BRAFV600E restructures cellular lactylation to promote anaplastic thyroid cancer proliferation.

Anaplastic thyroid cancer (ATC) is a rare but fatal cancer with BRAF mutation ranging from 30 to 50%. Histone lysine lactylation represents a novel epigenetic mark that translates cellular metabolic signals into transcriptional regulation. It is not clear whether the Warburg effect can promote the proliferation of ATC with BRAFV600E mutation via metabolite-mediated histone lactylation. Our study aimed at illustrating how BRAFV600E restructures the cellular protein lactylation landscape to boost ATC proliferation, and determining whether blockade of protein lactylation can sensitize mutant ATC to BRAFV600E inhibitors. Western blotting was used to evaluate lactylation status. Aerobic glycolysis was intervened by adding cell-permeable ethyl lactate or using metabolic inhibitors. Chromatin immunoprecipitation and RT-qPCR were applied to analyze the expression of growth-related genes. Different chemical inhibitors were used to inhibit BRAFV600E and other enzymes. ATC cell line-derived xenograft model was employed to examine the efficacy of mono and combinatorial therapies. The results showed that aerobic glycolysis in ATC increased global protein lactylation via improving cellular lactate availability. In particular, lactylation on Histone 4 Lysine 12 residue (H4K12La) activated the expression of multiple genes essential for ATC proliferation. Furthermore, oncogenic BRAFV600E boosted glycolytic flux to restructure the cellular lactylation landscape, leading to H4K12La-driven gene transcription and cell cycle deregulation. Accordingly, the blockade of cellular lactylation machinery synergized with BRAFV600E inhibitor to impair ATC progression both in vitro and in vivo. Our results demonstrated an extra beneficial effect of aerobic glycolysis on ATC, revealing a novel metabolism-epigenetics axis suitable for combinatorial therapy with BRAFV600E inhibition.

Genome-Wide DNA Methylation and Gene Expression Profiling Characterizes Molecular Subtypes of Esophagus Squamous Cell Carcinoma for Predicting Patient Survival and Immunotherapy Efficacy.

BACKGROUND: Immunotherapy is recently being used to treat esophageal squamous cell carcinoma (ESCC); however, response and survival benefits are limited to a subset of patients. A better understanding of the molecular heterogeneity and tumor immune microenvironment in ESCC is needed for improving disease management. METHODS: Based on the DNA methylation and gene expression profiles of ESCC patients, we identify molecular subtypes of patients and construct a predictive model for subtype classification. The clinical value of molecular subtypes for the prediction of immunotherapy efficacy is assessed in an independent validation cohort of Chinese ESCC patients who receive immunotherapy. RESULTS: We identify two molecular subtypes of ESCC (S1 and S2) that are associated with distinct immune-related pathways, tumor microenvironment and clinical outcomes. Accordingly, S2 subtype patients had a poorer prognosis. A 15-gene expression signature is developed to classify molecular subtypes with an overall accuracy of 94.7% (89/94, 95% CI: 0.880-0.983). The response rate of immunotherapy is significantly higher in the S1 subtype than in the S2 subtype patients (68.75% vs. 25%, p = 0.028). Finally, potential target drugs, including mitoxantrone, are identified for treating patients of the S2 subtype. CONCLUSIONS: Our findings demonstrated that the identified molecular subtypes constitute a promising prognostic and predictive biomarker to guide the clinical care of ESCC patients.

Complete Response to Immunotherapy Combined With Chemotherapy in a Patient With Gynecological Mixed Cancer Mainly Composed of Small Cell Neuroendocrine Carcinoma With High Tumor Mutational Burden: A Case Report.

