Preclinical evaluation of 68 Ga-labeled peptide CK2 for PET imaging of NRP-1 expression in vivo.

PURPOSE: Neuropilin-1 (NRP-1) is a multifunctional protein involved in a variety of biological processes such as angiogenesis, tumorigenesis and immunomodulation. It was usually overexpressed in many cancer cell lines and correlated with poor prognosis of breast cancer. Positron emission tomography (PET) is an advanced imaging technique for detecting the function and metabolism of tumor-associated molecules in real time, dynamically, quantitatively and noninvasively. To improve the level of early diagnosis and evaluate the prognosis of breast cancer, an NRP-1 targeting peptide-based tracer [68 Ga]Ga-NOTA-PEG4-CK2 was designed to sensitively and specifically detect the NRP-1 expression in vivo via PET imaging. METHODS: In silico modeling and microscale thermophoresis (MST) assay were carried out to design the NRP-1 targeting peptide NOTA-PEG4-CK2, and it was further radiolabeled with 68 Ga to prepare the tracer [68 Ga]Ga-NOTA-PEG4-CK2. The radiochemical yield (RCY), radiochemical purity (RCP), molar activity (Am), lipid-water partition coefficient (Log P) and stability of [68 Ga]Ga-NOTA-PEG4-CK2 were assessed. The targeting specificity of the tracer for NRP-1 was investigated by in vitro cellular uptake assay and in vivo PET imaging as well as blocking studies. The sensitivity of the tracer in monitoring the dynamic changes of NRP-1 expression induced by chemical drug was also investigated in vitro and in vivo. Ex vivo biodistribution, autoradiography, western blot, and immunofluorescence staining were also performed to study the specificity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1. RESULTS: [68 Ga]Ga-NOTA-PEG4-CK2 was designed and synthesized with high RCY (> 98%), high stability (RCP > 95%) and high affinity to NRP-1 (KD = 25.39 ± 1.65 nM). In vitro cellular uptake assay showed that the tracer [68 Ga]Ga-NOTA-PEG4-CK2 can specifically bind to NRP-1 positive cancer cells MDA-MB-231 (1.04 ± 0.04% at 2 h) rather than NRP-1 negative cancer cells NCI-H1299 (0.43 ± 0.05%). In vivo PET imaging showed the maximum tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 in MDA-MB-231 xenografts (4.16 ± 0.67%ID/mL) was significantly higher than that in NCI-H1299 xenografts (1.03 ± 0.19%ID/mL) at 10 min post injection, and the former exhibited higher tumor-to-muscle uptake ratio (5.22 ± 0.18) than the latter (1.07 ± 0.27) at 60 min post injection. MDA-MB-231 xenografts pretreated with nonradioactive precursor NOTA-PEG4-CK2 showed little tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 (1.67 ± 0.38%ID/mL at 10 min post injection). Both cellular uptake assay and PET imaging revealed that NRP-1 expression in breast cancer MDA-MB-231 could be effectively suppressed by SB-203580 treatment and can be sensitively detected by [68 Ga]Ga-NOTA-PEG4-CK2. Ex vivo analysis also proved the high specificity and sensitivity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1 expression in MDA-MB-231 xenografts. CONCLUSION: A promising NRP-1 targeting PET tracer [68 Ga]Ga-NOTA-PEG4-CK2 was successfully prepared. It showed remarkable specificity and sensitivity in monitoring the dynamic changes of NRP-1 expression. Hence, it could provide valuable information for early diagnosis of NRP-1 relevant cancers and evaluating the prognosis of cancer patients.

Ultrasonographic, clinical, and pathological features of papillary thyroid carcinoma in children and adolescents with or without Hashimoto's thyroiditis.

