Study design and inclusion criteria
This study was approved by the Ethics Committee of Sendai Kousei Hospital following ethical guidelines of the 1975 Declaration of Helsinki, and written informed consent was obtained from each individual.. The patients with advanced non-small cell lung cancer (NSCLC) who were treated with anti-PD-1 (nivolumab (3mg/kg every 2 weeks) (186 patients) or pembrolizumab (200mg every 3 weeks) (66 patients)) monotherapy at Sendai Kousei Hospital between January 2016 and June 2018 were enrolled in this study. We focused on the patients treated with monotherapy of PD-1 antibody (nivolumab or pembrolizumab) to exclude other factors potentially inducing liver damage such as molecular targeting agents, other iCIs and cell-killing anticancer drugs. In addition to the regimen of treatment, we focused on NSCLC patients to exclude the effects of primary cancer influencing the immune reactions.
Biochemical analysis and clinical factors analysis
Patients with iCIs-related hepatitis were diagnosed by the following criteria. Alanine aminotransferase (ALT) elevations were consistent at least two weeks after using PD-1 antibody. The ALT elevations were dominant compared with aspartate aminotransferase (AST) and alkaline phosphatase (ALP) elevations. The patients were excluded if they had liver metastasis, disseminated intravascular coagulation, multiple organ failure, alcohol drinking (>30g / day), chronic hepatitis C. All HBsAg positive patients (3 patients) were inactive carriers. Age, sex and tissue type of lung cancer were compared between patients with iCIs-related hepatitis and those without iCIs-related hepatitis. The grade of liver damage was analyzed by using common terminology criteria for adverse events (CTCAE) v5.0. We evaluated iCIs-related hepatitis together with ALT elevation using the grading of CTCAE v5.0. Some of biochemical markers and immunological markers were analyzed during PD-1-antibody treatment with or without iCIs-related hepatitis.
Histological evaluation
Liver biopsies were carried out immediately after consulting the Department of Hepatology about liver damage in some patients treated with PD-1 antibody. The selection of patients who should be carried out liver biopsy were evaluated by two hepatologists to exclude the risk of bleeding and the progression of lung cancer. All biopsy specimens were fixed in formalin and paraffin embedded (FFPE). Sections (3µm) were then stained with hematoxylin and eosin. Histopathological features were reviewed by a single expert of hepatic pathology focusing on the following features: fibrosis according to Brunt’s criteria (G1-G4)10, portal inflammation (0-3), lobular inflammation (0-3), lobular necrosis (0-3). The formation of macro- and micro- granulomas was also evaluated.
Immunohistochemical evaluation
Immunohistochemical staining for T-cell markers (CD3, CD4, CD8), B-cell marker (CD20), and plasma cell marker (CD138) were performed by an automatic immunostainer (Ventana BenchMark GX, Roche). The antibodies and sources were as follows: CD3 (mouse monoclonal, Leica, Germany), CD4 (mouse monoclonal, Nichirei, Japan), CD8 (mouse monoclonal, DAKO, Denmark), CD20 (mouse monoclonal, DAKO, Denmark) and CD138 (mouse monoclonal, Nichirei, Japan). Immunohistochemical analyses were performed in all of the patients and compared to those with typical auto-immune hepatitis (AIH) (n=3) and classical drug-induced liver injury (DILI) (n=3).
Statistical analysis
χ2 test or independent t test was employed to compare the differences between the patients treated by nivolumab without liver damage and those treated by nivolumab with liver damage using JMP Pro 15.0 (Table 1). We also compared the clinical factors of irAEs induced by pembrolizumab with liver damage or without liver damage in the same way. Independent t test was employed to compare the frequencies of immune cell subsets between the patients treated with PD-1 antibody and control subjects (Table 3).