Small cell neuroendocrine carcinoma (SCNEC) is rare in the gynecologic tract, which has high invasive and metastatic ability. Due to the aggressive behavior and lack of treatment, patients have an extremely poor prognosis. Here we report a 66-year-old female diagnosed with SCNEC in the gynecologic tract, mixed with endometrioid adenocarcinoma, squamous cell, and adenosquamous carcinoma. A tumor mutational burden of 13.14 Muts/Mb was detected by next-generation sequencing. The patient underwent a palliative operation of total hysterectomy with bilateral adnexectomy but suffered from disease progression in a short time after the operation. Chemotherapy (paclitaxel + carboplatin) combined with immunotherapy (toripalimab) was conducted every 3 weeks, achieving a partial response after 2 cycles of treatment. After 5 cycles of combined treatment, the patient consolidated with monotherapy of toripalimab for about half a year and achieved a complete response. Until December 2021, the patient has achieved 27 months of progression-free survival and maintains a continued complete response. This case is presented due to the rare combination of pathological types and durable response to treatment especially immunotherapy, suggesting the potential value of immunotherapy in SCNEC of the gynecologic tract.

Preoperative Thyroid Peroxidase Antibody Predicts Recurrence in Papillary Thyroid Carcinoma: A Consecutive Study With 5,770 Cases.

Background: Thyroid autoimmunity is common in papillary thyroid carcinoma (PTC) and was believed to confer a better prognosis; however, controversy still remains. This study aimed to investigate the prognostic value of chronic lymphocytic thyroiditis (CLT) and preoperative thyroid peroxidase antibody (TPOAb) in PTC patients. Methods: A retrospective analysis was performed on 5,770 PTC patients who underwent surgical treatment with pathologically confirmed PTC in our institution between 2012 to 2016. The patients were divided into groups with respect to the coexistence of CLT or preoperative TPOAb levels. The clinicopathological characteristics and disease-free survival (DFS) rates were compared between the groups. Results: The coexistence of CLT was likely to have bilateral, multifocal tumors. Particularly, PTC patients with TPOAb++ (>1,000 IU/L) had a larger tumor size (p = 0.007) and higher rates of bilaterality and multifocality than those with TPOAb- (TPOAb< 100 IU/L), while for lymph node metastasis and extrathyroidal extension, there is no statistical difference. Tumor recurrence was found in 15 of 425 (3.5%), 9 of 436 (2.1%), and 56 of 3,519 (1.6%) patients with TPOAb++, TPOAb+, and TPOAb-, respectively (p = 0.017). On univariate analysis, TPOAb++ was correlated with tumor recurrence, with a hazard ratio of 2.20 [95% confidence interval (CI), 1.25-3.89], which remained as an independent risk factor at 1.98 (95% CI, 1.10-3.55) on multivariate analysis. PTC patients with TPOAb++ had the lowest DFS rates (96.5 vs. 97.9 vs. 98.4%, p = 0.020). Conclusion: CLT is not a protective factor in PTC patients. We provide initial evidence that the preoperative TPOAb instead predicts recurrence in papillary thyroid carcinoma.

VCAM-1 Upregulation Contributes to Insensitivity of Vemurafenib in BRAF-Mutant Thyroid Cancer.

Vemurafenib, an inhibitor of mutant BRAF activity, is a promising anticancer agent for patients with BRAF-mutant metastatic melanoma. However, it is less effective in BRAF-mutant thyroid cancer, and the reason for this discrepancy is not yet fully elucidated. By RNA sequencing analysis, we identified vascular cell adhesion molecular-1 (VCAM-1) to be highly upregulated in both time- and dose-dependent manners during BRAF inhibition (BRAFi) in a BRAF-mutant papillary thyroid cancer cell line (BCPAP). Cell cytotoxicity and apoptosis assays showed that knockdown of the induced VCAM-1 in BCPAP cells augmented the antitumor effects of vemurafenib, with decreased IC50 values of 1.4 to 0.8 µM. Meanwhile, overexpression of VCAM-1 in a BRAF-mutant anaplastic thyroid cancer cell line (FRO) reduced the sensitivity to vemurafenib, with increased IC50 values of 1.9 to 5.8 µM. Further investigation showed that PI3K-Akt-mTOR pathway was activated during BRAFi. Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAFi. This combination further improved the efficacy of vemurafenib. Moreover, VCAM-1 promoted migration and invasion in thyroid cancer cells in vitro, which was also indicated in thyroid cancer patients. The present study is the first to demonstrate that VCAM-1 is upregulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells. Targeting the PI3K-Akt-mTOR pathway-mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.

Thyroid Function Abnormalities in COVID-19 Patients.

