Application of StrucGP in medical immunology: site-specific N-glycoproteomic analysis of macrophages.

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc4Hex5Fuc1Neu5Ac1, N4H5F1S1) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.

Evaluation of absorbent cotton for glycopeptide enrichment.

Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200µL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.

Proteomic characterization of four subtypes of M2 macrophages derived from human THP-1 cells.

Macrophages are widely distributed immune cells that contribute to tissue homeostasis. Human THP-1 cells have been widely used in various macrophage-associated studies, especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes. However, the molecular characterization of four M2 subtypes (M2a, M2b, M2c, and M2d) derived from THP-1 has not been fully investigated. In this study, we systematically analyzed the protein expression profiles of human THP-1-derived macrophages (M0, M1, M2a, M2b, M2c, and M2d) using quantitative proteomics approaches. The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated. The results showed that M2a and M2b, and M2c and M2d have very similar protein expression profiles. These data could serve as an important resource for studies of macrophages using THP-1 cells, and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.

Precision Structural Interpretation of Site-Specific N-Glycans in Seminal Plasma.

N-Linked glycoproteins are rich in seminal plasma, playing various essential roles in supporting sperm function and the fertilization process. However, the detailed information on these glycoproteins, particularly site-specific glycan structures, is still limited. In this study, a precision site-specific N-glycoproteome map of human seminal plasma was established by employing the site-specific glycoproteomic approach and a recently developed glycan structure interpretation software, StrucGP. A total of 9567 unique glycopeptides identified in human seminal plasma were composed of 773 N-linked glycan structures and 1019 N-glycosites from 620 glycoproteins. These glycans were comprised of four types of core structures and 13 branch structures. The majority of identified glycoproteins functioned in response to stimulus and immunity. As we reported in human spermatozoa, heavy fucosylation (fucose residues ≥6 per glycan) was also detected on seminal plasma glycoproteins such as clusterin and galectin-3-binding protein, which were involved in the immune response of biological processes and reactome pathways. Comparison of site-specific glycans between seminal plasma and spermatozoa revealed more complicated glycan structures in seminal plasma than in spermatozoa, even on their shared glycoproteins. These present data will be greatly beneficial for the in-depth structural and functional study of glycosylation in the male reproduction system.

Transcription Regulation of Tceal7 by the Triple Complex of Mef2c, Creb1 and Myod.

Tceal7 has been identified as a direct, downstream target gene of MRF in the skeletal muscle. The overexpression of Tceal7 represses myogenic proliferation and promotes cell differentiation. Previous studies have defined the 0.7 kb upstream fragment of the Tceal7 gene. In the present study, we have further determined two clusters of transcription factor-binding motifs in the 0.7 kb promoter: CRE#2-E#1-CRE#1 in the proximal region and Mef2#3-CRE#3-E#4 in the distal region. Utilizing transcription assays, we have also shown that the reporter containing the Mef2#3-CRE#3-E#4 motifs is synergistically transactivated by Mef2c and Creb1. Further studies have mapped out the protein-protein interaction between Mef2c and Creb1. In summary, our present studies support the notion that the triple complex of Mef2c, Creb1 and Myod interacts with the Mef2#3-CRE#3-E#4 motifs in the distal region of the Tceal7 promoter, thereby driving Tceal7 expression during skeletal muscle development and regeneration.

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa.

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.

Biological Functions and Large-Scale Profiling of Protein Glycosylation in Human Semen.

Glycosylation is one of the most important post-translational modifications of proteins and plays an essential role in spermatogenesis, maturation, extracellular quality control, capacitation, sperm-egg recognition, and final fertilization. Spermatozoa are synthesized in the testes inactively with a thick glycocalyx and passed through the epididymis for further modification by glycosylation, deglycosylation, and integration to reach maturation. Subsequently, sperm capacitation and further fertilization require redistribution of glycoconjugates and dramatic glycocalyx modification of the spermatozoa surface. Furthermore, glycoproteins and glycans in seminal plasma are functional in maintaining spermatozoa structure and stability. Therefore, aberrant glycosylation may cause alteration of semen function and even infertility. Currently, mass spectrometry-based technologies have allowed large-scale profiling of glycans and glycoproteins in human semen. Quantitative analysis of semen glycosylation has also indicated many involved glycoproteome issues in male infertility and the potential biomarkers for diagnosis of male infertility in clinical. This review summarizes the role of glycosylation during spermatozoa development, the large-scale profiling of glycome and glycoproteome in human semen, as well as the association of aberrant glycosylation with infertility.

