Radiol Oncol 2025; 59(1): 43-53. doi: 10.2478/raon-2025-0014 43 research article Early-time-point 18F-FDG-PET/CT and other prognostic biomarkers of survival in metastatic melanoma patients receiving immunotherapy Nezka Hribernik1,2, Katja Strasek3, Andrej Studen3,6, Katarina Zevnik7, Katja Skalic7, Robert Jeraj3,4,5,6, Martina Rebersek1,2 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia 4 University of Wisconsin Carbone Cancer Centre, Madison, WI, USA 5 Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA 6 Experimental Particle Physics Department, Jozef Stefan Institute, Ljubljana, Slovenia 7 Department of Nuclear Medicine, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2025; 59(1): 43-53. Received 12 December 2024 Accepted 4 January 2025 Correspondence to: Assoc. Prof. Martina Reberšek, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: mrebersek@onko-i.si Disclosure: Robert Jeraj is a cofounder and CSO of AIQ Solutions, Madison, WI, USA. Funding. No potential conflicts of interest are disclosed by the other co-authors. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. A considerable proportion of metastatic melanoma (mM) patients do not respond to immune checkpoint inhibitors (ICIs). There is a great need to develop noninvasive biomarkers to detect patients, who do not respond to ICIs early during the course of treatment. The aim of this study was to evaluate the role of early [18F]2fluoro- 2-deoxy-D-glucose PET/CT (18F-FDG PET/CT) at week four (W4) and other possible prognostic biomarkers of survival in mM patients receiving ICIs. Patients and methods. In this prospective noninterventional clinical study, mM patients receiving ICIs regularly underwent 18F-FDG PET/CT: at baseline, at W4 after ICI initiation, at week sixteen and every 16 weeks thereafter. The tu- mor response to ICIs at W4 was assessed via modified European Organisation for Research and Treatment of Cancer (EORTC) criteria. Patients with progressive metabolic disease (PMD) were classified into the no clinical benefit group (no-CB), and those with other response types were classified into the clinical benefit group (CB). The primary end point was survival analysis on the basis of the W4 18F-FDG PET/CT response. The secondary endpoints were survival analysis on the basis of LDH, the number of metastatic localizations, and immune-related adverse events (irAEs). Kaplan-Meier analysis and univariate Cox regression analysis were used to assess the impact on survival. Results. Overall, 71 patients were included. The median follow-up was 37.1 months (95% CI = 30.1–38.0). Three (4%) patients had only baseline scans due to rapid disease progression and death prior to W4 18F-FDG-PET/CT. Fifty-one (72%) patients were classified into the CB group, and 17 (24%) were classified into the no-CB group. There was a statisti- cally significant difference in median overall survival (OS) between the CB group (median OS not reached [NR]; 95% CI = 17.8 months – NR) and the no-CB group (median OS 6.2 months; 95% CI = 4.6 months – NR; p = 0.003). Univariate Cox analysis showed HR of 0.4 (95% CI = 0.18 – 0.72; p = 0.004). median OS was also significantly longer in the group with normal serum LDH levels and the group with irAEs and cutaneous irAEs. Conclusions. Evaluation of mM patients with early 18F-FDG-PET/CT at W4, who were treated with ICIs, could serve as prognostic imaging biomarkers. Other recognized prognostic biomarkers were the serum LDH level and occurrence of cutaneous irAEs. Key words: early time-point 18F-FDG-PET/CT; prognostic biomarkers; immune-related adverse effects metastatic melanoma; immune-checkpoint inhibitor Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI44 Introduction Immunotherapy with immune checkpoint in- hibitors (ICIs) has greatly impacted the treatment landscape of metastatic melanoma (mM) patients. Final, 10-year results from pivotal randomized clinical trial have shown ongoing benefits, with 10-year overall survival (OS) rates of 34%, 37%, and 43% in mM patients receiving pembrolizumab, nivolumab, and ipilimumab/nivolumab, respec- tively.1,2 However, a considerable proportion of mM patients do not respond to ICIs. Normal serum levels of lactate dehydroge- nase (LDH) and a small number of organs with metastatic involvement are two very strong and well-recognized prognostic biomarkers of better survival in mM patients.3-5 In addition, immune- related adverse events (irAEs) have been proven in some studies to be biomarkers of improved re- sponse