Ultrasound-based radiomics model for predicting molecular biomarkers in breast cancer.

Background: Breast cancer (BC) is the most common cancer in women and is highly heterogeneous. BC can be classified into four molecular subtypes based on the status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and proliferation marker protein Ki-67. However, they can only be obtained by biopsy or surgery, which is invasive. Radiomics can noninvasively predict molecular expression via extracting the image features. Nevertheless, there is a scarcity of data available regarding the prediction of molecular biomarker expression using ultrasound (US) images in BC. Objectives: To investigate the prediction performance of US radiomics for the assessment of molecular profiling in BC. Methods: A total of 342 patients with BC who underwent preoperative US examination between January 2013 and December 2021 were retrospectively included. They were confirmed by pathology and molecular subtype analysis of ER, PR, HER2 and Ki-67. The radiomics features were extracted and four molecular models were constructed through support vector machine (SVM). Pearson correlation coefficient heatmaps are employed to analyze the relationship between selected features and their predictive power on molecular expression. The receiver operating characteristic curve was used for the prediction performance of US radiomics in the assessment of molecular profiling. Results: 359 lesions with 129 ER- and 230 ER+, 163 PR- and 196 PR+, 265 HER2- and 94 HER2+, 114 Ki-67- and 245 Ki-67+ expression were included. 1314 features were extracted from each ultrasound image. And there was a significant difference of some specific radiomics features between the molecule positive and negative groups. Multiple features demonstrated significant association with molecular biomarkers. The area under curves (AUCs) were 0.917, 0.835, 0.771, and 0.896 in the training set, while 0.868, 0.811, 0.722, and 0.706 in the validation set to predict ER, PR, HER2, and Ki-67 expression respectively. Conclusion: Ultrasound-based radiomics provides a promising method for predicting molecular biomarker expression of ER, PR, HER2, and Ki-67 in BC.

Toll-Like Receptor 9 Inactivation Alleviated Atherosclerotic Progression and Inhibited Macrophage Polarized to M1 Phenotype in ApoE-/- Mice.

OBJECTIVE: Toll-like receptor 9 (TLR9) is involved in many inflammatory diseases, but its role in atherosclerosis remains controversial. This study aimed to investigate the role of TLR9 in atherosclerosis development and macrophage polarization. METHODS: ApoE(-/-) mice were treated with vehicle or IRS869 for 12 weeks. Plaque vulnerability was assessed with immunohistochemical analysis, picro-sirius red, and oil red O staining. The expressions of M1- and M2-associated markers in plaques were detected by RT-PCR and immunofluorescence. The aorta TLR9 and its downstream molecules including myeloid differentiation protein 88 (MyD88), phosphorylated nuclear factor-kappa B (p-NF-κB), and interferon regulatory factor 7 (IRF7) were determined by western blot analysis. The frequency of M1 and M2 subtype in RAW264.7 cells treated with IRS869 and/or ODN1826 was evaluated with flow cytometry. RESULTS: In ApoE(-/-) mice, functional inactivation of TLR9 pathway resulted in attenuated atherosclerosis development, as manifested by reduced plaque burden and by decreased plaque vulnerability. Mechanistically, TLR9 inhibition prevented the activation of MyD88/NF-κB pathway and shifted the balance of M1/M2 toward M2 macrophages that were involved. CONCLUSIONS: Our data indicated that TLR9 inactivation ameliorated atherosclerosis via skewing macrophage plasticity to M2 phenotype in ApoE-deficient mice. These findings may provide a promising therapeutic strategy for atherosclerosis.

Apolipoprotein E deficiency and high-fat diet cooperate to trigger lipidosis and inflammation in the lung via the toll-like receptor 4 pathway.

Apolipoprotein E deficiency (ApoE(-/-)) combined with a high-fat Western-type diet (WD) is known to activate the toll-like receptor (TLR4) pathway and promote atherosclerosis. However, to date, the pathogenic effects of these conditions on the lung have not been extensively studied. Therefore, the present study examined the effects of ApoE(-/-) and a WD on lung injury and investigated the underlying mechanisms. ApoE(-/-) and wild-type mice were fed a WD or normal chow diet for 4, 12 and 24 weeks. Lung inflammation, lung cholesterol content and cytokines profiles in broncho-alveolar lavage fluid (BALF) were determined. TLR4 and its main downstream molecules were analyzed with western blot analysis. In addition, the role of the TLR4 pathway was further validated using TLR4-targeted gene silencing. The results showed that ApoE(-/-) mice developed lung lipidosis following 12 weeks of receiving a WD, as evidenced by an increased lung cholesterol content. Moreover, dependent on the time period of receiving the diet, those mice exhibited pulmonary inflammation, which was manifested by initial leukocyte recruitment (at 4 weeks), by increased alveolar septal thickness and mean linear intercept as well as elevated production of inflammation mediators (at 12 weeks), and by granuloma formation (at 24 weeks). The expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B were markedly upregulated in ApoE(-/-) WD mice at week 12. However, these effects were ameliorated by shRNA-mediated knockdown of TLR4. By contrast, ApoE(-/-) ND or wild-type WD mice exhibited low-grade or no inflammation and mild lipidosis. The levels of TLR4 and MyD88 in those mice showed only minor changes. In conclusion, ApoE deficiency acts synergistically with a WD to trigger lung lipidosis and inflammation at least in part via TLR4 signaling.

