The idea to provoke an antitumor immunity by modifying the patients’ immune system has been proposed over 100 years ago.1 Today, immunotherapy has become an essential part of treatment regimens for several oncologic diseases.2–5 Checkpoint inhibitor therapy (CIT), like humanized monoclonal antibodies against the programmed-death-1 receptor or against the cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), have proven to potentially improve outcome of patients with melanoma in the long term.6 7 In brief, their mechanism of biological action is based on provoking an antitumor immune response.8 Besides tremendous therapy success in individual patients, CIT comes together with high costs and dangerous side effects while overall only 20%–40% of patients with melanoma benefit from CIT.9–12 Moreover, some non-responders even show an acceleration of tumor growth rate ≥2 under CIT which is defined as hyperprogression.13 Compared with CIT, hybrid imaging is far less expensive and comes with fewer side effects. By the early identification of responders to CIT, hybrid imaging might help to avoid ineffective or even harmful treatment regimes over a time period of usually 3 months. Early response evaluation by cross-sectional imaging (CT or MRI) or [¹⁸F]-labeled fluorodeoxyglucose-positron emission tomography (¹⁸F-FDG-PET) focusing on tumor assessment remains challenging as the mechanism of action of CIT differs from conventional chemotherapy.14–16 Blood immune biomarkers may potentially predict treatment outcome, since antitumor response requires an effective systemic immunity.17 From those biomarkers, the relative eosinophil count (REC), relative lymphocyte count (RLC) and the neutrophile-lymphocyte ratio (NLR) have been evaluated in larger melanoma patient cohorts.18–21

¹⁸F-FDG-PET is a non-invasive and quantitative imaging biomarker for glucose consumption, covering the whole body. In a recently published translational study, the potential of ¹⁸F-FDG-PET to evaluate primary (bone marrow) and secondary (spleen) lymphoid organs could be demonstrated by our group.22 While in the preclinical experiments fewer T cells and a higher number of neutrophils in the spleen led to a significantly higher ¹⁸F-FDG uptake in CIT-treated mice, the observed changes in the clinical data were small, and the examination time point varied due to the retrospective study setting. Preclinical studies in mice indicated that an antitumor immune response can be observed early after therapy start using the PET tracer (1-(2-deoxy-2-[¹⁸F]fluoroarabinofuranosyl) cytosine, which is targeting the deoxyribonucleotide salvage pathway.23 Furthermore, we were able to show in clinical PET data that a complete response to CIT in patients with melanoma can be observed already after the first cycle of therapy.16 Besides morphological data, MRI provides additional functional imaging techniques such as diffusion-weighted imaging (DWI), which monitors the diffusivity of water molecules, playing a central role in quantitative oncologic MRI.24

In this study, we aimed to evaluate whether early changes of lymphoid organs 14 days after initiation of CIT by multiparametric ¹⁸F-FDG-PET/MRI might be possible predictors of treatment response. Furthermore, correlation to baseline blood immune biomarkers was performed.

An overview of the finally included 17 patients, the results of the treatment response assessment and the baseline biomarkers are shown in table 1.

After 3 months of treatment, 9/17 patients were categorized as non-responders (PMD/iPD). Examples of metabolic and morphological treatment response are shown in figure 3.

In this study of multiparametric evaluation of lymphoid organs, we found that a systemic immune response in patients responding to CIT can be observed already 14 days after treatment initiation by ¹⁸F-FDG-PET, appearing as increased metabolic activity in the spleen. Changes of the volume of the spleen and the ¹⁸F-FDG-uptake of bone marrow showed the same tendency, but less pronounced. The ADC map did not add a noticeable value to early treatment response evaluation in our patient cohort.

