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Magnetic Resonance Spectroscopy-Detected Change in Marrow Adiposity Is Strongly Correlated to Postmenopausal Breast Cancer Risk
Original Study
Magnetic Resonance Spectroscopy-Detected Change in Marrow Adiposity Is Strongly Correlated to Postmenopausal Breast Cancer Risk Guanwu Li,1 Zheng Xu,2 Alex Zhuang,3 Shixin Chang,1 Lingmi Hou,4 Yongsheng Chen,3 Maki Polat,5 Dongmei Wu6 Abstract Previous ex vivo studies described marrow adipocytes influencing cancer bone metastasis. The prognosis of marrow adiposity on breast cancer risk remains elusive. We hypothesized that marrow adiposity and leptin levels would be associated with breast cancer risk. We found marrow fat expansion, but not circulating leptin is a predictor of postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Purpose: To determine whether marrow fat fraction (FF) is correlated with postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Methods: Fifty-six patients with newly diagnosed and histologically confirmed postmenopausal breast cancer and 56 healthy controls underwent serologic test and magnetic resonance spectroscopyebased FF measurements. Data were analyzed by logistic multivariate regression models to determine the independent predictors of breast cancer risk and clinicopathological characters of breast cancer. Results: Patients with breast cancer had higher FF than that of the controls. Marrow FF showed positive association with serum leptin levels (r ¼ 0.607, P < .001) in the cases, but no relationship was found in the controls. In the univariate analysis, both levels of leptin and marrow FF were significantly associated with breast cancer risk and clinicopathological characteristics of breast cancer. In the multivariable model with adjustment for established breast cancer risk factors, serum leptin was a significant predictor of breast cancer risk (OR 1.746; 95% CI, 1.226-2.556) and clinicopathological characteristics of breast cancer including TNM, tumor size, lymph node status, and histological grade (OR 1.461-1.695); but when marrow FF was additionally added to the regression model, marrow FF but not leptin levels was observed to be an independent risk factor for breast cancer risk (OR 1.940; 95% CI, 1.306-2.910) and clinicopathological characteristics of breast cancer (OR 1.770-1.903). Conclusion: Marrow adiposity is a predictor of postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Clinical Breast Cancer, Vol. -, No. -, --- ª 2017 Elsevier Inc. All rights reserved. Keywords: Bone metastasis, Leptin, Marrow fat content, Menopause
Introduction G.L. and Z.X. contributed equally to this study as first authors. 1 Department of Radiology, Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China 2 Xinzhuang Community Health Center, Shanghai, China 3 Department of Radiology, Wayne State University, Detroit, MI 4 Breast Surgery, Affiliated Hospital of North Sichuan Medical College, Sichuan, China 5 School of Medicine and Public Health, University of Wisconsin, Madison, WI 6 Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai, China
Submitted: Sep 7, 2016; Revised: Dec 30, 2016; Accepted: Jan 9, 2017 Addresses for correspondence: Guanwu Li, MD, Department of Radiology, Yueyang Hospital, Shanghai University of TCM, 110 Ganhe Rd, Shanghai 200437, China E-mail contact: [email protected]
1526-8209/$ - see frontmatter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2017.01.004
Breast cancer metastasis is responsible for most breast cancer mortality. Bone is the most preferred site for distant metastasis of breast cancer and bone metastasis is an incurable complication of breast cancer, affecting 70% to 80% of advanced patients.1 Both marrow adipocytes and osteoblasts arise from common multipotent progenitor cells, known as bone marrow mesenchymal stromal cells (MSCs). The associations of marrow fat with other adipose tissue depots are complicated. Marrow fat may play crucial roles in modulation of hemopoiesis, metabolic homoeostasis, and osteogenesis.2,3 Previous study showed that marrow fat cells appear to be capable of translocating stored lipids to the metastatic tumor cells. These adipocyte-supplied lipids serve as an energy source for cancer cells, a process suggested to increase tumor cell proliferation,
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Association of Marrow Adiposity and Breast Cancer Risk motility, and invasion.4,5 Recent ex vivo studies describe marrow fat cells influencing cancer bone metastasis, particularly breast and prostate cancer6-8; however, the prognosis of marrow adiposity on modulating tumor colonization and macro-metastasis in the skeleton remains largely unexplored. Marrow adipocytes store quantities of fat and produce adipokines, such as leptin and adiponectin, which are known for their roles in the regulation of energy metabolism through endocrine-, paracrine-, and autocrine-mediated pathways.9 Adipokines also have been studied on cancer cells and are implicated in proliferation of breast, prostate, and lung cancers10-12; however, there have been some controversies in the currently available data. Some,13-15 but not all studies16,17 showed that there exist associations between breast cancer incidence and adipokines, in particular leptin. Because there exists a clear association between levels of leptin and marrow adiposity,9,18 we hypothesized that elevated marrow fat content as measured by magnetic resonance spectroscopy (MRS) and serum leptin would be associated with increased risk for breast cancer and clinicopathological characteristics of breast cancer in postmenopausal females.
Material and Methods Study Populations
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Fifty-six postmenopausal patients (mean age, 63.0 8.5 years) with more than 1 year since menopause (YSM) who were newly diagnosed and histologically confirmed breast cancer with no prior surgical, chemotherapy, or radiotherapy treatment between May 2013 and June 2016 were enrolled in this study. Diagnosis of breast cancer was confirmed histologically in each case and estrogen receptor status was determined. Tumors were classified as receptorpositive status according to the following criteria: 10% of positive histologically stained cells, an any “plus-system” description, 20 fmol/mg, an Allred score of 3, an immunoreactive score of 2, or an H-score of 10.19 The staging of breast cancer was determined according to the TNM system, and histological grade was determined according to the modified ScarffeBloomeRichardson criteria.20 The healthy control individuals (n ¼ 56) were age and body mass index (BMI) matched with the cases. All control participants were confirmed free from benign or malignant breast diseases by physical examination and mammography. Furthermore, the key exclusion criteria were as follows: (1) any disease known to affect bone metabolism, such as previous or current malignant tumor (except for breast cancer), exposure to chemo-radiotherapy, diabetes mellitus, chronic renal failure, thyroid and parathyroid diseases, vitamin D deficiency; (2) any previous or current use of medications known to affect bone metabolism, such as bisphosphonate, glucocorticoid, hormone replacement therapy, or calcium or vitamin D supplementation; and (3) any confounding factor that had the potential to interfere with the interpretation of the findings, such as hip or lumbar spine fracture. For all participants, comprehensive questionnaires were used to collect medical information, including demographics (such as age, YSM, height, body weight, and BMI), alcohol consumption, smoking status, physical activity, detailed medical history, use of medications, as well as family history of breast cancer and other cancers. Physical activity was assessed using the International Physical Activity Questionnaire short form, with data reported as
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Metabolic Equivalent of Task hours per week.21 Written informed consent was obtained for all individual participants included in the study according to a protocol approved by a local research ethics committee and in accordance with the 2008 Helsinki declaration.
