Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.

Liquid biopsy of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) is gaining attention as a method for real‐time monitoring in cancer patients. Conventional methods based upon epithelial cell adhesion molecule (EpCAM) expression have a risk of missing the most aggressive CTC subpopulations due to epithelial‐mesenchymal transition and may, thus, underestimate the total number of actual CTC present in the bloodstream. Techniques utilizing a label‐free inertial microfluidics approach (LFIMA) enable efficient capture of CTC without the need for EpCAM expression. In this study, we optimized a method for analyzing genetic alterations using next‐generation sequencing (NGS) of extracted ctDNA and CTC enriched using an LFIMA as a first‐phase examination of 30 patients with head and neck cancer, esophageal cancer, gastric cancer and colorectal cancer (CRC). Seven patients with advanced CRC were enrolled in the second‐phase examination to monitor the emergence of alterations occurring during treatment with epidermal growth factor receptor (EGFR)‐specific antibodies. Using LFIMA, we effectively captured CTC (median number of CTC, 14.5 cells/mL) from several types of cancer and detected missense mutations via NGS of CTC and ctDNA. We also detected time‐dependent genetic alterations that appeared during anti–EGFR therapy in CTC and ctDNA from CRC patients. The results of NGS analyses indicated that alterations in the genomic profile revealed by the liquid biopsy could be expanded by using a combination of assays with CTC and ctDNA. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (ID: UMIN000014095).

Recently, we identified unique processing patterns of apolipoprotein A2 (ApoA2) in patients with pancreatic cancer. This study provides a first prospective evaluation of an ApoA2 isoform (“ApoA2-ATQ/AT”), alone and in combination with carbohydrate antigen 19-9 (CA19-9), as an early detection biomarker for pancreatic cancer. We performed ELISA measurements of CA19-9 and ApoA2-ATQ/AT in 156 patients with pancreatic cancer and 217 matched controls within the European EPIC cohort, using plasma samples collected up to 60 months prior to diagnosis. The detection discrimination statistics were calculated for risk scores by strata of lag-time. For CA19-9, in univariate marker analyses, C-statistics to distinguish future pancreatic cancer patients from cancer-free individuals were 0.80 for plasma taken ≤6 months before diagnosis, and 0.71 for >6-18 months; for ApoA2-ATQ/AT, C-statistics were 0.62, and 0.65, respectively. Joint models based on ApoA2-ATQ/AT plus CA19-9 significantly improved discrimination within >6-18 months (C = 0.74 vs. 0.71 for CA19-9 alone, p = 0.022) and ≤18 months (C = 0.75 vs. 0.74, p = 0.022). At 98% specificity, and for lag times of ≤6, >6-18 or ≤18 months, sensitivities were 57%, 36% and 43% for CA19-9 combined with ApoA2-ATQ/AT, respectively, vs. 50%, 29% and 36% for CA19-9 alone. Compared to CA19-9 alone, the combination of CA19-9 and ApoA2-ATQ/AT may improve detection of pancreatic cancer up to 18 months prior to diagnosis under usual care, and may provide a useful first measure for pancreatic cancer detection prior to imaging.

Despite complete resection of the early stage of oral tongue cancer by partial glossectomy, late cervical lymph node metastasis is frequently observed. Gene amplification of ACTN4 (protein name: actinin-4) is closely associated with the metastatic potential of various cancers. This retrospective study was performed to demonstrate the potential usefulness of ACTN4 gene amplification as a prognostic biomarker in patients with stage I/II oral tongue cancer. Fifty-four patients with stage I/II oral tongue cancer were enrolled retrospectively, in accordance with the reporting recommendations for tumour marker prognostic studies (REMARK) guidelines. The copy number of ACTN4 and the protein expression of actinin-4 were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. The overall survival time of patients with gene amplification of ACTN4 was significantly shorter than that of patients without gene amplification (P=0.0010, log-rank test). Gene amplification of ACTN4 was a significant independent risk factor for death in patients with stage I/II oral tongue cancer (hazard ratio 6.08, 95% confidence interval 1.66-22.27). Gene amplification of ACTN4 is a potential prognostic biomarker for overall survival in oral tongue cancer.

