Nuclear medicine imaging of multiple myeloma, particularly in the relapsed setting

Multiple myeloma (MM) is characterized by a monoclonal plasma cell population in the bone marrow. Lytic lesions occur in up to 90 % of patients. For many years, whole-body X-ray (WBX) was the method of choice for detecting skeleton abnormalities. However, the value of WBX in relapsing disease is limited because lesions persist post-treatment, which restricts the capacity to distinguish between old, inactive skeletal lesions and new, active ones. Therefore, alternative techniques are necessary to visualize disease activity. Modern imaging techniques such as magnetic resonance imaging, positron emission tomography and computed tomography offer superior detection of myeloma bone disease and extramedullary manifestations. In particular, the properties of nuclear imaging enable the identification of disease activity by directly targeting the specific cellular properties of malignant plasma cells. In this review, an overview is provided of the effectiveness of radiopharmaceuticals that target metabolism, surface receptors and angiogenesis. The available literature data for commonly used nuclear imaging tracers, the promising first results of new tracers, and our pilot work indicate that a number of these radiopharmaceutical applications can be used effectively for staging and response monitoring of relapsing MM patients. Moreover, some tracers can potentially be used for radio immunotherapy.


Introduction Multiple myeloma, diagnosis and treatment
Multiple myeloma (MM) is a disease characterized by a monoclonal plasma cell population in bone marrow. In most cases, the diagnosis of MM is based on the presence of a monoclonal M-protein or free-light chain in the blood and at least 10 % plasma cells in the bone marrow. Treatment is initiated when MM is symptomatic according to CRAB features: hypercalcemia (C), renal impairment (R), anemia (A) or bone lesions (B). Currently, patients with more than 60 % monoclonal plasma cells in their bone marrow, a free-light chain ratio greater than 100, or more than 1 focal bone or bone marrow lesion on the magnetic resonance imaging (MRI) are classified as high risk for development of MM, and should also receive treatment [1].
First-line treatment for patients younger than 70 years and eligible for autologous stem cell transplantation (ASCT) consists of induction chemotherapy, including a proteasoom inhibitor or an immunomodulator agent like bortezomib, lenalidomide or thalidomide, followed by ASCT [2,3]. Patients ineligible for ASCT are treated with a combination of melphalan, prednisolone and a novel agent, or with the combination of lenalidomide and dexamethasone [4,5]. detects bone lesions earlier than the myeloma-related bone destruction has occurred. In case of monoclonal gammapathy of unknown significance (MGUS), asymptomatic MM or solitary plasmacytoma of the bone, MRI can be used to distinguish a high-risk patient for developing symptomatic MM. Patients with more than one focal lesion on the MRI are classified as high risk for development of MM [10,11].
An alternative manner of imaging MM activity is to target cellular properties of MM cells or micro-environment which can be accomplished by using different radio-labeled compounds to visualize the affected skeleton areas.
Target mechanism in nuclear imaging PET imaging is now widely used for the detection and follow-up of malignant disorders. In MM, PET tracers are used especially for detecting medullary and extramedullary disease. These nuclear tracers can also provide information about the degree of uptake by the lesions of interest, which is indicated by calculation of the standardized uptake value (SUV). In addition, PET scanning has shown promise for monitoring treatment response, since increase or decrease can be visualized and/or calculated compared to a baseline scan [12]. Recently, various specific PET tracers have been developed which might be useful in the workup of patients with newly diagnosed and relapsing MM. In the present review, we evaluate several nuclear tracers and nuclear imaging techniques as defined by their primary target (shown in Table 1 and Fig. 1).

