HBV-host cfDNA as Minimal Residual Tumor Marker for HBV-related HCC

Development stages:

Stage I: Identification of integrated HBV DNA sites in HBV-related HCC tissues. the investigators will develop either HBV-specific inverse PCRs or capture-sequencing protocols to identify HBV integrations sites in the tumor chromosomes. The viral-host junction sequences of individual HCC identified and used as the template for developing assays for detecting the same HBV-host chimera DNA fragment in the circulation.

Stage II: New platforms to accurately detect or even quantitate the circulating vh-chimera DNA fragment.

These tumor-specific vh-chimera DNA will only represent a tiny fraction of total cfDNAs. However, as these sequences of the tumor-specific vh-chimera DNA have been known from stage 1, the investigators may develop better and more specific assays for quantitation.

Stage III: Assays for cell-free tumor specific vh-chimera DNA applied to the blood samples from post-curative HCC patients.

To demonstrate the efficacy of these assays, blood samples obtained at 4 weeks after curative therapies from 50 HBV-related HCC patients, and tested for the presence of cf tumor-specific vh-chimera DNAs. The presence or absence of such vh-chimera DNA will be correlated with the early HCC recurrence within the first year to determine any clinical significance. There will be one blood sampling at the time of HCC recurrence.

Sample collection In stage I and II, the investigators will set up methods for chimera DNA identification and quantification, which need tumor samples to support the development and evaluation of the feasibility for each assay. Therefore, the investigators will apply the approval for use the tissues from the Taiwan Liver Cancer Network (TLCN), including 20 pairs of HBV-related male HCC, 20 pairs of HBV-related female HCC (for positive control), 20 pairs of HCV related male HCC, 20 pairs of HCV related female HCC (for negative control), 20 pairs of HBV- and HCV-related male HCC, and 20 pairs of HBV- and HCV-related female HCC (to see if vh-chimera DNA is also applicable for HBV- and HCV-related HCC). Both the genomic DNA and RNA will be applied for these 120 patients in total. Genomic DNA will be used in assay development, including identification, detection and quantification; whereas RNA will be used in validation of the insertional mutagenic RNA transcripts, which will provide supporting evidence for tumor specific integration from transcription level.

In stage III, the investigators will investigate whether there is any correlation between tumor-specific vh-chimera DNA level and recurrence-free survival using the assays the investigators developed in stage I and II. Therefore, the investigators will collect the Tumor (T) and non-Tumor (NT) tissue pairs from 50 HBV-related HCC patients that receive surgical removal of tumor, and also the peripheral blood at 4w after surgery. T and NT tissues are for vh-chimera DNA identification, peripheral blood will be used for vh-chimera DNA detection and quantification. On the other hand, clinical information including tumor size, tumor grade, serum markers from routine liver function test (ALT, AST), current HCC marker (AFP), and recurrence-free survival time will be collected for correlation study. All data will be stored in computer with password protection.