The prediction of treatment response to CIT especially a very early response assessment is of paramount interest for oncologic treatment regimes in several fields. The biological mechanism of action makes the response evaluation of solid tumors to immunotherapy a challenging task for imaging modalities focusing on changes in metastatic lesions. In this connection, the role of ¹⁸F-FDG-PET including the evaluation of different response criteria has been investigated in several studies with small patient cohorts and equivocal results: Kaira et al 27 found PERCIST criteria to be more predictive in patients with NSCLC treated with immunotherapy as compared with RECIST 1.1.28 Cho et al 29 proposed a combination of anatomical and functional imaging parameters as a possible future approach. Anwar et al 30 even proposed new criteria for the response evaluation to CIT in PET/CT (PERCIMT). In our study, metabolic (PERCIST) and morphological (iRECIST) response evaluation was overall in good agreement. A significant difference was only found for patient no. 2 who showed a response to CIT which was rated as PMD with PERCIST and iSD with iRECIST at t₂. This patient was rated as ‘responder’ according to the decision of the interdisciplinary tumor board 3 months after treatment initiation. In patients no. 3, 14 and 15, PERCIST showed a clear trend of treatment (non-)response already at t₁ while iRECIST was stable (example is given in figure 3). In patients no. 7 and 14, remnants of metastases without specific tracer uptake were seen at t₂ (CMR vs iPR). Previous studies could observe changes in the ¹⁸F-FDG-uptake of the spleen 3 months or later after treatment initiation in patients responding to CIT.31 32 Moreover, a recently published report on the immunotherapy symposium held on the annual congress of the European Association of Nuclear Medicine takes the spleen for response assessment of CIT in PET into consideration.33 Nevertheless, the reported changes in FDG-uptake were small, the evaluation time points vary and although it may appear likely, the link between the metabolic activity of immune organs and an immune response has not been proven yet. The recently published translational study by Schwenck et al 22 and the correlation of the metabolic activity early after treatment initiation to baseline immune biomarkers observed in our study support the hypothesis, that ¹⁸F-FDG-PET is a reliable tool to monitor an effective systemic immune response in patients treated with CIT. A further important observation from this longitudinal patient study is that the systemic immune response seems a highly dynamic process, also in ¹⁸F-FDG-PET. This may explain the differing results to our previous retrospective study with examination time points between 50 days before and 125 days after treatment initiation.22 Invasive biopsies of the spleen or bone marrow under therapy are ethically not feasible and a detailed explanation of the observations in lymphoid organs regarding changes in cellular components of the immune system is beyond the scope of our work. In this connection, anti-CD8 immuno-PET might help gain a deeper understanding of the process of immune response under CIT in the future.34 In 8/17 patients, no clear trend could be observed at t₁ with PERCIST criteria (SMD) alone. In patients thereof finally responding to therapy, the ¹⁸F-FDG-uptake of the spleen as well as the relative change of ¹⁸F-FDG-uptake at t₁ was considerably higher compared with the patients finally not responding to CIT (responder/non-responder with SMD at t₁: SULmean 1.25/0.90; +62%/−6%). Especially in this group of patients, the supportive role of spleen evaluation might be of value and enable a early response assessment with hybrid imaging. Nevertheless, there is an overlap of the SULmean values and the relative changes of metabolic activity in the spleen t₁-t₀ between the responder and non-responder group, more pronounced in the SULmean values (online supplementary figure S1). Although the patient cohort is small, an increase of +25% and a SULmean >1 after 2 weeks of CIT might be reasonable first approaches for cut-off-values. Focusing on the group of patients treated with PD1 antibodies only, we also found a significant difference between the responder and the non-responder group regarding the change t₀-t₁ of ¹⁸F-FDG-uptake in the spleen. The predictive value of baseline blood immune biomarkers was confirmed in our study with significant differences between the two groups. Nonetheless, they have limitations and although there is a correlation between the metabolic activity of the spleen early after treatment initiation and baseline immune biomarkers, they do not necessarily mean the same thing: patient no. 12 shows a baseline NLR of >4 and a RLC of <17.5% but a complete response to CIT after 3 months; the relative differences in ¹⁸FDG-uptake t₁-t₀ were as follows: spleen, +25%; bone marrow, +107%. Patient no. 8 shows a baseline NLR <4, a RLC >17.5% and REC >1.5% but progressive disease after 3 months; the relative differences in ¹⁸F-FDG-uptake t₁-t₀ were as follows: spleen, −20%; bone marrow, −3%. Therefore, baseline immune biomarkers indicating a good treatment outcome do not necessarily translate in an increased ¹⁸F-FDG-uptake of lymphoid organs early after treatment initiation and good treatment response and vice versa. Maybe blood immune biomarkers indicate a general immune competence of a patient, but the increase of ¹⁸F-FDG-uptake in lymphoid organs confirms the actual process of CIT-induced immune response. Therefore, the