Comprehensive analysis of cancer of unknown primary and recommendation of a histological and immunohistochemical diagnostic strategy from China.

BACKGROUND: Previous studies on cancer of unknown primary (CUP) mainly focus on treatment and prognosis in western populations and lacked clinical evaluation of different IHC markers, so this study aimed to evaluate characteristics of CUP and recommend a diagnostic strategy from a single center in China. METHODS AND RESULTS: Data of 625 patients with CUP were retrospectively collected and reviewed. The patients ranged in age from 20 to 91 years, with a female-to-male ratio of 1.3:1. The predominant histological type was poor or undifferentiated adenocarcinomas (308; 49.3%). The results of Canhelp-Origin molecular testing for the identification of the tissue of origin in 262 of 369 patients (71.0%) were considered predictable (similarity score > 45), with the most common predicted primary tumor site being the breast (57, 21.8%). Unpredictable molecular results correlated with more aggressive clinical parameters and poor survival. Thee positivity rates of several targeted antibodies (GATA3, GCDFP15, TTF1, Napsin A, and PAX8), based on the clinically predicted site, were lower than those reported for the corresponding primary tumors. Nonetheless, TRPS1 and INSM1 were reliable markers of predicted breast carcinoma (75.0%) and neuroendocrine tumors (83.3%), respectively. P16 expression, as well as HPV and EBER testing contributed significantly to the diagnosis of squamous cell carcinomas. Survival analysis revealed that older ages (> 57), ≥ 3 metastatic sites, non-squamous cell carcinomas, bone/liver/lung metastases, unpredictable molecular results, and palliative treatment correlated with poor overall survival. CONCLUSIONS: We recommend a CUP diagnostic strategy involving the use of targeted antibody panels as per histological findings that is potentially applicable in clinical practice. The markers TRPS1, INSM1, and P16 expression, as well as HPV and EBER testing are particularly valuable in this aspect. Molecular testing is also predictive of survival rates.

EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-ß/SMAD signalling.

BACKGROUND: Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). METHODS: Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein-protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). RESULTS: In this study, we report that EEF1A2 mediates the epithelial-mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFß Receptor (TßR)-I, and TßRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. CONCLUSIONS: These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

The implication of integrative multiple RNA modification-based subtypes in gastric cancer immunotherapy and prognosis.

Previous studies have focused on the impact of individual RNA modifications on tumor development. This study comprehensively investigated the effects of multiple RNA modifications, including m6A, alternative polyadenylation, pseudouridine, adenosine-to-inosine editing, and uridylation, on gastric cancer (GC). By analyzing 1,946 GC samples from eleven independent cohorts, we identified distinct clusters of RNA modification genes with varying survival rates and immunological characteristics. We assessed the chromatin activity of these RNA modification clusters through regulon enrichment analysis. A prognostic model was developed using Stepwise Regression and Random Survival Forest algorithms and validated in ten independent datasets. Notably, the low-risk group showed a more favorable prognosis and positive response to immune checkpoint blockade therapy. Single-cell RNA sequencing confirmed the abundant expression of signature genes in B cells and plasma cells. Overall, our findings shed light on the potential significance of multiple RNA modifications in GC prognosis, stemness development, and chemotherapy resistance.

Evaluation of the Idylla™ EGFR Mutation Test on formalin-fixed, paraffin-embedded tissue of human lung cancer.