Purpose: The novel coronavirus COVID-19, has caused a worldwide pandemic, impairing several human organs and systems. Whether COVID-19 affects human thyroid function remains unknown. Methods: Eighty-four hospitalized COVID-19 patients in the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) were retrospectively enrolled in this study, among which 22 cases had complete records of thyroid hormones. In addition, 91 other patients with pneumonia and 807 healthy subjects were included as controls. Results: We found that levels of total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) were lower in COVID-19 patients than healthy group (p < 0.001). Besides, TSH level in COVID-19 patients was obviously lower than non-COVID-19 patients (p < 0.001). Within the group of COVID-19, 61.9% (52/84) patients presented with thyroid function abnormalities and the proportion of thyroid dysfunction was higher in severe cases than mild/moderate cases (74.6 vs. 23.8%, p < 0.001). Patients with thyroid dysfunction tended to have longer viral nucleic acid cleaning time (14.1 ± 9.4 vs. 10.6 ± 8.3 days, p = 0.088). To note, thyroid dysfunction was also associated with decreased lymphocytes (p < 0.001) and increased CRP (p = 0.002). The correlation between TT3 and TSH level seemed to be positive rather than negative in the early stage, and gradually turned to be negatively related over time. Conclusion: Thyroid function abnormalities are common in COVID-19 patients, especially in severe cases. This might be partially explained by nonthyroidal illness syndrome.

Nomogram-Based New Recurrence Predicting System in Early-Stage Papillary Thyroid Cancer.

BACKGROUND AND OBJECTIVES: The clinicopathological risk factors to predict recurrence of papillary thyroid cancer (PTC) patients remain controversial. METHODS: PTC patients treated with thyroidectomy between January 1997 and December 2011 at the First Affiliated Hospital of Zhejiang University (Zhejiang cohort) were included. Multivariate Cox regression analysis was conducted to identify independent recurrence predictors. Then, the nomogram model for predicting probability of recurrence was built. RESULTS: According to Zhejiang cohort (N = 1,697), we found that the 10-year event-free survival (EFS) rates of PTC patients with early-stage (TNM stages I, II, and III) were not well discriminated (91.6%, 89.0%, and 90.7%; P=0.768). The multivariate Cox model identified age, bilaterality, tumor size, and nodal status as independent risk factors for tumor recurrence in PTC patients with TNM stages I-III. We then developed a nomogram with the C-index 0.70 (95% CI, 0.64 to 0.76), which was significantly higher (P < 0.0001) than the AJCC staging system (0.52). In the validation group, the C-index remained at a similar level. CONCLUSIONS: In this study, we build up a new recurrence predicting system and establish a nomogram for early-stage PTC patients. This prognostic model may better predict individualized outcomes and conduct personalized treatments.

Clonal analysis of early-stage bilateral papillary thyroid cancer identifies field cancerization.

INTRODUCTION: Bilaterality is a newly identified indicator for aggressive tumor behavior and poor outcome in papillary thyroid cancer. However, the clonal origin of these bilateral tumors remains unclear. METHODS: Here we analyzed 28 pairs of early-stage papillary thyroid cancers (stage I-II without extra-thyroidal extension, lymph node metastasis or distant metastasis) that underwent surgery at First Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China). Genomic DNA was extracted from paraffin-embedded tissues after microdissection and analyzed for BRAF mutation and X-chromosome inactivation. RESULTS: A total of 16 patients (16/28, 57.1%) harbored different BRAF status in bilateral tumors. Fourteen patients were available for X-chromosome inactivation assay and 10 of them achieved informative results. Bilateral tumors from four cases had distinct patterns of X-chromosome inactivation. Combining the results of X-chromosome inactivation and BRAF analysis, we demonstrated that at least 64.3% (18/28) cases harbored discordant X-chromosome inactivation or BRAF status, indicating their independent clonal origin in bilateral tumors. CONCLUSIONS: The present study confirms "field cancerization" in early-stage bilateral thyroid cancers, suggesting that these subtype papillary thyroid cancers should be treated as independent and localized tumors.

GPRC5A: An Emerging Biomarker in Human Cancer.