Serologic Analysis Blood samples were collected from an overnight fast of at least 8 hours. Routine biochemical parameters, such as fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, were determined immediately after blood was drawn and plasma samples were stored at 20 C for further analyses. Serum leptin was determined by an enzyme-linked immunosorbent assay kit (Diagnostic Systems Laboratories Inc, Webster, TX). Levels of estradiol and insulin were assayed using a radioimmunoassay kit (Union Medical & Pharmaceutical Technology Tianjin Ltd, Tianjin, China); the intraassay and interassay coefficients of variation were less than 10%, respectively.
Marrow Fat Measurements Magnetic resonance imaging (MRI) examinations were performed on a MAGNETOM Verio 3T scanner (Siemens Medical Solutions, Erlangen, Germany). The images were acquired with the participant in the supine position using the standard spine-array receive coil. Sagittal T1- and T2-weighted turbo spin-echo sequences were used for morphological assessments of the lumbar region to exclude any confounders such as wedging of vertebrae, vertebral hemangiomas, end plate depression, and silent compression fractures. To quantify marrow fat fraction (FF), a tri-plane gradient echo localizer pulse sequence of the lumbar spine was used to guide positioning of a volume of interest (1.5 1.5 1.5 cm3) within the third vertebral body, and then single-voxel proton MRS data were acquired using the PRESS pulse sequence without water suppression with the following image parameters: repetition time, 3000 ms; echo time (TE), 30 ms; bandwidth, 2000 Hz; flip angle, 90 ; data points, 1024; number of acquisitions, 64. Six outer volume saturation bands were used to eliminate unwanted signal contamination from outside the voxel. Moreover, we applied default autoshimming as provided by the manufacturer. The acquisition time for the bone marrow MRS scan was 3 minutes 24 seconds. A commercially available imaging workstation (Siemens Syngo B17) was used for postprocessing of MRS data. Spectral assignments were based on previous studies,22,23 and only clearly identifiable peaks were measured. All spectra have shown a water peak located at 4.65 ppm and a lipid peak (bulk methylene) located at 1.30 ppm. FF value was calculated according to the following equation: FF ¼ (AUClipid/[AUClipid þ AUCwater]) 100%, where AUClipid and AUCwater referred to the peak area under the lipid and water curve, respectively.
Statistical Analysis Categorical data are described using frequencies and percentages. Continuous data are described using means SD. Normality of data was assessed using the Shapiro-Wilk test. Differences in the baseline characteristics between groups were evaluated using the c2 test for categorical data and Student’s t-test or Wilcoxon rank sum test for
Guanwu Li et al continuous data where appropriate. Pearson or Spearman correlation tests were used to determine the relationships of serum leptin, marrow FF, and clinicopathological characteristics of breast cancer (TNM, tumor size, lymph node metastasis, and histological grade) where appropriate. To determine independent predictors of clinicopathological characteristics of breast cancer and breast cancer risk, we performed multivariate logistic regression analysis to estimate odds ratios (ORs; OR of large tumor size [> 2.0 cm] against small tumor size [ 2.0 cm], histological grade III against grade I and II, lymph node positive against lymph node negative, TNM stage I against stage II-III17,24) and 95% confidence intervals (CIs), with adjustment for confounder-related breast cancer risk including age, YSM, BMI, smoking status, alcohol consumption, physical activity, regular nonsteroidal anti-inflammatory drug use, family history of breast cancer, glucose, insulin, endogenous estradiol levels, and blood lipids.13,25 Data were analyzed using IBM SPSS v. 23.0 (IBM SPSS, Armonk, NY). Statistical significance was set to P < .05.
leptin, estradiol, triglyceride, and high- and low-density lipoprotein cholesterol between groups; but no significant differences were found in total cholesterol, insulin, and fasting plasma glucose. As expected, patients with breast cancer had significantly higher marrow FF than that of the controls (P < .001) (Table 1 and Figure 1). This indicated significant accumulation of marrow fat in patients with breast cancer.
Relationships of Leptin, Marrow FF, and Clinicopathological Characteristics of Breast Cancer In the breast cancer group, marrow FF showed positive association with levels of leptin (r ¼ 0.607, P < .001); but no relationship was found in the controls. Marrow FF showed positive correlations with lymph node metastasis (r ¼ 0.701), TNM (r ¼ 0.611), tumor size (r ¼ 0.574), and histological grade (r ¼ 0.683). Serum leptin also was positively correlated with lymph node metastasis (r ¼ 0.530), TNM (r ¼ 0.585), tumor size (r ¼ 0.492), and histological grade (r ¼ 0.461, all P < .05).
