Musculoskeletal tumors are frequent recording an incidence of 3/1,000 in USA, but only 0.67% are malignant, that pose a serious public health problem in the modern world (1). Malignant musculoskeletal tumors are associated with high mortality and disability rates (2–4). Although it is not possible to determine the exact cost of the burden of malignant musculoskeletal tumors, a good example comes from a model for the productivity costs of cancer mortality that projected the annual expenses for USA at approximately $115.8 billion in the year 2000, and $147.6 billion for 2020 (5). It is very important to raise global awareness of the growing burden of malignant musculoskeletal tumor. Benign and malignant musculoskeletal tumours are notoriously difficult to differentiate and represent a clinical, radiological and histological challenge. Meanwhile, accurate prediction of the grade of tumor cell before surgical removal could affect the type of surgery and need for adjuvant therapy, but it is not always possible with the current imaging methods. Typical imaging characteristics exist for common benign lesions such as lipomas and hemangiomas. In contrast, no specific imaging features and clinical manifestations exist for certain musculoskeletal tumors. Therefore, distinguishing between malignant tumors and benign tumors is difficult using common methods (6,7). At present, pathological inspection is clearly the ‘gold standard’ for identifying pathological changes, and histopathology is the most objective method because it involves the direct examination of pathological changes. However, the inherent shortcoming of histopathology is that it involves invasive inspection. Therefore, there is an urgent need for clinicians to identify non-invasive approaches that are comparable to biopsies to qualitatively describe lesions.

Functional magnetic resonance imaging has become commonly used worldwide and has great potential for characterizing pathological changes (8,9). Functional magnetic resonance imaging includes perfusion-weighted imaging, diffusion-weighted imaging and magnetic resonance spectroscopy.

Tumor angiogenesis is the formation of new blood vessels by budding or sprouting from the existing vasculature (10). Angiogenesis plays a key role in the tumorigenesis, invasion and metastasis of solid tumors (11,12) and is considered a sign of tumor invasion because the resulting rich vascular network provides tumor cells with sufficient oxygen and nutrients and contributes to tumor metastasis (13). The microvessel density (MVD) count is the standard method used to analyze tumor angiogenesis (14). Dynamic observations of angiogenesis and the functional status in living organisms cannot be performed due to the invasiveness and in vitro of such procedures.

Three-dimensional arterial spin labeling (3D-ASL) can perform using a pseudo-continuous arterial spin labeling sequence that uses blood as an endogenous contrast agent, allowing noninvasive perfusion measurements to be performed without gadolinium administration. The objective of our study is to evaluate 3D-ASL perfusion imaging in discriminating between benign, intermediate and malignant musculoskeletal tumors as well as to analyze the correlation between tumor blood flow (TBF) and MVD.

There were 12 subjects in the benign group, 10 in the intermediate group and 22 in the malignant group. Detailed diagnoses are reported in Table I. Routine pathological sections were obtained in all subjects (Figs. 1A, 2A and 3A). All tumor sections were positive for CD34 (Figs. 1B, 2B and 3B). The MVD counts were 10.38±4.58 and 14.64±6.69 in the benign and intermediate groups, respectively, and 32.97±11.61 in the malignant group.

Good inter-observer agreement was found for the TBF measurements made by the two observers as assessed by the intra-class correlation coefficient test (ICC=0.891, P<0.05). The brightly colored regions in TBF images obtained by 3D-ASL represent areas of high perfusion. The solid-appearing parts of malignant tumors were brighter in color than the benign and intermediate tumors (Figs. 1C, 2C and 3C), which can be observed more obviously in fusion images (Figs. 1D, 2D and 3D). Additionally, necrotic or cystic areas demonstrated perfusion defects. Significant differences were found in the MVD and TBF among the three groups (F=28.33, P<0.05, F=32.34, P<0.05, respectively). The MVD and TBF results are displayed in Table II. The MVD and TBF values were higher for malignant tumors than for benign and intermediate tumors (P<0.05), and TBF was not significantly different between benign and intermediate tumors.

The ROC curve of malignant musculoskeletal tumors plotted in Fig. 4 demonstrates that the TBF AUC_ROC is 0.951 and that the ROC cut-off value of TBF is 45 ml·min⁻¹·100 g⁻¹ (P<0.05). The diagnostic sensitivity and specificity are 90.5% and 100%, respectively.

According to Pearson's correlation analysis, a significant positive correlation was found between TBF and MVD (r=0.784, P<0.05), as demonstrated by the approximately linear positive correlation shown in Fig 5.