Aberrant expression of G protein-coupled receptors (GPCRs) is frequently associated with tumorigenesis. G Protein-coupled receptor class C group 5 member A (GPRC5A) is a member of the GPCR superfamily, is expressed preferentially in lung tissues, and is regulated by various entities at multiple levels. GPRC5A exerts a tumor suppressive role in lung cancer and GPRC5A deletion promotes lung tumor initiation and progression. Recent advances have highlighted that GPRC5A dysregulation is found in various human cancers and is related to many tumor-associated signaling pathways, including the cyclic adenosine monophosphate (cAMP), nuclear factor (NF)-κB, signal transducer and activator of transcription (STAT) 3, and focal adhesion kinase (FAK)/Src signaling. This review aimed to summarize our updated view on the biology and regulation of GPRC5A, its expression in human cancers, and the linked signaling pathways. A better comprehension of the underlying cellular and molecular mechanisms of GPRC5A will provide novel insights into its potential diagnostic and therapeutic value.

Association of telomerase reverse transcriptase promoter mutations with clinicopathological features and prognosis of thyroid cancer: a meta-analysis.

The clinicopathological and prognostic significance of telomerase reverse transcriptase (TERT) promoter mutations have been widely investigated in thyroid cancer; however, the results are still discrepant. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, and the Cochran Library databases for relevant articles prior to April 2016. Mutation rates were synthesized by R statistical software. The odds ratio or standardized mean difference with 95% confidence interval was pooled by Stata. A total of 22 studies with 4,907 cases were included in this meta-analysis. TERT promoter mutations tended to present in aggressive histological types including poorly differentiated thyroid cancer (33.37%), anaplastic thyroid cancer (38.69%), and tall-cell variant papillary thyroid cancer (30.23%). These promoter mutations were likely to exist in older patients and males and were well associated with larger tumor size, extrathyroidal extension, vascular invasion, lymph node metastasis, distant metastasis, advanced tumor stage, disease recurrence/persistence, and mortality. In addition, TERT promoter mutations (especially C228T) tended to coexist with BRAFV600E mutation, which indicated more aggressive tumor behavior. Therefore, TERT promoter mutations may be promising biomarkers for early diagnosis, risk stratification, prognostic prediction, and management of thyroid cancer.

A Comprehensive Characterization of Mitochondrial Genome in Papillary Thyroid Cancer.

Nuclear genetic alterations have been widely investigated in papillary thyroid cancer (PTC), however, the characteristics of the mitochondrial genome remain uncertain. We sequenced the entire mitochondrial genome of 66 PTCs, 16 normal thyroid tissues and 376 blood samples of healthy individuals. There were 2508 variations (543 sites) detected in PTCs, among which 33 variations were novel. Nearly half of the PTCs (31/66) had heteroplasmic variations. Among the 31 PTCs, 28 specimens harbored a total of 52 somatic mutations distributed in 44 sites. Thirty-three variations including seven nonsense, 11 frameshift and 15 non-synonymous variations selected by bioinformatic software were regarded as pathogenic. These 33 pathogenic mutations were associated with older age (p = 0.0176) and advanced tumor stage (p = 0.0218). In addition, they tended to be novel (p = 0.0003), heteroplasmic (p = 0.0343) and somatic (p = 0.0018). The mtDNA copy number increased in more than two-third (46/66) of PTCs, and the average content in tumors was nearly four times higher than that in adjacent normal tissues (p < 0.0001). Three sub-haplogroups of N (A4, B4a and B4g) and eight single-nucleotide polymorphisms (mtSNPs) (A16164G, C16266T, G5460A, T6680C, G9123A, A14587G, T16362C, and G709A) were associated with the occurrence of PTC. Here we report a comprehensive characterization of the mitochondrial genome and demonstrate its significance in pathogenesis and progression of PTC. This can help to clarify the molecular mechanisms underlying PTC and offer potential biomarkers or therapeutic targets for future clinical practice.

Diagnostic value of BRAF (V600E)-mutation analysis in fine-needle aspiration of thyroid nodules: a meta-analysis.

