Immune Checkpoint Inhibitors in Advanced NSCLC: [18F]FDG PET/CT as a Troubleshooter in Treatment Response

Introduction: The aim of this study was to investigate whether [18F]FDG PET/CT-derived semi-quantitative parameters can predict immunotherapy treatment response in non-small cell lung cancer (NSCLC) patients. Secondly, immune-related adverse events (irAEs) and lymphoid cell-rich organs activation were evaluated. Materials and Methods: Twenty-eight patients who underwent [18F]FDG PET/CT scans before and at first restaging therapy with immuno-checkpoint inhibitors (ICIs) were retrospectively analyzed. PET-based semi-quantitative parameters extracted from both scans were respectively: SUVmax and SUVpeak of the target lesion, whole-body metabolic tumor volume (MTVWB), and whole-body total lesion glycolysis (TLGWB), as well as their interval changes (ΔSUVmaxTL, ΔSUVpeakTL, ΔMTVWB, ΔTLGWB). These PET-derived parameters were correlated to controlled disease (CD) assessed by RECIST 1.1. IrAEs, if present, were also described and correlated with clinical benefit (CB). SUVmax of the spleen and bone marrow at restaging scans were also correlated to CB. Results: The CD was achieved in 54% of patients. Out of 28 eligible patients, 13 (46%) experienced progressive disease (PD), 7 showed SD, 7 had PR, and only in one patient CR was achieved. ΔSUVmaxTL (p = 0.002) and ΔSUVpeakTL (p < 0.001) as well as ΔMTVWB (p < 0.001) and ΔTLGWB (p < 0.005) were significantly associated with PD vs. non-PD. IrAEs and lymphoid cell-rich organs activation did not correlate with CB. Conclusions: [18F]FDG PET/CT by using interval changes of PET-derived semi-quantitative parameters could represent a reliable tool in immunotherapy treatment response evaluation in NSCLC patients.


Introduction
In the last decade, the advent of immunotherapy has paved the way for new treatment options for patients with advanced non-small cell lung cancer (NSCLC) [1]. Immunocheckpoint inhibitors (ICIs) therapy exploits the use of antibodies that target specific molecules involved in tumor signaling, determining the suppression of cytotoxic T lymphocytes [2]. In addition, by releasing the brakes of the host-immune system, ICIs may alter the physiological homeostasis of immune response, thus leading to the development of immune-related adverse events (irAEs) [3]. To date, a standard method to evaluate the success of these innovative therapies and to identify patients who may benefit from them remains undetermined. Morphological imaging by using Response Evaluation Criteria in Solid Tumors (RECIST1.1) represents the standard modality to cytotoxic therapies response assessment [4]. To face this new clinical issue, the immune-related Response Evaluation Criteria in Solid Tumor (irRECIST) was developed [5], but their efficacy in early treatment response evaluation is still limited [6]. Fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([ 18 F]FDG PET/CT) represents an essential diagnostic tool in the management of NSCLC patients, from staging to treatment response evaluation. Even in the new scenario of immunotherapy, [ 18 F]FDG PET/CT, thanks to the semi-quantitative analysis and PETderived parameters, could represent a reliable diagnostic technique offering additional information to standard modalities [7,8]. Several studies investigated the role of PET extracting data before and/or during immunotherapy [9]. Among them, standardized uptake value (SUV), the most commonly used, was reported to be correlated to response rate. Takada et al., in 89 patients with advanced or recurrent NSCLC, showed that patients with a baseline SUVmax ≥ 11.16 had a significantly higher response rate compared to patients with lower SUVmax values [10]. A plethora of evidence is already available on the predictive role of volume-based PET-parameters. Some authors highlighted the predictive value of metabolic tumor volume (MTV) as well as the total lesion glycolysis (TLG) in NSCLC patients treated with ICIs [11]. Despite the increased amount of evidence, a common agreement has still not been reached.
Interestingly, [ 18 F]FDG PET/CT could reveal irAEs before their clinical manifestation and/or laboratory test positivity. However, few literature data are still available regarding the irAEs imaging features and radiological description. Consequently, the relation between irAEs and treatment response needs further investigation [12].
This study aims to evaluate the role of [ 18 F]FDG PET/CT-derived semi-quantitative parameters in the immunotherapy response assessment. Secondly, the detection of immunerelated adverse events (irAEs) and lymphoid cell-rich organs activation were investigated in order to evaluate their possible predictive value.

Subject
A single-center database was retrospectively interrogated to identify patients with a history of advanced NSCLC treated with ICIs who underwent [ 18 F]FDG PET/CT. The inclusion criteria were: (a) histologically/cytologically proven NSCLC; (b) [ 18 F]FDG PET/CT scans before and at first restaging after the start of immunotherapy; (c) minimum follow-up of 3 months after treatment initiation; (d) radiological assessment during treatment every 8-12 weeks with CT scans, for good clinical practice; and (e) the availability of information on the best clinical response to immunotherapy.
All patients were observed for at least 6 months after the first restaging [ 18 F]FDG PET/CT, except for those who died. Age, sex, histological subtypes, previous surgery, prior lines, and type of therapy and molecular profile (if available) were also collected.
All patients had already given their consent for the use of their data for clinical research. Our Institutional Review Board does not require the Ethical Committee's approval for review of patients' files.