Cell metabolism
Glucose [18 F]-fluorodeoxyglucose PET ([18 F]-FDG-PET) uses enhanced glucose metabolic activity to visualize areas of interest [13]. [18 F]-FDG, a glucose analogue, is actively transported into cells mediated by a group of structurally related glucose transporter proteins (GLUT). Tumor cells, including MM cells, show increased numbers of these glucose transporters, particularly GLUT-1 and GLUT-3 [14]. Glucose and [18 F]-FDG are phosphorylated intracellularly by hexokinase. In contrast to glucose, [18 F]-FDG undergoes no further metabolism in the glucose pathway and becomes trapped intracellularly as [18 F]-FDG-6-phosphate [15]. The use of [18 F]-FDG-PET as a baseline scan has been studied extensively in newly diagnosed MM patients. Based on the number of focal lesions and the maximum SUV (SUV max ), [18 F]-FDG-PET detects more lesions compared to WBX [9]. In addition, follow-up [18 F]-FDG-PET scans can be used to monitor treatment response and are of prognostic value for survival [12]. Complete normalization of the [ PET scan before and after ASCT correlates with both improved progression-free survival (PFS) and overall survival (OS) [12]. This imaging technique may also be useful in patients with relapsing MM since it is not hampered by the presence of pre-existing skeletal defects due to it visualizing areas of enhanced metabolic activity [9,[16][17][18][19] (Table 2A) [12]. This is probably due to a diffuse distribution of malignant plasma cells in the skeleton. Skull lesions smaller than 1 cm are especially difficult to detect with [18 F]-FDG-PET [19] because of the high physiological uptake in the brain tissue. Improvements have been made regarding uptake measurements with [18 F]-FDG-PET. Total lesion glycolysis (TLG) and metabolic tumor volume (MTV) can be calculated from [18 F]-FDG-PET and might be a more accurate measurement than the conventional measurements predicting overall tumor burden of focal lesions in MM. In a recent study of newly diagnosed MM patients, [18 F]-FDG-PET was performed at baseline (Table 2A). In this study, a TLG >620 g and MTV >210 cm [3] was found to be a significant predictor for a poorer PFS and OS [23]. Combining TLG and MTV with other risk factors like gene expression profiling and International Staging System (ISS) stage resulted in the identification of a high-risk subgroup [23].
The Italian group presented guidelines for uniforming [18 F]-FDG-PET quantification by using a five-point scale including: the metabolic state of the bone marrow, number and site of focal [18 F]-FDG-PET-positive lesions with or without osteolytic characteristics, presence and site of extramedullary disease, presence of paramedullary disease (a bone lesion involving surrounding soft tissues with bone cortical interruption), and presence of fractures. The visual degree of uptake is defined for the target lesion and extramedullary lesions according to the Deauville criteria [24]. In a small series, this five-point scale seemed reproducible and can be used for comparing [18 F]-FDG-PET scans from baseline to  scans after treatment; furthermore, it provides a guideline to uniform research data [24].

Amino acids
Methionine, an amino acid required for protein synthesis, can be used for PET scanning.
[11C]-Methionine ([11C]-MET) is frequently used for imaging brain tumours, since the physiological background uptake of [11C]-MET is low in the brain. Due to the active protein synthesis by the malignant plasma cells, this might also be a useful tracer for MM. In several studies, [11C]-MET was compared with [18 F]-FDG-PET (Table 2B) [25,26].  (Table 2) [27].

DNA proliferation marker
Thymidine, a DNA nucleoside required for DNA synthesis, can also be used as a tracer. Fluorothymidine is an analogue of the nucleoside thymidine (deoxythymidine) and can be labelled to produce [18 F]-fluoro-3-deoxy-L-thymidine ([18 F]-FLT). FLT is transported into the cell and is a substrate for thymidine kinase-1 (TK1), which is related to DNA synthesis and is, therefore, a surrogate marker for cell proliferation [28]. Using [18 F]-FLT PET scanning for solid malignancy, a high bone marrow background activity has been demonstrated, which indicates the proliferative activity of the hematopoietic cells in the bone marrow cavity [29]. Patients with MM were included in a pilot study using [18 F]-FLT with haematological malignancies. Areas with skeletal lesions showed a very low uptake of [18 F]-FLT compared to non-affected areas, which is in line with the low proliferative activity of MM cells [30]. Follow up scans with this tracer may be difficult since it has been shown that bone marrow cells undergo a phenotypic shift after chemotherapy and autologous stem cell transplantation, thereby altering their cycling time and the uptake seen on the [18 F]-FLT-PET scan [31].  (Table 2B) [25].