Angiogenesis is a dynamic process that is related to many factors. Under physiological conditions, angiogenesis is strictly controlled by various factors to maintain the delicate balance between angiogenic and anti-angiogenic factors (16). Under pathological conditions, in which this regulation is out of balance, angiogenic factors surpass anti-angiogenic factors, and control is lost (17). The presence of many immature new blood vessels is an important feature of malignant tumors. Angiogenesis in tumors is closely related to the tumor growth rate, the grade of malignancy, the possibility of invasion or metastasis and the prognosis (18–20). Quantitatively analyzing angiogenesis is therefore critical. Since Weidner et al (14) first used MVD to evaluate tumor angiogenesis, many scholars (21,22) have investigated and analyzed tumor angiogenesis in a variety of solid tumors using the MVD. At present, the MVD is the ‘gold standard’ for assessing tumor angiogenesis because it is an objective and sensitive measure. In the present study, differences in the MVD between the benign and malignant groups and between the intermediate and malignant groups were statistically significant (P<0.05). Thus, the MVD may reflect the nature and the degree of differentiation of musculoskeletal tumors to a certain extent. Luczynska and colleagues reached a similar conclusion in their study of prostate tumors (23). In our study, no significant difference in the MVD was found between the benign and intermediate groups. There are a couple possible explanations for this result. First, although we investigated various musculoskeletal tumor types, the sampling capacity of the study was limited. Second, tumor angiogenesis is not only related to the number of capillaries but also to the function of capillaries, which includes vascular endothelial cell and basement membrane integrity, blood vessel permeability and capillary flow. Therefore, further research on the subject is warranted. Although we agree that the MVD is the ‘gold standard’ for identifying angiogenesis in tumors, MVD counts cannot be extensively performed due to invasiveness as well as to in vitro assay defects (24).

Functional imaging can evaluate tumor angiogenesis based on the level of perfusion. CT perfusion imaging (25) and MR perfusion-weighted imaging (26) can both measure perfusion, but both modalities require the intravenous injection of contrast medium, which may be associated with contrast medium allergies or side effects to the viscera. CT examination is also associated with a risk of radiation exposure. Loizides et al (27) showed by Contrast Enhanced Ultrasound that the enhancement curves of benign and malignant tumors are different. This difference may contribute to the ability to distinguish between benign and malignant tumors of a similar size and location. However, the results were based on the analysis of semi-quantitative data; therefore, the reliability was not very good.

In this study, 3D-ASL was performed using a pseudo-continuous arterial spin labeling sequence that uses blood as an endogenous contrast agent, allowing noninvasive perfusion measurements to be performed without gadolinium administration. Inflowing blood is selectively labeled with the opposite magnetization of the destination tissue. The difference between a labeled image (tag) and an unlabeled image (control) can be used to calculate tissue perfusion. 3D-ASL uses a spin echo sequence as an acquisition approach to obtain a wide range of perfusion with a low specific absorption rate (SAR) and a high signal-to-noise ratio and tag rate. 3D-ASL, first applied clinically to examine the brain, has recently demonstrated some clinical value in body examinations (28). To date, ASL perfusion weighted-imaging has rarely been performed on musculoskeletal tumors in humans. However, a few studies using a rabbit VX2 tumor model have used 3D-ASL to evaluate tumor angiogenesis (29). In the present study, the TBF values of benign and intermediate tumors were lower than the TBF value of malignant tumors; this finding is consistent with the MVD results. Not only the organ injury risk but also the higher cost and poor repeatability due to intravenous injection limit the use of MR perfusion-weighted imaging with contrast agents, although CT perfusion imaging and MR perfusion-weighted imaging have achieved similar positive results (30,31). Although both TBF and MVD relate to angiogenesis, how close the relationship is between them remains unknown. According to our research, TBF is significantly positively correlated with MVD. This conclusion strengthens the theory that TBF indicates angiogenesis by reflecting tissue perfusion (32). Therefore, we believe that TBF obtained from 3D-ASL is an effective biomarker of tumor angiogenesis.

According to the ROC analysis, TBF has good sensitivity and specificity in the characterization musculoskeletal tumors. We consider 3D-ASL an important sequence in discriminating between benign, intermediate and malignant musculoskeletal tumors used in combination with routine imaging methods. Additionally, TBF imaging combined with traditional structural imaging can provide direct tumor perfusion information for future operation or further treatment.

This paper has following limits: The tumors are very heterogenous. Many of these are represented by a single case. Furthermore, 3D-ASL is operator-dependent as it is subjective the choice of ROIs in the quantitative analysis. Another problem is that TBF image cannot match the structure image perfectly due to technique reasons, that will cause the deviation in measurement. Nevertheless, we believe that the inclusion of 3D-ASL in the work-up of musculoskeletal masses and the Correlation with MVD can serve as a new, fast-forward diagnostic tool for distinguishing malignant musculoskeletal tumors from their benign counterparts, the more so as 3D-ASL is a broadly available, non-invasive technique compared with contrast enhanced MRI and CT perfusion, for example.

In conclusion, the MVD represents musculoskeletal tumor angiogenesis and may reflects their biological behavior. Therefore, the MVD has clinical value in discriminating between types of tumors. Furthermore, a correlation exists between TBF and the MVD. 3D-ASL could be useful for pathological examination to a certain extent and is promising as a repeatable, completely non-invasive technique to evaluate musculoskeletal tumor angiogenesis in vivo. It has some application to determination of malignancy, but that it is premature to state that it can be used as a sole, or even dominant criterion. At least, the inclusion of 3D-ASL in established perfusion-based assessment protocols improves the evaluation of musculoskeletal masses. By the analysis of tumour perfusion, an important aspect of tumour pathophysiology-which is beyond the scope of grey-scale and conventional contrast enhanced MRI-becomes assessable. This has practical consequences for patient management: Better detection of malignant tumours by 3D-ASL and thus timely, fast-forward institution of further patient work-up, biopsy and treatment.

For the near future, a multicenter study is hoped in order to increase the number of the subject and the type of the musculoskeletal tumor, that will help the persuasion of the research.