Abstract
The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.
Highlights
Genetic diversity is characteristic among tumors of the same entity and within the tumor itself
Prior to a clinical implementation of human papillomaviruses (HPV)-integrates as biomarkers for disease monitoring it is essential to clarify the issue of intra-tumor heterogeneity. Our study addresses this topic systematically by analyzing DNA from micro-dissected tissue of tumors by amplification of the viral-cellular junction
We aimed to evaluate the influence of intra-tumor heterogeneity on biomarker performance
Summary
Genetic diversity is characteristic among tumors of the same entity and within the tumor itself The latter is often referred to as intra-tumor heterogeneity and reflects the co-existence of different tumor sub-clones. In the absence of a common genetic aberration it would be necessary to perform multiple biopsies to identify a biomarker specific for each sub-clone in order to reliably detect persistent or recurrent disease based on liquid biopsies. This illustrates clearly the importance of a profound knowledge of the tumor’s genomic landscape and mutations common to all cells for optimal biomarker design
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