rates and longer survival in patients treated with ICIs.6,7 Specifically, endocrine and cutaneous irAEs were associated with favourable survival outcomes compared with patients without this type of irAE in a large retrospective multicohort study.6 There is a great need to develop other non- invasive imaging biomarkers (IBM) to detect mM patients who do not respond to ICIs early in the course of treatment. Positron emission tomography/computed to- mography with [18F]2fluoro-2-deoxy-D-glucose PET/CT (18F-FDG PET/CT) is a noninvasive method that combines anatomical and functional data. It is generally used as a diagnostic tool for the staging of melanoma patients and is now gaining valuable value in immunotherapy treatment response eval- uation and prognosticating outcomes.7,8 In addi- tion to staging and response monitoring, 18F-FDG PET/CT has also shown some potential for detect- ing immune-related side effects (irAEs), such as the use of organ 18F-FDG uptake, quantified by percentiles of standardized uptake values (SUV) distribution as a quantitative IBM of irAEs.9-11 The optimal timing of the first on-treatment 18F-FDG PET/CT evaluation of mM patients on ICIs is a matter of ongoing investigations. Joint EANM/ SNMMI/ANZSNM practice guidelines/procedure standards recommended an interim 18F-FDG PET/ CT scan during immunomodulatory treatment in patients with solid tumors at 8–12 weeks after the start of treatment (i.e., after 3–4 cycles of immu- notherapy).7 However, there are studies analysing 18F-FDG PET/CT scans performed earlier after ICI initiation. In a prospective study with 20 mM pa- tients treated with ipilimumab, 18F-FDG PET/CT was performed between 21-28 days after treatment start. They concluded that the combination of changes in lesion dimensions along with changes in 18F-FDG uptake may be associated with immune activation and a favourable outcome.12 We previously reported the results of our pro- spective study regarding the role of quantitative IBM in early 18F-FDG PET/CT, which was per- formed four weeks after ICI initiation, for the de- tection of immune-related adverse events in mela- noma patients.10 Here, we evaluated the role of ear- ly 18F-FDG-PET/CT at week four and other possible prognostic biomarkers of survival in mM patients receiving ICIs. Patients and methods Patients We enrolled patients 18 years of age or older who had histologically confirmed, unresectable, ad- vanced melanoma and were planned to be treated per standard of care with ICIs with anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) and/or anti-programmed death-1 (anti-PD-1) treatment in the first or second line of systemic treatment at the Institute of Oncology Ljubljana, Slovenia. The key exclusion criteria included symptomatic brain metastases and malignant dis- eases other than melanoma. Trial design In this noninterventional, prospective study, pa- tients underwent baseline 18F-FDG PET/CT within four weeks before treatment initiation and were monitored regularly with serial 18F-FDG PET/CT: at week four (W4) (+/- 5 days), week 16 (W16) (+/- 7 days), and week 32 (W32) (+/- 7 days) after treat- ment initiation and every 16 weeks thereafter. The first follow-up 18F-FDG PET/CT at W4 was per- formed for investigational purposes and was not necessarily used to guide treatment decisions. The clinical data and images included in this analysis were obtained from disease diagnosis up to 1st September 2024. All 18F-FDG PET/CT data were acquired before and during ICI treatment, and all clinical data were collected for review. The irAE grade was as- signed prospectively and scored with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v.5.0).13 The irAE were classified as serous irAE in case of high- er grade of irAE of 3 and above. Imaging and clini- Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI 45 cal data were anonymized and stored in a secure LabKey database server.14 The clinical protocol was approved by the Ethics Committee ERIDEK-0034/2020 and the Clinical Trials Protocol Review Committee ERID- KSOPKR-0032/2020 at the Institute of Oncology Ljubljana and by the Commission of the Republic of Slovenia for Medical Ethics (approval number: 0120-256/2020-14, September 15th 2020). It was con- ducted following the ethical standards defined by the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. The study was registered with ClinicalTrials.gov under the registration number NCT06207747. The study was conducted with the acknowledg- ment and consent of the subjects. All patients pro- vided signed informed consent for treatment and consent allowing the use of their data for scientific purposes. 