Fine-needle aspiration (FNA) is a reliable method for preoperative diagnosis of thyroid nodules; however, about 10%-40% nodules are classified as indeterminate. The BRAF (V600E) mutation is the most promising marker for thyroid FNA. This meta-analysis was conducted to investigate the diagnostic value of BRAF (V600E) analysis in thyroid FNA, especially the indeterminate cases. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, Elsevier, and the Cochrane Library databases for relevant studies prior to June 2015, and a total of 88 studies were ultimately included in this meta-analysis. Compared with FNA cytology, the synergism of BRAF (V600E) testing increased the diagnostic sensitivity from 81.4% to 87.4% and decreased the false-negative rate from 8% to 5.2%. In the indeterminate group, the mutation rate of BRAF (V600E) was 23% and varied in different subcategories (43.2% in suspicious for malignant cells [SMC], 13.77% in atypia of undetermined significance/follicular lesion of undetermined significance [AUS/FLUS], and 4.43% in follicular neoplasm/suspicious for follicular neoplasm [FN/SFN]). The sensitivity of BRAF (V600E) analysis was higher in SMC than that in AUS/FLUS and FN/SFN cases (59.4% vs 40.1% vs 19.5% respectively), while specificity was opposite (86.1% vs 99.5% vs 99.7% respectively). The areas under the summary receiver-operating characteristic curve also confirmed the diagnostic value of BRAF (V600E) testing in SMC and AUS/FLUS rather than FN/SFN cases. Therefore, BRAF (V600E) analysis can improve the diagnostic accuracy of thyroid FNA, especially indeterminate cases classified as SMC, and select malignancy to guide the extent of surgery.

Galectin-1 mediates TGF-ß-induced transformation from normal fibroblasts into carcinoma-associated fibroblasts and promotes tumor progression in gastric cancer.

Rcinoma-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment. Cancer cells can induce the transformation from normal fibroblasts (NFs) into CAFs, reciprocally, CAFs promote tumor invasion and proliferation. TGF-ß has been the mostly accepted factor to fuel NFs transformation into CAFs. Galectin-1 (Gal1) is highly upregulated in CAFs of multiple human cancers, and overexpression of Gal1 in CAFs promotes tumor progression. The effect of Gal1 on TGF-ß-induced CAFs activation has not yet been established in gastric cancer (GC). In this study, we show that Gal1 expression in stroma is positively related to TGF-ß in epithelial cells by retrospective analysis of GC patient samples. Meanwhile, conditioned media (CMs) from gastric cancer cells induce expression of both Gal1 and the CAFs marker alpha smooth muscle actin (α-SMA) in NFs via TGF-ß secretion. Knockdown of Gal1 prevents TGF-ß-induced the conversion of NFs to CAFs. CMs from fibroblasts overexpressing Gal1 inhibits cancer cells apoptosis, promotes migration and invasion in vitro. Thus, Gal1 is significantly involved in the development of tumor-promoting microenvironment by enhancing TGF-ß signaling in a positive feedback loop. Targeting Gal1 in tumor stroma should be considered as a potential therapeutic target for GC.

Comparison of the clinicopathologic features and prognosis of bilateral versus unilateral multifocal papillary thyroid cancer: An updated study with more than 2000 consecutive patients.

BACKGROUND: Bilaterality is common in papillary thyroid cancer (PTC), but its clinical and prognostic implications are still controversial, and it remains unclear whether its behavior is more aggressive than multifocality. METHODS: The clinicopathologic features of 2211 consecutive patients with PTC who underwent surgical treatment at the authors' institute between 1997 and 2011 were reviewed. Among these surgical patients, 425 (19.2%) had bilateral PTCs, and 1786 had unilateral PTCs. The patients who had unilateral PTCs were subdivided into a group with unilateral-multifocal PTCs (210 patients) and a group with solitary PTCs (1576 patients). The 10-year disease-free survival (DFS) rates were calculated to compare the prognosis between groups. B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutation status was examined by direct DNA sequencing. RESULTS: Patients who had bilateral PTCs were likely to have larger tumors, higher rates of extrathyroid extension and lymph node metastasis, and more advanced tumor stage than those who had unilateral-multifocal PTCs. Multivariate analysis identified only lymph node metastasis as an independent risk factor for PTC recurrence (P < .001). The 10-year DFS rate for patients with bilateral PTCs was much lower than that for those with unilateral-multifocal and solitary PTCs (78.8% vs 85.7% and 89.3%, respectively; P = .005). It is noteworthy that patients who had bilateral PTCs with lymph node metastasis had the worst prognosis in terms of DFS. Incidence of the BRAF V600E mutation (valine to glutamic acid mutation at position 600) was higher in the bilateral PTC group than that in the unilateral and unilateral-multifocal PTC groups. CONCLUSIONS: The current results provide initial evidence that bilateral PTCs are more aggressive than unilateral-multifocal PTCs, and patients who have bilateral disease have more advanced stage and shorter DFS. The poorer outcome of patients with bilateral PTCs may be caused in part by their high incidence of lymph node metastasis. Cancer 2016;122:198-206. © 2015 American Cancer Society.

