Feasibility of CTCs Isolation for Transcriptomes and Genome Wide Associated Analysis in Post-OP High Risk OSCC Patients.

Oral Cavity Squamous Cell Carcinoma

Oral Cavity Cancer, Circulating Tumor Cells, Whole-genome analysis, transcriptomes, distant metastasis, locoregional recurrence, second primary tumor

Anti-cancer treatments have been thought to be closely related to their unique genetic alterations. In the past few years, the investigator have used cDNA microarray to delineate the transcriptome profiles of differentially-expressed genes between OSCC tumors and normal epithelium. By supervised hierarachical clustering analysis, the investigator further analyzed and validated the differentially-expressed genes for OSCC tumors. In the investigators' previous research, the investigators have used this strategy to analyze the potential tissue proteins associated with OSCC tumors, indicating the feasibility of this strategy. However, gene detection is a great limitation and challenge in CTCs researches owing to the small number of isolated cells by traditional methods. Fortunately, by means of the investigators' developing high-purity CTCs isolation techniques, some preliminary data implied that isolated CTCs by this method could achieve the criteria of Whole-genome analysis (WGA), which brings the investigators' passion for further investigation.

First Year 1\. Negative Selection Method 2. Positive Selection method (Dynabeads) 3. ODEP devices 4. Flow Cytometry and Sorting System 5. Cell lines for Positive control Second Year &Third year 1. Clinical Trial, Patient Enrollment and Design of Schedule The Inclusion criteria and aims are as followings: 1. Patients with oral cavity squamous cell carcinoma (n=180), with age at diagnosis ≥ 20 years, will be enrolled before surgery at Ear-Nose-Throat department inpatient or outpatient service by Dr. Liao, Dr. Hsieh, Dr. Wang and co-PIs. 2. All patients will be subgrouped by pathologic features and risk factors Group A (n=90): OSCC, post-surgery, no need for any adjuvant therapy Group B (n=90): OSCC, post-surgery, with major risk factor(s) or 2 minor risk factors Stratification by Risk factors, alcohol, betel nut chewing and cigarette use status 3. Testing: Circulating tumor cells (CTCs) (x4) and NGS for WGA and transcriptomes (x4) 4. Endpoints: Loco-regional recurrence, one-year distant metastasis rate, death, second primary tumor occurrence. 5. Exclusion criteria are: (1) Patient's refusal; (2) Poor compliance, cannot cooperate for blood sampling for CTCs isolation as time schedule or clinical treatment or follow-up (3) Difficult blood sampling; (4) No more needs for CTCs evaluation, decided by clinicians. 2. Whole-Genome Analysis for Cancer Tissue Samples/Circulating Tumor Cells When needed, OncoScan™ FFPE Express Extraction will be performed Requirements need to be observed when submitting FFPE slices: Slices should be sent in 1.5mL conical vials Tumor samples in sections should have a surface area of 100 square mm (Minimum) to 1000 square mm (Maximum). Normal samples in sections should have a surface area of 200 square mm (Minimum) to 1000 square mm (Maximum). Section thickness should be 10 micron. The range of sections acceptable per sample is 2-6. Slices for OncoScan analysis and immunohistochemistry study are separated; and will be doubly charged by the Dep. of Clinical Pathology. The QIAamp DNA FFPE Tissue Kit is optimized for purification of DNA from FFPE tissue sections. OncoScan™ Express Samples will be performed in Chang Gung Memorial Hospital 1J GMRCL CoreLab or Chang Gung University Labs for whole-genome analysis. 3. Negative Selection Method 4. Positive Selection method (Dynabeads) 5. ODEP devices 6. Flow Cytometry and Sorting System

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