Results Baseline Characteristics The baseline characteristics of the patients with breast cancer and their respective controls are presented in Table 1. Compared with the controls, patients with breast cancer had smaller YSM, greater alcohol consumption, a higher frequency of nonsteroidal antiinflammatory drug use, and were more likely to have a family history of breast cancer and more often reported being insufficient exercisers and smokers. No differences in other demographic variables, such as age and BMI, were observed between groups. For serologic variables, there were significant differences in levels of
Table 1 Baseline Clinical and Laboratory Characteristics of Participants
Age (y) Years since menopause (y) Body mass index (kg/m2) Alcohol consumption, n (%) Smokers, n (%) Physical activity (METs/wk) Regular NSAID use, n (%) Family history of BC, n (%) Triglyceride (mmol/L) Total cholesterol (mmol/L) LDL-c (mmol/L) HDL-c (mmol/L) Insulin (mIU/L) Glucose (mmol/L) Serum estradiol (pg/mL) Leptin (ng/mL) Marrow fat fraction (%)
Cases (n [ 56)
Controls (n [ 56)
P
63.0 8.5 11.2 7.1 24.2 3.4 5 (8.9) 4 (7.1) 9.2 4.6 4 (7.1) 3 (5.4) 2.89 1.21 4.33 1.62 3.56 0.83 1.02 0.34 7.37 2.50 4.51 0.81 13.24 4.09 19.89 5.53 72.7 8.4
62.1 7.8 14.5 6.4 23.3 3.0 2 (3.6) 2 (3.6) 14.5 5.8 1 (1.8) 1 (1.8) 2.01 1.14 4.00 1.37 2.37 0.70 1.50 0.39 7.01 2.16 4.40 0.68 10.06 3.78 15.02 4.84 60.1 7.9
>.05 <.05 >.05 >.05 >.05 <.05 >.05 >.05 <.05 >.05 <.05 <.05 >.05 >.05 <.05 <.05 <.001
Continuous data are presented as means (with SDs) and were compared using Student’s t-test or Wilcoxon rank sum test. Categorical data are presented as numbers (with % values) and were compared using the c2 test. Abbreviations: BC ¼ breast cancer; HDL-c ¼ high-density lipoprotein cholesterol; LDL-c ¼ low-density lipoprotein cholesterol; METs ¼ Metabolic Equivalent of Tasks; NSAID ¼ nonsteroidal anti-inflammatory drug.
Risk Factors for Breast Cancer and Clinicopathological Characteristics of Breast Cancer Table 2 depicts the univariate and multivariate analysis for the breast cancer risk and clinicopathological characteristics of breast cancer. In the univariate analysis, both serum leptin and marrow FF were significantly associated with risk of breast cancer and clinicopathological characteristics of breast cancer, including TNM, tumor size, lymph node metastasis, and histological grade. To determine independent predictors of the breast cancer risk and clinicopathological characteristics of breast cancer, we performed logistic regression analysis with adjustment for the established breast cancer risk factors. In the multivariable model, serum leptin was a significant predictor of breast cancer risk and clinicopathological characteristics of breast cancer. However, when marrow FF was additionally added to the regression model, marrow FF but not leptin level was observed to be an independent risk factor for breast cancer risk and clinicopathological characteristics of breast cancer (Table 2).
Discussion Marrow fat is an endocrine organ with the potential to affect bone remodeling and hematopoiesis. Although expansion of marrow fat may be driving bone loss in some clinical conditions such as osteoporosis, anorexia nervosa, and diabetes mellitus,2,26 compelling evidence suggests that marrow adiposity is not casually related to loss of skeletal integrity.27,28 Adipocytes, one of the major components of the breast microenvironment, have been shown to provide protumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo.29 Adipocyte-secreted factors, such as leptin and interleukin-6, have a paracrine effect on breast cancer cells. Several ex vivo studies demonstrated that leptin promotes the proliferation, migration, and survival of human breast cancer cell lines via leptin receptoreactivated signaling pathways.30,31 Leptin may enhance aromatase activity, which results in increasing the amount of estrogen in fat tissue, including intratumoral sites in breast tissue.32 Additionally, leptin levels also correlated with higher recurrence rates in estrogen- and progesteronereceptorepositive cancers, underscoring its role in increased
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Association of Marrow Adiposity and Breast Cancer Risk Figure 1 Representative Magnetic Resonance Spectroscopy (MRS). MRS Performed on the L3 Vertebral Body of a 68.3-Year-Old Woman With TNM Stage I Breast Cancer ([A] Fat Fraction [FF], 67.9%), a 67.5-Year-Old Woman With TNM Stage III Breast Cancer ([B] FF, 75.6%) and a 70-Year-Old Healthy Woman ([C] FF, 58.2%)
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invasiveness.33 These data provide support for the potential use of leptin-signaling inhibition as a novel treatment for breast cancer. Our data indicated serum leptin was positively related to postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer, a finding that is in line with previous data indicating breast cancer cells migrate to human bone tissueeconditioned medium in relation to elevated levels of leptin.8,13 Additionally, leptin receptors are expressed by breast cancer tissue and high expression of leptin receptor mRNA correlated with tumor size and poor prognosis in patients with hyperleptinemia.24 Conversely, some studies did not find an association between circulating inflammatory adipokines, such as leptin, and breast cancer risk.16,34 Such controversy may be attributable to different experimental designs, such as the measurement of leptin after diagnosis, varying levels of controlling for confounding factors, variation in sample sizes, differences in sample collection and measurement techniques, and inhomogeneity of associations with pre- and postmenopausal breast cancer. Interestingly, the association of circulating leptin with breast cancer risk was attenuated and lost statistical significance following inclusion of marrow fat content in the multivariable model, which may be inferred that any effects of serum leptin on breast cancer risk may be mediated, at least in part, through marrow adipose tissue. A potential limitation in the assessment of leptin in relation to breast
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cancer risk is that leptin primarily functions in a paracrine manner and plasma levels may be a poor surrogate for local levels.16 Circulating levels of leptin, thus, may not accurately reflect processes at the levels in breast or adipose tissue. Bone is a major site of breast cancer metastasis, indicating the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells in bone. Najar and colleagues35 demonstrated that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues. Breast cancer causes osteolytic lesions. One important factor in this process is the need for energy. Marrow fat tissue is metabolically distinct from peripheral and visceral fat depots. Marrow fat cells are filled with plenty of lipid droplets that may serve as effective sources of fatty acids in response to increased metabolic demand.36 Although there are several in vitro studies depicting these potentially dangerous effects of marrow adipocytes on prostate and breast cancer in particular,6-8 there are no reports assessing the effect of marrow fat content on breast cancer in vivo. The key finding of the present study is that marrow fat content, but not serum leptin, was positively associated with breast cancer incidence and clinicopathological characteristics of breast cancer. Our data provide in vivo imaging evidence supporting previous ex vivo study indicating fatty marrow is a depot of host-derived factors that prepare the “soil,” making it a more favorable environment for implantation and propagation of the
P
Abbreviations: CI ¼ confidence interval; FF ¼ fat fraction; OR ¼ odds ratio; TNM ¼ tumor-node-metastasis. a Univariate analysis. b Multivariable model adjusted for age, years since menopause, body mass index, smoking status, alcohol consumption, physical activity, regular nonsteroidal anti-inflammatory drug use, family history of breast cancer, blood lipid levels, glucose, endogenous estradiol levels.