18F-FDG PET/CT acquisition and analysis All 18F-FDG PET/CT scans were obtained on Biograph mCT PET/CT (Siemens, Knoxville, TN). Imaging protocol required patients to fast for 6 hours prior to injection of the radiotracer and have a blood glucose level below 10 mmol/L at the time of the scan. Patients were required to hold all diabetic medication, including metformin, for 6 hours prior to radiotracer injection. All scans were acquired per standard of care. CT that meets response evaluation criteria in solid tumours (RECIST) analysis needs was acquired according to adjusted protocol including sinogram affirmed iterative reconstruction (SAFIR) to minimize dose. Following reconstruction, PET images were nor- malized by patient weight and injected dose to compute SUV. More details about image acquisi- tion can be found in our previous paper, where the same cohort of patients was used for analysis.11 The tumor response to ICIs on 18F-FDG PET/ CT was evaluated by a nuclear medicine special- ist combining the European Organisation for Research and Treatment of Cancer (EORTC) crite- ria and visual response assessment.15 The SUVmax and size of the lesions were measured in all most representative tumor lesions, which are the largest lesions of a certain area or organ with the high- est FDG uptake at baseline, W4 and all consecu- tive 18F-FDG PET/CT scans. Patients were classified into four major categories on the basis of the tu- mor response to ICIs: complete metabolic response (CMR), partial metabolic response (PMR), stable TABLE 1. Patient demographics, cancer staging, treatment details, and outcomes Characteristics No = 71 (%) Age; mean (+/-SD) (yr) 62 ± 12 Gender Male 43 (61) Female 28 (39) ECOG performance status 0 30 (42) 1 41 (58) AJCC III.D 1 (1) M1a 16 (23) M1b 10 (14) M1c 32 (45) M1d 12 (17) Anatomic site of primary Cutaneous 58 (82) Ocular 4 (6) Mucosal 3 (4) Unknown primary 6 (8) Line of systemic treatment for metastatic disease 1st line 63 (89) 2nd line 8 (11) Baseline LDH Elevated 23 (32) Normal 49 (68) Number of organs with metastatic involvement 1 25 (35) 2 21 (30) 3 11 (15) > 3 14 20) Actionable mutation BRAF wild type 21 (30) BRAF V600E 28 (39) BRAF V600K 10 (14) BRAF V600 - others 1 (1) NRAS 11 (16) Type of systemic treatment PD-1 inhibitors 47 (66) Combination of PD-1 and CTLA-4 inhibitors 24 (34) Tumor response on week 4 18F-FDG PET/CT Complete metabolic response 3 (4) Partial metabolic response 12 (17) Stable metabolic disease 10 (14) Heterogenous response 6 (8) Possible pseudoprogression 20 (28) Progressive metabolic disease 17 (24) AJCC = American Joint Classification of Cancer; BRAF = V-Raf murine sarcoma viral oncogene homolog B; CTLA-4 = Cytotoxic T lymphocyte-associated antigen 4; ECOG = Eastern Cooperative Oncology Group; ICI = Immune checkpoint inhibitors; No = number of patients; NRAS = neuroblastoma RAS viral homolog; PD-1 = programmed death-1; SD = standard deviation Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI46 metabolic disease (SMD) and progressive meta- bolic disease (PMD).15 According to the informa- tion gathered after the whole -body 18F-FDG PET/ CT visual assessment, two new categories, of het- erogenous response (HGR) and possible pseudo- progressive disease (PPD), were added for tumor response evaluation. HGR was assigned when multiple lesions were variably meeting the crite- ria of PMD, SMD, PMR and CMR and could not be classified into only one response evaluation category. PPD was assigned in the case of moder- ate metabolic progression of the baseline tumor lesions with few locally distributed new lesions. Obvious progression with multiple new tumor le- sion sites was classified as true progression (PMD). The patients were further stratified into two groups: patients with PMD were classified into the no clinical benefit (no-CB) group and patients with other response categories into the clinical benefit (CB) group. A summary of the response criteria is presented in Supplementary Table 1. Outcomes and statistical analysis The primary end point of this study was the analy- sis of median OS based on the W4 18F-FDG PET/CT response. The secondary endpoint was the median OS, which was analysed on the basis of the level of LDH, the number of organs with metastatic in- volvement at the beginning of ICI treatment, oc- currence of irAE, higher irAE, cutaneous