Clinical value of multi-gene testing in distinguishing benign and malignant thyroid nodules.

BACKGROUND: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC). METHODS: A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET. RESULTS: Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%). CONCLUSIONS: Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.

Significant dysregulation of lipid metabolism in patients with papillary thyroid carcinoma after thyroidectomy.

Introduction: Thyroidectomy and thyrotropin suppressive therapy is the widely used surgical treatment for papillary thyroid carcinoma (PTC) patients. However, systematic metabolic changes of post-operative PTC patients were rarely reported. Methods: Here, untargeted metabolomic detection of cohorts from PTC before (t0) and 1-month-after (t1) thyroidectomy, were performed to characterize circulating metabolic signatures after surgical treatment. Results: Our results showed PTC patients exhibited lower thyroid stimulating hormone degree, higher total thyroxine, and significant lipid-related metabolic alternations after thyroidectomy, which included 97 upregulations (including 93 lipids) and 5 downregulations (including 2 lipids and 3 nucleotides). Enrichment of metabolic pathways mainly included biosynthesis of fatty acids, purine metabolism, and linoleic acid metabolism. We also demonstrated that differential surgical approaches (hemi- and total thyroidectomy) and post-operative complication phenotypes (insomnia, fatigue), might lead to characteristic metabolic signatures. Discussion: This study revealed dynamic changes of metabolite characteristics of PTC patients after surgical treatment, which were associated with clinical thyroid function parameters, surgical approaches, and complication occurrence. It enlightened us to pay more attention on the post-operative metabolic dysregulation of PTC patients and their long-term qualities of life, so as to provide cautious clinical decisions on surgical choices, treatments, and follow-up details.

A Combination of Pathological Features Identifies Crohn's Disease in Surgical Bowel Resection Specimens.

BACKGROUND: Diagnosis of Crohn's disease is challenging. This study aims to compare the histological features of Crohn's disease and non-Crohn's disease (other intestinal inflammatory diseases) in surgical specimens to identify a set of histologic features distinguishing Crohn's disease from non-Crohn's disease. METHODS: Patients with Crohn's disease (N = 171) and patients with non-Crohn's disease (N = 215) diagnosed between 2010 and 2015 who had surgical bowel resection were identified. The frequency of histological features in surgical resection specimens was compared between these two groups. RESULTS: Univariate analysis revealed that transmural inflammation, subserosal lymphoid aggregates, fissures or sinus-like structures, granulomas or granuloma-like nodules, abnormalities of the enteric nervous system, and mucosa structure alterations (muscularis mucosa thickening or mucosal atrophy with pseudopyloric gland metaplasia) were more frequent in Crohn's disease than non-Crohn's disease cases (p < 0.001 for all). Some of the above histologic features were further grouped as chronic inflammatory change which includes granulomas or granuloma-like nodules, lymphoid aggregates in the muscularis propria or subserosa, fissures or sinus-like structures, and architectural abnormality which is defined as the presence of abnormal enteric nervous system and/or mucosa structural alterations (muscularis mucosa thickening or mucosal atrophy with pseudopyloric gland metaplasia). A combination of transmural inflammation, chronic inflammatory change, and architectural abnormality had a sensitivity of 92.4% and a specificity of 97.7% for Crohn's disease. CONCLUSIONS: In surgical bowel resection specimens, a combination of transmural inflammation, chronic inflammatory change, and architectural abnormality help diagnose Crohn's disease.

Corrigendum: Molecular Portrait of GIST Associated With Clinicopathological Features: A Retrospective Study With Molecular Analysis by a Custom 9-Gene Targeted Next-Generation Sequencing Panel.

[This corrects the article DOI: 10.3389/fgene.2022.864499.].

Molecular Portrait of GISTs Associated With Clinicopathological Features: A Retrospective Study With Molecular Analysis by a Custom 9-Gene Targeted Next-Generation Sequencing Panel.

Objectives: The study aims to investigate genetic characterization of molecular targets and clinicopathological features with gastrointestinal stromal tumors based on targeted next-generation sequencing. Materials and Methods: We selected 106 patients with GISTs from Sir Run Run Shaw Hospital between July 2019 and March 2021. FFPE samples and paired blood samples were obtained from these patients who underwent excision of the tumor. A customized targeted-NGS panel of nine GIST-associated genes was designed to detect variants in the coding regions and the splicing sites of these genes. Results: In total, 106 patients with a GIST were included in the study which presented with various molecular driver alterations in this study. KIT mutations occurred most often in GISTs (94/106, 95.92%), followed by point mutations in PDGFRA. KIT or PDGFRA mutations were detected to be mutually exclusive in the GIST. A total of eight patients with wide-type KIT/PDGFRA were characterized as WT-GISTs, according to clinical diagnosis which included six quadruple-WT GISTs, 1 BRAF-mutant, and 1 NF1-mutant GIST. In KIT exon 11, the most common mutation type was the codon Mutation (in-frame deletion or indels), whereas the missense mutation was the dominant type in KIT exon 13 and KIT exon 17. All variations in KIT exon 11 observed in this study were concentrated at a certain position of codon 550 to codon 576. Mutation in KIT exon 9 was mostly located at codon 502-503. Two germline pathogenic mutations were detected: NF1-R681* and KRAS-T58I. NF1-L591P was a germline mutation to be identified for the first time and is not recorded in the database. The frequency of driving mutations differed between the primary anatomical site in the GIST (p = 0.0206). KIT exon 11 mutants had a lower proliferation index of Ki67 (68.66%,≤5%), while 50.00% of KIT exon 9 mutants had the Ki67 status greater than 10%. Conclusion: The occurrence and development of a GIST is driven by different molecular variations. Resistance to TKIs arises mainly with resistance mutations in KIT or PDGFRA when they are the primary drivers. Targeted NGS can simultaneously and efficiently detect nine GIST-related gene mutations and provide reference for clinicians' individualized diagnosis and treatment. Our results have important implications for clinical management.

