Functional imaging with dual-energy computed tomography for supplementary non-invasive assessment of mast cell burden in systemic mastocytosis

Systemic mastocytosis (SM) is characterized by multifocal accumulation of neoplastic mast cells (MCs), predominately affecting the bone marrow (BM). Imaging with computed tomography (CT) is used for assessment of bone mineral density and structure. However, the value of functional imaging with dual-energy CT (DECT) and the assessment of virtual-non-calcium attenuation values (VNCa-AV) for visualization of BM disease burden in SM has not yet been assessed. DECT of the axial skeleton was performed in 18 patients with SM (indolent SM [ISM], n = 6; smoldering SM [SSM]/advanced SM [AdvSM], n = 12) and 18 control subjects. VNCa-AV were obtained in 5 representative vertebraes per patient and correlated with laboratory, morphologic and molecular parameters. VNCa-AV strongly correlated with quantitative BM MC infiltration (r = 0.7, R2 = 0.49, P = 0.001) and serum tryptase levels (r = 0.7, R2 = 0.54, P < 0.001). Mean VNCa-AV were significantly higher in SSM/AdvSM as compared to ISM (− 9HU vs. − 54HU, P < 0.005) and controls (− 38HU, P < 0.005). Nine of 10 (90%) patients with a VNCa-AV >  − 30HU and 7/7 (100%) patients with a VNCa-AV >  − 10HU had SSM or AdVSM. BM VNCa-AV provide information about the MC burden of SM patients and correlate with SM subtypes. DECT may therefore serve as a supplementary tool for SM diagnosis, subclassification and monitoring in a one-stop-shop session.

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by neoplastic expansion and accumulation of clonal mast cells (MCs), predominantly affecting bone marrow (BM), skin and visceral organs [1][2][3][4][5] . SM is subcategorized in indolent SM (ISM), smoldering SM (SSM) and advanced SM (AdvSM), the latter comprising aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL) 6 . While ISM patients benefit from an almost normal life expectancy, AdvSM is associated with poor prognosis and a median overall survival of less than 3-4 years 1,7-14 . BM histology with qualitative and quantitative assessment of MC and AHN is of fundamental importance for diagnosis and subclassification of SM. In addition to blood counts and serum chemistry, radiological imaging including X-ray, computed tomography (CT) and magnetic resonance imaging (MRI) are pivotal supplementary tools for visualizing anatomical disease extent and specifically bone and BM involvement 9,[15][16][17][18] . In a recent study on measurement of bone mineral density (BMD) through CT, an increased BMD/osteosclerosis was associated with a more aggressive phenotype and inferior survival 19 . Moreover, an activated BM, reflected as BM edema in MRI, is indicative for a high MC burden, organ damage and adverse survival 20 . However, BM edema can only be visualized but not quantified by MRI and decreased/increased BMD can only be quantified by CT.
To overcome those individual limitations of CT and MRI, functional imaging through dual-energy CT (DECT) and analysis of virtual-non-calcium attenuation values (VNCa-AV) has recently been established for detection of BM edema in trauma patients 21,22 and discrimination between various infiltration patterns in multiple myeloma 23 . We therefore sought to investigate whether functional imaging with DECT may also be useful in patients with SM for visualization of BM disease burden.

Methods
Patients and control group. This retrospective analysis included 18 patients with SM (female, n = 8 [44; median age 63 years, range 45-86). Detailed demographic and SM-associated disease characteristics are presented in Tables 1 and 2. Eighteen control patients (female [n = 7, 39%]; median age 61 years, range 41-83) were included using the same DECT protocol. All control patients were diagnosed with non-metastatic malignant melanoma; other hematologic neoplasms were not present. The analysis adhered to the tenets of the Declaration of Helsinki and was approved by the relevant institutional review board (Medical Faculty Mannheim, University of Heidelberg). All patients gave written informed consent.
Diagnosis and subclassification. Diagnosis and subclassification were performed according to the revised world health organization (WHO) 2017 classification: ISM in 6/18 (33%) and SSM/AdvSM in 12/18 (67%) patients (Tables 1, 2) 1,2,24 . Diagnosis of SSM was established through presence of at least two of three B-findings (MC infiltration > 30% and serum tryptase level > 200 ng/mL; signs of dysplasia or myeloproliferation in non-MC lineage compartments of the BM, but no AHN; hepatomegaly without impairment of liver function and/or splenomegaly and/or lymphadenopathy). Diagnosis of ASM was based on the presence of one or more C-findings (cytopenia with neutropenia < 1 × 10 9 /L, anemia <10 g/dL or thrombocytopenia < 100 × 10 9 /L, palpable hepatomegaly with impaired liver function, palpable splenomegaly with signs of hypersplenism, malabsorption with weight loss due to gastrointestinal MC infiltrates or skeletal involvement with large osteolytic lesions and/or pathological fractures). SM-AHN met the criteria for SM and for an AHN (e.g. chronic myelomonocytic leukemia, myelodysplastic/myeloproliferative neoplasm unclassified [MDS/MPNu] or chronic eosinophilic leukemia). MCL was diagnosed based on the presence of at least ≥ 20% MCs in a BM smear.