Membrane
Acetate is an important metabolite that is used to synthesize amino acids, nucleotides and fatty acids. When ligated to coenzyme A (acetyl-CoA), it can be converted into fatty acids [32].  [34,35].
Cluster of differentiation 38 (CD38), which is a glycoprotein functioning in cell adhesion, signal transduction and calcium signaling [36], is highly expressed on myeloma cells, but at relatively low levels on normal hematopoietic cells and in some tissues of non-hematopoietic origin, like lung tissue. Currently, an anti-CD38 human monoclonal antibody, daratumab, has been developed. This drug is promising in the treatment of MM. Treatment with daratumumab monotherapy in relapsed and refractory MM patients did have a significant effect on PFS [36]. Phase 3 trials combining daratumumab with conventional treatments such as bortezomib and lenalidomide are ongoing. Use of radiolabeled anti-CD38 could be a promising approach not only for diagnostic purposes and treatment monitoring, but it might also be used in the context of radio-immunotherapy due to the restricted expression of CD38 on normal tissues. Preclinical studies have already been performed in xenograft mice models with radio-immunoconjugates consisting of the αemitter [213Bi] coupled to the anti-CD38 monoclonal antibody, with a significant targeting of CD38 [37].
CD138, or syndecan-1, is a member of the syndecan family, expressed by epithelial cells, precursor B cells, and normal plasma cells. CD138 is highly expressed on myeloma cells. Phase I-II studies have been initiated with anti-CD138, called Indatuximab [38,39]. Alpha-radio-immunotherapy treatment using a [213Bi]-labelled anti-mouse CD138 antibody has been performed in mouse models, showing promising results. Mice treated with [213Bi]-CD138 had a longer median survival than the control group [40].

Receptor targeting
In vitro studies with plasma cell lines have shown that somatostatin receptors, especially subtypes 1, 2 and 5, are highly expressed on MM cells [41]. Somatostatin receptor scintigraphy (SRS) using the single-photon emission computed tomography (SPECT) tracer [111In]-pentetreotide is able to visualize somatostatin receptors, especially subtypes 2 and 5 [42]. SPECT has, by definition, a lower spatial resolution and less quantification possibilities, compared to PET. Studies with SRS in MM patients have been performed specifically in relapsing MM patients. A positive SRS scan was reported in 83 % of the patients, which is significantly higher than the results obtained with WBX [43,44]. However, when SRS was compared to [18 F]-FDG-PET, the latter identified significantly more focal lesions [44], which may be explained by the better resolution of PET compared to SPECT. SRS is also used in the workup of neuroendocrine tumors (NET). In this context, [68Ga]-DOTA-TOC/TATE/NOC/lanreotide PET/CT is used, which has superior resolution and, hence, better sensitivity than the conventional SRS, thereby replacing SRS for staging NET [45]. Considering these properties, [68Ga]-DOTA subtypes might also be useful for diagnostic purposes in MM.
Chemokine receptor 4 (CXCR4) is expressed on hematopoietic stem and progenitor cells residing in the bone marrow niche, which has an important function in the homing of these cells to the bone marrow compartment. Disrupting this interaction by Plerixafor, a CXCR4 antagonist, results in mobilization of stem cells out of the bone marrow niche, a property used for stem cell mobilization. CXCR4 is also expressed on other cell types of the hematopoietic system, including plasma cells. Approximately 50 % of the MM cells express CXCR4 in high density [46]. [68Ga]-pentixafor is a labelled peptide with high affinity for CXCR4, which has been studied in relapsing MM patients in comparison to [ [47]. Based on these pilot studies, [68Ga]-pentixafor scanning might be used to select patients treatment with radiolabeled [68Ga]-pentixafor and to monitor the effects of therapy [47].
Another membrane receptor is the very late antigen-4 (VLA-4, α4β1integrin, CD49d/CD29), which is a transmembrane adhesion receptor. VLA-4 is over-expressed on MM cells and plays a key role in the adhesion and spreading through the bone marrow compartment. VLA-4 binds to vascular cell adhesion molecule-1 (VCAM-1) and fibronectin of bone marrow stromal cells. The PET tracer [64Cu]-CB-TE1A1P-LLP2 is now under investigation in preclinical models [48].