[ 18 F]FDG PET/CT Examination and Analysis
[ 18 F]FDG PET/CT scans were performed with a Discovery 710 PET/CT scanner (GE, Healthcare Technologies, Milwaukee, WI, USA), and the same scanner was used for baseline and first post-treatment evaluation. All patients fasted for at least 6 h and presented a blood glucose levels less than 200 mg/dL. An intravenous injection of 3.0 MBq/kg of [ 18 F]FDG was administered and PET/CT scanning was performed 60 min after injection. Non-contrast CT images and subsequent PET images were acquired from the skull base to the upper thigh in the supine position with the arms raised. PET image acquisition was performed for 4 min per bed in 3-dimensional acquisition mode using 7 to 10 beds. Image review and analysis were conducted on a dedicated workstations and software (AW Server 4.7; GE, Healthcare Technologies, Milwaukee, WI, USA).
All PET/CT scans were reviewed by a nuclear medicine expert who performed visual interpretation and semiquantitative analysis, documenting all pathological foci of FDG uptake as well as the appearance of abnormal metabolism (non-physiologic) in organs possibly activated by immune-system response at first restaging PET/CT images.
Over PET parameters, the standardized uptake value (SUV) was evaluated in terms of SUV max and SUV peak . In addition, volume-based PET parameters, MTV and TLG were obtained and used for further analysis.

Response Evaluation
The diagnostic assessment was performed according to Response Evaluation Criteria for Solid Tumors criteria version 1.1 (RECIST1.1), in terms of complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) at first radiological restaging. The controlled disease (CD) was defined as the achievement of CR, PR, and SD. All forementioned PET-derived parameters were correlated to treatment response.
Moreover, clinical benefit (CB) was determined considering patients' clinical course after the last follow-up: (a) continued/stopped immunotherapy, (b) disease control/ exacerbation, (c) changing to chemotherapy, or (d) death. IrAEs, if present, were also described and correlated, together with SUV max of lymphoid cell-rich organs, to CB at follow up.

Statistical Analysis
Categorical variables were described using absolute and relative frequencies; continuous variables were described using median range. To assess the correlation between each PET/CT parameters and response groups (PD vs. no-PD), the Mann-Whitney test was used, and the results were represented graphically by box-plots. The multivariate Cox model was adopted to assess the association of PET-parameters, adjusting for gender, drugs, line of therapy, and previous lung surgery, both for CD and CB.
Finally, the association between the presence of irAEs at first restaging [ 18 F]FDG PET/CT exams and CD as well as CB, was investigated using Chi-square and Fisher's exact test. A p value less than 0.05 was considered statistically significant. All statistical analysis was performed using IBM SPSS Statistic Version 28 (IBM, Armonk, NY, USA).
The median time between the baseline PET/CT exam and the start of immunotherapy was 38 days (range: 2-90 days), whereas the median time between PET/CT scans was 4 months (range: 2-11 months).
Thirteen (46%) eligible patients, experienced progressive disease (PD) at first restaging, 7 (25%) patients showed SD, 7 (25%) had PR, and only in one patient (4%) CR was obtained. The CD was achieved in 15/28 (54%) patients ( Figure 1).    None of parameters extracted from the PET/CT before starting immunotherapy (preSUV maxTL , preSUV peakTL , preMTV WB , preTLG WB ) showed a significant correlation with radiological response. Conversely, ∆SUV maxTL (p = 0.002) and ∆SUV peakTL (p < 0.001) as well as ∆MTV WB (p < 0.001) and ∆TLG WB (p < 0.005) were significantly associated with PD vs. non-PD. No difference was showed between the pembrolizumab and nivolumab treated-groups. In the subgroup with available PD-L1 status, the ligand expression was shown to be statistically correlated with baseline SUV max (p = 0.017) and baseline SUV peak (p = 0.03). The relation between CD and ∆SUV maxTL , ∆SUV peakTL , ∆MTV WB , ∆TLG WB is reported in the box plots in Figure 2. ΔMTVWB (p < 0.001) and ΔTLGWB (p < 0.005) were significantly associated with PD vs. non-PD. No difference was showed between the pembrolizumab and nivolumab treatedgroups. In the subgroup with available PD-L1 status, the ligand expression was shown to be statistically correlated with baseline SUVmax (p = 0.017) and baseline SUVpeak (p = 0.03). The relation between CD and ΔSUVmaxTL, ΔSUVpeakTL, ΔMTVWB, ΔTLGWB is reported in the box plots in Figure 2. Five patients showed immuno-related findings on PET/CT. Notably, in two out of the five patients, diffuse thyroid [ 18 F]FDG uptake, as for thyroiditis, was detected. PET/CT finding of colitis was observed in one patient only, who reported persistent diarrhea during pembrolizumab treatment. A pattern of immunotherapy-related arthritis and pneumonitis associated with sarcoid reaction was shown on PET/CT scans of the last two patients, respectively. Table 2 shows the demographics of patients who developed irAEs and the most representative cases are depicted in Figure 3. Five patients showed immuno-related findings on PET/CT. Notably, in two out of the five patients, diffuse thyroid [ 18 F]FDG uptake, as for thyroiditis, was detected. PET/CT finding of colitis was observed in one patient only, who reported persistent diarrhea during pembrolizumab treatment. A pattern of immunotherapy-related arthritis and pneumonitis associated with sarcoid reaction was shown on PET/CT scans of the last two patients, respectively. Table 2 shows the demographics of patients who developed irAEs and the most representative cases are depicted in Figure 3.   Three out of the five patients with irAEs PET/CT findings reached CD (1 SD; 2 PR), while the other two showed PD on morphological imaging. No statistically significant correlation was reached between irAEs and CD (p = 0.429). Similarly, three out of five patients showed CB at follow-up but no significant correlation was found (p = 0.229).
None of lymphoid cell-rich organs metabolic PET-parameters was significantly correlated to CB. All statistical results are detailed in Tables 3 and 4.

