Mapping heterogeneity in glucose uptake in metastatic melanoma using quantitative 18F-FDG PET/CT analysis

Background Metastatic melanoma patients can have durable responses to systemic therapy and even long-term survival. However, a large subgroup of patients does not benefit. Tumour metabolic alterations may well be involved in the efficacy of both targeted and immunotherapy. Knowledge on in vivo tumour glucose uptake and its heterogeneity in metastatic melanoma may aid in upfront patient selection for novel (concomitant) metabolically targeted therapies. The aim of this retrospective study was to provide insight into quantitative 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) parameters and corresponding intra- and inter-patient heterogeneity in tumour 18F-FDG uptake among metastatic melanoma patients. Consecutive, newly diagnosed stage IV melanoma patients with a baseline 18F-FDG PET/CT scan performed between May 2014 and December 2015 and scheduled to start first-line systemic treatment were included. Volume of interests (VOIs) of all visible tumour lesions were delineated using a gradient-based contour method, and standardized uptake values (SUVs), metabolically active tumour volume (MATV) and total lesion glycolysis (TLG) were determined on a per-lesion and per-patient basis. Differences in quantitative PET parameters were explored between patient categories stratified by BRAFV600 and RAS mutational status, baseline serum lactate dehydrogenase (LDH) levels and tumour programmed death-ligand 1 (PD-L1) expression. Results In 64 patients, 1143 lesions ≥ 1 ml were delineated. Median number of lesions ≥ 1 ml was 6 (range 0–168), median maximum SUVpeak 9.5 (range 0–58), median total MATV 29 ml (range 0–2212) and median total TLG 209 (range 0–16,740). Per-patient analysis revealed considerable intra- and inter-patient heterogeneity. Maximum SUVs, MATV, number of lesions and TLG per patient did not differ when stratifying between BRAFV600 or RAS mutational status or PD-L1 expression status, but were higher in the patient group with elevated LDH levels (> 250 U/l) compared to the group with normal LDH levels (P < 0.001). A subset of patients with normal LDH levels also showed above median tumour 18F-FDG uptake. Conclusions Baseline tumour 18F-FDG uptake in stage IV melanoma is heterogeneous, independent of mutational status and cannot be fully explained by LDH levels. Further investigation of the prognostic and predictive value of quantitative 18F-FDG PET parameters is of interest. Electronic supplementary material The online version of this article (10.1186/s13550-018-0453-x) contains supplementary material, which is available to authorized users.


Background
Novel therapies, especially immunotherapy, have revolutionized the treatment of stage IV metastatic melanoma over the past decade. One-year overall survival (OS) rates have improved to 50-75% and a subset of patients shows durable responses [1]. Still, a considerable number of patients do not respond, especially those with elevated serum lactate dehydrogenase (LDH) levels [1].
The metabolic reprogramming that characterizes cancer cells may well be involved in the efficacy of antitumour immune responses [2,3]. Cancer cells metabolize a substantial amount of the consumed glucose through glycolysis only-even under aerobic conditions-in order to generate sufficient biomass for rapid cellular proliferation [4,5]. Novel therapeutic agents interfering with this altered glucose metabolism have shown hints of anticancer activity in (pre)clinical studies, for example in breast cancer, non-small cell lung cancer and glioblastoma [6,7]. Additionally, preclinical data suggest metabolically targeted therapies can improve antitumour immune response and susceptibility to adjuvant chemo-and radiotherapy [8][9][10]. In patients, however, such treatments can result in toxicity in highly glucose-dependent healthy tissues, such as the kidney [7,11]. Furthermore, recent in vitro studies demonstrate that not all melanomas rely on altered glucose metabolic pathways to the same extent [12,13]. This underlines the need for upfront selection of patients with highly glucose-dependent tumours in order to maximize the benefit of (concomitant) metabolic therapies and ensure a sufficiently broad therapeutic window.
Metastatic melanoma is clinically renowned for its high uptake of the glucose analogue 18 F-fluorodeoxyglucose ( 18 F-FDG) on positron emission tomography/computed tomography (PET/CT) scans. Whole-body 18 F-FDG PET/ CT is therefore part of standard care staging procedures at baseline in stage IV disease, where it is used in a qualitative fashion to provide information on the presence and location of metastases. However, quantitative 18 F-FDG PET/CT scan analysis has been completely unvisited in stage IV melanoma so far and could provide a wealth of knowledge on quantitative tumour glucose uptake in vivo, potentially useful for upfront patient selection for metabolically targeted therapies. The aim of this retrospective study was to provide an overview of tumour 18 F-FDG uptake and corresponding intra-and inter-patient heterogeneity in metastatic melanoma patients using quantitative 18 F-FDG PET/CT scan analysis.