irAEs, endocrine irAEs and immune-related thyroiditis (irThyroiditis). Patient characteristics were summarized via descriptive statistics. Survival analysis was per- formed via the Kaplan‒Meier method, and 95% confidence intervals (CIs) were calculated. The associations of each of the eight metrics with OS were assessed with a univariate Cox proportional hazard model. With the use of the Bonferroni cor- rection for testing multiple hypothesis, probability values p < 0.006 were considered statistically sig- nificant. Results From September 2020 through September 2022, a total of 71 patients were enrolled. The character- istics of the patients are summarized in Table 1. At the cut-off date of the observational period for this analysis on 1st September 2024, the median follow-up was 37.1 months (95% CI = 30.1-38.0). The median duration of ICI therapy was 10.2 months (range: 1-39.4 months). Three (4%) patients had on- ly baseline scans due to rapid disease progression and death prior to W4 18F-FDG-PET/CT. The timing of 18F-FDG PET/CT relative to ICI treatment initia- tion and the number of 18F-FDG-PET/CT images are shown in the Supplementary Table 2. FIGURE 1. Kaplan-Meier curves show overall survival (OS) probability (A) and progression-free survival (PFS) probability (B) over time in patients with metastatic melanoma treated with immune checkpoint inhibitors. The grey shading reflects the 95% confidence interval. Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI 47 FIGURE 2. Swimmer plot shows individual patient’s treatment progression in each horizontal line. Colourful bars and lines indicate type and duration of treatment, while dots indicate specific action - 18F-FDG-PET/CT imaging or reason for end-of-study (if applicable). Vertical dashed lines indicate a time when 18F-FDG-PET/CT scan should be performed for patients in this study. Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI48 reached [NR]) and 8.1 months (95% CI = 4.3–26.3 months), respectively (Figure 1). Among the whole group, 39 (55%) patients died, and 44 (62%) patients progressed to immunotherapy before the cut-off date. On Figure 2, the swimmer plot displays data for individual patients, where each horizontal line or bar shows type and duration of treatment, and each point represents either 18F-FDG-PET/CT or end-of-study reason (if applicable). W4 18F-FDG PET/CT and survival outcomes Among the 68 (96%) patients who underwent W4 18F-FDG PET/CT, 51 (72%) patients were classified into the CB group, and 17 (24%) were classified into the no-CB group. The median OS was not reached (NR) (95% CI = 17.8 months - NR) in the CB group and was 6.2 months (95% CI = 4.6 months - NR) in the no-CB group (Figure 3). In univariate Cox analysis classification was statistically significant- ly correlated to OS with hazard ratio (HR) of 0.4 (95% CI = 0.18–0.72; p = 0.004). Among the 17 patients with PMD, who were classified into the no-CB group, 7 (42%) died be- fore the W16 18F-FDG PET/CT scan. Three (18%) pa- tients had PMD on W16 18F-FDG PET/CT, 6 (35%) had PMR, and one (6%) patient had CMR. Two (12%) patients with PMD on W4 18F-FDG PET/CT changed systemic therapy from ICI therapy to tar- geted therapy because of clinical and radiological signs of rapid progression affecting vital organs. Three patients were classified as CMR on W4 18F-FDG PET/CT. One patient achieved a durable response with CMR, one patient experienced fatal grade 5 immune-related encephalitis during treat- ment and one experienced local progression in soft tissues 25 months after CMR imaging on W4 18F-FDG PET/CT. The site of progression was ame- nable for local treatment with radiotherapy, and a complete metabolic response was achieved. In Figure 4, the alluvial plot shows the respons- es on 18F-FDG PET/CT scans for each patient at week 4, 16, 48 and 96. Other biomarkers and survival outcomes LDH and the number of organs with metastatic involvement Twenty-three (32%) patients had elevated serum LDH at ICI initiation. The median OS of patients with normal LDH levels was NR (95% CI = 17.8 months - NR), and that of patients with elevated TABLE 2. Clinical diagnosis of immune-related adverse events Immune-related adverse event Any grade No (%) Grade 3-5 No (%) Time to onset of irAE (mean ± SD) [weeks] No. of pts with at least one irAE 56 (79) 13 (18) - Number of all irAE events 144 14 144 ± 161 Gastrointestinal Diarrhea 14 (20) 2 (3) 31.7 ± 30.6 Colitis 7 (10) 2 (3) 39 ± 32 Xerostomia 2 (3) 0 30.1 ± 4.4 Gastritis 2 (3) 0 