(1.098-2.139) (1.304-4.410) (0.660-2.218) (1.137-3.018) 1.461 2.402 1.214 1.856
OR (95% CI) P
.004 <.001 .687 <.001 (1.054-2.168) (1.275-3.225) (0.593-2.306) (1.110-3.036) 1.513 2.035 1.168 1.903
OR (95% CI) P
<.001 <.001 .504 <.001 (1.196-2.548) (1.270-3.773) (0.717-3.405) (1.190-2.886) 1.695 2.193 1.326 1.804
OR (95% CI) P
.007 <.001 .832 <.001 (1.003-2.549) (1.261-3.584) (0.486-3.082) (1.118-2.847) 1.590 2.120 1.290 1.770
OR (95% CI) P
<.001 <.001 .415 <.001 (1.226-2.556) (1.543-4.251) (0.837-3.870) (1.306-2.910)
OR (95% CI)
1.764 2.562 1.440 1.940 Serum leptina Marrow FFa Serum leptinb Marrow FFb
Histological Grade Tumor Size TNM Lymph Node Status Risk for Breast Cancer
Table 2 Factors Associated With Breast Cancer Risk and Clinicopathologic Characteristics of Breast Cancer Assessed by Logistic Regression Analysis
.012 <.001 .581 <.001
Guanwu Li et al migrating metastatic cells.8 This is particularly evident for cancers that grow in adipocyte-rich microenvironments, such as breast cancer, or metastasize to the predominantly adipocyte-dominated host sites, such as metabolically active red bone marrow (eg, prostate, gastric, and ovarian cancers).37 Fatty acid binding protein 4 (FABP4) is a mediator of marrow adipocyte-tumor cell interactions in the bone metastatic niche. FABP4 inhibitor can suppress lipid trafficking between cancer cells and marrow fat cells, thus reducing the formation of the metastatic tumor cells in bone.7 In other words, marrow adipocyte FABP4 may serve as a potential therapeutic target for skeletal metastasis of breast cancer. The major limitation of our study is that the sample is crosssectional; therefore, we cannot directly assess the association between marrow fat content and postmenopausal breast cancer risk. Second, we did not conduct separate analyses for estrogen receptorepositive and enegative, hormone receptorepositive and enegative breast cancers. Third, despite careful controlling for potential confounding factors, such as YSM, BMI, physical activity, and family history of breast cancer, we did not adjust for bone mineral density, a well-established determinant influencing marrow fat volume. Finally, we did not perform a T2 correction of the MRS data, thus yielding the T2-weighted FF rather than the proton density FF.22 In comparison with the proton density FF, the T2-weighted FF overestimates the FF value. Further studies are needed to use multi-TE MRS to remove the T2 decay effects. In conclusion, our study suggests that marrow adiposity is an independent risk factor for breast cancer and clinicopathological characteristics of breast cancer in postmenopausal women. Given the potential tumor-supporting role of adipocytes, interventions aiming at the regulations of the balance between marrow adipogenesis and osteogenesis may open up new therapeutic avenues for bone metastasis of breast cancer and cancer-induced bone disease.
Clinical Practice Points Marrow fat tissue influences cancer bone metastasis. Effect of circulating leptin on breast cancer risk may be mediated
through marrow adiposity. Marrow adiposity is an independent risk factor for post-
menopausal breast cancer and clinicopathological characteristics of breast cancer. The removal of marrow fat expansion may open up new therapeutic avenues for bone metastasis of breast cancer.
Acknowledgments The authors thank Dr Liping Yang, Yuliang Zhang, and Ming Gu for their work in study coordination, data collection, and biostatistical support. This study was funded by the National Natural Science Foundation of China (NSFC) (81202809 and 81373856).
Disclosure The authors have stated that they have no conflicts of interest.