Patients
Patients for this retrospective study were selected from a prospectively maintained database containing all melanoma patients registered at the Department of Medical Oncology of the University Medical Center Groningen (UMCG), the Netherlands, from 2012 onwards. All patients ≥ 18 years of age with histologically proven cutaneous or mucosal metastatic melanoma (American Joint Committee on Cancer [AJCC] 7th edition stage IV melanoma [14]) without prior systemic treatment and with a baseline 18 F-FDG PET/CT scan performed between May 2014 and December 2015 were eligible for inclusion (n = 108). Exclusion criteria were unknown or inadequate adherence to European Association of Nuclear Medicine (EANM) PET/CT scan acquisition guidelines [15] (e.g. PET/CT scan not performed at our hospital) (n = 26), no indication for start of first-line systemic treatment within 2 months of baseline PET/CT scan (n = 10), concurrent malignancy or other malignancy within the previous 10 years (n = 5) and/or no PET-positive lesions (n = 3). Ultimately, 64 patients were included (Additional file 1: Figure S1). The Medical Ethics Committee approved the study. Consultation of the local objection registry verified that none of the selected patients had objected to use of their personal data for research purposes. Patients were pseudonymized, and data were stored on a secured server following local data management regulations. 18 F-FDG PET/CT imaging 18 F-FDG PET/CT scans were acquired using a Siemens Biograph mCT PET/CT system (Siemens/CTI, Knoxville, TN) accredited by the European Association of Nuclear Medicine (EANM) Research Limited (EARL). Scan acquisition and reconstructions were performed following the recommendations of the EANM guideline for oncology 18 F-FDG imaging [15]. Patients were instructed to fast and avoid exercise at least 4-6 h prior to intravenous 18 F-FDG injection at an activity of 3 MBq/kg. Serum glucose levels before tracer injection were < 8.3 mmol/l. Whole-body PET/CT scanning (from the top of the skull to the bottom of the feet) was performed 60 min after 18 F-FDG injection with 1-3 min per bed position. Prior to the PET acquisition, patients underwent a low-dose CT (LD-CT) scan during tidal breathing for attenuation correction (80-140 kVp, quel. ref. 30 mAs and pitch of 1). 18

F-FDG PET/CT scan analysis and volume of interest delineation
All PET/CT scans were initially reported by a nuclear medicine physician as part of routine patient care. Quantitative scan analysis and identification and delineation of all tumour lesions for this study were performed by one investigator (EH) and verified by a board-certified nuclear medicine physician with expertise in melanoma (AB).
PET(/CT) and gradient PET images were displayed side-to-side, and volume of interests (VOIs) were delineated on the gradient PET images using a gradient-based manual contouring method (in-house developed software program). Gradient PET images are derived directly from reconstructed PET images and depict the relative change in counts between neighbouring voxels (Δ standardized uptake value [SUV]), which is typically the highest around tumour borders. Gradient PET images consequently provide an image where the borders of the lesion are most intense. Use of gradient PET data enables a (manual) VOI delineation method where lesion border location is independent of colour scale, in contrast to manual contouring on regular PET images. Additional motives for choosing gradient-based delineation were a lack of systematic delineation studies in metastatic melanoma and inaccuracy of EARL-recommended semiautomatic delineation methods for delineation of large heterogeneous tumour lesions or small yet highly 18 F-FDG-avid lesions [15].
A region of interest (ROI) was manually drawn around each tumour lesion on consecutive transaxial slices. Subsequently, the observer adjusted a %-threshold based on maximum SUV (SUV max ) until the VOI borders optimally corresponded with the location of the steepest gradient on the gradient PET images as judged visually. SUV max , mean SUV (SUV mean ), peak SUV (SUV peak , i.e. a 1.2-cm 3 spheric region positioned to yield the highest average value), metabolically active tumour volume (MATV) and total lesion glycolysis (TLG, the product of SUV mean and MATV) were determined for each VOI. SUVs were corrected for serum glucose level and lean body mass according to the Janmahasatian formula [15].
Lesions with an MATV < 1 ml were excluded from the final quantitative analysis to prevent partial volume effects. PET parameters were analysed on a per-patient, per-location and per-lesion basis. Patient's maximum SUV and median SUV reflect respectively the highest and median value derived from all lesions ≥ 1 ml within that patient. Interquartile range (IQR) SUV peak was derived from the SUV peak s of all individual lesions delineated in one patient as a measure for intra-patient 18 F-FDG uptake heterogeneity. Total MATV or total TLG equals the sum of respectively MATV or TLG of all lesions ≥ 1 ml within that patient.