Objective: To compare the ultrasonographic, clinical, and pathological features of children and adolescents with papillary thyroid carcinoma (PTC) with and without Hashimoto's thyroiditis (HT). Materials and methods: A total of 52 children and adolescent patients surgically diagnosed with PTC between 2017 and 2022 were included; 14 children and adolescent patients with PTC were diagnosed with HT via pathological examination. The preoperative ultrasonographic, postoperative histological, and molecular and clinical characteristics were retrospectively analyzed. Results: The prevalence rate of PTC in patients with HT was 27%. Papillary thyroid microcarcinomas were found in 11 of 38 patients without HT, but none in patients with HT (p = 0.023). Extrathyroidal extension, capsular invasion, and lymph node metastases were more frequent in patients with PTC and HT than in patients with PTC alone (p < 0.05 for both). The ultrasonographic features of nodule composition, echogenicity, shape, margin, Thyroid Imaging Reporting and Data System categories, and total points were similar. The patterns of echogenic foci were more prominent in the nodules of patients with HT than in those of patients without HT (p = 0.016). Conclusion: The frequency of papillary thyroid microcarcinomas in patients with PTC and HT was less, whereas that of extrathyroidal extension, capsular invasion, and lymph node metastasis was significantly higher in patients with PTC and HT than in those with PTC alone. The patterns of echogenic foci on ultrasonography may represent a risk for PTC.

Comparison of ultrasonography and pathology features between children and adolescents with papillary thyroid carcinoma.

Objective: To compare the ultrasonography and pathology features between children and adolescents with papillary thyroid carcinoma (PTC). Methods: A total of 53 patients who were surgically diagnosed with childhood or adolescent PTC between 2017 and 2022 were included in this study. The pre-operative ultrasonography, post-operative histology, and molecular and clinical characteristics were retrospectively analyzed. Results: No differences were observed in composition, echogenicity, and shape using ultrasonography. Moreover, there was a significantly higher rate of extrathyroidal extension, punctate echogenic foci, and lymph node metastases in children compared to adolescents. The molecular analysis showed that BRAFV600E mutations are the most prevalent abnormality in adolescent PTC (12/20, 60.0%). However, they are less in childhood PTC (7/23, 30.4%). In addition, using next-generation sequencing, three cases with oncogenic fusion (one TRIM33-RET case, one CCDC6-RET case, and one STRN-ALK case) were identified in childhood PTC. Conclusion: The frequency of extrathyroidal extension, punctate echogenic foci, and lymph node metastases were higher in childhood PTC, while BRAFV600E mutations were higher in adolescent PTC.

Integrin α6 Targeted Near Infrared Fluorescent Imaging and Photoacoustic Imaging of Hepatocellular Carcinoma in Mice.

Background and Aims: Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death and ranks sixth in terms of incident cases worldwide. The purpose of this study was to develop an effective and sensitive method to distinguish liver cancer tissues from normal tissues in HCC patients. Integrin α6 is a promising cell surface target for molecular imaging of HCC, where it is overexpressed and is a prognostic biomarker. We previously identified an integrin α6-targeted peptide CRWYDENAC (RWY) that has been used for positron emission tomography (PET) imaging of HCC in mouse models. Methods: We labeled the integrin α6-targeted RWY peptide with cyanine 7 (Cy7) to form an optical probe (Cy7-RWY) for near infrared fluorescent (NIRF) and photoacoustic (PA) imaging in HCC. Mice transplanted with subcutaneous HCC-LM3 or orthotopic HCC-H22 cells that overexpressed integrin α6 were intravenously injected with Cy7-RWY and its corresponding Cy7-control. NIRF and PA images of mice were collected from 0 to 48 h after injection. Results: Both NIRF and PA signals started to accumulate in the tumor 2 h after injection of Cy7-RWY and peaked at 24 h. Conclusions: Cy7-RWY is a promising optical probe for NIRF and PA imaging of HCC in mice, and has potential clinical application for HCC detection.

A Practical CEUS Thyroid Reporting System for Thyroid Nodules.

