11C-acetate positron emission tomography is more precise than 18F-fluorodeoxyglucose positron emission tomography in evaluating tumor burden and predicting disease risk of multiple myeloma

The optimal method of tumor burden evaluation in newly diagnosed multiple myeloma (NDMM) is yet to be determined. This study aimed to compare the value of 11C-acetate positron-emission tomography (PET)/computed tomography (CT) (AC-PET and 18F-fluorodeoxyglucose PET/CT (FDG-PET) in the assessment of tumor burden in NDMM. This study evaluated 64 NDMM patients between February 2015 and July 2018. AC-PET and FDG-PET were used to assess myeloma lesions. The clinical data, imaging results, and their correlations were analyzed. Diffuse bone marrow uptake in AC-PET was significantly correlated with biomarkers for tumor burden, including serum hemoglobin (P = 0.020), M protein (P = 0.054), the percentage of bone marrow plasma cells (P < 0.001), and the Durie–Salmon stage of the disease (P = 0.007). The maximum standard uptake value (SUVmax) of focal lesions and high diffuse bone marrow uptake in AC-PET showed stronger correlations with high-risk disease (P = 0.017, P = 0.013) than those in FDG-PET. Moreover, the presence of diffuse bone marrow uptake, more than ten focal lesions, and an SUVmax of focal lesions of > 6.0 in AC-PET, but not in FDG-PET, predicted a higher probability of disease progression and shorter progression-free survival (P < 0.05). AC-PET outperformed FDG-PET in tumor burden evaluation and disease progression prediction in NDMM.

Treatment. The patients received frontline chemotherapy, which mainly included bortezomib, lenalidomide, and thalidomide. Nine patients also received autologous hematopoietic stem cell transplantation (ASCT) following chemotherapy. Follow-up. All patients were followed up via outpatient visits or telephone calls once every three months until September 2019. Treatment responses, disease progression, and survival were recorded. The treatment response was assessed according to the IMWG consensus criteria 23 . Progression-free survival (PFS) and overall survival (OS) were defined as the time from the date of diagnosis until the date of disease progression or any cause of death, respectively. Data collection. The serum levels of beta 2-microglobulin (β2-MG), lactate dehydrogenase (LDH), M protein according to serum protein electrophoresis, and the 24-h light chain urine level were determined. International staging system (ISS) and D-S staging were determined according to the IMWG criteria 20 . Chromosome aberrations of CD138-sorted bone marrow plasma cells were examined by fluorescence in situ hybridization 24 . High-risk disease was defined as the presence of any of the following: 1q21 amplification, 17p deletion, and t (4; 14) or t (14; 16) translocations. www.nature.com/scientificreports/ Statistical analysis. The detection value and its correlation with tumor burden and the cases of high-risk cytogenetic abnormalities, were compared. Associations between image characteristics and treatment response, PFS, OS were also analyzed. All data were analyzed using SPSS 24.0 software (IBM Corp., Armonk, NY, USA).

Comparison of AC-PET and FDG-PET in the detection of NDMM.
AC-PET showed greater sensitivity than FDG-PET for the detection of MM. The strong physiological uptake by the brain on FDG-PET interfered with the detection of adjacent skull lesions, leading to a high rate of false negatives. In contrast, the background uptake by the brain was very weak on AC-PET; therefore, skull deformation and skull lesions with high SUV were more easily detected by AC-PET ( Fig. 2A-C). In addition, FDG-PET has a high rate of false www.nature.com/scientificreports/ positives at fracture sites. AC-PET was more capable than FDG-PET in distinguishing an old fracture from an active tumor lesion ( Fig. 2D-J). As shown in Fig. 3, the positive rate of marrow involvement detected by AC-PET was 93.8% (60/64), including 34 patients with both diffuse and focal hypermetabolic lesions, 21 patients with diffuse lesions only, and 5 patients with focal lesions only. The focal lesions showed a median SUV max of 5.68 (range 1.29-47.60) and were mainly located in the pelvis (41.7%), followed by the long bones of the lower limbs (25%), sternum and ribs (20.8%), craniofacial bones (8.3%), and vertebral bones (4.2%). FDG-PET detected marrow involvement in only 65.6% of patients (42/64), a significantly lower rate than that in AC-PET (P = 0.004). Of the 42 cases of marrow involvement detected by FDG-PET, 14 were both diffuse and focal hypermetabolic lesions (P = 0.001), 14 were diffuse involvement only (P = 0.001), and 14 were focal lesions only (P = 0.007). In AC-PET, 18.8% of patients (12/64) presented with more than 20 focal lesions, while in FDG-PET, only 6.3% (4/64) were determined to have more than 20 focal lesions (P = 0.001).
AC-PET and FDG-PET detected extramedullary plasmacytomas in the same eight patients with similar sensitivity. The images of a typical patient with extramedullary plasmacytomas are shown in Fig. 2K-Q. Fig. 4, in the AC-PET group, patients with diffuse high marrow uptake presented with lower hemoglobin (92.49 ± 25.03 vs. 120.44 ± 20.42 g/L, P = 0.002), higher M protein in IgG or IgA patients (35.71 ± 23.20 vs. 16.07 ± 18.40 g/L, P = 0.05), and a higher percentage of bone marrow plasma cells (36.88 ± 23.44 vs 11.74 ± 8.72%, P < 0.001), but with similar calcium and LDH levels (data not shown), compared to patients with negative marrow uptake. In the FDG-PET group, the M protein levels in IgG or IgA subtypes (34.33 ± 20.34 vs. 32.31 ± 25.72 g/L, P = 0.778) and myeloma cell densities in the bone marrow (37.43 ± 22.15 vs. 30.17 ± 24.54%, P = 0.226) were similar between patients with diffuse high marrow uptake and those with negative uptake. However, a significantly lower hemoglobin level was detected in those with diffuse high bone marrow uptake than those with negative uptake (86.79 ± 19.56 vs. 103.92 ± 28.42 g/L, P = 0.008).