Mitochondrial activity
[99mTc]-sestamibi and [99mTc]-tetrofosmin have been developed for myocardial perfusion SPECT imaging. These compounds consist of a lipophilic monovalent cation, which is sequestered in the mitochondria by the large negative membrane potential [ (Table 2B) [49], probably due to better spatial resolution and different target imaging. Furthermore, [18 F]-FDG-PET and [99mTc]-sestamibi detected more lesions, including extramedullary disease, than MRI [49].

Angiogenesis & hypoxia
The increased FDG uptake by malignant MM cells is related to higher metabolic activity. This might be a consequence of tumour hypoxia resulting from increased O 2 consumption by the malignant MM cells or due to altered micro-vascularisation of the bone marrow containing plasma cells. Due to hypoxia, hypoxia inducible factor (HIF)-1α and HIF-2α are produced by the malignant cells or by the surrounding endothelial cells, which triggers the production of vascular endothelial growth factor (VEGF), resulting in increased micro-vessel density (MVD) around the malignant plasma cells. Several studies have shown that increased MVD correlates with tumor progression [50][51][52][53]. A relatively low MVD has been observed in smouldering MM, which increases significantly during disease progression [51]. The PET tracer 1-α-D: −(5-deoxy-5-[18F]-fluoroarabino furanosyl)-2-nitroimidazole (18F-FAZA), has been developed for visualizing in vivo tumor hypoxia. Under hypoxic conditions, two nitroimidazole compounds undergo reduction, forming highly reactive oxygen radicals and binding to macromolecules inside the cell.
[18F]-FAZA has been used in patients with solid tumors, such as head and neck cancers and non-small cell lung cancers [54,55]. At our institute, five patients diagnosed with relapsing MM based on a positive [18 F]-FDG-PET scan underwent [18 F]-FAZA scanning. However, enhanced uptake of [18 F]-FAZA was not demonstrated in any of the patients, despite the presence of focal disease ( Fig. 2a and b) [14]. These findings suggest that the degree of hypoxia is not substantially different in MM spots compared to the surrounding bone marrow compartment. An alternative approach might be to use tracers that bind to the VEGF produced by MM cells. This can be achieved by labeling bevacizumab, a recombinant, humanised monoclonal antibody that binds with high affinity to all isoforms of free human VEGF. Treatment with bevacizumab is well-established in solid tumors such as colon cancers and renal cell carcinomas.
[89]Zr-bevacizumab PET scanning has been used to detect solid tumors like breast cancer and demonstrated positive scans in 96 % of the patients [56].
[89Zr]-Bevacizumab PET scanning was also performed in five relapsed MM patients ( Fig. 2c and d