Background: Epidermal growth factor receptor (EGFR) mutation detection is essential for the therapy of lung cancer. A sensitive, specific, and cost-effective standardized method to quickly and accurately detect EGFR mutations is urgently needed. Methods: We evaluated the Idylla™ EGFR Mutation Assay for EGFR mutations in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 232 lung cancer patients, and compared the results with amplification refractory mutation system (ARMS) (n=146) and next-generation sequencing (NGS) (n=86). The surgical tumor sections and cell blocks derived from the same FFPE section were compared. Overall concordance, specificity, sensitivity, cost-effectiveness and turnaround time were compared among the three methods. Results: The overall concordance between Idylla and ARMS was 89.51% [95% confidence interval (CI): 83.31% to 93.64%] and the specificity of Idylla was 88.68% (95% CI: 80.69% to 93.76%). A concordance of 97.67% (95% CI: 91.41% to 99.86%) was obtained between Idylla and NGS, the specificity of Idylla was 96.30% (95% CI: 86.16% to 99.36%). Compared to the ARMS and NGS, the Idylla™ system significantly reduces the turnaround time. Combining labor, equipment, reagents and time costs, Idylla is more affordable. Conclusions: Clinically urgent cases with adequate cellularity, can first perform Idylla to detect critical markers, then perform NGS for a comprehensive mutation analysis. Besides, with limited molecular expertise or infrastructure, the Idylla has the potential to extend EGFR testing to more pathology laboratories in primary hospitals.

Comprehensive analysis of next generation sequencing and ARMS-PCR for detecting EGFR exon 20 insertion (ex20ins) mutations in Chinese non-small cell lung cancer patients.

Background: Amivantamab (JNJ-372) and mobocertinib (TAK-788) have been reported to have favorable therapeutic effect for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations. Thus, accurate detection of EGFR ex20ins mutations is crucial for subsequent individualized therapy. The aim of this study was to compare the two common methods of next generation sequencing (NGS) and amplification refractory mutation system polymerase chain reaction (ARMS-PCR) for detecting EGFR ex20ins mutations in Chinese NSCLC patients. Methods: We retrospectively analyzed EGFR mutations, especially for ex20ins, in 3,606 NSCLC patients detected by NGS and 1,785 patients by ARMS. Results: Among the 3,606 NGS patients, a total of 2,077 EGFR mutations and 95 EGFR ex20ins were identified, accounting for 57.6% and 2.6%, respectively. While 48.4% of EGFR mutations and 1.1% of ex20ins were detected in 1,785 ARMS patients, which were significantly lower than those of NGS (P<0.01). Thirty-four unique ex20ins variants were identified by NGS, and eight of them was reported for the first time. However, ARMS was designed to detect only several known EGFR ex20ins variants, and even did not include the most common variants in Chinese NSCLC patients. Conclusions: NGS is more advantageous and strongly recommended for the detection of EGFR ex20ins mutations. Considering the fast and cost-effective ARMS detection method, it is suggested that the primers design should be updated according to the characteristics of EGFR ex20ins mutations in Chinese NSCLC patients.

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer.

The genetic diversity of human papillomavirus (HPV) 16 within cervical cells and tissue is usually associated with persistent virus infection and precancerous lesions. To explore the HPV16 mutation patterns contributing to the cervical cancer (CC) progression, a total of 199 DNA samples from HPV16-positive cervical specimens were collected and divided into high-grade squamous intraepithelial lesion (HSIL) and the non-HSIL(NHSIL) groups. The HPV16 E6 region (nt 7125-7566) was sequenced using next-generation sequencing. Based on HPV16 E6 amino acid mutation features selected by Lasso algorithm, four machine learning approaches were used to establish HSIL prediction models. The receiver operating characteristic was used to evaluate the model performance in both training and validation cohorts. Western blot was used to detect the degradation of p53 by the E6 variants. Based on the 13 significant mutation features, the logistic regression (LR) model demonstrated the best predictive performance in the training cohort (AUC = 0.944, 95% CI: 0.913-0.976), and also achieved a high discriminative ability in the independent validation cohort (AUC = 0.802, 95% CI: 0.601-1.000). Among these features, the E6 D32E and H85Y variants have higher ability to degrade p53 compared to the E6 wildtype (P < 0.05). In conclusion, our study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting HSIL. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of CC. IMPORTANCE The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high-grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

TBC1D8 Amplification Drives Tumorigenesis through Metabolism Reprogramming in Ovarian Cancer.