Prenatal nicotine exposure induces gender-associated left ventricular-arterial uncoupling in adult offspring.

Alterations in vascular or myocardial structure and function have been demonstrated in offspring subjected to prenatal nicotine exposure (PNE), however, limited data are available on how these changes interact. The present study assessed the hypothesis that prenatal nicotine exposure induced gender-specific alterations of left ventricular-arterial coupling indices in adult offspring. Female Sprague-Dawley rats were exposed to either nicotine (8 mg/kg/day) or saline via subcutaneous osmotic mini-pumps throughout gestation. Male and female offspring, aged 12 months, underwent non-invasive echocardiography and invasive left ventricular cannulation. Left ventricular-arterial coupling was analysed as the ratio of effective arterial elastance (Ea) to ventricular end-systolic elastance (Ees). The left ventricular myocardium and aorta were stained with hematoxylin and eosin and the myocardial cell cross-sectional area was calculated. Simultaneously, the ratio of medium thickness to internal diameter in the aorta and mesenteric artery was determined. The fibrosis component of left ventricle myocardium was analyzed by Sirius-red staining and further confirmed by hydroxyproline determination. The elastic properties of the aortic wall were analyzed by van Gieson staining. PNE caused significant increases in pulse pressure (56.36 ± 7.41 vs. 50.16 ± 4.94 mmHg; P<0.05) and left ventricular meridional wall stress (78.25 ± 9.12 vs. 69.64 ± 7.58 kdyne/cm(2); P<0.05) in male offspring compared with the control. Conversely, no similar effect was observed in female offspring. An elevated augmentation index was noted in male and female pups. Additionally, Ea/Ees was reduced in PNE males compared with control males, due to a disproportionate increase in Ees vs. Ea whereas in females, Ea/Ees did not differ significantly due to tandem increase in Ea and Ees. In addition, collagen cross-linking was markedly higher in male offspring, whereas it was unaltered in females compared with their respective controls. Fragmentation of the elastic network in the aorta and the increased ratio of medial thickness to internal diameter in the mesenteric artery were more evident in male offspring when compared with female offspring. PNE caused combined ventricular-arterial stiffening in male and female offspring, with lower Ea/Ees in males, while Ea/Ees was preserved in females. Enhanced collagen cross-linking in the myocardium, underdeveloped elastic fibers in the aorta and remodeled resistance vessels were associated with pathological ventricular arterial mismatching. The results of the present study indicated that male offspring were more susceptible to the development of ventricular and arterial dysfunction in response to PNE compared with female offspring.

Effects of pristane alone or combined with chloroquine on macrophage activation, oxidative stress, and TH1/TH2 skewness.

We investigated the protective role of chloroquine against pristane-induced macrophage activation, oxidative stress, and Th1/Th2 skewness in C57BL/6J mice. Those mice were treated with pristane alone or combined with chloroquine. Hematological and biochemical parameters, macrophage phagocytic function, the oxidant/antioxidant index, cytokine for IFN-γ, TNF-α, IL-4, and IL-6, and the isotypes of IgG2a and IgG1 were determined. And the expression of T-bet/GATA-3 and IL-12/IL-10 mRNA in spleen were analyzed by real-time PCR. We found that pristane treatment for a period of 12 or 24 weeks triggered macrophage activation syndrome, characterized by hemophagocytosis in spleen and peripheral blood, enhanced lipid phagocytosis by peritoneal macrophages in vitro, erythropenia and leucopenia, increased anti-Smith, lactic dehydrogenase, triglyceride, and ferritin, as well as hypercytokinemia of IFN-γ, TNF-α, IL-4, and IL-6. In parallel, a significant increase in lipid peroxidation and a decrease in superoxide dismutase, glutathione, and catalase activity, as well as a skewed Th1/Th2 balance in spleen, were observed. However, chloroquine supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that pristane administration induces macrophage activation, oxidative stress, and Th1/Th2 skewness, which can be attenuated by chloroquine.

Role of toll-like receptor 4 on lupus lung injury and atherosclerosis in LPS-challenge ApoE⁻/⁻ mice.