Correlations Among Ultrasound-Guided Diffuse Optical Tomography, Microvessel Density, and Breast Cancer Prognosis.

OBJECTIVES: To investigate the correlation among ultrasound-guided diffuse optical tomography (DOT), microvessel density, and breast cancer prognosis. METHODS: Before surgery, the total hemoglobin (Hb) concentrations of 184 female patients with breast cancer with only a single lesion were measured. During follow-up, 23 patients had recurrence or metastatic disease after surgery. Among these patients, 18 with recurrence or metastatic disease within 3 years after surgery were paired with 18 patients without recurrence or metastatic disease. We retrospectively reviewed the pathologic sections of those 36 patients, conducted immunohistochemical staining, and counted the microvessel densities. Then we analyzed the correlation between microvessel density and total Hb, compared total Hb and microvessel density among breast cancers with different prognoses, and tested the value of DOT in predicting the prognosis of breast cancer. RESULTS: Microvessel density and total Hb were linearly correlated (r = 0.584; P < .001). Total Hb and microvessel density were significantly increased in the metastasis group (P = .001 and .027, respectively). A receiver operating characteristic curve analysis showed that at a total Hb cutoff value of 221.7 µmol/L, the sensitivity, specificity, and area under the curve of DOT for predicting recurrence or metastasis were 0.826, 0.516, and 0.660, respectively. CONCLUSIONS: The total Hb concentration can reflect a tumor's blood supply. Patients with a high total Hb concentration and microvessel density have a higher risk for a poorer prognosis. Total Hb can be used as an indicator of breast cancer prognosis. Diffuse optical tomography can help physicians identify patients with a high risk of metastasis and make clinical decisions.

Ultrasound Identifies Broken Drainage Tube Postthyroidectomy.

Drains are routinely used after thyroidectomy and lateral neck dissection. In rare cases, the drainage tube is broken and retained in the neck. It is difficult to identify the sonographic features of the remnant tube due to variable reasons. However, through comparative and phantom observations, we noticed the double-tract sign of the tube wall and drainage holes as the key points to solving this problem. We report such a case of the ultrasound-guided localization of a broken drainage tube that was retained in the patient.

Tumor biology, clinicopathological characteristics and prognosis of screen detected T1 invasive non-palpable breast cancer in asymptomatic Chinese women (2001-2014).

BACKGROUND: Mammography screening usually detects low-risk breast cancer in the western world. However, little is known about the ultrasound and mammography screen-detected T1 invasive non-palpable breast cancer (NPBC) in asymptomatic Chinese women. RESULTS: With the increase of tumor size (T1a, b, c), lymph node positivity (8.7%, 18.3%, 26.0%, p = 0.018), pN (p = 0.028) and TNM stage (p = 0.035) increased accordingly. Tumor size (T1a, b, c) was correlated with high Ki-67 index (defined as ≥ 14%, 37.9%, 45.8%, 56.2%, p = 0.017), chemotherapy (20.4%, 35.2%, 57.3%, p < 0.001) and targeted therapy (2.9%, 9.9%, 15.1%, p = 0.008). T1a disease had higher chance of being luminal A and accompanied with ductal carcinoma in situ (DCIS), while T1c tumor being triple-negative and without DCIS. The 5-year disease free survival (DFS) of T1a, b, c NPBC were 99.0%, 96.9% and 92.9%, whereas the 5-year overall survival (OS) were 100.0%, 100.0% and 97.9% respectively. There was no significant difference in 5-year DFS or OS among the T1 NPBC subgroups or subtypes/immunophenotypes. PATIENTS AND METHODS: From 2001 to 2014, 4,574 screening positive women received biopsies in Peking Union Medical College (PUMC) Hospital, and 729 NPBC including 437 T1 unilateral invasive NPBC were diagnosed. With a median follow-up time of 32 months (6-163 months), the clinicopathological characteristics, treatment choice, 5-year DFS and OS were compared between T1a, T1b and T1c NPBC. The DFS and OS prognostic factors were identified. CONCLUSION: Screen-detected T1 invasive NPBC could be regarded as low-risk cancer in Chinese women. TNM stage and LN metastasis instead of molecular subtype was identified as the DFS prognostic factors while radiotherapy as the OS predictor.

Clinicopathological characteristics and long-term prognosis of screening detected non-palpable breast cancer by ultrasound in hospital-based Chinese population (2001-2014).