64.4 ± 7.7 Stomatitis 1 (1) 0 3 ± 0 Respiratory Pneumonitis 5 (7) 0 (0) 40.9 ± 45.7 Sarcoid reaction 2 (3) 0 (0) 6.3 ± 0.3 Hepatic Increased AST/ALT 16 (23) 4 (6) 7.7 ± 8.3 Endocrine Hypothyroidism 10 (14) 0 12.3 ± 6 Hyperthyroidism 7 (10) 0 7.3 ± 7.1 Adrenal insufficiency 2 (3) 2 (3) 34.4 ± 20.1 Diabetes mellitus 1 (1) 1 (1) 72.7 ± 0 Pancreatitis 1 (1) 1 (1) 32.4 ± 0 Cutaneous Pruritus 23 (32) 0 10.7 ± 10 Skin rash 16 (23) 0 14 ± 16.3 Vitiligo 9 (13) 0 37.4 ± 31.6 Poliosis of hair 1 (1) 0 34.7 ± 0 Musculoskeletal Arthritis 10 (14) 0 28.7 ± 27.3 Myalgia 2 (3) 0 16.7 ± 12.3 Arthralgia 1 (1) 0 14 ± 0 Synovitis 1 (1) 0 65.4 ± 0 Neurological Encephalitis 2 (3) 1 (1) 36.4 ± 32.9 Psychosis 1 (1) 0 25.4 ± 0 Other Fatigue 6 (8) 0 6.5 ± 4.9 Hypophosphatemia 1 (1) 0 17 ± 0 AST/ALT = aspartate transaminase / alanín aminotransferaza; irAE = immune-related adverse events Survival outcomes The Kaplan–Meier estimated OS and progression- free survival (PFS) for the whole patient group were 18.5 months (95% CI = 14.4 months – not Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI 49 LDH levels was 6.5 months (95% CI = 4.0 months - NR). The difference in OS was statistically sig- nificant between these two groups (p = 0.004) (Figure 5A); Univariate Cox analysis showed a HR = 0.4 (95% CI = 0.21–0.76; p = 0.005). The difference in OS based on the number of or- gans with metastatic involvement was not statisti- cally significant (p = 0.094) (Figure 5B). Immune-related adverse events (irAEs) Among the 71 included patients, 56 (79%) devel- oped irAEs, including 13 (18%) with grade 3 or higher irAEs. All irAE, their number and time to onset, are presented in Table 2. One (2%) patient died of immune-related encephalitis. Due to irAEs, 7 (10%) patients were hospitalized. Three (4%) pa- tients were diagnosed with autoimmune disease prior to ICI initiation: one had vitiligo, one had scalp psoriasis, and one was diagnosed with rheu- matoid arthritis at the time of ICI initiation. None of them experienced an exacerbation of their au- toimmune disease or needed special treatment for that reason. The median OS was 25.3 months (95% CI = 17 months - NR) in patients with irAEs and 4.6 months (95% CI = 3.7 months - NR) in patients without irAEs (p = 0.004) (Figure 6A). Univariate Cox analysis showed a HR 0.9 (95% CI = 0.18–0.75; p = 0.006). The median OS was not reached (95% CI = 23.7 months - NR) in patients who experi- enced cutaneous irAEs and was 8.2 months (95% CI = 4.6–17.8) in patients without cutaneous irAEs (p < 0.0001) (Figure 6C); Cox analysis showed a HR 0.36 (95% CI = 0.19–0.66; p = 0.001). Using Kaplan- Meier analysis, a significant statistical difference in OS was not observed between patients with and without higher-grade irAEs (p = 0.783) (Figure 6B), endocrine irAEs (p = 0.7) (Figure 6D) or immune- related thyroiditis (p = 0.711) (Supplementary Figure 1). Discussion The evaluation of mM pts with early 18F-FDG-PET/ CT at W4, when treated with ICIs, can serve as a survival imaging biomarker (IBM). Based on our results, patients with no-CB at W4 had a shorter survival compared with the CB group (p = 0.001). This was also observed in the study by Cho et al., where early 18F-FDG-PET/CT scans of 20 patients 21–28 days after treatment started showed a pre- dictive role for response. Unlike in our group, pa- FIGURE 3. Kaplan-Meier curves showing probability of median overall survival (OS) between clinical benefit (CB) and no-CB group as classified by findings on week four (W4) 18F-FDG PET/CT. The curves are statistically significantly different (p = 0.03). FIGURE 4. Alluvial plot illustrates the flow of patients between different response categories on 18F-FDG PET/CT scan across four evaluation time points: at week 4 (W4), week 16 (W16), week 48 (W48) and week 96 (W96). CMR = complete metabolic response; HGR = heterogeneous response; PMD = progressive metabolic disease; PPD = pseudoprogressive disease; SMD = stable metabolic disease; PMR = partial metabolic response; n = number of patients tients in their study were mostly treated with the CTLA-4 inhibitor ipilimumab and tumor response was assessed according to RECIST, immune-related response criteria, PERCIST and EORTC criteria.12 In another study by Anderson et al., 18F-FDG-PET/ CT was performed after a single dose of pembroli- zumab, at a median of 7 days (range: 3–21 days) after the start of treatment. They concluded that early scan could identify metabolic changes in me- tastases that are potentially predictive