The data of 172 patients treated with ipilimumab plus nivolumab therapy (1 mg/kg ipilimumab and 240 mg/body nivolumab on day 1, every 3 weeks) for RCC between October 2015 and June 2023 at eight hospitals in Japan (Nagoya City University Hospital, Wakayama Medical University Hospital, Kobe University Hospital, Tokushima University Hospital, Kindai University Hospital, Nara Medical University Hospital, Japanese Red Cross Wakayama Medical Center, and Tottori University Hospital) were retrospectively obtained and analyzed. Eleven patients with eosinophilic disorders or those whose eosinophil proportion was not measured before treatment initiation were excluded. Therefore, eventually, 161 patients were evaluated ( Figure 1 ). All patients were followed up until death or loss of contact. Eosinophil proportions 1 week before (baseline sample), 2 weeks after one course of treatment (2-week sample), and 3 weeks after one course of treatment (3-week sample) were determined according to our previous studies (15, 16). In addition, to determine the predictors of irAE occurrence, the systemic neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and C-reactive protein-to-albumin ratio (CAR) were analyzed, as in our previous study (16).

The best clinical response according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 (17), including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), was determined. Overall survival (OS) was defined as the period from the start of ipilimumab plus nivolumab therapy to death. Progression-free survival 2 (PFS2) refers to the period from the start of ipilimumab plus nivolumab therapy to second-line therapy.

Symptoms that activated the immune system and/or required treatment with immunosuppressants were categorized as irAEs. We divided irAEs into six disease groups, such as endocrine, skin, gastrointestinal, pulmonary, and others (arthritis, demyelinating polyneuropathy, dysgeusia, encephalitis, elevation of serum creatine kinase, fever, hyponatremia, hypokalemia, infusion reaction, myocarditis, myasthenia gravis, myositis, myoclonus, optic neuritis, and renal disorder). Grading of irAEs was based on the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. Grade ≥3 irAEs were defined as severe irAEs. The patients were divided into two groups based on whether or not they experienced irAEs (irAE and non-irAE groups, respectively). The irAE group was further subdivided based on irAE severity: Patients who experienced <grade 2 and ≥grade 3 irAEs were categorized into non-severe and severe irAE groups, respectively.

A P-value of <0.05 was considered statistically significant. Fisher’s exact test was used to assess differences in patient characteristics. Differences in the quantified data between groups were compared using the Welch t-test followed by Bonferroni test in the case of non-homoscedasticity or one-way analysis of variance (ANOVA) followed by Tukey’s test in the case of homoscedasticity. Because the number of patients was sufficient, normality was observed. All reported P-values were two-sided. The optimal cutoff values were determined from receiver operating characteristic (ROC) curves. The median OS (mOS), PFS (mPFS), and PFS2 (mPFS2) were calculated using the Kaplan–Meier method and log-rank tests. Statistical analyses were performed using GraphPad Prism 9 software and EZR [Saitama Medical Center, Jichi Medical University, Saitama, Japan (18)].

Patient characteristics are shown in Table 1 . Among the 161 patients enrolled in the current study, 33.5% (n = 54), 29.8% (n = 48), and 36.7% (n = 59) belonged to the non-irAE, non-severe irAE, and severe irAE groups, respectively. The proportions of patients aged >65 and <65 years were 64.0% (n = 103) and 34.2% (n = 55), respectively. Of the patients, 81.4% (n = 131) were male and 18.6% (n = 30) were female. Regarding International Metastatic RCC Database Consortium (IMDC) risk, 57.1% (n = 92) and 42.9% (n = 69) of the patients were classified into the intermediate and poor IMDC risk groups, respectively. Among the components of IMDC risk classification, diagnosis-to-treatment time of <1 year (73.3%, n = 118) was associated with the highest positivity rate, followed by hemoglobin level < the upper limit of normal (ULN; 62.7%, n = 101), neutrophil count > ULN (27.3%, n = 44), Karnofsky Performance Status score < 80 (22.4%, n = 36), platelet count > ULN (19.3%, n = 31), and calcium level > ULN (9.9%, n = 16). The proportions of patients diagnosed with clear-cell or non-clear-cell carcinoma (papillary, chromophobe, Bellini duct, spindle, and others) were 67.1% (n = 108) and 24.2% (n = 39), respectively. Tissues that were difficult to classify pathologically were diagnosed as RCC after consultation with an experienced urologist and radiologist (8.7%, n = 14). The proportion of patients with sarcomatoid changes was 11.2% (n = 18). The metastasis sites were as follows: bone, 31.7% (n = 51); liver, 14.9% (n = 24); lung, 59.0% (n = 95); and others (abdominal wall, adrenal glands, brain, contralateral kidney, dorsal muscles, gluteal muscle, inferior vena cava, lymph node, pancreas, peritoneal dissemination, subcutaneous tissue, tooth ridge, urethra, iliopsoas muscle, and pleura), 55.3% (n = 89). At the start of ipilimumab plus nivolumab therapy, 1.2% (n = 2) of the patients continuously used steroids. Proportion of patients who treated steroid or steroid pulse therapy due to irAEs was 38.5% (n = 62) and 11.2% (n = 18), respectively. The ratios of IMDC risk classification, Karnofsky Performance Status, histological subtypes, sarcomatoid changes, and proportion of patients who treated steroid or steroid pulse therapy differed significantly among the irAE, non-severe irAE, and severe irAE groups.