Knockdown of Golgi phosphoprotein 2 inhibits hepatocellular carcinoma cell proliferation and motility.

Golgi phosphoprotein 2 (GP73) is highly expressed in hepatocellular carcinoma (HCC) cells, where it serves as a biomarker and indicator of disease progression. We used MTS assays, anchorage-independent cell colony formation assays and a xenograft tumor model to show that GP73-specific siRNAs inhibit HCC proliferation in HepG2, SMMC-7721, and Huh7 cell lines and in vivo. Following GP73 silencing, levels of p-Rb, a factor related to metastasis, were reduced, but cell cycle progression was unaffected. Our results suggest that GP73 silencing may not directly suppress proliferation, but may instead inhibit cell motility. Results from proliferation assays suggest GP73 reduces expression of epithelial mesenchymal transition (EMT)-related factors and promotes cell motility, while transwell migration and invasion assays indicated a possible role in metastasis. Immunofluorescence co-localization microscopy and immunoblotting showed that GP73 decreases expression of N-cadherin and E-cadherin, two key factors in EMT, which may in turn decrease intracellular adhesive forces and promote cell motility. This study confirmed that GP73 expression leads to increased expression of EMT-related proteins and that GP73 silencing reduces HCC cell migration in vitro. These findings suggest that GP73 silencing through siRNA delivery may provide a novel low-toxicity therapy for the inhibition of tumor proliferation and metastasis.

PCR-sequencing is a complementary method to amplification refractory mutation system for EGFR gene mutation analysis in FFPE samples.

Amplification Refractory Mutation System (ARMS) is the most popular technology for EGFR gene mutation analysis in China. Cutoff Ct or ΔCt values were used to differentiate low mutation abundance cases from no mutation cases. In this study, all of 359 NSCLC samples were tested by ARMS. Seventeen samples with larger Ct or ΔCt than cutoff values were retested by PCR-sequencing. TKI treatment responses were monitored on the cases with ARMS negative and PCR-sequencing positive results. One exon 18 G719X case, 67 exon 19 deletion cases, 2 exon 20 insertion cases, 1 exon 20 T790M case, 60 exon 21 L858R cases, 5 exon 21 L861Q cases and 201 wild type cases were identified by ARMS. Another 22 cases were evaluated as wild type but had later amplification fluorescent curves. Seventeen out of these 22 cases were retested by PCR-sequencing. It turns out that 3 out of 3 cases with exon 19 deletion later amplifications, 2 out of 2 cases with L858R later amplifications and 4 out of 12 cases with T790M later amplifications were identified as mutation positive. Two cases with exon 19 deletion and L858R respectively were treated by TKI and got responses. Our study indicated that PCR-sequencing might be a complementary way to confirm ARMS results with later amplifications.

Somatic mutation in synchronous primary adenocarcinomas of the left lung: a case report.

Multiple lung adenocarcinomas (AC) are uncommon. We herein report a case of multiple AC. A 62-year-old Chinese woman was admitted to our hospital because of her chest enhanced computed tomography (ECT) finding. Her ECT revealed a suspected lung cancer in the left lower lung and ill-defined weak ground-glass opacity in the left upper lobe. Upon operation, however, both were pathologically diagnosed as AC, an invasive one and an in-situ one, respectively. Only the invasive AC presented epidermal growth factor receptor (EGFR) mutation, while anaplastic lymphoma kinase (ALK) rearrangement was detected in none of them. To not miss rare invasion-related mutations, the whole exome sequencing of the in-situ and invasive AC was conducted subsequently matched the adjacent normal tissue of this patient. On sequencing the invasive tumor sample was found to have 27 exclusive somatic mutations as compared with in-situ and adjacent normal tissues. Our case exemplifies the need for deep sequencing and the discovery of new potential driver mutations.

Effect of Golgi phosphoprotein 2 (GOLPH2/GP73) on autophagy in human hepatocellular carcinoma HepG2 cells.