CT and brain MRI scan analysis
Previously, PET-negative (i.e. with SUV max < 1.5) melanoma metastases have been described, and we excluded three eligible patients upfront due to the presence of only PET-negative lesions [16]. Therefore, we aimed to evaluate the first 20 included patients for the presence of PET-negative lesions with a diameter ≥ 1 cm on baseline contrast-enhanced CT (ce-CT) scan performed within 1 month of the baseline PET/CT. ce-CT scan was available in 12 of the 20 patients and revealed only 2 additional 18 F-FDG PET-negative lesions ≥ 1 cm on top of the total of 491 PET-positive lesions > 1 ml in these patients (0.4%). Due to this limited additional value, ce-CT analysis was omitted for the remaining patients.
High physiological background 18 F-FDG uptake prevents accurate detection and quantification of brain metastases. Therefore, the presence of brain lesions was additionally evaluated on baseline cerebral MRI scans or cerebral ce-CT. Quantitative data from brain lesions were not incorporated in per-patient PET parameters. When brain lesions were measurable (longest axis on MRI > 1 cm according to Response Assessment in Neuro-Oncology Brain Metastases [RANO-BM] criteria [17]) and 18 F-FDG-avid, SUV peak and SUV max were measured.

Data acquisition
Patient and tumour characteristics, baseline serum LDH levels and respectively tumour BRAF and RAS mutation status were retrospectively determined from the electronic patient file. Pre-treatment serum LDH levels were derived from the date closest to the baseline PET/CT scan. When pre-treatment archival tumour biopsies for a distant metastasis were available, PD-L1 immunohistochemistry (IHC) was performed as previously described elsewhere using the 22C3 anti-PD-L1 antibody (DAKO, Merck) on Ventana BenchMark ULTRA platform [18]. Tissue derived from primary melanomas, local recurrences, in-transit cutaneous metastases or lymph node metastases was excluded. Scoring was performed by two board-certified pathologists (GFHD, NAH) and performed according to the manufacturer's instructions.

Statistical analysis
Variables were assessed for normal distribution by Q-Q plots. Independent Mann-Whitney U tests were used to assess differences in PET parameters between LDH, BRAF and RAS groups, respectively, and Kruskal-Wallis tests for differences between metastatic locations and PD-L1 expression groups. Spearman's rank correlation was used for the correlation between lesion MATV and SUV peak . A P value < 0.05 (two-sided) was considered statistically significant. Statistical analysis was performed using SPSS Statistics, version 23.0 (IBM Corp., Armonk, NY).

Overall survival
Following the baseline PET/CT scan, patients commenced standard systemic treatment consisting of either immune checkpoint inhibition, BRAF(/MEK) inhibition and/or dacarbazine chemotherapy. Given the various systemic treatments used, overall survival analysis was performed for exploratory purposes only (Kaplan-Meier overall survival curves in Additional file 5: Figure S4).