Table 2 shows the therapeutic history of the patients enrolled in this study. Regarding the number of courses of ipilimumab plus nivolumab therapy, 12.4% (n = 20), 13.7% (n = 22), 16.1% (n = 26), and 57.8% (n = 93) of the patients received 1, 2, 3, and 4 courses, respectively. The percentages of patients who underwent nephrectomy before or after ipilimumab plus nivolumab therapy were 52.8% (n = 85) and 5.6% (n = 9), respectively. Nivolumab monotherapy after ipilimumab plus nivolumab therapy was administered to 60.2% (n = 97) of the patients. The median number of courses of nivolumab monotherapy was 8 (range: 1-66). Regarding additional treatment, 49.1% (n = 79) of the patients received second-line therapy, such as axitinib (19.3%, n = 31), cabozantinib (14.3%, n = 23), pazopanib (8.7%, n = 14), sunitinib (4.3%, n = 7), temsirolimus (1.2%, n = 2), and sorafenib (0.6%, n = 1) therapies. The percentage of patients who received third-line therapy was 20.5% (n = 33). Consistent with the numbers associated with second-line therapy, the proportion of patients who received axitinib (8.0%, n = 13) as third-line therapy was the highest, followed by those of patients who received cabozantinib (6.2%, n = 10), pazopanib (3.1%, n = 5), sunitinib (1.2%, n = 2), everolimus (0.6%, n = 1), nivolumab monotherapy (0.6%, n = 1), and sorafenib (0.6%, n = 1). The proportions of patients who received fourth- and fifth-line therapies were 5.6% (n = 9) and 1.8% (n = 3), respectively. The percentages of treatment courses, nephrectomy before or after ipilimumab plus nivolumab therapy, and nivolumab monotherapy after ipilimumab plus nivolumab therapy significantly differed among the irAE, non-severe irAE, and severe irAE groups.

The mOS and mPFS in the current study were 32.6 and 8.4 months, respectively ( Supplementary Figures 1A, B ). Regarding responses to ipilimumab plus nivolumab therapy, complete response, partial response, stable disease, and progressive disease were achieved in 10.5% (n = 17), 34.2% (n = 55), 26.1% (n = 42), and 23.0 (n = 37) of the patients ( Table 3 ). The overall response rate (ORR) and disease control rate (DCR) were 44.7% (n = 72) and 70.8% (n = 114), respectively ( Table 3 ). The ORR and DCR were significantly higher in the non-severe and severe irAE groups than in the non-irAE group (ORR: 50.0% vs. 57.6% vs. 25.9%; DCR: 77.1% vs. 81.4% vs. 53.7%; P < 0.05; Table 3 ).