To investigate the pathologic mechanisms of toll-like receptor 4 (TLR4) in lung injury and atherosclerosis, ApoE⁻/⁻ or wild-type mice were intraperitoneally administered saline, lipopolysaccharides (LPS), or LPS plus TAK-242 (TLR4 inhibitor), respectively, twice a week for 4 weeks. Serum autoantibody of antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA), and cytokines of interferon-gamma (IFN-γ), tumor necrosis factor (TNF-α ), and interleukin-1 (IL-1ß) were assessed by ELISA. Hematoxylin and eosin (HE) and Perl's stains for lung pathomorphology as well as HE staining for atherosclerosis were employed. TLR4 in macrophages was detected by double immunofluorescent staining. While protein expressions of TLR4, nuclear factor-kappa B p65 (NF-κB p65), and B cell activating factor belonging to the TNF family (BAFF) were examined by immunohistochemistry. We found that serum autoantibody (ANA and anti-dsDNA), cytokines (IFN-γ, TNF-α, IL-1ß), lung inflammation, and intima-media thickness in brachiocephalic artery were obviously increased after LPS challenge in both genotypes, but to a lesser extent in wild-type strains. And those alterations were alleviated by coadministration of LPS and TAK-242. Mechanistically, upregulation of TLR4, NF-κb, and BAFF was involved. We concluded that TLR4/NF-κb/BAFF in macrophages might be a possible common autoimmune pathway that caused lung injury and atherosclerosis. TLR4 signal will be a therapeutic target in atherosclerosis and immune-mediated lung injury.

Detecting metastasis of lymph nodes and predicting aggressiveness in patients with breast carcinomas.

OBJECTIVE: The purpose of this study was to evaluate the contrast-enhanced ultrasonographic (CEUS) characteristics of metastatic lymph nodes (LNs) and to determine the correlation of CEUS parameters with the tumor aggressiveness in patients with breast cancer. METHODS: Real-time gray scale CEUS of axillary LNs was preoperatively performed in 51 consecutive patients with breast carcinoma who were scheduled for axillary lymph node dissection. The CEUS characteristics assessed by a direct visualization method and quantification software were compared with pathologic findings. Expression of human epidermal growth factor receptor 2 (HER-2/neu) in the primary tumor was detected by immunohistochemical analysis. Correlation analysis of CEUS parameters with HER-2/neu expression and the LN stage was performed. RESULTS: Of the LNs examined, 27 were metastatic, and 25 were diagnosed as reactive hyperplasia. Lymph nodes with metastasis were characterized by centripetal progress (66.7%) and a heterogeneous pattern (55.6%) or no or scarce perfusion (25.9%). However, LNs with nonmetastases were characterized by with centrifugal enhancement (56.0%) and a homogeneous pattern (80.0%). The difference between the hyperintense and hypointense regions was higher in metastatic LNs than nonmetastatic ones (P < .001). No significant differences were found in the arrival time, time to peak intensity, and peak intensity between the two groups. A histopathologic diagnosis could be predicted with sensitivity, specificity, and accuracy of 92.6%, 76.0%, and 84.6% respectively, by a standardized difference between maximum and minimum signal intensity (SI(max)-SI(min)) value of 28. Human epidermal growth factor receptor 2 expression and the LN histopathologic stage were significantly associated with the SI(max)-SI(min). In metastatic LNs, the relationship between the diagnostic sensitivity of CEUS and the transverse diameter of LNs remained statistically significant (P < .05). CONCLUSIONS: Noninvasive CEUS can play a role in discriminating metastatic from nonmetastatic LNs and predicting the aggressiveness in patients with breast cancer.

Contrast-enhanced ultrasound is helpful in the differentiation of malignant and benign breast lesions.

OBJECTIVE: To evaluate the significance of contrast-enhanced ultrasound (CEUS) examination in differential diagnosis of malignant and benign breast lesions. METHODS: Seventy-one patients with seventy-six breast tumors are selected randomly. CEUS examinations were performed before and after bolus injection of the contrast agent SonoVue (Bracco, Milan, Italy). Specific sonographic quantification software, Qontrast, was adopted to determine the morphology of vessels. Wash-in and wash-out parameters of each lesion were assessed for both procedures. RESULTS: The final histopathological findings distinguished 45 malignant and 31 benign from all of the lesions. Following SonoVue administration different perfusion phases could be identified: early (0-1min), mid (1-4min) and late (4-6min) phases. In the early phase, CEUS identified 91.1% of malignant tumors characterized by a claw-shaped enhancement, while 83.9% of benign tumors had a homogeneous enhancement, with a statistically significant difference between the two enhancement patterns (chi(2)=43.16, P<0.01). Moreover, contrast medium persistence in the late phase was helpful in the identification of benign and malignant tumors (chi(2)=46.88, P<0.01): contrast medium was present in 88.9% of malignant tumors, while in only 9.7% of the benign tumors. The study showed that various parametric imaging color maps for peak intensity and time to peak were mostly suggestive of malignancy, while quite uniform peak intensity and time to peak of color maps were the characteristic of benign tumors. The study also found that malignant lesions presented with a higher maximum intensity signal than benign ones (P<0.05) on the time-intensity curves. CONCLUSIONS: CEUS cooperating with conventional US shows improved accuracy in differentiating between malignant and benign breast tumors. It could be a reliable diagnostic method of breast lesions.