Impact of Omega-3 Fatty Acid Supplements on Gastrointestinal Cancer Patients after Surgery: Beneficial or Useless?

Omega-3 polyunsaturated fatty acids (w-3 PUFAs) are essential nutrients for human beings and their potential roles against cancer development and progression have become of wide concern recently. Some studies have suggested that perioperative supplementation with omega-3 fatty acids may have beneficial effects in gastrointestinal cancer patients undergoing surgery, while other researchers reported contrary results. This paper reviews recent research to establish therapeutic effects as well as possible underlying mechanisms of ????PUFA actions, and to help explain possible reasons for inconsistent results from different institutions.

Establishment and characterization of GCSR1, a multi-drug resistant signet ring cell gastric cancer cell line.

Signet ring cell gastric cancer (SRCGC) has very poor prognosis worldwide, and studying its molecular characteristics is urgent for improving the outcome. However, few well-characterized SRCGC cell lines are available for research. Therefore, we established a novel cell line GCSR1, from a Chinese male SRCGC patient. Cell morphology of GCSR1 in culture, maintained in vitro for over 90 passages, is similar to the cells from the patient. GCSR1 cells proliferated in vitro with a doubling time of 67.65 h. Karyotyping showed they were aneuploid. Missense mutation occurred in codon 193 of P53 and deletion occurred in exons 1 and 3 of P16. Results of CCK8 assay revealed that GCSR1 was more resistant to 5-fluorouracil (5-FU) and mitomycin (MMC) than other gastric cancer cell lines. Stem cell marker assay by flow cytometry showed that GCSR1 had high proportion of CD44+ and/or CD133+ cells. It formed colonies easily in soft agar and generated xenograft tumors in nude mice. In conclusion, GCSR1 is a well-established, well-characterized multi-drug resistant cell line with abundant cancer stem cells.

ER-α36: a novel biomarker and potential therapeutic target in breast cancer.

Estrogen receptor-alpha36 (ER-α36) is a 36-kDa variant of estrogen receptor-alpha (ER-α) firstly identified and cloned by Wang et al in 2005. It lacks both transactivation domains (activation function 1 and activation function 2) and has different biological characteristics compared to traditional ER-α (ER-α66). ER-α36 primarily locates on plasma membrane and cytoplasm and functions as a mediator in the rapid membrane-initiated non-genomic signaling pathway. Additionally, it inhibits the traditional genomic signaling pathway mediated by ER-α66 in a dominant-negative pattern. Accumulating evidence has demonstrated that ER-α36 regulates the physiological function of various tissues. Thus, dysregulation of ER-α36 is closely associated with plenty of diseases including cancers. ER-α36 is recognized as a molecular abnormality which solidly correlates to carcinogenesis, aggressiveness, and therapeutic response of breast cancer. Additionally, special attention has been paid to the role of ER-α36 in endocrine therapy resistance. Therefore, ER-α36 provides a novel biomarker of great value for diagnosis, prognosis, and treatment of breast cancer. It may also be a potential therapeutic target for breast cancer patients, especially for those who are resistant to endocrine therapy. In this review, we will overview and update the biological characteristics, underlying mechanism, and function of ER-α36, focusing on its biological function in breast cancer and endocrine therapy resistance. We will evaluate its application value in clinical practice.