of response to ICIs.16 Additionally, in recent studies on neoad- Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI50 FIGURE 5. Kaplan-Meier curves show the median overall survival (OS) probability of patients with metastatic melanoma treated with immune checkpoint inhibitors according to (A) LDH level and (B) the number of organs with metastatic involvement. The shading reflects the 95% confidence interval. A B FIGURE 6. Kaplan-Meyer curves of the median overall survival (OS) over time in patients with metastatic melanoma treated with immune checkpoint inhibitors according to (A) occurrence of immune-related adverse events, (B) occurrence of serious immune-related adverse events, (C) cutaneous immune-related side effects, (D) immune-related endocrine immune-related side effects. The blue and pink shading reflects the 95% confidence intervals for respecting groups. A B C D Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI 51 juvant immunotherapy in melanoma patients, re- sponses were reported as early as two weeks after ICI initiation, and pathological responses were reported at 4-6 weeks after treatment start.17,18 The use of this strategy with early evaluation is not yet fully understood, but it may lead to new imaging evaluation strategies for patients undergoing im- munotherapy. Seven (10%) patients in our study who were clas- sified as PMD on W4 18F-FDG-PET/CT showed a later response, as seen on subsequent 18F-FDG PET/ CT scans (Figure 4). For this subgroup of patients, FIGURE 7. A 67-year-old male patient, diagnosed with metastatic NRAS-mutated cutaneous melanoma with lung metastases in January 2021, was treated with pembrolizumab in the first-line setting. Serial 18F-FDG PET/CT scans were obtained per the study protocol. The images above show the maximal intensity projection (MIP) on the baseline PET/CT (left), on the week 4 evaluation PET/CT (middle) and at the endpoint of the study (right). The images below show transverse sections of the lungs in different planes, revealing three FDG-avid metastatic nodules in the right lung (lower left images, red arrows), only small nodules with no FDG uptake on week 4 PET/CT (lower middle images, yellow arrows), a complete metabolic response, and no residual nodules found at the end point of the PET/CT images with persistent complete remission (lower right images). Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI52 early cessation of ICI therapy has a detrimental ef- fect. More analysis is needed to identify optimal early timepoint scans and to find more specific biomarkers, perhaps using artificial intelligence with automated deep learning-based lesion seg- mentation, to distinguish patients and lesions that are in progress from those patients who are just late responders to ICIs.19,20 18F-FDG-PET/CT seems to play an important role not only at the early beginning of treatment with ICIs at W4 but also later during treatment. In a retrospective analysis of 104 patients with base- line and 1-year CT and PET/CT scans, Dimitriou et al. reported that almost all patients with CMR at one year had an ongoing response to ICIs thereaf- ter.8 In our cohort of patients, most of the patients with CMR at week 42 remained in remission, as shown in the alluvial plot in Figure 4. The case presentation in Figure 5 shows a patient with early and long-lasting CMR. In our cohort, occurrence of cutaneous irAEs was clearly associated with longer survival. Cutaneous irAEs, especially vitiligo, are more common in patients with melanoma than in other cancers. The higher frequency can be explained by shared immunogenic antigens between healthy tissue and tumors.21,22 Cutaneous irAEs are usual- ly lower grade and vigorous immunosuppressive management is not necessary; therefore, there is no unfavorable effect of irAE management on ICI efficacy or survival.22 Contrary to the results from a large retrospective multicohort study6, our study proved no survival benefit for patients with irThy- roiditis or endocrine irAEs, possibly due to the low number of patients with this type of adverse event. Further studies will clarify the prognostic role of this type of irAE. Our study is limited by not performing analy- sis of circulating tumor DNA in plasma, not avail- able at Institute of Oncology Ljubljana when the study started, and not using other volumetric PET parameters, like metabolic tumor volume or total lesion glycolysis.16,23,24 Another limitation of this study is that we did not perform lesion-level response