Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma.

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.

Heat shock protein 90 as a molecular target for therapy in oral squamous cell carcinoma: Inhibitory effects of 17­DMAG and ganetespib on tumor cells.

Heat shock protein 90 (HSP90) expression is upregulated in numerous types of cancer. However, its role as a candidate for molecular targeted therapy in oral squamous cell carcinoma (OSCC) cells is poorly understood. In the present study, a common upstream search was performed using molecular network analysis software for proteins with expression abnormalities that were found in a proteomic analysis of six OSCC cell lines. HSP90 was identified as a target protein. In clinical samples, high frequencies of HSP90­high expression were detected via immunohistochemistry (26/58; 45%). Furthermore, the HSP90 expression status was associated with cervical lymph node metastasis (P=0.015). Furthermore, the potential of HSP90 as a candidate for molecular targeted therapy in OSCC cells was investigated using the HSP90 inhibitors 17­dimethylaminoethylamino­17­demethoxygeldanamycin (17­DMAG) and ganetespib. KON cells, which strongly express HSP90, were treated with the HSP90 inhibitors. The numbers of living cells in the 17­DMAG and ganetespib­treated groups were lower than those in the non­treated group. The cells treated with the inhibitors demonstrated reduced cell viability and migration, and this was associated with markedly decreased levels of the HSP90 target proteins EGFR, phospho­EGFR, phospho­MEK and phospho­MAPK in the treated groups compared with the non­treated group. To the best of our knowledge, this was the first study to investigate the effects of 17­DMAG and ganetespib on OSCC cells. The present results indicated the potential of HSP90 as a useful candidate for molecular targeted therapy in OSCC. However, additional studies with larger sample sizes are required to confirm these findings.

BP180 Is a Prognostic Factor in Head and Neck Squamous Cell Carcinoma.

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. Overall 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.

Monitoring of cancer patients via next-generation sequencing of patient-derived circulating tumor cells and tumor DNA.

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).

CA19-9 and apolipoprotein-A2 isoforms as detection markers for pancreatic cancer: a prospective evaluation.

Recently, we identified unique processing patterns of apolipoprotein A2 (ApoA2) in patients with pancreatic cancer. Our 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.

Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells.

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.