PURPOSE: The mainstay modality of breast cancer screening in China is the hospital-based opportunistic screening among asymptomatic self-referred women. There is little data about the ultrasound (US) detected non-palpable breast cancer (NPBC) in Chinese population. METHODS: We analyzed 699 consecutive NPBC from 1.8-2.3 million asymptomatic women from 2001 to 2014, including 572 US-detected NPBC from 3,786 US-positive women and 127 mammography (MG) detected NPBC from 788 MG-positive women. The clinicopathological features, disease-free survival (DFS) and overall survival (OS) were compared between the US- and MG-detected NPBC. Prognostic factors of NPBC were identified. RESULTS: Compared to MG, US could detect more invasive NPBC (83.6% vs 54.3%, p<0.001), lymph node positive NPBC (19.1% vs 10.2%, p=0.018), lower grade (24.8% vs 16.5%, p<0.001), multifocal (19.2% vs 6.3%, p<0.001), PR positive (71.4% vs 66.9%, p=0.041), Her2 negative (74.3% vs 54.3%, p<0.001), Ki67 high (defined as >14%, 46.3% vs 37.0%, p=0.031) cancers and more NPBC who received chemotherapy (40.7% vs 21.3%, p<0.001). There was no significant difference in 10-year DFS and OS between US-detected vs MG-detected NPBC, DCIS and invasive NPBC. For all NPBC and the US-detected NPBC, the common DFS-predictors included pT, pN, p53 and bilateral cancers. CONCLUSION: US could detect more invasive, node-positive, multifocal NPBC in hospital-based asymptomatic Chinese female, who could achieve comparable 10-year DFS and OS as MG-detected NPBC. US would not delay early detection of NPBC with improved cost-effectiveness, thus could serve as the feasible initial imaging modality in hospital-based opportunistic screening among Chinese women.

Correlation between Ultrasound-guided Diffuse Optical Tomography and Hypoxia-inducible Factor-1Α of Breast Cancer.

Objective To investigate the correlation between ultrasound-guided diffuse optical tomography (US-DOT) and hypoxia-inducible factor-1Α (HIF-1Α) of breast cancer. Methods Totally 69 patients with pathologically confirmed breast cancer underwent preoperative conventional breast ultrasonography examinations and US-DOT at Peking Union Medical College Hospital From October 2007 to February 2010 were enrolled in this study.After surgery,immunohistochemical staining of HIF-1Α and CD34 were performed,and the differences of total hemoglobin concentration (THC) and microvessel density (MVD) between HIF-1Α positive and negative groups were analyzed. Results HIF-1Α was positive in 12 cases (17.4%) and negative in 57 cases (82.6%). The average THC and MVD of HIF-1Α-positive cases were (274.763±77.661) Μmol/L and (33.8±10.8)/0.2 mm(2) respectively. The average THC and MVD of HIF-1Α-negative cases were (228.059±65.760)Μmol/L and (28.4±7.4)/0.2 mm(2). MVD(t=2.049,P=0.04) and THC(t=2.167,P=0.034) of HIF-1Α-positive group were significantly higher than those of HIF-1Α-negative group. Conclusions HIF-1Α can promote tumor angiogenesis and thus increase the blood supply and THC. As an indicator of tumor blood supply,THC can indirectly reflect the angiogenic activity of breast cancer.

Rapid Detection and Characterisation of Triterpene Saponins from the Root of Pulsatilla chinensis (Bunge) Regel by HPLC-ESI-QTOF-MS/MS.

INTRODUCTION: Triterpene saponins are the major bioactive components in the root of Pulsatilla chinensis (Bunge) Regel (RPC), and have been reported to possess antitumor and immunological adjuvant activities. However, the isolation, purification and elucidation procedures of triterpene saponins from RPC are difficult and time consuming due to high polarity and structural similarity. OBJECTIVES: To develop an analytical strategy for discovering and elucidating triterpene saponins in RPC. METHODS: Methanolic extract of RPC is analysed by high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight-mass spectrometry (HPLC-ESI-QTOF-MS/MS). The MS and MS/MS experiments are conducted using the negative-ionisation mode, in order to provide molecular-mass information and production spectra for the structural elucidation of compounds. RESULTS: Based on retention times, accurate mass and mass spectrometric fragmentation, 24 triterpene saponins are identified or tentatively elucidated from RPC, of which nine triterpene saponins were not reported previously. CONCLUSION: The HPLC-ESI-QTOF-MS/MS could be employed as a rapid, effective technique to screen and identify triterpene saponins in RPC without tedious and time-consuming isolation of pure constituents. Copyright © 2016 John Wiley & Sons, Ltd.