analysis, which would provide even better insights into lesion-level and patient-level response patterns. Regarding response criteria to ICIs, a wide range of criteria have been proposed and compared in recent years: PERCIST, PECRIT, PERCIMT iPERCIST and imPERCIST5.7,24-26 As fur- ther evaluation of these newly proposed criteria is still warranted, our decision was to use standard EORTC criteria, adapted based on recommenda- tions from the EANM/SNMMI/ANZSNM. Whole-body PET imaging has great potential for future work, especially the use of artificial in- telligence.27 In line with this, our future work will include segmentation of all disease with lesion- by-lesion analysis on W4 and later 18F-FDG-PET/ CT images in our cohort of patients. With more in- depth analysis, we hope to identify specific lesions that do not respond to treatment early in the start of the treatment and offer our patients more per- sonalized treatment. Larger, possibly multicenter studies using same steps in analysis are needed to develop new biomarkers, including imaging bio- markers, to guide patient and treatment selection.28 Conclusions The evaluation of mM patients with early 18F-FDG- PET/CT at W4 who were treated with ICIs revealed a strong prognostic IBM. To obtain more informa- tion from early 18F-FDG-PET/CT, artificial intelli- gence will likely play an important role. Acknowledgments The research was financially supported by The Slovenian Research Agency (ARIS), grant numbers P3-0321 and P1-0389. The manuscript was edited by AJE Digital/ Curie. References 1. Wolchok JD, Chiarion-Sileni V, Rutkowski P, Cowey CL, Schadendorf D, Wagstaff J, et al; CheckMate 067 Investigators. Final, 10-Year outcomes with nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 2024; 392: 11-22. doi: 10.1056/NEJMoa2407417 2. Long GV, Carlino MS, McNeil C, Ribas A, Gaudy-Marqueste C, Schachter J, et al. Pembrolizumab versus ipilimumab for advanced melanoma: 10-year follow-up of the phase III KEYNOTE-006 study. Ann Oncol 2024; 35: 1191-9. doi: 10.1016/j.annonc.2024.08.2330 3. Pires da Silva I, Ahmed T, McQuade JL, Nebhan CA, Park JJ, Versluis JM, et al. Clinical models to define response and survival with anti-PD-1 antibodies alone or combined with ipilimumab in metastatic melanoma. J Clin Oncol 2022; 40: 1068-80. doi: 10.1200/JCO.21.01701 4. Schadendorf D, Livingstone E, Zimmer L. Treatment in metastatic mela- noma-time to rethink. Ann Oncol 2019; 30: 501-3. doi: 10.1093/annonc/ mdz050 5. Hribernik N, Boc M, Ocvirk J, Knez-Arbeiter J, Mesti T, Ignjatovic M, et al. Retrospective analysis of treatment-naive Slovenian patients with metastat- ic melanoma treated with pembrolizumab - real-world experience. Radiol Oncol 2020; 54: 119-27. doi: 10.2478/raon-2020-0003. 6. Wan G, Chen W, Khattab S, Roster K, Nguyen N, Yan B, et al. Multiorgan immune-related adverse events from immune checkpoint inhibitors and their downstream implications: a retrospective multicohort study. Lancet Oncol 2024; 25: 1053-69. doi: 10.1016/S1470-2045(24)00278-X Radiol Oncol 2025; 59(1): 43-53. Hribernik N et al. / 18F-FDG-PET/CT and other prognostic biomarkers in melanoma patients on ICI 53 7. Lopci E, Hicks RJ, Dimitrakopoulou-Strauss A, Dercle L, Iravani A, Seban RD, et al. Joint EANM/SNMMI/ANZSNM practice guidelines/procedure standards on recommended use of [18F] FDG PET/CT imaging during im- munomodulatory treatments in patients with solid tumors version 1.0. Eur J Nucl Med Mol Imaging 2022; 49: 2323-41. doi: 10.1007/s00259-022- 05780-2 8. Dimitriou F, Lo SN, Tan AC, Emmett L, Kapoor R, Carlino MS, et al. FDG-PET to predict long-term outcome from anti-PD-1 therapy in metastatic mela- noma. Ann Oncol 2022; 33: 99-106. doi: 10.1016/j.annonc.2021.10.003 9. Santo G, Cucè M, Restuccia A, Del Giudice T, Tassone P, Cicone F, et al. Immune-related [18F]FDG PET findings in patients undergoing checkpoint inhibitors treatment: correlation with clinical adverse events and prognostic implications. Cancer Imaging 2024; 24: 125. doi: 10.1186/s40644-024- 00774-9 10. Hribernik N, Strasek K, Huff DT, Studen A, Zevnik K, Skalic K, et al. Role of quantitative imaging biomarkers in an early FDG-PET/CT for detection of immune-related adverse events in melanoma patients: a prospective study. Radiol Oncol 2024; 58: 335-47. doi: 10.2478/raon-2024-0045 11. Hribernik N, Huff DT, Studen A, Zevnik K, Klaneček Ž, Emamekhoo H, et al. Quantitative imaging biomarkers of immune-related adverse events in immune-checkpoint blockade-treated metastatic