Background The role of contrast-enhanced US (CEUS) in reducing unnecessary biopsies of thyroid nodules has received little attention. Purpose To construct and externally validate a thyroid imaging reporting and data system (TI-RADS) based on nonenhanced US and CEUS to stratify the malignancy risk of thyroid nodules. Materials and Methods This retrospective study evaluated 756 patients with 801 thyroid nodules who underwent nonenhanced US, CEUS, and fine-needle aspiration and received a final diagnosis from January 2018 to December 2019. Qualitative US features of the thyroid nodules were analyzed with univariable and multivariable logistic regression to construct a CEUS TI-RADS. The CEUS TI-RADS was validated with use of internal cross-validation and external validation. Results A total of 801 thyroid nodules in 590 female (mean age, 44 years ± 13) and 166 male (mean age, 47 years ± 13 [SD]) patients were included. Independent predictive US features included nodule composition at CEUS, echogenicity, nodule shape, nodule margin, echogenic foci, extrathyroidal extension, enhancement direction, peak intensity, and ring enhancement. The CEUS TI-RADS showed a higher area under the receiver operating characteristic curve of 0.93 (95% CI: 0.92, 0.95; P < .001 in comparison with all other systems), a biopsy yield of malignancy of 66% (157 of 239 nodules), and an unnecessary biopsy rate of 34% (82 of 239 nodules). In the external validation, the area under the receiver operating characteristic curve, biopsy yield of malignancy, and unnecessary biopsy rate of CEUS TI-RADS were 0.89 (95% CI: 0.84, 0.92), 61% (65 of 106 nodules), and 39% (41 of 106 nodules) for the first external validation set and 0.90 (95% CI: 0.85, 0.94), 57% (56 of 99 nodules), and 43% (43 of 99 nodules) for the second external validation set. Conclusion A contrast-enhanced US (CEUS) thyroid imaging reporting and data system was created with thyroid nodule malignancy risk stratification according to the simplified regression coefficients of nonenhanced US and qualitative features of CEUS. Clinical trials registration no. ChiCTR2000028712 Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Comparison of the diagnostic performances of US-guided fine needle aspiration cytology and thyroglobulin measurement for lymph node metastases in patients with differentiated thyroid carcinoma: a meta-analysis.

OBJECTIVES: Ultrasound (US)-guided fine needle aspiration cytology (FNAC) and thyroglobulin measurement (FNA-Tg) are two common methods for confirming lymph node metastases (LNM) in patients with differentiated thyroid carcinoma (DTC). This study aimed at comparing the diagnostic performance of FNAC, FNA-Tg alone, and in combination by means of a meta-analysis. METHODS: Eligible articles were selected according to predefined criteria, and their quality was evaluated as per the QUADAS-2 checklist. We calculated pooled sensitivity (Se), specificity (Sp), positive/negative likelihood ratio, and diagnostic odds ratio (DOR), and plotted the summary receiver operating characteristic (SROC) curve using the Meta-DiSc1.4 software. RESULTS: Twenty-one studies pooling 1662 malignant and 1279 benign LNs from 2712 patients with DTC were included. The results showed that FNAC was more specific (pooled Sp, 0.98) while FNA-Tg was more sensitive (pooled Se, 0.94). FNAC and FNAC+FNA-Tg performed better postoperatively than FNA-Tg, while FNA-Tg performed better preoperatively. The combination of FNAC and FNA-Tg could achieve a better diagnostic performance than each alone (DOR 446.00, area under the curve [AUC] 0.9862), no matter preoperatively (DOR 378.14, AUC 0.9879) or postoperatively (DOR 788.72, AUC 0.9930). Besides, the combination of FNAC and FNA-Tg/serum-Tg ratio obtained a higher Sp (0.98) than the combination of FNAC and FNA-Tg. CONCLUSION: The addition of FNA-Tg, especially the FNA-Tg/serum-Tg ratio, to FNAC could increase the diagnostic performance of LNM in both preoperative and postoperative patients with DTC. Since one test or test combinations could perform differently according to the clinical situation, the best-fitting test should be chosen accordingly. KEY POINTS: • FNAC is more specific than FNA-Tg while FNA-Tg is more sensitive than FNAC. • The combination of FNAC and FNA-Tg could achieve a better diagnostic performance than either alone, no matter preoperatively or postoperatively. • The combination of FNAC and FNA-Tg/serum-Tg ratio could reach a higher Sp than the combination of FNAC and FNA-Tg.

NK homeobox 2.2 functions as tumor suppressor in colorectal cancer due to DNA methylation.