relative changes of lymphoid organs in ¹⁸F-FDG-PET might add an independent value to response evaluation in CIT. However, the results of our study do not allow a general statement regarding the predictive capacity of early changes in lymphoid organs as compared with baseline immune biomarkers. As anatomic CT images today represent the standard of care for therapy monitoring, we also evaluated the volume of the spleen to investigate if this might be of help for CIT response assessment. We observed an increase of the spleen volume under therapy in most of our patients, especially in the first follow-up scan 2 weeks after therapy start. This peak was overall more pronounced in patients responding to CIT, however, the difference between the two groups did not reach significance, probably due to the small patient cohort. In patients with iSD at t₁, the increase of spleen volume t₁-t₀ was higher in patients finally responding to CIT as compared with non-responders (30% vs 11%), however, less pronounced as the changes observed in ¹⁸F-FDG-PET. Besides anatomic images, MRI provides several functional imaging parameters and nowadays, DWI plays a pivotal role in oncologic whole-body imaging.24 For the evaluation of a systemic immune response, the role of ADC has not been evaluated previously. Based on our results, ADC values of the spleen do not seem to be of value for an early response assessment, but there was a trend towards higher ADC values of the spleen in the responder group at t₂.The ADC map in our study was calculated based on to b values: b=50 and 800 s/mm². This protocol is aimed for oncologic whole-body DWI. The spleen is a highly perfused organ and with a low b value of 50 s/mm² and two acquired b values, the ADC is influenced by perfusion effects. Although there might also be true changes in the diffusivity of the spleen (eg, caused by changes in the cellularity), a main part of the increase of ADC might be caused by changes in the perfusion. In order to eliminate this effect, the acquisition of more and higher b values is necessary which is associated with longer acquisition times. We evaluated the changes in metabolic activity in the spleen and the bone marrow (and metastases) using the SULmean and did not compute a ratio to the liver as it is one of the most commonly affected organs in metastasized melanoma and might also play a role in the systemic immune response.35 Besides the spleen and the bone marrow, draining and distant lymph nodes also play a significant role in the immune system and changes under CIT were reported in the study by Tsai et al.32 An example from our patient cohort of an intensive increase of tracer uptake in the hilar lymph nodes at t₁ is shown in online supplementary figure S2, known as sarcoid-like reaction.36 Although this is likely to also represent a CIT-induced systemic immune response, we did not systematically evaluate lymph nodes in this work for the following reasons: first, we cannot differentiate by imaging if an increase of tracer uptake in (draining) lymph nodes is caused by a metastatic involvement or a response to CIT. A systematic evaluation using fine needle aspirations is ethically not feasible. On the contrary, metastases in the spleen and the bone can be excluded by multiparametric PET/MRI. Second, healthy lymph nodes are (without immune activation) small and do not show a relevant tracer uptake; changes in size, ADC value or tracer accumulations are therefore susceptible to the methodology used. Future oncologic hybrid imaging will ask for quantitative and robust imaging biomarkers, which can be added to clinical routine image evaluation. By segmenting the spleen and the spine based on anatomical images, we want to propose a reproducible approach for the evaluation of primary and secondary lymphoid organs in hybrid imaging which can easily be applied. Against this background, a visual and non-quantitative evaluation of lymph nodes might not to be robust enough. The major limitation of our study is the relatively low number of patients, however, the special values of the study are the prospective design and the strict inclusion criteria. The included patients were treated solely with CIT and the lymphoid organs were monitored in a complex whole-body PET/MRI setting which is unique in the literature so far. Although the statistical power is therefore limited, the findings of our study strongly support the role of the evaluation of lymphoid organs in hybrid imaging for treatment assessment. There was a high number of patients who quit the study (figure 1), mostly due to long examination time of 1.5 hours and the resulting limited patient comfort.37 Shorter examination times can be achieved by reducing the number of MRI sequences and the use of accelerated imaging techniques38 in the future. A large proportion of non-responders can be expected in patients who have abandoned the study or who have had changes in therapy during the 3 months; this also explains the balanced proportion of responders and non-responders in the finally evaluated patient cohort.

In conclusion, our findings indicate that an effective systemic immune response in patients undergoing CIT can be observed in the spleen in ¹⁸F-FDG-PET, especially early after treatment initiation. Spleen volume and metabolic activity of bone marrow seem also be valuable in this regard but need to be confirmed in studies with larger patient cohorts in the future. These findings might help to support the evaluation of treatment response by hybrid imaging in the future.