melanoma patients: a pilot study. Eur J Nucl Med Mol Imaging 2022; 49: 1857-69. doi: 10.1007/ s00259-021-05650-3 12. Cho SY, Lipson EJ, Im HJ, Rowe SP, Gonzalez EM, Blackford A, et al. Prediction of response to immune checkpoint inhibitor therapy using early-time-point 18F-FDG PET/CT imaging in patients with advanced melanoma. J Nucl Med 2017; 58: 1421-8. doi: 10.2967/jnumed.116.188839 13. National Cancer Institute (NCI). NCI Common Terminology Criteria for Adverse Events (CTCAE). Version 5.0.2021. [cited 2023 Oct 23]. Available at: https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ docs/ctcae_v5_quick_reference_8.5x11.pdf 14. Nelson EK, Piehler B, Eckels J, Rauch A, Bellew M, Hussey P, et al. LabKey Server: an open source platform for scientific data integration, analysis and collaboration. BMC Bioinformatics 2011; 12: 71. doi: 10.1186/1471- 2105-12-71 15. Young H, Baum R, Cremerius U, Herholz K, Hoekstra O, Lammertsma AA, et al. Measurement of clinical and subclinical tumor response using [18F]-fluorodeoxyglucose and positron emission tomography: review and 1999 EORTC recommendations. European Organization for Research and Treatment of Cancer (EORTC) PET Study Group. Eur J Cancer 1999; 35: 1773- 82. doi: 10.1016/s0959-8049(99)00229-4 16. Anderson TM, Chang BH, Huang AC, Xu X, Yoon D, Shang CG, et al. FDG PET/ CT imaging 1 week after a single dose of pembrolizumab predicts treatment response in patients with advanced melanoma. Clin Cancer Res 2024; 30: 1758-67. doi: 10.1158/1078-0432.CCR-23-2390 17. Reijers ILM, Menzies AM, van Akkooi ACJ, Versluis JM, van den Heuvel NMJ, Saw RPM, et al. Personalized response-directed surgery and adjuvant therapy after neoadjuvant ipilimumab and nivolumab in high-risk stage III melanoma: the PRADO trial. Nat Med 2022; 28: 1178-88. doi: 10.1038/ s41591-022-01851-x 18. Blank CU, Lucas MW, Scolyer RA, van de Wiel BA, Menzies AM, Lopez-Yurda M, et al. Neoadjuvant nivolumab and ipilimumab in resectable stage III mel- anoma. N Engl J Med 2024; 391: 1696-708. doi: 10.1056/NEJMoa2402604 19. Taghanaki SA, Zheng Y, Kevin Zhou S, Georgescu B, Sharma P, Xu D, et al. Combo loss: handling input and output imbalance in multiorgan segmenta- tion. Comput Med Imaging Graph 2019; 75: 24-33. doi: 10.1016/j.comp- medimag.2019.04.005 20. Huff DT, Ferjancic P, Namías M, Emamekhoo H, Perlman SB, Jeraj R. Image intensity histograms as imaging biomarkers: application to immune-related colitis. Biomed Phys Eng Express 2021; 7: 065019. doi: 10.1088/2057-1976/ ac27c3 21. Suijkerbuijk KPM, van Eijs MJM, van Wijk F, Eggermont AMM. Clinical and translational attributes of immune-related adverse events. Nat Cancer 2024; 5: 557-71. doi: 10.1038/s43018-024-00730-3 22. Verheijden RJ, van Eijs MJM, May AM, van Wijk F, Suijkerbuijk KPM. Immunosuppression for immune-related adverse events during checkpoint inhibition: an intricate balance. NPJ Precis Oncol 2023; 7: 41. doi: 10.1038/ s41698-023-00380-1 23. Wong A, Callahan J, Keyaerts M, Neyns B, Mangana J, Aberle S, et al. 18F-FDG PET/CT based spleen to liver ratio associates with clinical outcome to ipili- mumab in patients with metastatic melanoma. Cancer Imaging 2020; 20: 36. doi: 10.1186/s40644-020-00313-2 24. Ayati N, Sadeghi R, Kiamanesh Z, Lee ST, Zakavi SR, Scott AM. The value of 18F-FDG PET/CT for predicting or monitoring immunotherapy response in patients with metastatic melanoma: a systematic review and meta-analysis. Eur J Nucl Med Mol Imaging 2021; 48: 428-48. doi: 10.1007/s00259-020- 04967-9 25. Kitajima K, Watabe T, Nakajo M, Ishibashi M, Daisaki H, Soeda F, et al. Tumor response evaluation in patients with malignant melanoma undergo- ing immune checkpoint inhibitor therapy and prognosis prediction using 18F-FDG PET/CT: multicenter study for comparison of EORTC, PERCIST, and imPERCIST. Jpn J Radiol 2022; 40: 75-85. doi: 10.1007/s11604-021-01174-w 26. Iravani A, Hicks RJ. Imaging the cancer immune environment and its re- sponse to pharmacologic intervention, Part 1: The role of 18F-FDG PET/CT. J Nucl Med 2020; 61: 943-50. doi: 10.2967/jnumed.119.234278 27. Shreve JT, Khanani SA, Haddad TC. Artificial intelligence in oncology: current capabilities, future opportunities, and ethical considerations. Am Soc Clin Oncol Educ Book 2022; 42: 1-10. doi: 10.1200/EDBK_350652 28. de Groot DJA, Lub-de Hooge MN, van Meerten T, Brouwers AH, de Vries EGE. Facts and hopes for PET imaging-derived immunotherapy biomarkers. Clin Cancer Res 2024; 30: 5252-9. doi: 10.1158/1078-0432.CCR-24-1427