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20. Le Doussal V, Tubiana-Hulin M, Hacéne K, et al. 666 Have histological grade nuclear components (MSBR) of scarff bloom richardson (SBR) a prognostic value for lobular invasive breast carcinoma? Eur J Cancer 1995; 31:140-1. 21. Craig CL, Marshall AL, Sjostrom M, et al. International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc 2003; 35:1381-95. 22. Li G, Xu Z, Gu H, et al. Comparison of chemical shift-encoded water-fat MRI and MR spectroscopy in quantification of marrow fat in postmenopausal females. J Magn Reson Imaging 2017; 45:66-73. 23. Singhal V, Miller KK, Torriani M, Bredella MA. Short- and long-term reproducibility of marrow adipose tissue quantification by 1H-MR spectroscopy. Skeletal Radiol 2016; 45:221-5. 24. Miyoshi Y, Funahashi T, Tanaka S, et al. High expression of leptin receptor mRNA in breast cancer tissue predicts poor prognosis for patients with high, but not low, serum leptin levels. Int J Cancer 2006; 118:1414-9. 25. Gunter MJ, Hoover DR, Yu H, et al. Insulin, insulin-like growth factor-I, and risk of breast cancer in postmenopausal women. J Natl Cancer Inst 2009; 101:48-60. 26. Li G, Xu Z, Chen Y, et al. Longitudinal assessment of marrow fat content using three-point Dixon technique in osteoporotic rabbits. Menopause 2016; 23: 1339-44. 27. Cawthorn WP, Scheller EL, Parlee SD, et al. Expansion of bone marrow adipose tissue during caloric restriction is associated with increased circulating glucocorticoids and not with hypoleptinemia. Endocrinology 2016; 157:508-21. 28. Doucette CR, Horowitz MC, Berry R, et al. A high fat diet increases bone marrow adipose tissue (MAT) but does not alter trabecular or cortical bone mass in C57BL/6J mice. J Cell Physiol 2015; 230:2032-7. 29. Wolfson B, Eades G, Zhou Q. Adipocyte activation of cancer stem cell signaling in breast cancer. World J Biol Chem 2015; 6:39-47. 30. Strong AL, Ohlstein JF, Biagas BA, et al. Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers. Breast Cancer Res 2015; 17:112. 31. Kim SH, Nagalingam A, Saxena NK, Singh SV, Sharma D. Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3. Carcinogenesis 2011; 32:359-67. 32. Magoffin DA, Weitsman SR, Aagarwal SK, Jakimiuk AJ. Leptin regulation of aromatase activity in adipose stromal cells from regularly cycling women. Ginekol Pol 1999; 70:1-7. 33. Rene Gonzalez R, Watters A, Xu Y, et al. Leptin-signaling inhibition results in efficient anti-tumor activity in estrogen receptor positive or negative breast cancer. Breast Cancer Res 2009; 11:R36. 34. Mantzoros CS, Bolhke K, Moschos S, Cramer DW. Leptin in relation to carcinoma in situ of the breast: a study of pre-menopausal cases and controls. Int J Cancer 1999; 80:523-6. 35. Najar M, Fayyad-Kazan H, Faour WH, Badran B, Journe F, Lagneaux L. Breast cancer cells and bone marrow mesenchymal stromal cells: a regulated modulation of the breast tumor in the context of immune response. Inflamm Res 2017; 66: 129-39. 36. Morris EV, Edwards CM. The role of bone marrow adipocytes in bone metastasis. J Bone Oncol 2016; 5:121-3. 37. Hardaway AL, Herroon MK, Rajagurubandara E, Podgorski I. Bone marrow fat: linking adipocyte-induced inflammation with skeletal metastases. Cancer Metastasis Rev 2014; 33:527-43.
Magnetic Resonance Spectroscopy-Detected Change in Marrow Adiposity Is Strongly Correlated to Postmenopausal Breast Cancer Risk Guanwu Li,1 Zheng Xu,2 Alex Zhuang,3 Shixin Chang,1 Lingmi Hou,4 Yongsheng Chen,3 Maki Polat,5 Dongmei Wu6 Abstract Previous ex vivo studies described marrow adipocytes influencing cancer bone metastasis. The prognosis of marrow adiposity on breast cancer risk remains elusive. We hypothesized that marrow adiposity and leptin levels would be associated with breast cancer risk. We found marrow fat expansion, but not circulating leptin is a predictor of postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Purpose: To determine whether marrow fat fraction (FF) is correlated with postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Methods: Fifty-six patients with newly diagnosed and histologically confirmed postmenopausal breast cancer and 56 healthy controls underwent serologic test and magnetic resonance spectroscopyebased FF measurements. Data were analyzed by logistic multivariate regression models to determine the independent predictors of breast cancer risk and clinicopathological characters of breast cancer. Results: Patients with breast cancer had higher FF than that of the controls. Marrow FF showed positive association with serum leptin levels (r ¼ 0.607, P < .001) in the cases, but no relationship was found in the controls. In the univariate analysis, both levels of leptin and marrow FF were significantly associated with breast cancer risk and clinicopathological characteristics of breast cancer. In the multivariable model with adjustment for established breast cancer risk factors, serum leptin was a significant predictor of breast cancer risk (OR 1.746; 95% CI, 1.226-2.556) and clinicopathological characteristics of breast cancer including TNM, tumor size, lymph node status, and histological grade (OR 1.461-1.695); but when marrow FF was additionally added to the regression model, marrow FF but not leptin levels was observed to be an independent risk factor for breast cancer risk (OR 1.940; 95% CI, 1.306-2.910) and clinicopathological characteristics of breast cancer (OR 1.770-1.903). Conclusion: Marrow adiposity is a predictor of postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer. Clinical Breast Cancer, Vol. -, No. -, --- ª 2017 Elsevier Inc. All rights reserved. Keywords: Bone metastasis, Leptin, Marrow fat content, Menopause
Introduction G.L. and Z.X. contributed equally to this study as first authors. 1 Department of Radiology, Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China 2 Xinzhuang Community Health Center, Shanghai, China 3 Department of Radiology, Wayne State University, Detroit, MI 4 Breast Surgery, Affiliated Hospital of North Sichuan Medical College, Sichuan, China 5 School of Medicine and Public Health, University of Wisconsin, Madison, WI 6 Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai, China
Submitted: Sep 7, 2016; Revised: Dec 30, 2016; Accepted: Jan 9, 2017 Addresses for correspondence: Guanwu Li, MD, Department of Radiology, Yueyang Hospital, Shanghai University of TCM, 110 Ganhe Rd, Shanghai 200437, China E-mail contact: [email protected]
1526-8209/$ - see frontmatter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2017.01.004
Breast cancer metastasis is responsible for most breast cancer mortality. Bone is the most preferred site for distant metastasis of breast cancer and bone metastasis is an incurable complication of breast cancer, affecting 70% to 80% of advanced patients.1 Both marrow adipocytes and osteoblasts arise from common multipotent progenitor cells, known as bone marrow mesenchymal stromal cells (MSCs). The associations of marrow fat with other adipose tissue depots are complicated. Marrow fat may play crucial roles in modulation of hemopoiesis, metabolic homoeostasis, and osteogenesis.2,3 Previous study showed that marrow fat cells appear to be capable of translocating stored lipids to the metastatic tumor cells. These adipocyte-supplied lipids serve as an energy source for cancer cells, a process suggested to increase tumor cell proliferation,
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Association of Marrow Adiposity and Breast Cancer Risk motility, and invasion.4,5 Recent ex vivo studies describe marrow fat cells influencing cancer bone metastasis, particularly breast and prostate cancer6-8; however, the prognosis of marrow adiposity on modulating tumor colonization and macro-metastasis in the skeleton remains largely unexplored. Marrow adipocytes store quantities of fat and produce adipokines, such as leptin and adiponectin, which are known for their roles in the regulation of energy metabolism through endocrine-, paracrine-, and autocrine-mediated pathways.9 Adipokines also have been studied on cancer cells and are implicated in proliferation of breast, prostate, and lung cancers10-12; however, there have been some controversies in the currently available data. Some,13-15 but not all studies16,17 showed that there exist associations between breast cancer incidence and adipokines, in particular leptin. Because there exists a clear association between levels of leptin and marrow adiposity,9,18 we hypothesized that elevated marrow fat content as measured by magnetic resonance spectroscopy (MRS) and serum leptin would be associated with increased risk for breast cancer and clinicopathological characteristics of breast cancer in postmenopausal females.