Discussion
The advent of immunotherapy in clinical practice and the approval of several drugs for advanced solid tumors led to the development of new challenges for imaging. In this scenario, [ 18 F]FDG PET/CT, a consolidate tool in the evaluation of lung cancer, was proposed as a promising marker for immunotherapy treatment response evaluation, providing useful and unique information [13].
In the last few years, there has been a growing interest in the evaluation of PET-derived parameters as predictors of response [14]. This study pointed out that metabolic changes during immunotherapy statistically correlate with treatment response as well as with CB. Notably, the interval changes of all PET-derived parameters showed a significant decrease in responders, potentially representing a marker of long-term clinical response. Similar results were reached by Nobashi et al., in their study on 40 patients affected by different cancer types, where they found a significant correlation between PET parameters' variation and best overall response at one year [15].
Other authors assessed the predictive role of [ 18 F]FDG PET/CT by semiquantitative analysis. In 2019, Evangelista et al. retrospectively studied 32 patients with metastatic lung cancer under nivolumab treatment. SUV maxWB was significantly higher in patients with PD compared with those with SD and PR, whereas a similar trend was shown for TLG WB and MTV WB , both higher in non-responders than responders, even without a statistical significance [16]. Conversely, in other studies, SUV max was unable to predict response. However, volume-based PET parameters were extracted. In this context, a bicentric Italian study performed an analysis in a bigger cohort of 92 patients, demonstrating that those with PD had higher MTV median values compared to those with controlled disease [17]. Similarly, Polverani and colleagues observed a significant association of MTV and TLG of the primary lesions with PD, since lower MTV and TLG values were associated with non-PD status [18]. In our study, we emphasized the variation of PET-derived parameters during immunotherapy in NSCLC patients as a potential biomarker of treatment response.
Even if PD-L1 status was not available for all patients included in our sample, the analysis revealed its significant correlation with baseline SUVs: increased values of SUVs were detected in patients with PD-L1 expression higher than 50%. Albeit with a limited number of cases, this finding, supported by the literature data, suggests a potential role of [ 18 F]FDG PET/CT in predicting PD-L1 status [19,20].
Interestingly, in the multivariate analysis, it was shown that immunotherapy employed as a second or more line of therapy was significantly correlated to a better response rate. We can speculate that this finding could be associated with the growing evidence that cytotoxic chemotherapy and radiotherapy could impact tumor ligand expression, determining changes in cell PD-L1 expression as well as in the tumor microenvironment [21,22].
Even if the occurrence of irAEs seems to be associated with better response and prolonged overall survival (OS) [26], few data are available in the literature about the prognostic role of irAEs detected on PET and response. In a study conducted by Sachpekidis et al. in a cohort of metastatic melanoma patients, irAEs on PET/CT correlated with response to immunotherapy-patients who developed at least one irAE had a significantly longer PFS than those without irAEs [27,28].
In our sample, any statistically significant correlation was reached, probably due to the few immune-related events frequency registered. However, three out of five patients who showed irAE on PET/CT experimented CD or CB. Moreover, as PET/CT could monitor the metabolic changes in peripheral lymphoid organs and related ones [29], we conducted a semi-quantitative evaluation on lymphoid cell-rich organs, such as the spleen and bone marrow but none of PET-derived parameters were shown to be statistically significant. Conversely, Nobashy et al. conducted a similar analysis reporting a significant increase of SUV max of the spleen in those patients who did not show any clinical benefit after the start of immunotherapy [15].
Despite the retrospective nature of the present study and the small population size that could impact the results, our study aimed to help highlight the emerging and promising role of PET imaging in predicting response in the field of immunotherapy.

Conclusions
[ 18 F]FDG PET/CT could represent a reliable and efficacious diagnostic tool in immunotherapy treatment response evaluation in advanced NSCLC patients. The decrease of all tumor parameters at first restaging PET/CT results in a predictive role for immunotherapy response and could represent a useful biomarker to estimate treatment response evaluation. Further research is needed to confirm these preliminary data and to explore the interesting field of irAEs findings on PET/CT and their correlation with response.