Local Recurrent Phyllodes Tumors of the Breast: Clinical and Sonographic Features.

OBJECTIVES: The purpose of this study was to investigate the clinical, pathologic, and sonographic features of local recurrent phyllodes tumors of the breast and provide evidence for diagnosing tumor recurrence. METHODS: A total of 127 patients with phyllodes tumors who underwent surgery from 1993 to 2012 were followed. The clinical, pathologic, and sonographic features of local recurrent tumors were analyzed. RESULTS: Of the 127 patients, pathologic diagnoses were benign, borderline, and malignant in 75 (59.1%), 41 (32.3%), and 11 (8.7%) patients, respectively. The patients were followed for a mean duration ± SD of 50.9 ± 42.1 months (range, 2-225 months). Twenty-one patients had local recurrence, for a recurrence rate of 16.5%. The benign, borderline, and malignant recurrence rates were 14.7%, 17.1%, and 27.3%. The local recurrence in the 21 patients occurred after a mean interval of 19.4 ± 13.4 months. The mean times to local recurrence were 20.2 ± 12.1, 16.9 ± 10.8, and 20.3 ± 19.0 months for benign, borderline, and malignant tumors. The histologic characteristics of the recurrent tumors were similar to those of the initial tumors in 14 patients (66.7%), whereas the characteristics changed in 7 (33.3%). The sonographic features of the recurrent tumors were as follows: 69.4% were lobulated; 88.9% appeared as well-defined lesions; 41.7% had posterior acoustic enhancement; 11.1% had intramural cystic areas; and 83.4% had moderate or marked blood flow. CONCLUSIONS: The recurrence rate for phyllodes tumors is high. One to 2 years after surgery is the key time for follow-up. The sonographic features contribute to recurrence diagnosis.

Diffuse optical tomography of breast carcinoma: can tumor total hemoglobin concentration be considered as a new promising prognostic parameter of breast carcinoma?

RATIONALE AND OBJECTIVES: Diffuse optical tomography (DOT) is an emerging functional modality, which can reflect tumor metabolic activity and angiogenesis. The purpose of this exploratory study was to correlate the total hemoglobin concentration (THC) measured by noninvasive DOT with prognostic factors in breast carcinomas. MATERIALS AND METHODS: We prospectively imaged 251 breast carcinomas in 229 consecutive women (mean age, 51.18 ± 12.32 years) using DOT from 2007 to 2010. Tumor angiogenesis and metabolic activity were assessed based on quantitatively measured THC. The THC was correlated with prognostic factors, including tumor size, histopathologic classification, histologic grade, estrogen receptor (ER), progesterone receptor (PR), c-erbB-2, and p53. RESULTS: In univariate analysis, THC was significantly correlated with the following prognostic factors: tumor size (P < .001), histologic grade (P < .001), ER (P < .05), PR (P < .001), and c-erbB-2 (P < .05). THC was not associated with histopathologic classification (P = .170) or p53 (P = .463). On the basis of a stepwise multiple regression analysis, THC of invasive ductal carcinoma was significantly correlated with tumor size (P < .001), histologic grade (P < .001), and PR (P < .05). CONCLUSIONS: THC was associated with prognostic factors of breast carcinoma. THC may be considered as a new prognostic parameter of breast carcinoma and a prediction of tumor behavior and biological activity.

[Automated breast volume scanning in the diagnosis of breast intraductal papilloma].

OBJECTIVE: To investigate the value of automated breast volume scanning (ABVS) in the diagnosis of breast intraductal papilloma (IDP). METHODS: Totally 239 lesions in 213 women who were scheduled for open biopsy were investigated in this study. The patients underwent both ABVS and conventional ultrasound (US). According to the imaging diagnostic standards of IDP, the diagnostic accuracy for ABVS was calculated, and then compared with US, using histopathological examination as the gold standard. RESULTS: Among the 239 breast lesions studied, 85 were pathologically proved to be malignancies and 154 benign lesions, among which there were 26 cases of IDP. ABVS found 41 cases of IDP (17.2%), with the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy being 88.5%, 91.5%, 56.1%, 98.5%, and 91.2%, respectively. In contrast, US found 32 cases of IDP (13.4%), with the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy being 73.1%, 93.9%, 59.4%, 96.6%, and 91.6%, respectively. CONCLUSION: ABVS coronal images can better display the structures of dilated lactiferous ducts and the intraluminal echoes, increase the diagnostic accuracy of IDP, and thus provide more information for the diagnosis and differential diagnosis of IDP.

