*3.2. E*ffi*cacy and Safety of ICI Treatments*

The initial ICI treatment consisted of nivolumab for 19 (54.3%) patients, pembrolizumab for 12 (34.3%) patients, and atezolizumab for four (11.4%) patients. The rechallenge treatment consisted of nivolumab for five (14.3%) patients, pembrolizumab for 7 (20.0%) patients, and atezolizumab for 23 (65.7%) patients. The patients were treated with different regimens of ICIs between the initial and rechallenge treatments. In the initial ICI treatment, no patients experienced a complete response (0%), 12 experienced a partial response (34.3%), 12 experienced stable disease (34.3%), 10 experienced progressive disease (28.6%), and one was non-evaluable (2.9%). The objective response rate was 34.3%, and the disease control rate was 68.6% (Figure 1a). The PFS and OS of the initial ICI treatments were 120 d (95% CI 84–139 d) and 596 d (95% CI 455–864 d), respectively (Figure 2a,b). In the ICI rechallenge treatment, no patients experienced a complete response (0%), one experienced a partial response (2.9%), 14 experienced stable disease (40.0%), 18 experienced progressive disease (51.4%), and two were non-evaluable (5.7%). The objective response rate was 2.9% and the disease control rate was 45.7% (Figure 1b). The PFS and OS of the ICI rechallenge were 81 d (95% CI 41–112 d) and 225 d (95% CI 106–361 d), respectively (Figure 2c,d).

**Figure 1.** Frequency of the best overall response to immune checkpoint inhibitors (ICIs). (**a**) Frequency of the best overall response to first ICI treatment. (**b**) Frequency of the best overall response to ICI rechallenge treatment. PD, progressive disease; PR, partial response; SD, stable disease; NE, not evaluated.

**Figure 2.** Kaplan–Meier survival curves of progression-free survival (PFS) and overall survival (OS) of patients who received immune checkpoint inhibitor (ICI) rechallenge treatment. (**a**) PFS of non-small cell lung cancer (NSCLC) patients (*n* = 35) on first ICI treatment. (**b**) OS of NSCLC patients (*n* = 35) on first ICI treatment. (**c**) PFS of NSCLC patients (*n* = 35) on ICI rechallenge treatment. (**d**) OS of NSCLC patients (*n* = 35) on ICI rechallenge treatment.

Univariate analyses of the patient data revealed that ECOG-PS ≥ 2 (HR 2.21, 95% CI 1.00–4.83, *p* = 0.048), BMI > 20 (HR 0.47, 95% CI 0.22–0.99, *p* = 0.047), NLR ≥ 5 (HR 2.22, 95% CI 1.02–4.84, *p* = 0.045), and LMR < 1.7 (HR 0.44, 95% CI 0.21–0.93, *p* = 0.032) were significantly associated with PFS of ICI rechallenge (Table 2). Moreover, multivariate analysis demonstrated that ECOG-PS ≥ 2 (HR 2.38, 95% CI 1.03–5.52, *p* = 0.043) and BMI > 20 (HR 0.43, 95% CI 0.19–0.95, *p* = 0.036) were significantly associated with PFS of ICI rechallenge (Table 3 and Figure 3).


**Table 2.** Cox proportional hazards and logistic regression models for progression-free survival (PFS) and overall survival (OS).



**Table 3.** Cox proportional hazards and logistic regression models for progression-free survival (PFS) and overall survival (OS).


**Figure 3.** Kaplan–Meier survival curves for progression-free survival (PFS) of patients who received immune checkpoint inhibitor (ICI) rechallenge treatment. (**a**) Eastern Cooperative Oncology Group (ECOG-PS) ≥ 2, (**b**) body mass index (BMI) ≤ 20, (**c**) neutrophil-to-lymphocyte ratio (NLR) > 5, and (**d**) lymphocyte-to-monocyte ratio (LMR) ≤ 1.7 were significantly associated with inferior PFS.

Univariate analyses of the patient data revealed that ECOG-PS ≥ 2 (HR 4.23, 95% CI 1.65–10.89, *p* = 0.0023), CRP > 1.0 (HR 2.92, 95% CI 1.10–7.76, *p* = 0.032), albumin > 3.5 (HR 0.37, 95% CI 0.15–0.90, *p* = 0.028), and PLR > 262 (HR 2.80, 95% CI 1.02–7.67, *p* = 0.045) were significantly associated with OS of ICI rechallenge (Table 2). Multivariate analysis demonstrated that ECOG-PS ≥ 2 (HR 3.01, 95% CI 1.10–8.24, *p* = 0.032) was significantly associated with OS of ICI rechallenge (Table 3).