Material and Methods Study Populations
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Fifty-six postmenopausal patients (mean age, 63.0 8.5 years) with more than 1 year since menopause (YSM) who were newly diagnosed and histologically confirmed breast cancer with no prior surgical, chemotherapy, or radiotherapy treatment between May 2013 and June 2016 were enrolled in this study. Diagnosis of breast cancer was confirmed histologically in each case and estrogen receptor status was determined. Tumors were classified as receptorpositive status according to the following criteria: 10% of positive histologically stained cells, an any “plus-system” description, 20 fmol/mg, an Allred score of 3, an immunoreactive score of 2, or an H-score of 10.19 The staging of breast cancer was determined according to the TNM system, and histological grade was determined according to the modified ScarffeBloomeRichardson criteria.20 The healthy control individuals (n ¼ 56) were age and body mass index (BMI) matched with the cases. All control participants were confirmed free from benign or malignant breast diseases by physical examination and mammography. Furthermore, the key exclusion criteria were as follows: (1) any disease known to affect bone metabolism, such as previous or current malignant tumor (except for breast cancer), exposure to chemo-radiotherapy, diabetes mellitus, chronic renal failure, thyroid and parathyroid diseases, vitamin D deficiency; (2) any previous or current use of medications known to affect bone metabolism, such as bisphosphonate, glucocorticoid, hormone replacement therapy, or calcium or vitamin D supplementation; and (3) any confounding factor that had the potential to interfere with the interpretation of the findings, such as hip or lumbar spine fracture. For all participants, comprehensive questionnaires were used to collect medical information, including demographics (such as age, YSM, height, body weight, and BMI), alcohol consumption, smoking status, physical activity, detailed medical history, use of medications, as well as family history of breast cancer and other cancers. Physical activity was assessed using the International Physical Activity Questionnaire short form, with data reported as
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Metabolic Equivalent of Task hours per week.21 Written informed consent was obtained for all individual participants included in the study according to a protocol approved by a local research ethics committee and in accordance with the 2008 Helsinki declaration.
Serologic Analysis Blood samples were collected from an overnight fast of at least 8 hours. Routine biochemical parameters, such as fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, were determined immediately after blood was drawn and plasma samples were stored at 20 C for further analyses. Serum leptin was determined by an enzyme-linked immunosorbent assay kit (Diagnostic Systems Laboratories Inc, Webster, TX). Levels of estradiol and insulin were assayed using a radioimmunoassay kit (Union Medical & Pharmaceutical Technology Tianjin Ltd, Tianjin, China); the intraassay and interassay coefficients of variation were less than 10%, respectively.
Marrow Fat Measurements Magnetic resonance imaging (MRI) examinations were performed on a MAGNETOM Verio 3T scanner (Siemens Medical Solutions, Erlangen, Germany). The images were acquired with the participant in the supine position using the standard spine-array receive coil. Sagittal T1- and T2-weighted turbo spin-echo sequences were used for morphological assessments of the lumbar region to exclude any confounders such as wedging of vertebrae, vertebral hemangiomas, end plate depression, and silent compression fractures. To quantify marrow fat fraction (FF), a tri-plane gradient echo localizer pulse sequence of the lumbar spine was used to guide positioning of a volume of interest (1.5 1.5 1.5 cm3) within the third vertebral body, and then single-voxel proton MRS data were acquired using the PRESS pulse sequence without water suppression with the following image parameters: repetition time, 3000 ms; echo time (TE), 30 ms; bandwidth, 2000 Hz; flip angle, 90 ; data points, 1024; number of acquisitions, 64. Six outer volume saturation bands were used to eliminate unwanted signal contamination from outside the voxel. Moreover, we applied default autoshimming as provided by the manufacturer. The acquisition time for the bone marrow MRS scan was 3 minutes 24 seconds. A commercially available imaging workstation (Siemens Syngo B17) was used for postprocessing of MRS data. Spectral assignments were based on previous studies,22,23 and only clearly identifiable peaks were measured. All spectra have shown a water peak located at 4.65 ppm and a lipid peak (bulk methylene) located at 1.30 ppm. FF value was calculated according to the following equation: FF ¼ (AUClipid/[AUClipid þ AUCwater]) 100%, where AUClipid and AUCwater referred to the peak area under the lipid and water curve, respectively.
Statistical Analysis Categorical data are described using frequencies and percentages. Continuous data are described using means SD. Normality of data was assessed using the Shapiro-Wilk test. Differences in the baseline characteristics between groups were evaluated using the c2 test for categorical data and Student’s t-test or Wilcoxon rank sum test for
Guanwu Li et al continuous data where appropriate. Pearson or Spearman correlation tests were used to determine the relationships of serum leptin, marrow FF, and clinicopathological characteristics of breast cancer (TNM, tumor size, lymph node metastasis, and histological grade) where appropriate. To determine independent predictors of clinicopathological characteristics of breast cancer and breast cancer risk, we performed multivariate logistic regression analysis to estimate odds ratios (ORs; OR of large tumor size [> 2.0 cm] against small tumor size [ 2.0 cm], histological grade III against grade I and II, lymph node positive against lymph node negative, TNM stage I against stage II-III17,24) and 95% confidence intervals (CIs), with adjustment for confounder-related breast cancer risk including age, YSM, BMI, smoking status, alcohol consumption, physical activity, regular nonsteroidal anti-inflammatory drug use, family history of breast cancer, glucose, insulin, endogenous estradiol levels, and blood lipids.13,25 Data were analyzed using IBM SPSS v. 23.0 (IBM SPSS, Armonk, NY). Statistical significance was set to P < .05.
leptin, estradiol, triglyceride, and high- and low-density lipoprotein cholesterol between groups; but no significant differences were found in total cholesterol, insulin, and fasting plasma glucose. As expected, patients with breast cancer had significantly higher marrow FF than that of the controls (P < .001) (Table 1 and Figure 1). This indicated significant accumulation of marrow fat in patients with breast cancer.