Overall, 107 patients experienced a total of 174 irAEs. Among the 174 events, 57.5% (100 events) and 42.5% (74 events) were grade <2 and grade ≥3 irAEs, respectively ( Supplementary Table 1 ). The rate of endocrinal irAEs was the highest (29.9%, 52 events), followed by skin-related (19.5%, 34 events), gastrointestinal (19.5%, 34 events), others (19.0%, 33 events), and pulmonary (12.1%, 21 events) irAEs ( Supplementary Table 1 ). The proportions of patients who experienced one and two or more irAEs was 59.8% (n = 64) and 40.2% (n = 43; Supplementary Table 2 ), respectively. The percentage of patients who experienced two or more irAEs was significantly higher in the severe irAE group than in the non-severe irAE group (52.5% vs. 25.0%, P < 0.05; Supplementary Table 2 ). The reasons for discontinuing ipilimumab plus nivolumab therapy were irAEs in 39.3% (n = 42) and non-irAEs (disease progression, death, complete remission, and others) in 38.3% (n = 41) of the patients ( Supplementary Table 3 ). The proportions of patients who discontinued treatment because of irAEs were not significantly different between the non-severe and severe irAE groups (27.1% vs. 49.2%, P = 0.17; Supplementary Table 3 ). The subgroup analysis showed discontinuation due to irAEs did not affect overall survival ( Supplementary Figure 2 ).

We investigated whether an increased eosinophil was related to irAE severity. No differences in eosinophil were observed between baseline samples (non-irAE: 2.5% vs. non-severe irAE: 3.1%, P = 0.26; non-irAE: 2.5% vs. non-severe irAE: 2.8%, P = 0.75; Figure 2A ). Notably, while the eosinophil in the 2-week samples did not differ between the non-irAE and non-severe irAE groups (non-irAE: 3.3% vs. non-severe irAE: 5.5%, P = 0.33), and the eosinophil in the severe irAE group was significantly higher than that in the non-irAE group (non-irAE: 3.3% vs. severe irAE: 6.6%, P < 0.05; Figure 2B ). Consistent with that in the 2-week samples, in the 3-week samples, the eosinophil in the severe irAE group tended to be higher than that in the non-irAE group (non-irAE group: 5.1% vs. severe irAE group: 7.1%, P = 0.06; Supplementary Figure 3 ). The NLR, PLR, and CAR in baseline samples did not differ among the three groups ( Supplementary Figure 4 ).

The optimal cut-off value of the eosinophil proportion in 2-week samples against the occurrence of severe irAEs was 3.0% (area under the curve = 0.653, 95% CI = 0.53–0.77, sensitivity = 0.79, specificity = 0.52; Figure 2C ). Notably, univariate and multivariate logistic regression analyses showed that an eosinophil proportion of ≥3.0% in the 2-week sample increased the risk for occurrence of severe irAEs (univariate analysis: OR = 4.19, 95% CI = 1.53–11.5; P < 0.05; multivariate analysis: OR = 6.01, 95% CI = 1.81–20.0; P < 0.05; Table 4 ).

The mOS and mPFS of the non-irAE group were associated with poor survival compared with those of the non-severe and severe irAE groups (mOS: 13.3 months (non-irAE) vs 52.3 months (non-severe irAE) vs 36.9 months (severe irAE); mPFS: 5.3 months (non-irAE) vs 14.2 months (non-severe irAE) vs 9.2 months (severe irAE); Figures 3A, B ). Kaplan–Meier analysis showed no statistically significant difference in the mPFS between the non-severe and severe irAE groups (non-severe irAE: 14.2 months, severe irAE: 9.2 months, P = 0.45; Figure 3B ), the mOS tended to be shorter for the severe irAE group than for the non-severe irAE group (non-severe irAE: 52.3 months, severe irAE: 36.9 months, P = 0.06; Figure 3A ).

The differences in the mOS and mPFS between the non-severe and severe irAE groups inspired us to investigate PFS2, which is the PFS until the second-line therapy after ipilimumab plus nivolumab therapy. Consistent with results obtained for mOS and mPFS, the mPFS2 of the non-irAE group was significantly shorter than that of the non-severe and severe irAE groups (non-irAE, 19.7 vs non-severe irAE, not reached vs severe irAE, 29.2 months, P < 0.05; Figure 3C ). Notably, the mPFS2 of the severe irAE group tended to be shorter than that of the non-severe irAE group (non-severe irAE, not reached vs severe irAE, 29.2 months, P = 0.10; Figure 3C ). These results indicated that severe irAE occurrence was associated with a poor prognosis.