*3.3. Survival and 18F-FDG-PET:*

The median OS and PFS were 716 and 161 days, respectively, and the 2-year OS rate was 44.8%. Among all patients, those with a low TLG exhibited significantly better OS than those with a high TLG. Similarly, the survival analysis of MTV on 18F-FDG uptake demonstrated a significantly worse OS and PFS for patients with a high MTV (Figure 2). No statistically significant difference in the OS and PFS was observed between patients with a low and high SUVmax (Figure 2). However, in 70 patients without an EGFR mutation, a statistically significant difference in OS and PFS was recognized between patients with low and high TLG or low and high MTV, but not between those with low and high SUVmax and low and high TB (Figure 3). Survival analysis results are listed in Table 2. Univariate analysis in all patients identified performance status (PS), TLG, and MTV as significant prognostic markers for OS; the significant predictors for PFS were PS and MTV. Subsequently, we performed a multivariate analysis according to TLG and MTV and confirmed that PS, TLG, and MTV were independent prognostic factors for poor OS and PFS; the significant prognostic marker for PFS was PS (Table 3). In patients without EGFR mutation, a multivariate analysis identified TLG and MTV as independent prognostic factors for predicting poor OS. Histological typing confirmed TLG and MTV as significant prognostic factors in patients with adenocarcinoma, but not in those with non-adenocarcinoma.

**Figure 2.** Discriminative value by log-rank test according to various TLG (**A**), MTV (**C**) and SUVmax (**E**) cutoff for OS and TLG (**B**), MTV (**D**) and SUVmax (**F**) cutoff for PFS in 18F-FDG-PET.

**Figure 3.** Kaplan–Meier curves according to various TLG (**A**), MTV (**B**), SUVmax (**C**) and TB (**D**) for OS and PFS. (**A**); OS (upper left) and PFS (lower left) in all patients and OS (upper right) and PFS (lower right) in those without EGFR mutation according to TLG. (**B**); OS (upper left) and PFS (lower left) in all patients and OS (upper right) and PFS (lower right) in those without EGFR mutation according to MTV. (**C**); OS (upper left) and PFS (lower left) in all patients and OS (upper right) and PFS (lower right) in those without EGFR mutation according to SUVmax. (**D**); OS (upper left) and PFS (lower left) in all patients and OS (upper right) and PFS (lower right) in those without EGFR mutation according to TB. (**E**); OS (upper left) and PFS (lower left) in patients with AC patients and OS (upper right) and PFS (lower right) in those with non-AC according to TLG. (**F**); OS (upper left) and PFS (lower left) in patients with non-AC patients and OS (upper right) and PFS (lower right) in those with non-AC according to MTV. Abbreviation: TLG, total lesion glycolysis; MTV, metabolic tumor volume; SUVmax: maximal standardized under value; TB, tumor burden; OS, overall survival; PFS, progression-free survival; AC, adenocarcinoma; non-AC, non-adenocarcinoma.


**Table 2.** Univariate analysis in overall survival and progression-free survival.

Abbreviations: TLG, total lesion glycolysis; MTV, metabolic tumor volume; SUVmax, the maximum of standardized uptake value; AC, adenocarcinoma; Non-AC, non-adenocarcinoma; PS, performance status; HR, hazard ratio; 95% CI, 95% confidence interval; NR, not reached; EGFR, epidermal growth factor receptor; TB, tumor burden; Bold character shows statistically significance.


**Table 3.** Multivariate analysis of overall survival and progression-free survival.

Abbreviations: TLG, total lesion glycolysis; MTV, metabolic tumor volume; PS, performance status; HR, hazard ratio; 95% CI, 95% confidence interval; EGFR, epidermal growth factor receptor.

#### **4. Discussion**

To the best of our knowledge, this is the first study to evaluate the prognostic significance of metabolic parameters measured by 18F-FDG-PET for predicting outcomes after the initiation of anti-PD-1 antibodies in patients with previously treated NSCLC. We found that the tumor metabolic volume assessed by TLG and MTV was an independent prognostic factor, but not SUVmax and TB. The roles of TLG and MTV as significant predictive markers after anti-PD-1 antibody therapy may be closely associated with patients with a histology of adenocarcinoma and without EGFR mutation. Although the value of SUVmax on 18F-FDG uptake within tumor cells was closely correlated with that of TLG and MTV, SUVmax on 18F-FDG uptake before anti-PD-1 antibody could not accurately predict the outcome after the initiation of treatment. Furthermore, we found that low uptake of 18F-FDG was closely associated with the occurrence of irAEs and the incidence of grade 3 or 4 irAEs exhibited no close correlation with the degree of 18F-FDG uptake on PET. In addition, there was no significant correlation between the response to the anti-PD-1 antibody and the degree of 18F-FDG uptake. However, we discovered that MTV of pretreatment 18F-FDG-PET plays a crucial role in the prognostic significance of anti-PD-1 antibody treatment. Unlike previous studies [12,13], our study indicated that TB determined by baseline tumor size was not a significant predictor of ICI or irAEs. Further studies are warranted to investigate whether pretreatment of MTV can predict the outcome of ICI plus ICI or the combination of ICI plus cytotoxic chemotherapy as first-line treatment.