Relationships of Leptin, Marrow FF, and Clinicopathological Characteristics of Breast Cancer In the breast cancer group, marrow FF showed positive association with levels of leptin (r ¼ 0.607, P < .001); but no relationship was found in the controls. Marrow FF showed positive correlations with lymph node metastasis (r ¼ 0.701), TNM (r ¼ 0.611), tumor size (r ¼ 0.574), and histological grade (r ¼ 0.683). Serum leptin also was positively correlated with lymph node metastasis (r ¼ 0.530), TNM (r ¼ 0.585), tumor size (r ¼ 0.492), and histological grade (r ¼ 0.461, all P < .05).
Results Baseline Characteristics The baseline characteristics of the patients with breast cancer and their respective controls are presented in Table 1. Compared with the controls, patients with breast cancer had smaller YSM, greater alcohol consumption, a higher frequency of nonsteroidal antiinflammatory drug use, and were more likely to have a family history of breast cancer and more often reported being insufficient exercisers and smokers. No differences in other demographic variables, such as age and BMI, were observed between groups. For serologic variables, there were significant differences in levels of
Table 1 Baseline Clinical and Laboratory Characteristics of Participants
Age (y) Years since menopause (y) Body mass index (kg/m2) Alcohol consumption, n (%) Smokers, n (%) Physical activity (METs/wk) Regular NSAID use, n (%) Family history of BC, n (%) Triglyceride (mmol/L) Total cholesterol (mmol/L) LDL-c (mmol/L) HDL-c (mmol/L) Insulin (mIU/L) Glucose (mmol/L) Serum estradiol (pg/mL) Leptin (ng/mL) Marrow fat fraction (%)
Cases (n [ 56)
Controls (n [ 56)
P
63.0 8.5 11.2 7.1 24.2 3.4 5 (8.9) 4 (7.1) 9.2 4.6 4 (7.1) 3 (5.4) 2.89 1.21 4.33 1.62 3.56 0.83 1.02 0.34 7.37 2.50 4.51 0.81 13.24 4.09 19.89 5.53 72.7 8.4
62.1 7.8 14.5 6.4 23.3 3.0 2 (3.6) 2 (3.6) 14.5 5.8 1 (1.8) 1 (1.8) 2.01 1.14 4.00 1.37 2.37 0.70 1.50 0.39 7.01 2.16 4.40 0.68 10.06 3.78 15.02 4.84 60.1 7.9
>.05 <.05 >.05 >.05 >.05 <.05 >.05 >.05 <.05 >.05 <.05 <.05 >.05 >.05 <.05 <.05 <.001
Continuous data are presented as means (with SDs) and were compared using Student’s t-test or Wilcoxon rank sum test. Categorical data are presented as numbers (with % values) and were compared using the c2 test. Abbreviations: BC ¼ breast cancer; HDL-c ¼ high-density lipoprotein cholesterol; LDL-c ¼ low-density lipoprotein cholesterol; METs ¼ Metabolic Equivalent of Tasks; NSAID ¼ nonsteroidal anti-inflammatory drug.
Risk Factors for Breast Cancer and Clinicopathological Characteristics of Breast Cancer Table 2 depicts the univariate and multivariate analysis for the breast cancer risk and clinicopathological characteristics of breast cancer. In the univariate analysis, both serum leptin and marrow FF were significantly associated with risk of breast cancer and clinicopathological characteristics of breast cancer, including TNM, tumor size, lymph node metastasis, and histological grade. To determine independent predictors of the breast cancer risk and clinicopathological characteristics of breast cancer, we performed logistic regression analysis with adjustment for the established breast cancer risk factors. In the multivariable model, serum leptin was a significant predictor of breast cancer risk and clinicopathological characteristics of breast cancer. However, when marrow FF was additionally added to the regression model, marrow FF but not leptin level was observed to be an independent risk factor for breast cancer risk and clinicopathological characteristics of breast cancer (Table 2).
Discussion Marrow fat is an endocrine organ with the potential to affect bone remodeling and hematopoiesis. Although expansion of marrow fat may be driving bone loss in some clinical conditions such as osteoporosis, anorexia nervosa, and diabetes mellitus,2,26 compelling evidence suggests that marrow adiposity is not casually related to loss of skeletal integrity.27,28 Adipocytes, one of the major components of the breast microenvironment, have been shown to provide protumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo.29 Adipocyte-secreted factors, such as leptin and interleukin-6, have a paracrine effect on breast cancer cells. Several ex vivo studies demonstrated that leptin promotes the proliferation, migration, and survival of human breast cancer cell lines via leptin receptoreactivated signaling pathways.30,31 Leptin may enhance aromatase activity, which results in increasing the amount of estrogen in fat tissue, including intratumoral sites in breast tissue.32 Additionally, leptin levels also correlated with higher recurrence rates in estrogen- and progesteronereceptorepositive cancers, underscoring its role in increased
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Association of Marrow Adiposity and Breast Cancer Risk Figure 1 Representative Magnetic Resonance Spectroscopy (MRS). MRS Performed on the L3 Vertebral Body of a 68.3-Year-Old Woman With TNM Stage I Breast Cancer ([A] Fat Fraction [FF], 67.9%), a 67.5-Year-Old Woman With TNM Stage III Breast Cancer ([B] FF, 75.6%) and a 70-Year-Old Healthy Woman ([C] FF, 58.2%)
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invasiveness.33 These data provide support for the potential use of leptin-signaling inhibition as a novel treatment for breast cancer. Our data indicated serum leptin was positively related to postmenopausal breast cancer risk and clinicopathological characteristics of breast cancer, a finding that is in line with previous data indicating breast cancer cells migrate to human bone tissueeconditioned medium in relation to elevated levels of leptin.8,13 Additionally, leptin receptors are expressed by breast cancer tissue and high expression of leptin receptor mRNA correlated with tumor size and poor prognosis in patients with hyperleptinemia.24 Conversely, some studies did not find an association between circulating inflammatory adipokines, such as leptin, and breast cancer risk.16,34 Such controversy may be attributable to different experimental designs, such as the measurement of leptin after diagnosis, varying levels of controlling for confounding factors, variation in sample sizes, differences in sample collection and measurement techniques, and inhomogeneity of associations with pre- and postmenopausal breast cancer. Interestingly, the association of circulating leptin with breast cancer risk was attenuated and lost statistical significance following inclusion of marrow fat content in the multivariable model, which may be inferred that any effects of serum leptin on breast cancer risk may be mediated, at least in part, through marrow adipose tissue. A potential limitation in the assessment of leptin in relation to breast