Aim: The role of NK homeobox 2.2 (NKX2.2) in human colorectal cancer (CRC) remains to be unveiled. This study was designed to explore the epigenetic regulation and function of NKX2.2 in human CRC. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were used to assess the methylation data of NKX2.2 in CRC. Six CRC cell lines (HCT116, SW480, HT29, LOVO, SW1116, SW640) and 20 pairs of primary CRC tumor and normal tissues were utilized to explore the function of NKX2.2 in CRC using Sequenom EpiTYPER®, verified by cloning-based bisulfite sequencing analysis, semi-quantitative reverse transcription PCR, western blot, cell viability assessment, plate clone formation assay , and transwell assays. Results: Bioinformatic analysis showed that NKX2.2 was significantly hypermethylated in primary tumors compared to normal tissues (p < 0.05). Our study also found that NKX2.2 methylation was upregulated (p<0.05) in tumors than normal tissues. In vitro experiments demonstrated that 5-aza-2'-deoxycytidine downregulated the methylation of NKX2.2 and retrieved its expression of mRNA and protein levels (p<0.05). No significant association was found between the NKX2.2 methylation and sex, age, tumor differentiation, TNM stage, CEA, CA199, and fecal occult blood (p>0.05). Kaplan-Meier analysis indicated that NKX2.2 hypermethylation showed a trend but not statistical significance for predicting poor overall survival in CRC patients (p=0.33). NKX2.2 overexpression suppressed cell proliferation, colony formation, and inhibited tumor invasion and migration in CRC cells (both p<0.05). Conclusions: This study indicates that NKX2.2 is a tumor suppressor in CRC due to hypermethylation.

A systematic review and quantitative assessment of methylation biomarkers in fecal DNA and colorectal cancer and its precursor, colorectal adenoma.

Colorectal cancer (CRC) arises from accumulated genetic and epigenetic alterations, which provide the possibility to identify tumor-specific biomarkers by analyzing fecal DNA. Methylation status in human genes from tumor tissue is highlighted as promising biomarker in the early detection of CRC. A number of studies have documented altered methylation levels in DNA extracted from stool samples, but generated heterogeneous results. We performed a systematic review and quantitative assessment of existing studies to compare levels of DNA methylation in most frequently studied genes and their diagnostic value in CRC and its precursor, colorectal adenoma, with their counterparts in healthy subjects. Robust searches of the literature were performed in our study with explicit strategies and definite inclusion/exclusion criteria. Pooled data revealed that methylation levels of SFRP2, SFRP1, TFPI2, BMP3, NDRG4, SPG20, and BMP3 plus NDRG4 genes exceeded a sensitivity of 70% and a specificity of 80% for CRC detection. The DOR of the seven candidate biomarkers ranged from 19.80 to 334.33, indicating a good diagnostic power in discriminating cancer from normal tissues. The AUC range was from 0.88 to 0.95, indicating a good or very good discriminatory performance. When test results for BMP3 and NDRG4 were combined, the DOR of CRC detection was 98.36, which was higher than that for BMP3 and NDRG4 separately. As for adenoma detection, the DOR of methylated NDRG4 is higher than that for CRC (CRC vs. adenoma: 54.86 vs. 57.22). Both the sensitivity and specificity of NDRG4 for adenoma detection exceeded 70%. These findings demonstrate the eligibility and feasibility of DNA methylation as a minimally invasive biomarker in feces in the diagnosis of CRC and adenoma. The use of DNA from human stools has the potential to be readily applicable to detect aberrant DNA methylation levels among many subjects for CRC early screening.

Fine needle aspiration biopsy indications for thyroid nodules: compare a point-based risk stratification system with a pattern-based risk stratification system.