Correlation between PET image characteristics and clinical parameters. As indicated in
Diffuse high marrow uptake in AC-PET was positively correlated with high-risk disease characterized by the presence of high-risk cytogenetic abnormalities (r = 0.29, P = 0.020) and the D-S stage (r = 0.39, P = 0.001).

Comparison of AC-PET and FDG-PET for determining treatment response and clinical outcome.
Of the 50 patients who received bortezomib-based regimens, 42 were treated with bortezomib, cyclophosphamide, and dexamethasone; four with bortezomib, lenalidomide, and dexamethasone; two with bortezomib and dexamethasone (BD); and two with BD-cisplatin, doxorubicin, cyclophosphamide, and etoposide (PACE). Of the nine patients who received thalidomide-based regimens, seven were treated with thalidomide, cyclophosphamide, and dexamethasone, and two with thalidomide, dexamethasone, cisplatin, doxorubicin, cyclophosphamide, and etoposide (DT-PACE). The remaining five patients received lenalidomide and dexamethasone. In addition to frontline chemotherapy, nine patients received ASCT following drug treatment. The responses to therapy included 15 cases of stringent complete response, seven cases of complete response, 19 cases of very good partial response, 16 cases of partial response, six cases of stable disease, and one case of progressive disease. Figure 5 and Supplemental Table 1 illustrate that the median follow-up duration was 19 months (range 1-52 months). By the end of the follow-up, 54.7% (35/64) patients remained responsive to therapy, and 35.9% (23/64) patients had died. The estimated 3-year survival rate was 58%. We found no significant correlation between the treatment response and diffuse high bone marrow uptake, the number of focal lesions, and the SUVmax of focal lesions on AC-PET. However, patients with diffuse high marrow uptake on AC-PET had a higher probability of disease relapse (50.9 vs. 11.1%, P = 0.033) and a shorter median PFS (21 months, 95% confidence interval [CI] 9.58-32.42 vs. not reached, P = 0.041). We also found a positive correlation between disease relapse and the number of focal lesions on AC-PET. The rates of disease relapse in patients with oligo (1-9)  www.nature.com/scientificreports/ (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and numerous (> 20) focal lesions were 30.0, 42.9, and 75.0%, respectively (P = 0.046). The median PFS of patients with multiple and numerous focal lesions (17 months, 95% CI 14.7-19.3) on AC-PET was shorter than that of patients with oligo lesions (29 months, 95% CI 9.1-48.9), but the difference was not significant (P = 0.104). Compared with patients who had an SUV max of focal lesions of ≤ 6.0 on AC-PET, patients with an SUV max of focal lesions of > 6.0 had a higher rate of disease relapse (71.4 vs 32.0%, P = 0.018) and a shorter median PFS (15 months, 95% CI 9.1-20.9 vs. 29 months, 95% CI 15.4-42.7, P = 0.017). In contrast, we found no significant correlation between disease relapse or the median PFS and diffuse bone marrow uptake, the number of focal lesions, or the SUV max of focal lesions on FDG-PET (all P > 0.05). Kaplan-Meier survival analysis of PFS in AC-PET and FDG-PET showed the presence of diffuse bone marrow uptake and an SUV max of focal lesions of > 6.0 in AC-PET, but not in FDG-PET, predicted a significantly shorter progression-free survival (P < 0.05) (Fig. 5).
The OS rate did not significantly differ according to diffuse bone marrow uptake (yes or no), the number of focal lesions (< 10 or ≥ 10), or the SUV max of focal lesions (> 6.0 or ≤ 6.0) on both AC-PET and FDG-PET during the 19 months median follow-up period (all P > 0.05).