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cancer risk is that leptin primarily functions in a paracrine manner and plasma levels may be a poor surrogate for local levels.16 Circulating levels of leptin, thus, may not accurately reflect processes at the levels in breast or adipose tissue. Bone is a major site of breast cancer metastasis, indicating the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells in bone. Najar and colleagues35 demonstrated that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues. Breast cancer causes osteolytic lesions. One important factor in this process is the need for energy. Marrow fat tissue is metabolically distinct from peripheral and visceral fat depots. Marrow fat cells are filled with plenty of lipid droplets that may serve as effective sources of fatty acids in response to increased metabolic demand.36 Although there are several in vitro studies depicting these potentially dangerous effects of marrow adipocytes on prostate and breast cancer in particular,6-8 there are no reports assessing the effect of marrow fat content on breast cancer in vivo. The key finding of the present study is that marrow fat content, but not serum leptin, was positively associated with breast cancer incidence and clinicopathological characteristics of breast cancer. Our data provide in vivo imaging evidence supporting previous ex vivo study indicating fatty marrow is a depot of host-derived factors that prepare the “soil,” making it a more favorable environment for implantation and propagation of the
P
Abbreviations: CI ¼ confidence interval; FF ¼ fat fraction; OR ¼ odds ratio; TNM ¼ tumor-node-metastasis. a Univariate analysis. b Multivariable model adjusted for age, years since menopause, body mass index, smoking status, alcohol consumption, physical activity, regular nonsteroidal anti-inflammatory drug use, family history of breast cancer, blood lipid levels, glucose, endogenous estradiol levels.
(1.098-2.139) (1.304-4.410) (0.660-2.218) (1.137-3.018) 1.461 2.402 1.214 1.856
OR (95% CI) P
.004 <.001 .687 <.001 (1.054-2.168) (1.275-3.225) (0.593-2.306) (1.110-3.036) 1.513 2.035 1.168 1.903
OR (95% CI) P
<.001 <.001 .504 <.001 (1.196-2.548) (1.270-3.773) (0.717-3.405) (1.190-2.886) 1.695 2.193 1.326 1.804
OR (95% CI) P
.007 <.001 .832 <.001 (1.003-2.549) (1.261-3.584) (0.486-3.082) (1.118-2.847) 1.590 2.120 1.290 1.770
OR (95% CI) P
<.001 <.001 .415 <.001 (1.226-2.556) (1.543-4.251) (0.837-3.870) (1.306-2.910)
OR (95% CI)
1.764 2.562 1.440 1.940 Serum leptina Marrow FFa Serum leptinb Marrow FFb
Histological Grade Tumor Size TNM Lymph Node Status Risk for Breast Cancer
Table 2 Factors Associated With Breast Cancer Risk and Clinicopathologic Characteristics of Breast Cancer Assessed by Logistic Regression Analysis
.012 <.001 .581 <.001
Guanwu Li et al migrating metastatic cells.8 This is particularly evident for cancers that grow in adipocyte-rich microenvironments, such as breast cancer, or metastasize to the predominantly adipocyte-dominated host sites, such as metabolically active red bone marrow (eg, prostate, gastric, and ovarian cancers).37 Fatty acid binding protein 4 (FABP4) is a mediator of marrow adipocyte-tumor cell interactions in the bone metastatic niche. FABP4 inhibitor can suppress lipid trafficking between cancer cells and marrow fat cells, thus reducing the formation of the metastatic tumor cells in bone.7 In other words, marrow adipocyte FABP4 may serve as a potential therapeutic target for skeletal metastasis of breast cancer. The major limitation of our study is that the sample is crosssectional; therefore, we cannot directly assess the association between marrow fat content and postmenopausal breast cancer risk. Second, we did not conduct separate analyses for estrogen receptorepositive and enegative, hormone receptorepositive and enegative breast cancers. Third, despite careful controlling for potential confounding factors, such as YSM, BMI, physical activity, and family history of breast cancer, we did not adjust for bone mineral density, a well-established determinant influencing marrow fat volume. Finally, we did not perform a T2 correction of the MRS data, thus yielding the T2-weighted FF rather than the proton density FF.22 In comparison with the proton density FF, the T2-weighted FF overestimates the FF value. Further studies are needed to use multi-TE MRS to remove the T2 decay effects. In conclusion, our study suggests that marrow adiposity is an independent risk factor for breast cancer and clinicopathological characteristics of breast cancer in postmenopausal women. Given the potential tumor-supporting role of adipocytes, interventions aiming at the regulations of the balance between marrow adipogenesis and osteogenesis may open up new therapeutic avenues for bone metastasis of breast cancer and cancer-induced bone disease.
Clinical Practice Points Marrow fat tissue influences cancer bone metastasis. Effect of circulating leptin on breast cancer risk may be mediated
through marrow adiposity. Marrow adiposity is an independent risk factor for post-
menopausal breast cancer and clinicopathological characteristics of breast cancer. The removal of marrow fat expansion may open up new therapeutic avenues for bone metastasis of breast cancer.
Acknowledgments The authors thank Dr Liping Yang, Yuliang Zhang, and Ming Gu for their work in study coordination, data collection, and biostatistical support. This study was funded by the National Natural Science Foundation of China (NSFC) (81202809 and 81373856).
Disclosure The authors have stated that they have no conflicts of interest.
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