Ultrasound-guided diffuse optical tomography (DOT) of invasive breast carcinoma: does tumour total haemoglobin concentration contribute to the prediction of axillary lymph node status?

OBJECTIVES: To prospectively study the ultrasound-guided near-infrared diffuse optical tomography (DOT) findings of the total haemoglobin concentration (THC) detected in invasive breast carcinomas and its contribution to the prediction of axillary lymph node (LN) status. METHODS: A total of 195 invasive breast carcinomas were prospectively studied with DOT before surgery. Lumpectomy or mastectomy with full axillary nodal dissection was performed. Tumour size and THC level were correlated with LN status by a logistic regression analysis. RESULTS: One hundred twenty-four patients (63.59%) was LN(-) and 71 (36.41%) was LN(+). The average THC was significantly higher in the LN(+) group than in the LN(-) group (252.94±69.19 µmol/L versus 203.86±83.13 µmol/L, P=0.01). A multivariate analysis showed an independent relationship between the probability of axillary metastasis, elevated THC level (P=0.01), and tumour size (P=0.001). The odds ratio with THC≥140 µmol/L was 13.651 (1.781-104.560), whereas that of tumour size with a 1cm increment was only 1.777 (1.283-2.246). CONCLUSIONS: The THC level and the tumour size are independent and preoperative predictors of axillary nodal status; these variables may improve the diagnosis of patients with lymph node metastasis.

Interobserver agreement for sonograms of breast lesions obtained by an automated breast volume scanner.

OBJECTIVE: To evaluate the interobserver agreement of radiologists in the description and final assessment of breast sonograms obtained using an automated breast volume scanner (ABVS) using a unique descriptor of three-dimensional ultrasound (3D US) and the Breast Imaging Reporting and Data System (BI-RADS) US lexicon. METHODS: From October to December 2010, 208 patients were subjected to an ABVS examination in the supine position, and data were automatically sent to the ABVS workstation. Two radiologists independently evaluated 234 breast masses (148 benign and 86 malignant masses) using a unique descriptor from the 3D US and the BI-RADS US lexicon. The reviewers were blinded to the patient's mammographic images, medical history, and pathologic findings. The interobserver agreement was measured using kappa statistics. RESULTS: Substantial agreement was obtained for lesion shape, orientation, margin, echo pattern, posterior acoustic features, calcification and final assessment (κ=0.79, 0.74, 0.76, 0.69, 0.68, 0.71 and 0.70, respectively). Fair agreement was obtained for retraction phenomenon and lesion boundary (κ=0.54 and 0.42, respectively). CONCLUSIONS: The interobserver agreement for breast sonograms obtained by ABVS is good, especially for lesion shape and margin; however, the interobserver agreement for the retraction phenomenon, which is a unique descriptor of coronal-plane 3D US, needs to be improved.

WITHDRAWN: Inter-observer variability in Breast Imaging Reporting and Data System (BI-RADS) ultrasound final assessments.

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Detecting angiogenesis in breast tumors: comparison of color Doppler flow imaging with ultrasound-guided diffuse optical tomography.

We investigated the correlation between color Doppler flow imaging (CDFI) and ultrasound (US)-guided diffuse optical tomography (DOT) for detection of breast tumor angiogenesis. Both CDFI and DOT were performed in 214 breast lesions scheduled for biopsy. The lesions were classified as vascular or nonvascular on CDFI and total hemoglobin concentration (THC) was measured by DOT. Sonographic results were correlated with the THC measurements. Pathologic examination showed 118 breast cancers and 96 benign breast masses. When vascularization on CDFI as a sign of malignancy and a cutoff of 140 µmol/L was used, the sensitivity, specificity and accuracy were 83.9, 50.0 and 68.7% for CDFI and 83.9, 66.7 and 76.2% for DOT, respectively. Thirteen (11.0%) nonvascular breast cancers presented high THC levels. Twenty-five (52.1%) vascular benign tumors demonstrated low THC levels. Mean THC did not differ significantly in malignancies with vascular or without vascular (228.14 ± 85.37 µmol/L vs. 191.42 ± 92.59 µmol/L; p > 0.05). Likewise, for benign lesions, the difference between THC values in vascular lesions and nonvascular lesions was not statistically significant (140.86 ± 79.63 µmol/L vs. 110.13 ± 85.05 µmol/L; p > 0.05). Our results suggest that the addition of DOT to CDFI could be helpful for characterizing CDFI nonvascular lesions that are suspicious for malignancy or vascular lesions that are probably benign.