Discussion
To the best of our knowledge, this is the largest prospective single-center study to compare dual-tracer PET/CT imaging techniques in NDMM patients. Compared with the more conventional FDG-PET, the novel AC-PET exhibited greater sensitivity for detecting bone marrow infiltration and skull lesions. Diffuse bone marrow uptake on AC-PET was significantly correlated with biomarkers that reflect the tumor burden, and the correlation was stronger than that on FDG-PET. Meanwhile, the SUV max of the focal lesion was significantly correlated with a high-risk disease on AC-PET but not on FDG-PET. Finally, the presence of diffuse bone marrow uptake, more than ten focal lesions, and an SUV max of focal lesions of > 6.0 on AC-PET were significantly associated with disease progression and PFS in this cohort.
The conventional FDG-PET is commonly used to evaluate the baseline tumor burden and therapeutic response 25 . However, it has limited sensitivity in detecting diffuse bone marrow infiltration and focal lesions because of the low uptake of fluorodeoxyglucose in myeloma cells 18 . Several alternate tracers, including 11 C-acetate, have been investigated to overcome the limitations of FDG-PET. Acetate is a precursor for lipid synthesis. It could be selectively taken up by certain tumor cells that rely more heavily on fatty acid metabolism than on glycolysis 26 . It is known that the energy production of MM predominantly occurs via aerobic glucose metabolism, which includes the tricarboxylic acid cycle 27 , and the production of abnormal immunoglobulins requires active lipid synthesis in plasma cells. Therefore, we observed high lesion uptake but low physiological brain, bone, and bone marrow uptake of the 11 C-acetate tracer on AC-PET, which also explains why AC-PET was significantly more sensitive than FDG-PET for detecting skull lesions, lesions at bone fracture sites, and bone marrow involvement of NDMM in this study.
There have been some reports comparing AC-PET and FDG-PET in the clinical examination of MM. In two single-case reports, AC-PET appeared to be more sensitive than FDG-PET in assessing the tumor burden and/ or response to therapy 16,17 . In addition, there only two cohort studies have been published to date, which compared these two imaging methods for this disease. Ho et al. 19 reported dual AC-PET and FDG-PET scanning of 35 patients, including 26 with MM, five with smoldering MM (SMM), and four with monoclonal gammopathy of undetermined significances (MGUS). In this cohort study, AC-PET outperformed FDG-PET with a higher sensitivity (84.6 vs. 57.7%) and specificity (100 vs. 93.1%) in distinguishing active MM from SMM or MGUS. In addition, the number of 11 C-acetate-avid bone lesions was highly correlated with serum β2-MG, and the resolution of 11 C-acetate marrow activity was correlated with the clinical response to chemotherapy. Another cohort study conducted by Lin et al. 18 with 15 NDMM patients who underwent both AC-PET and FDG-PET scanning reported that bone marrow 11 C-acetate uptake in these patients was positively correlated with bone marrow plasma cell infiltration. The study also revealed a significantly higher mean SUV max on AC-PET than on FDG-PET. Our findings verified that AC-PET is more sensitive than FDG-PET for detecting both diffuse and focal MM lesions. Of note, the present study further analyzed the associations between AC-PET image characteristics and clinical outcomes in MM patients. We found diffuse high uptake of bone marrow, more than ten focal lesions, and an SUV max of focal lesions of > 6.00 on AC-PET, but not FDG-PET, were associated with disease progression and PFS. However, such associations were not significant in predicting OS for MM patients. Overall, our results indicate that AC-PET is a sensitive method for evaluating the tumor burden of active MM and is critical for risk stratification as well as disease prognosis, leading to more effective disease intervention and management.
This study had a few limitations. Firstly, this was a single-center observational study with bias inherent to the study's design. Further, although we have included as many patients as possible in our analysis, the sample size of 64 patients was relatively small. Finally, the median follow-up duration of 19 months was not sufficiently long to allow a thorough analysis of OS. Future multicenter studies with larger sample sizes and longer follow-up durations are needed to confirm the findings of this study.

Conclusion
In summary, AC-PET outperformed FDG-PET in tumor burden evaluation and risk stratification of NDMM, as well as disease progression prediction. AC-PET is thus a more suitable method for the evaluation of MM.

Data availability
Data sharing does not apply to this article as no datasets were generated or analyzed during the current study. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.