Discussion
We show major intra-and inter-patient heterogeneity in tumour lesion 18 F-FDG uptake among metastatic melanoma patients. Presence of tumours with above median 18 F-FDG uptake was independent of tumour mutational status and did not fully coincide with high serum LDH level. This suggests that tumour 18 F-FDG uptake is an independent feature and that 18 F-FDG PET parameters might be suitable as a selection tool for novel metabolic therapies.
This is the first large study providing an overview of intra-and inter-patient differences in tumour glucose consumption in metastatic melanoma patients using quantitative whole-body imaging of 18 F-FDG uptake. Previous melanoma studies on 18 F-FDG PET/CT imaging focused on its diagnostic accuracy for qualitative lesion detection and/or used quantitative parameters derived from the primary melanoma or only a limited number of (the most intense) lesions for response evaluation or prognostic models. By performing quantitative evaluation of all tumour lesions, we highlight the utility of 18 F-FDG PET/CT in demonstrating heterogeneity of glucose uptake among metastatic melanoma patients. Preliminary estimates of the influence of tumour 18 F-FDG uptake on survival support further prospective investigation as a prognostic biomarker.
Compared to previous studies, we found a higher proportion of bone metastases and a lower incidence of lung and soft tissue metastases. Two previous studies in metastatic melanoma using 18 F-FDG PET/CT ± other Fig. 1 Individual tumour lesions (≥ 1 ml) and their SUV peak displayed per patient. For each patient (x-axis; n = 64), individual tumour lesions are plotted against their SUV peak (left y-axis). Grey shaded bars represent the patient's total MATV (right y-axis). The heatmap displays respectively the patient's LDH level and tumour BRAF and NRAS status and PD-L1 expression. Three patients are not displayed since they only had lesions < 1 ml, which resulted in SUVs, a MATV and TLG of 0. LDH lactate dehydrogenase, LN lymph node, PD-L1 programmed death-ligand 1, ULN upper limit of normal imaging methods qualitatively report metastases predominantly to the lung, liver, lymph nodes and skin/soft tissue [19,20]. This difference might be explained by differing patient populations, especially since our study also included patients with an unknown primary melanoma and subsequent widespread (skeletal) metastases (n = 8), as opposed to the study performed by Schoenewolf et al. [19]. Furthermore, we excluded lesions with an MATV < 1 ml to minimize partial volume effects. Three patients had only lesions with an MATV < 1 ml, which all concerned metastases in the lymph nodes, lung, subcutis and/or muscles. The small MATV at these locations, resulting in the exclusion of these lesions for the analysis, further explains the smaller fraction of soft tissue, lymph node and subcutaneous lesions in our PET-based study.
BRAF V600 mutant melanoma cells rely heavily on glycolysis with high glycolytic rates induced by activation of the mitogen-activated protein kinase (MAPK) pathway [21,22]. BRAF V600 wild-type melanomas (approximately 50% of melanomas) often have alternative mutations in the MAPK-pathway including RAS or MEK1/2 that are also associated with glycolytic dependency and increased glucose uptake [23][24][25]. In thyroid carcinoma, BRAF V600E tumours show increased expression of glucose transporter (GLUT) and higher SUVs compared to BRAF V600 wild-type tumours [26,27]. We found no difference in tumour glucose uptake and MATV between patients with and without a BRAF V600 or RAS mutation. Overexpression of other proteins Table 2 [25], and mTOR (10.4% of primary melanomas) or PDK1, involved in the PI3K-Akt-mTOR pathway [28,29]. Furthermore, patient's BRAF status is determined based on one tumour tissue sample, not uncommonly the (excised) primary melanoma, and consequently does not necessarily represent the mutational status of all metastases within a patient [30].
Patients with an elevated serum LDH level-a well-established prognostic biomarker for both worse survival and poor treatment response-had higher tumour 18 F-FDG uptake as well as higher metabolic tumour volume compared to those with normal LDH levels. However, we also observed tumours with high 18 F-FDG in patients with (still) relatively low MATV and normal LDH levels. Moreover, several patients with an elevated LDH level had only tumour lesions with relatively low 18 F-FDG uptake. LDH is a cytoplasmic enzyme that catalyses the interconversion of pyruvate and lactate downstream of glycolysis. A high LDH serum level is generally regarded as a marker of cell damage or necrosis, but the exact source of serum LDH levels is unknown. The biological role of LDH in glucose metabolism has also been suggested as an underlying mechanism and in vitro data suggest differential reliance on aerobic glycolysis and oxidative phosphorylation between patients with normal and elevated serum LDH [3,13]. Unfortunately, meaningful multivariate approaches to unravel the interrelations between tumour volume, tumour glucose consumption and a proposed metabolic factor underlying serum LDH levels were prohibited by collinearity in our data.
Metabolic targeting may constitute a promising novel approach for patients with tumours with high glucose uptake identified by 18 F-FDG PET. Furthermore, glycolysis results in extracellular accumulation of lactate and low pH, which are known to impair immune cell function and contribute to an immunosuppressive tumour microenvironment [3,5]. Metabolic interference combined with immunotherapy might thus be attractive for improving immunotherapy response, for instance in the poorly responding group of metastatic melanoma patients with elevated LDH levels. Metabolic cancer therapies have numerous specific metabolic targets and so far, studies into the correlation between melanoma expression of specific glycolytic transporters and enzymes, such as GLUT1 and hexokinase (HK), and 18 F-FDG uptake are limited and contradictive [31,32]. New studies are needed to integrate tumour 18 F-FDG uptake and other clinical biomarkers with tumour dependence upon specific metabolic pathways and targetable metabolic transporters and enzymes.
Limitations of our study include its retrospective nature and patient heterogeneity in treatment, which allowed only preliminary estimates of the influence of tumour PET parameters on survival. The lack of ce-CT in several patients and its more detailed anatomical lesion information could have resulted in erroneous inclusion of physiological PET-positive lesions or exclusion of malignant PET-positive lesions, respectively. Since tumour measurements were performed on PET images only, necrotic areas (observed in three patients) and brain metastases (n = 22) are not incorporated in the MATV.

Conclusions
Tumour 18 F-FDG uptake is heterogeneous within and among metastatic melanoma patients. High 18 F-FDG uptake is independent of BRAF/RAS mutation status and does not fully correlate with serum LDH levels. This suggests 18 F-FDG PET metabolic parameters could serve as an (additional) selection tool for melanoma patients potentially benefiting from metabolic therapies. Further investigation of the prognostic and predictive value of quantitative 18 F-FDG PET parameters is warranted.

Additional files
Additional file 1: Figure S1. Patient selection. Flow diagram showing the selection of eligible patients. (DOCX 547 kb) Additional file 2: Table S1. 18