OBJECTIVES: We aim to compare the diagnostic performance to assess thyroid nodules and reliability for recommending fine needle aspiration biopsy (FNAB) between American College of Radiology thyroid image reporting and data system (ACR TI-RADS) and American Thyroid Association (ATA) guidelines. METHODS: In total, this retrospective study included 1001 consecutive thyroid nodules in 918 patients from May 2016 to December 2017. US features of the thyroid nodules, including composition, echogenicity, shape, margins, echogenic foci, and size, were reviewed and were classified according to ACR TI-RADS and ATA guidelines, respectively. The diagnostic performance to assess thyroid nodules and reliability for recommending fine needle aspiration biopsy were compared between ACR TI-RADS and ATA guidelines. RESULTS: Of the 1001 thyroid nodules, 609 (60.8%) were benign and 392 (39.2%) were malignant. The sensitivity, specificity, PPV, NPV, and accuracy were 96.7%, 77.3%, 73.3%, 97.3%, and 84.9%, respectively, for ACR TI-RADS and 99.2%, 16.1%, 43.2%, 97.0%, and 48.7%, respectively, for ATA guidelines. AUC of ACR TI-RADS was significantly greater than ATA guidelines (0.935 (0.918, 0.949) vs 0.884 (0.862, 0.903), p < 0.001). Biopsy yield of malignancy, biopsy rate of malignancy, and unnecessary FNAB rate were 59.5%, 91.3%, and 40.5%, respectively, for ACR TI-RDS and 38.5%, 97.4%, and 61.5%, respectively, for ATA guidelines. CONCLUSIONS: ACR TI-RADS was more accurate than ATA guidelines for differentiating malignant thyroid nodules from benign nodules and more reliable than ATA guidelines for recommending thyroid nodules for FNAB. KEY POINTS: • Malignant risk of thyroid nodules can be stratified by ultrasound. • American College of Radiology guidelines were more accurate for differentiating malignant thyroid nodules from benign nodules. • American College of Radiology guidelines were more reliable for recommending thyroid nodules for biopsy.

Advance in plasma SEPT9 gene methylation assay for colorectal cancer early detection.

This review article summarizes the research advances of the plasma-based SEPT9 gene methylation assay for the clinical detection of colorectal cancer and its limitations. Colorectal cancer is a common malignancy with a poor prognosis and a high mortality, for which early detection and diagnosis are particularly crucial for the high-risk groups. Increasing evidence supported that SEPT9 gene methylation is associated with the pathogenesis of colorectal cancer and that detecting the level of methylation of SEPT9 in the peripheral blood can be used for screening of colorectal cancer in susceptible populations. In recent years, the data obtained in clinical studies demonstrated that the SEPT9 gene methylation assay has a good diagnostic performance with regard to both sensitivity and specificity with the advantage of better acceptability, convenience and compliance with serological testing compared with fecal occult blood tests and carcinoembryonic antigen for colorectal cancer (CRC). Furthermore, the combination of multiple methods or markers has become a growing trend for CRC detection and screening. Nevertheless, the clinical availability of the methylated SEPT9 assay is still limited because of the large degree of sample heterogeneity caused by demographic characteristics, pathological features, comorbidities and/or technique selection. Another factor is the cost-effectiveness of colorectal cancer screening strategies that hinders its large-scale application. In addition, improvements in its accuracy in detecting adenomas and premalignant polyps are required.

Meta-analysis of the clinical characteristics and prognostic relevance of NOTCH1 and FBXW7 mutation in T-cell acute lymphoblastic leukemia.

The NOTCH1 signaling pathway is crucial for T-cell development, and NOTCH1 and/or FBXW7 mutations are frequently detected in T-cell acute lymphoblastic leukemia (T-ALL). We performed a systematic review and meta-analysis of 18 randomized controlled trials (RCTs) to assess the prognostic impact of mutations in the NOTCH1 pathway. After retrieving relevant articles from PubMed, EMBASE, and the Cochrane Library, we investigated overall survival (OS) and event-free survival (EFS) with hazard ratios (HRs) using fixed-effects or random-effects models and conducted subgroup analyses based on population and mutation status. NOTCH1/FBXW7 mutations correlated significantly with better prognosis (5-year EFS: HR, 0.57; 95% confidence interval [CI], 0.46 to 0.68; P < 0.001 and 5-year OS: HR, 0.61; 95% CI, 0.51 to 0.74; P < 0.001). The HR for 5-year EFS and OS with NOTCH1 mutations were 0.63 (95% CI, 0.53 to 0.75) and 0.76 (95% CI, 0.60 to 0.95), respectively; with FBXW7 mutations, they were 0.82 (95% CI, 0.60 to 1.11) and 0.79 (95% CI, 0.55 to 1.12), respectively. However, differences between children and adults showed no significance. We conclude that the presence of NOTCH1/FBXW7 mutations is an independent prognostic factor for 5-year EFS and 5-year OS.