Treatment.
Patients with ISM received a symptom-directed conventional therapy including H1-and H2-antagonists, cromolyn acid, proton pump inhibitors and corticosteroids. One patient with SSM was treated with hydroxurea. All patients with AdvSM were treated with the multikinase inhibitor midostaurin; 3 patients further received the purine analogue cladribine as second-line treatment. The use of bisphosphonates was not documented in any patient at time of DECT.
DECT scan protocol, image reconstruction and postprocessing. All examinations were performed on a dual-source CT system in dual-energy mode (SOMATOM Force; Siemens Healthineers, Forchheim, Germany). Median time between diagnosis and DECT was 1.0 years (range 0-11.0). For evaluation of osteosclerosis, weighted-average coronal and sagittal multiplanar reformations were calculated. For DECT postprocessing, axial slices with a thickness of 1.0 mm were reconstructed. Postprocessing was performed on a dedicated dualenergy software (Syngo.via; version VB30A; Siemens Healthineers) with a three-material decomposition algorithm for bone mineral, yellow marrow and red marrow 25 . For further assessment, DECT images were viewed as weighted-average CT merged with a colour-coded VNCa overlay using the BM setting (Siemens Healthineers). Quantitative image analysis. All images were separately analyzed in a randomized order by two readers (J.R. and P.R.) with 10 years of experience each in oncologic imaging. The readers were blinded to clinical data and VNCa-AV measurements were performed in consensus. In patients with SM and patients from the control group, five circular region of interest (ROI) measurements of at least 100 mm 2 were obtained between Th11-12 and L1-3. ROI borders were maintained 2 mm away from adjacent cortical bone in order to only include BM in Table 1
In patients with SSM/AdvSM diffuse BM infiltration patterns have also been described using MRI technique 20 , however no data is available about the applicability of DECT in these patients. We therefore evaluated DECTgenerated VNCa-AV in clinically and morphologically well characterized patients with ISM and SSM/AdvSM. VNCa-VA were strongly correlated with quantitative bone marrow MC infiltration and serum tryptase levels but also other characteristics, e.g. levels of alkaline phosphatase, albumin or KIT D816V variant allele frequency, indicating a strong correlation with an advanced phenotype. Mean VNCa-AV were significantly higher in patients with SSM/AdvSM with values > − 30HU almost exclusively and values > − 10HU exlusively found in SSM/AdvSM. Within the SSM/AdvSM patient group, patients with an elevated VNCa-AV revealed a more aggressive phenotype. Of note, two AdvSM patients with regular bone structure by conventional CT showed markedly elevated VNCa-AV. Both patients were associated with a poor clinico-genetic risk profile, e.g. KIT D816V negative MCL and multimutated SM-MDS/MPNu progressing to secondary acute myeloid leukemia even more indicating DECT as a supplementary tool for identification of high-risk disease.
The increased VNCa-AV may be best explained by variable displacement of healthy adipose marrow by tumor cells or rather MCs. Because an AHN is present in 70-80% of AdvSM patients, it could be argued that the AHN also contributes to the pathologically elevated VNCa-AV, e.g. through hypercellularity. However, the strong association between VNCa-AV and several SM-specific factors outside of the bone marrow such as serum tryptase levels and to a lesser extent alkaline phosphatase, albumin and KIT D816V are clearly in favor of SM.
Several limitations of this study have to be addressed. First, the control group consisted of patients with non-metastatic malignant melanoma and not healthy probands. This patient group was chosen, because (i) the retrospective design of this study did not allow to enroll healthy probands, (ii) scans for metastatic melanomatous deposits in patients with malignant melanoma were mandatory and (iii) the absence of metastases in patients with malignant melanoma and otherwiese "healthy" BM comprised a control group similar to what we expect in a general sex-and age-matched population. Further, the small sample size of 18 patients derived from a single center limits statistical power, especially for subgroup analysis. However, SM is a very rare disease and the application of DECT is not yet part of the routine clinical work-up. Larger patient populations and a prospective study design would be desirable.
Last, to underline our results, the correlation of VNCa-AV with the BM morphology on MRI might be of interest. Unfortunately, due to the retrospective study design timely spine MRI was not available for the patients included in this study. This aspect should also be highlighted in future studies.
Imaging techniques such as DECT are appreciated for its remarkable value in biological characterization of tissue involvement through generating functional information upon the tissue microstructure. Radiological biomarkers, which allow a quantitative and objective analysis of these images, are of increasing importance in this context. We conclude, that DECT represents an excellent supplementary tool for one-stop-shop imaging of organomegaly, osteopenia, osteoporosis, osteosclerosis and bone marrow edema in patients with SM. The technique allows non-invasive assessment of the mast cell burden and may therefore serve as supplementary tool for diagnosis, subclassification and monitoring of the SM disease course. Hemoglobin, g/dL (mean ± SD) 11.6 ± 1.5 9.5 ± 2.7 11.7 ± 3.0 n.s. n.s. www.nature.com/scientificreports/

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.