Cancer cells undergo metabolic reprogramming to support their energy demand and biomass synthesis. However, the mechanisms driving cancer metabolism reprogramming are not well understood. Methods: The differential proteins and interacted proteins were identified by proteomics. Western blot, qRT-PCR and IHC staining were used to analyze TBC1D8 levels. In vivo tumorigenesis and metastasis were performed by xenograft tumor model. Cross-Linking assays were designed to analyze PKM2 polymerization. Lactate production, glucose uptake and PK activity were determined. Results: We established two aggressive ovarian cancer (OVCA) cell models with increased aerobic glycolysis. TBC1D8, a member of the TBC domain protein family, was significantly up-regulated in the more aggressive OVCA cells. TBC1D8 is amplified and up-regulated in OVCA tissues. OVCA patients with high TBC1D8 levels have poorer prognoses. TBC1D8 promotes OVCA tumorigenesis and aerobic glycolysis in a GAP activity-independent manner in vitro and in vivo. TBC1D8 bound to PKM2, not PKM1, via its Rab-GAP TBC domain. Mechanistically, TBC1D8 binds to PKM2 and hinders PKM2 tetramerization to decreases pyruvate kinase activity and promote aerobic glycolysis, and to promote the nuclear translocation of PKM2, which induces the expression of genes which are involved in glucose metabolism and cell cycle. Conclusions:TBC1D8 drives OVCA tumorigenesis and metabolic reprogramming, and TBC1D8 serves as an independent prognosis factor for OVCA patients.

Long noncoding RNA CAR10 promotes lung adenocarcinoma metastasis via miR-203/30/SNAI axis.

Long noncoding RNAs (lncRNAs) play an important role in lung adenocarcinoma (LUAD) metastasis. Here, we found that lncRNA chromatin-associated RNA 10 (CAR10) was upregulated in the tumor tissue of patients with LUAD and enhanced tumor metastasis in vitro and in vivo. Mechanistically, CAR10 induced epithelial-to-mesenchymal transition (EMT) by directly binding with miR-30 and miR-203 and then regulating the expression of SNAI1 and SNAI2. CAR10 overexpression was positively correlated with a poor prognosis in LUAD patients, whereas overexpression of both CAR10 and SNAI was correlated with even worse clinical outcomes. In conclusion, the CAR10/miR-30/203/SNAI axis is a novel and potential therapeutic target for LUAD.

MiR-34b-3 and miR-449a inhibit malignant progression of nasopharyngeal carcinoma by targeting lactate dehydrogenase A.

MicroRNA expression profiling assays have shown that miR-34b/c and miR-449a are down-regulated in nasopharyngeal carcinoma (NPC); however, the targets and functions of miR-34b/c and miR-449a in the pathologenesis of NPC remain elusive. In this study, we verified miR-34b/c and miR-449a were significantly reduced with the advance of NPC. Overexpression of miR-34b-3 and miR-449a suppressed the growth of NPC cells in culture and mouse tumor xenografts. Using tandem mass tags for quantitative labeling and LC-MS/MS analysis to investigate protein changes after restoring expression of miR-34b-3, 251 proteins were found to be down-regulated after miR-34b-3 transfection. Through 3 replicate experiments, we found that miR-34b-3 regulated the expression of 15 potential targeted genes mainly clustered in the key enzymes of glycolysis metabolism, including lactate dehydrogenase A (LDHA). Further investigation revealed that miR-34b-3 and miR-449a negatively regulated LDHA by binding to the 3' untranslated regions of LDHA. Furthermore, LDHA overexpression rescued the miR-34b-3 and miR-449a induced tumor inhibition effect in CNE2 cells. In addition, miR-34b-3 and miR-449a suppressed LDH activity and reduced LD content, which were directly induced by downregulation of the LDHA. Our findings suggest that miR-34b-3 and miR-449a suppress the development of NPC through regulation of glycolysis via targeting LDHA and may be potential therapeutic targets for the treatment of NPC.