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The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2–inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.

Background/Aim: Prognosis plays a vital role in head and neck squamous cell carcinoma (HNSCC) patient management and decision-making. This study aimed to identify the role of BP180 as a prognostic factor in HNSCC. Patients and Methods: Protein expression of bullous pemphigoid antigen II (BP180) was verified by immunohistochemistry (IHC) in a tissue microarray study of 202 cases. Results: IHC analysis revealed that protein expression of BP180 among HNSCC patients differed significantly in the presence and absence of neural invasion, and according to T status in laryngeal and pharyngeal cancer subgroups. Οverall survival and multivariate analysis showed that positive BP180-IHC and advanced clinical stage were significant independent positive predictors of mortality in HNSCC patients. In addition, in the oral cancer subgroup, independent positive predictors were positive BP180-IHC, advanced N status and neural invasion. In laryngeal and pharyngeal cancer subgroups, predictors were positive BP180-IHC and advanced clinical stage. Conclusion: BP180 is a prognostic factor in head and neck squamous cell carcinoma.Head and neck cancer was reported as the 7 th most common cancer worldwide in 2018, and the vast majority of malignant head and neck cancers are head and neck squamous cell carcinomas (HNSCCs) (1). Squamous cell carcinoma (SCC) affects the oral cavity, nasopharynx, oropharynx, hypopharynx and larynx. Early-stage patients with stage I or II disease can be cured with surgery alone and/or adjuvant therapies, improving long-term survival rates in approximately 70-90% of patients (2). About 60% of HNSCC patients present with stage III or IV disease (3). Such locally advanced disease shows high malignancy, easily develops early metastasis, and carries a poor prognosis with an overall 5-year survival rate below 50%. This pathology also displays a local recurrence rate of 15-40% and frequent distant metastasis (4).Bullous pemphigoid antigen II (BP180; also called collagen XVII, BPA-2 or BPAg2) is not only an epithelial transmembrane protein, but also a hemidesmosome transmembrane adhesion molecule, and likely participates in keratinocyte-matrix interactions in both physiological and pathological conditions (5,6). The intracellular domain contains binding sites for plectin, integrin b4, and BP230 (7). Hemidesmosomes are adhesion complexes connecting keratin intermediate filaments of stratified and complex epithelia to extracellular matrix components (EMCs) (8, 9). EMCs are responsible for cell communications, adhesion and proliferation, and has also been recognized as playing key roles in both progression and tumor initiation (10). Aberrant expression of BP180 has been reported in many malignant tumors, such as skin (11,12), esophageal (13), oral ( 14),

Introduction Factors associated with prognosis and molecular mechanisms leading to a malignant phenotype in head and neck squamous cell carcinoma (HNSCC) need to be identified. The focus was on the biological function and clinical impact of hepatitis B X‐interacting protein (HBXIP) in HNSCC. Methods mRNA expression profiles of HBXIP were compared between tumor and normal tissues in head and neck cancer cases from an online database. The protein expressions of candidate genes were verified by immunohistochemistry (IHC) with tissue microarray blocks designed with 221 cases. Prognostic significance was analyzed using the Kaplan–Meier method and Cox regression analysis. The molecular function of malignant phenotypes was investigated by generating knockdown cell lines of HNSCC with RNA interference. Results Analysis of the online database showed that the mRNA of HBXIP was significantly higher in the HNSCC group than in normal specimens. IHC analysis showed significant differences in the protein expression of HBXIP by clinical stage, T status, and recurrence. Multivariate analysis showed that, in HBXIP IHC‐positive patients, N status, T status, and neural invasion were significantly associated with overall survival. In addition, downregulation of the HBXIP gene in SAS, a tongue squamous cancer cell line, inhibited proliferation and the invasion phenotype. Conclusions By increasing the malignant phenotypes of cell proliferation and invasion, upregulation of HBXIP was a prognostic factor in HNSCC.