observation on three practical cases (Table 3), we found that ICIs induced AIHA mostly harasses the aged patients with melanoma or NSCLC. But it should be aware of the bias of higher usage rate in melanoma and NSCLC patients. Anti-PD-1 antibodies, particularly pembrolizumab and nivolumab, induce more AIHA than anti-PD-L1 antibodies or other ICIs.
It may be explained by their molecular mechanism of interrupting wider signal pathways including PD-L1 and PD-L2.
AIHA is an autoimmune disorder in which RBCs are destroyed prematurely due to the abnormal production of antibodies directly against the patient's own RBCs.  (Mqadmi et al., 2005) found that depletion of CD25 + regulatory T cells using anti-CD25 increased the incidence of AIHA in C57/Bl6 mice from 30% to 90%. The adoptive transfer of puri ed CD4 + CD25 + regulatory T cells from immunized mice prevented the induction of AIHA. Furthermore, serum IL-33 was discovered elevated in patients with AIHA and positively correlated with AIHA disease activity. Enforced IL-33 promoted AIHA incidence and accelerated disease activity, while blockade of IL-33 interfered with AIHA incidence and ameliorated disease activity. (Bu et al., 2015) In our report (Table 3), three patients previously with anemia were treated with three different anti-PD-1 antibodies, which were pembrolizumab, camrelizumab, and nivolumab. Their anemia was all successively exacerbated and developed AIHA. Likewise, 43.3% patients in Table 1 had anemia before ICIs treatment. Thereupon, we suspect that cancer patients with anemia may be a special group either with high erythrocyte fragility, existing special antibody, or with activated immune system of high tendency to produce abnormal antibody. If pre-existing anemia is a risk factor of developing AIHA, the risk assessment has to be necessarily done prior to initiating ICIs treatment. Hemolysis related tests, such as direct and indirect Coombs tests, could be performed to exclude pretreatment hemolytic anemia. Besides, screening pre-existing autoimmune disorders is also important because autoimmune status undoubtedly relates to AIHA when AIHA is an new anti-CD20 monoclonal antibody ofatumumab and anti-CD52 monoclonal antibody alemtuzumab are optional therapeutic approaches. (Zanella and Barcellini, 2014) RBCs transfusion is bene cial in increasing oxygen-carrying capacity provided by the transfused RBCs as a supportive treatment, however, it may increase the risk of further hemolysis and its application require extra cautiousness. In our second case, the patient's anemia did not improve after RBC transfusion, instead, her hemoglobin deteriorate to 40 g/ L. It is methylprednisolone that brought her hemoglobin back to normal gradually and saved her from severe anemia. Our other two cases, the same as the previously reported 28 cases shown in Table 1, responded well to glucocorticoids and recover from AIHA.  (Table 1) reused ICIs and did not subsequently report uncontrolled AIHA. The safety of re-challenging ICIs is uncertain based on mere experience from limited clinical cases. Further prospective trials are required to test whether or not it is worthy to take such big a risk.

Conclusion
ICIs induced AIHA requires more consciousness due to its severity and high fatality. Early recognition and prompt treatment are important in mitigating its severity and improving the recovery of AIHA. Previously reported cases and our three cases remind us that risk assessments, especially hemolysis related tests, are necessary and needed to be done prior to initiating ICIs treatment for patients previously with anemia. Because of the rarity of AIHA, guidelines for treatment, monitoring and re-challenging are all immature, which demands further prospective trials to optimize public understanding on AIHA.