Serum microRNA profiles as diagnostic biomarkers for HBV-positive hepatocellular carcinoma.

BACKGROUND & AIMS: The discovery of effective and reliable biomarkers to detect hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC) at an early stage may improve the survival of HCC. The aim of this study was to establish serum microRNA (miRNA) profiles as diagnostic biomarkers for HBV-positive HCC. METHODS: We used deep sequencing to screen serum miRNAs in a discovery cohort (n=100). Quantitative polymerase chain reaction (qPCR) assays were then applied to evaluate the expression of selected miRNAs. A diagnostic 2-miRNA panel was established by a logistic regression model using a training cohort (n=182). The predicted probability of being detected as HCC was used to construct the receiver operating characteristic (ROC) curve. Area under the ROC curve (AUC) was used to assess the diagnostic performance of the selected miRNA panel. RESULTS: The predicted probability of being detected as HCC by the 2-miRNA panel was calculated by: logit P=-2.988 + 1.299 × miR-27b-3p + 1.245 × miR-192-5p. These results were further confirmed in a validation cohort (n=246).The miRNA panel provided a high diagnostic accuracy of HCC (AUC=0.842, P<.0001 for training set; AUC=0.836, P<.0001 for validation set respectively). In addition, the miRNA panel showed better prediction of HCC diagnosis than did alpha-foetoprotein (AFP). The miRNA panel also differentiated HCC from healthy (AUC=0.823, P<.0001), and cirrhosis patients (AUC=0.859, P<.0001) respectively. CONCLUSIONS: Differentially expressed serum miRNAs may have considerable clinical value in HCC diagnosis, and be particularly helpful for AFP-negative HCC.

Serum microRNA profiles as prognostic biomarkers for HBV-positive hepatocellular carcinoma.

To establish serum microRNA profiles as prognostic biomarkers in hepatocellular carcinoma patients (HCCs), we used deep sequencing to screen serum microRNAs in a discovery set .Twelve up-regulated serum miRNAs were selected for qPCR analysis in a training set. MiR-192-5p and miR-29a-3p were identified and associated with HCC prognosis. HCCs with high concentrations of miR-192-5p and miR-29a-3p had poorer overall survival (OS) and progression-free survival (PFS) than those with low concentrations. We calculated a prognostic index (PI) score and classified patients into low-, medium- and high-risk groups. OS and PFS among the 3 groups from the training set were significantly different (all P < 0.05). PI (PIOS, PIPFS) score was the only independent prognostic predictor for OS and PFS of HCCs in the training set. These results were further confirmed in a validation set. In conclusion, differentially expressed serum miRNAs can be helpful for predicting survival in HCCs.

SPECT and near-infrared fluorescence imaging of breast cancer with a neuropilin-1-targeting peptide.

Breast cancer is the most common malignant cancer and is the leading cause of cancer death among females. Molecular imaging is a promising approach for the early detection and staging of breast cancer as well as for assessing therapeutic responses. Tumor-targeting peptides are effective targeting vehicles for molecular imaging. Here, we identified a breast cancer-targeting peptide CLKADKAKC (CK3) contains a cryptic C-end rule motif that may mediate its binding to neuropilin-1 (NRP-1), an attractive therapeutic target which expression was associated with poor outcome of the patients with breast cancer. Phage CK3 bound to NRP-1-positive breast cancer cells, which could be inhibited by peptide CK3 in a dose-dependent manner or by knock-down NRP-1 expression. Consistently, NRP-1 overexpression in cells increased the binding of phage CK3. Furthermore, peptide CK3 co-localized with NRP-1. Importantly, unlike previously reported NRP-1-targeting peptides with exposed C-end rule motifs, peptide CK3 did not penetrate into lungs and heart in vivo, which could make it more clinically applicable. Single-photon emission CT (SPECT) and near-infrared fluorescence (NIRF) imaging showed enrichment of peptide CK3 to the xenograft tumors in nude mice. In conclusion, as a novel NRP-1-targeting peptide, peptide CK3 could be used for breast cancer molecular imaging, which may represent a new avenue for breast cancer diagnostics, staging and assessments of therapeutic response.