### **4. Discussion**

PD-L1 expression in tumors has been used clinically as a positive predictive biomarker for the effective initial ICI treatment of patients with NSCLC [16]. However, clinically useful biomarkers have not yet been identified for predicting the efficacy of ICI rechallenge. Fujita et al. reported that objective response rate (ORR), disease control rate (DCR), and PFS values of pembrolizumab rechallenge after refractory nivolumab for 12 patients with NSCLC were 8.3%, 41.7%, and 3.1 months, respectively [8]. In addition, ORR, DCR, and PFS of atezolizumab rechallenge after refractory anti-PD-1 antibodies for 18 patients with NSCLC were 0%, 38.9%, and 2.9 months, respectively [17]. Another report showed that ORR, DCR, and PFS values of ICI rechallenge in 14 patients with ICI refractory tumors were 7.1%, 21.4%, and 1.6 months, respectively [7]. Our current observations showed that ORR, DCR, PFS, and OS values of ICI rechallenge in 35 patients with NSCLC were 2.9%, 42.9%, 2.7 months, and 7.5 months, respectively. These reproducible findings suggest that refractory NSCLC tumors for initial ICI treatments may exhibit poor responses to ICI rechallenge treatments, and the clinical benefits may be limited compared with those of the initial ICI treatment. However, a subset of patients with NSCLC demonstrate good outcomes with ICI rechallenge treatments. Therefore, there is a need for the elucidation of predictive clinical factors for re-treatment of ICI responders among patients with NSCLC.

Our multivariate analysis identified ECOG-PS and BMI as independent factors associated with poorer PFS of ICI rechallenge treatment in patients with NSCLC who were refractory to initial ICI treatment. This is the first report that identifies predictive clinical factors for the efficacy of ICI rechallenge in patients with NSCLC. The general and nutritional status of patients with NSCLC are closely related to the effects of ICI treatment. Several studies have demonstrated that poor ECOG-PS is a predictive negative factor related to clinical outcomes of initial ICI treatment in patients with NSCLC [10,13,18,19]. ECOG-PS is one of the factors that determines the tumor immune environment, and it has been reported that an imbalance of circulating T-lymphocyte subpopulations in patients with gastric cancer correlates with ECOG-PS [20]. BMI is widely used for relating weight to height, defining body size, and indicating nutritional status. In addition, a lower BMI is associated with increased mortality risk [21–24]. Our previous clinical study demonstrated that NSCLC patients with sarcopenia exhibit a significantly shorter median PFS following ICI treatment compared to that of non-sarcopenia patients [25]. Given these observations, a poor ECOG-PS and a low BMI at the time of ICI treatment intervention may be useful for predicting non-responders to initial ICI treatment, as well as ICI rechallenge treatment among patients with NSCLC. Recent clinical trials demonstrated that the ghrelin/growth hormone secretagogue receptor agonist anamorelin increases lean body mass and improves the performance status in NSCLC patients with cachexia [26,27]. Therefore, the administration of anamorelin may improve the effect of ICI rechallenge treatment.

The effectiveness of initial ICI treatments has been reported to be associated with PD-L1 expression in NSCLC tumors [28]. Our current observations show that the patients with PD-L1 expression tended to have longer PFS. This suggests that PD-L1 expression levels in pre-treatment tumors may be a factor to consider when with regard to ICI rechallenge treatment. Regardless, based on our observations, the values of blood NLR, LMR, and PLR at baseline may be useful tools for predicting responders to the ICI rechallenge treatment, which is consistent with the initial ICI treatment [10,13,15,29]. Thus, when considering ICI rechallenge treatment in patients with NSCLC, the inflammation markers, such as NLR, LMR, and PLR, may be useful to some extent for identifying responders to ICI rechallenge.

A previous report suggests that the response to initial ICI treatments correlates with the clinical response to ICI rechallenge treatment in patients with melanoma [30]. However, our results failed to indicate a relationship between clinical outcomes of initial ICI treatment and ICI rechallenge treatment in patients with NSCLC. This suggested that there may be differences between the immunological properties of NSCLC and melanoma.

The current study had several limitations. First, it consisted of a small retrospective sample. Therefore, a further large-cohort study is warranted to identify the predictive markers of ICI rechallenge

treatment. Second, although the treatment was administered at multiple centers, there may have been bias in terms of the timing of evaluating the patients using CT scanning, even though it was performed every 1–3 months after treatment.
