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H.S. Leong

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    MINI 22 - New Technology (ID 134)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI22.12 - Molecular Characterisation of SCLC Using Both Circulating Tumour DNA and Circulating Tumour Cells Isolated from the Same Whole Blood Sample (ID 251)

      16:45 - 18:15  |  Author(s): H.S. Leong

      • Abstract
      • Presentation
      • Slides

      Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Response rates to first line chemotherapy are generally high, but progression free survival is short due to development of chemotherapy resistance via mechanisms not well understood. Due to the difficulty in collecting tissue biopsies in SCLC, blood, which can be sampled simply and routinely, provides a means of inferring the current genetic status of a patients tumour via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). These offer a minimally invasive opportunity to study drug resistance mechanisms, evaluate tumour heterogeneity and potentially reveal new drug targets in this disease. However, accurate assessment of both CTCs and ctDNA requires all blood cells be maintained intact until samples are processed, particularly when analytes present are at very low concentrations. Here we describe and validate a blood collection protocol that does not require on-site processing, and which is amenable for analysis of both CTCs and ctDNA following storage at ambient temperature in CellSave vacutainers for up to 96 hours after blood collection.

      To evaluate the suitability of using CellSave preserved samples for circulating free DNA (cfDNA) analysis, we undertook a 20 healthy normal volunteers (HNV) study and 45 patient sample study, with parallel EDTA and CellSave bloods collected. For each sample cfDNA was isolated between 4 hours and 96 hours post-draw and cfDNA yields determined. A potential issue with using CellSave blood was that the CellSave preservative could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, the EDTA and CellSave cfDNA samples were subjected to next generation sequencing (NGS) to estimate the overall mutation burden. In addition, the utility of CellSave ctDNA for targeted NGS was also determined. Finally, SCLC-specific copy number aberrations (CNA) were analysed in ctDNA and CTCs isolated from the same CellSave blood sample from individual SCLC patients.

      We demonstrate that yields of cfDNA obtained from 96-hour whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 hours of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples, thereby validating CellSave blood as a viable source of ctDNA. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA allowing direct comparison of the genetic status of patients’ tumours.

      In summary, we have demonstrated the suitability of whole blood CellSave samples for both CTC and ctDNA molecular analysis in SCLC. The ability to generate informative molecular profiles of both CTCs and ctDNA from a simple whole blood sample, up to 4 days post-draw represents a significant methodological improvement for clinical benefit. We posit that as minimally invasive, liquid biopsies become increasingly employed for cancer patient management, the ability to routinely and simply draw blood and ship samples to accredited biomarker assessment laboratories will greatly facilitate the delivery of personalised cancer medicines.

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