Discussion
Various nuclear imaging techniques and tracers have been used for detecting disease activity and bone lesions in newly diagnosed MM patients, but few studies have been conducted in relapsed MM patients. Nuclear imaging can be helpful, especially in relapsed MM because it is frequently difficult to detect new disease activity with WBX, MRI or bone marrow biopsy due to its scattered growth pattern. Additionally, nuclear imaging can be helpful in determining the response to treatment, which might have also prognostic significance for clinical outcome. As shown in Table 1 and Fig. 1 A drawback of these tracers is the necessity of an on-site cyclotron, which prevents wide distribution since it has to be applied shortly following production. [18 F]-Choline, has a longer half-life, is commercially available, and could be a good alternative.
Angiogenesis plays a distinct role in disease progression in MM [50][51][52]. Therefore, it seems promising to use tracers related to this process. Immunohistochemical staining of bone marrow biopsies of MM patients show increased MVD, increased VEGF expression by plasma cells and increased expression of HIF-2α by the endothelial cells. However, no It has been suggested that HIF-1α increases immediately in response to hypoxia, whereas HIF-2α responds to prolonged periods of hypoxia [57]. The negative results might be related to technical or biological issues, for example, due the fact that the bone marrow itself is in a state of low hypoxia, which might hamper detection by [18 F]-FAZA.
With immunohistochemical staining we detected high levels of intracellular VEGFa and clear alteration in the microvasculature, suggesting that the observed VEGF is secreted by the malignant plasma cells. However, this VEGFa could not be visualized by [89Zr]-bevacizumab. This is probably due to the limited concentration resulting from rapid binding to plasma cells or platelets and the limitations in spatial resolution with the use of 89Zr-tracers. Based on these first findings, both [18 F]-FAZA and [89Zr]-bevacizumab do not appear to be useful in the work-up of relapsing MM patients.

Future perspectives
Stand-alone MRI is frequently used in the diagnosis of MM, especially for the spine and pelvis. PET/MRI appears to be a promising new technique for diagnosis and follow up of myeloma patients. It is now possible to perform PET/MRI studies in which metabolic components are combined with the anatomic components of the MRI. In MM patients, [18 F]-FDG-PET/CT has been compared to FDG-PET/MRI. Almost all lesions detected by [18 F]-FDG-PET/CT were also detected by FDG-PET/MRI [58]. Further investigation and optimalization of the protocol for PET/MRI is needed to provide more information about the role of PET/MRI for diagnostic purposes and response monitoring in MM. Novel MRI sequences are now available. For example, diffusion weighted imaging (DWI) and delayed contrast enhancement (DCE) seem to improve the diagnostic properties of MRI in MM patients [59,60]. Combining PET with these novel MRI techniques may be valuable for optimal diagnosis and evaluation of relapsed MM activity.
The tracers discussed in this review might also be useful in the radio-immunotherapy setting, as described for [68Ga]pentixafor. However, a drawback might be the negative effects on normal hematopoietic cells due to the high expression of CXCR4 on these cells. These negative effects on normal hematopoietic cells might be circumvented if this type of radioimmunotherapy is used in the context of autologous stem cell transplantation.
In the future, radiolabeled daratumumab might be very interesting; preclinical data is promising and treatment with daratumumab is well-tolerated with encouraging results [36,37]. Also, anti-CD138 might be a promising approach [38].
CD319 also called signaling lymphocytic activation molecule (SLAM) F7 is a receptor present on immune cells including plasma cells. The antibody against SLAMF7 is called elotuzumab. Treatment with this antibody demonstrates promising results in relapsing MM patients [39]. Like anti-CD38 and anti-CD138, labeling of this antibody might provide disease information and perhaps to be a new target for radioimmunotherapy. Furthermore, labeling with [64Cu]-chelates might also be used in future research. A mice study with [64Cu]-anti-CD138 showed promising results [61].
Nuclear imaging is of growing importance in the diagnostic process and follow up of MM patients. It provides important information regarding the anatomical localization and the metabolic activity of the areas of interest. Combining both pieces of information according to uniform scoring systems will provide important information for the individual patient but also for the comparison of future study protocols. Examples are the five-point scaling score shown by the Italian group [24] and the combined use of TLG and MTV [23].
In conclusion, nuclear imaging is an important tool for diagnostic purposes and response monitoring, particularly in relapsing MM patients. Various nuclear tracers have been developed to detect bone, bone marrow and extramedullary involvement. In the future, these tracers might also be used for treatment monitoring, including detection of minimal residual disease.

Conflict of interest None
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