This study aims to investigate the effect of Golgi Protein 73 (GP73) on autophagy in human hepatoma line cells HepG2. We investigated the functional effects of GP73 on autophagy in hepatoma cell line HepG2 using immunofluoscence staining, Western blotting and real-time PCR. Our data showed that specific small interference RNA (siRNA) notably induced formation of autophagic vacuoles. In addition, upregulation of GP73 significantly inhibited formation of starvation-induced LC3-positive structures. We provide the first experimental evidence to show that GP73 may play an important role in the inhibitory regulation of autophagy. Therefore, our data suggest a new molecular mechanism for GP73-related hepatoma progression.

Association of Merkel cell polyomavirus infection with EGFR mutation status in Chinese non-small cell lung cancer patients.

OBJECTIVES: Female lung cancer patients with no smoking habit and non-mucinous adenocarcinoma have a higher rate of epidermal growth factor receptor (EGFR) gene mutations, which is related to tyrosine kinase inhibitors (TKIs) sensitivity. Unfortunately the cause of EGFR gene mutations is still elusive. In this study, we search for the association between Merkel cell polyomavirus (MCPyV) infection and EGFR gene mutations. MATERIALS AND METHODS: We studied 189 non-small cell lung cancer (NSCLC) samples for the presence of MCPyV large T (LT) DNA, LT antigen and EGFR hotspot mutations. Clinicopathological parameters of this cohort were also analyzed. RESULTS: Thirty out of 163 adenocarcinoma and 2 out of 18 squamous cell carcinoma were found to have MCPyV LT DNA by PCR. Immunostaining also showed LT protein expression in most of the DNA positive samples. EGFR mutations were more frequently detected in female (P=0.009) and non-smoking patients (P=0.0001). Furthermore, a significant association between MCPyV infection and EGFR mutations was found (P=0.001). CONCLUSION: Our study shows that MCPyV LT DNA is present in a subgroup of NSCLC, which is significantly correlated with EGFR mutations. To the best of our knowledge, this is the first study to find an association between MCPyV infection and EGFR hotspot mutations. These results support the possibility that MCPyV has a partial role in the carcinogenesis of NSCLC in a subgroup of patients.

Cardiac-specific overexpression of farnesyl pyrophosphate synthase induces cardiac hypertrophy and dysfunction in mice.

AIMS: Farnesyl pyrophosphate synthase (FPPS) is a key enzyme in the mevalonate pathway. In our previous study, we found that inhibition of FPPS attenuates cardiac hypertrophy in spontaneously hypertensive rats (SHRs) and prevents angiotensin (Ang) II-induced hypertrophy in cardiomyocytes. Here, we further investigate the role of FPPS in cardiac hypertrophy and heart failure (HF) using a transgenic (Tg) model, and its mechanisms. METHODS AND RESULTS: Tg mice with cardiac-specific expression of FPPS were studied as an experimental model. The results showed that Tg mice with overexpression of FPPS exhibited cardiac hypertrophy, fibrosis, and HF, as well as increased synthesis of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate in heart tissue. These pathological changes were associated with the activation of RhoA and other known kinases in the hypertrophic signalling pathway, such as extracellular signal-related kinases 1/2 and p38. Adenoviral infection of FPPS in cultured neonatal cardiomyocytes induced a hypertrophic response characterized by an increased cell size and an increased extent of sarcomeric organization, as well as an increased activation profile of small GTPases and downstream protein kinases concordant with those seen in vivo. Further investigation showed a marked increase of FPPS protein levels in hypertrophic ventricles of patients with valvular heart disease. CONCLUSION: Taken together, these results suggest that FPPS may function as a potent regulator in myocardial remodelling. The FPPS-regulated signalling pathway is relevant to the pathological changes in cardiac hypertrophy and HF.

[Advances on Golgi glycoprotein 73 and its association with diseases].

Golgi glycoprotein 73(GP73) is a transmembrane glycoprotein residing in the cis-Golgi complex, which is strongly expressed in hepatocellular carcinoma (HCC) and secreted into the blood. It has been regarded as a promising serum tumor marker for the detection of HCC with higher sensitivity and specificity than AFP. GP73 is also significantly elevated in kidney cancer, prostate cancer, lung adenocarcinoma, esophageal cancer and seminomas; therefore, it would be helpful for the diagnosis of these diseases. However, the function of GP73 and the regulatory mechanism for its expression are unclear. In this article, the physical-chemical properties, the regulation of its expression, the relation with various cancers and the clinical applications of GP73 are reviewed.

Establishment of transgenic mice carrying the gene for farnesyl pyrophosphate synthase.

Farnesyl pyrophosphate synthase (FPPS) is an essential enzyme in the mevalonate pathway and might be relevant to hypertension and other cardiovascular diseases. FPPS transgenic mice were produced by microinjecting a construct with the FPPS gene into fertilized eggs derived from an inbred C57BL/6 strain. Three mice were identified as carrying copies of the transgene using the PCR. Reverse transcription PCR and Western blotting showed that the transgene was expressed in heart, liver, lung, ear, brain, thymus, and blood vessels in the transgenic mouse. Pathological analysis (hematoxylin and eosin staining) showed that FPPS expression did not cause obvious pathological changes in multiple tissues of 6-week-old transgenic mice. This FPPS transgenic mouse model, may therefore, facilitate the investigation of the biological functions of FPPS in vivo.