A recent study reported that the metabolic response to 18F-FDG uptake measured by TLG or MTV was a stronger biomarker for the prediction of efficacy and survival at one month after the initiation of nivolumab than SUVmax [10]. The predictive probability of partial response (100% versus 29%, *p* = 0.021) and progressive disease (100% versus 22.2%, *p* = 0.002) at one month after nivolumab treatment was significantly higher in 18F-FDG-PET than in CT. Moreover, multivariate analysis confirmed the 18F-FDG uptake measurement by TLG or MTV as an independent prognostic factor. Thus, 18F-FDG-PET may be a useful radiographic modality for immune monitoring to predict the efficacy of ICI. However, a large-scale prospective study is required to confirm these results [10,18]. A recent meta-analysis suggested that in lung cancer, metabolic parameters such as TLG and MTV are better prognostic predictors after any treatment when compared with SUVmax [11].

Recently, Jreige et al. described that the metabolic-to-morphological volume ratio (MMVR)—calculated by dividing MTV by morphological tumor volume (MoTV)—was able to predict the radiological response to anti-PD-1/PD-L1 blockade and was negatively correlated with tumor PD-L1 expression and tumor necrosis [18]. Although their study analyzed only 17 patients who received anti-PD-1 therapy, they suggested that anti-PD-1 therapy is effective for tumor tissues with a low ratio of MTV, and the presence of a low MTV contributes to the upregulation of PD-L1 expression by the hypoxic environment in the tumor arising from necrosis and inflammation. However, it remained unclear whether MMVR could predict the outcome after anti-PD-1 therapy. Our study, however, indicated that patients with a low MTV exhibited a favorable prognosis after anti-PD-1 therapy. Several studies have demonstrated that tumor necrosis triggers inflammation, which facilitates the influx of lymphocytes and the upregulation of PD-L1, related to tumor necrosis factor-α, and that necrosis or inflammation was closely linked to tumor recurrence and worse survival [19,20]. Our study could not investigate the relationship between necrosis or inflammation and tumor metabolic volume; therefore, it remains unclear whether low MTV and TLG are associated with necrosis, inflammation, and hypoxic environment in the tumor. Further investigation is warranted to elucidate the prognostic significance of MTV and TLG as predictive markers of anti-PD-1 therapy based on the biological aspect.

The mechanism by which tumor cells uptake 18F-FDG requires glucose metabolism, hypoxia, and angiogenesis, and 18F-FDG is closely associated with the expression of these markers [6]. The expression of PD-L1 is significantly related to Glut1, HIF-1α and SUVmax on 18F-FDG uptake in lung cancer [7–9]. However, it is reported that TLG and MTV on 18F-FDG uptake were not closely correlated with PD-L1 expression levels [18], but Foxp3-Tregs are described to be positively associated with TLG and MTV [21]. Considering these evidences, the active environment with high MTV may form an immunosuppressive state, contributing to the resistant situation to ant-PD-1 blockage.

Our study has several limitations. First, we employed a retrospective approach and a small cohort, which may have introduced bias in our results. More than half of the patients with previously treated NSCLC had not undergone 18F-FDG-PET just before the initiation of anti-PD-1 antibody treatment. Therefore, we think that the number of eligible patients was slightly limited. However, the current study is meaningful for the prediction of anti-PD-1 therapy. Second, our study suggests that MTV could be a predictive marker for ICI treatment compared to the morphological tumor volume. However, the

appropriateness of morphological assessment used for calculating the optimal tumor volume of all metastatic lesions using CT scan is debatable. Thus, it may be difficult to perform a concise comparison between metabolic and morphological tumor volumes. Moreover, we had no information about the calculation of MMVR in our study, thus, there were some limitations in discussing the prognostic significance of metabolic tumor volume compared to MMVR. Lastly, an the immunohistochemical analysis of PD-L1, expression within tumor cells using tumor specimens was not performed before anti-PD1 antibody treatment. At our institution, re-biopsy was not done routinely before ICI treatment except for the initial diagnosis. Further study is warranted to investigate the relationship between MTV and the immune environment.

In conclusion, TLG and MTV on 18F-FDG uptake may predict the prognosis after anti-PD-1 antibodies in patients with previously treated NSCLC, but not SUVmax on 18F-FDG uptake. Although MTV may indirectly reflect the presence of morphological tumor volume with active tumor cells, the assessment of TLG and MTV on 18F-FDG uptake is easily executable in daily practice. A prospective study is needed to confirm these results.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/3/725/s1.

**Author Contributions:** K.H., A.S., K.K. (Kunihiko Kobayashi) and H.K.: conception and preparation of the manuscript. A.M., A.S., O.Y., K.H., Y.M. (Yu Miura), and Y.M. (Yoshitake Murayama): management of the patient. O.Y.: statistical analysis and patient's data collection. A.S., A.M., O.Y., K.K. (Kyoichi Kaira), I.K. and H.K.: revising the manuscript. All authors contributed and agreed with the content of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

**Conflicts of Interest:** A.M., K.K., O.Y. and H.K. have received research grants and a speaker honorarium from Ono Pharmaceutical Company and Bristol-Myers Company. All remaining authors have declared no conflicts of interest.
