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N. Chen



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    MINI 26 - Circulating Tumor Markers (ID 148)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI26.07 - Circulating Tumor Cells (CTC) Enrichment as Liquid-Biopsy for Molecular and Genomic Characterization in ALK-Rearranged (ALK+) Lung Cancer (ID 3132)

      17:15 - 17:20  |  Author(s): N. Chen

      • Abstract
      • Presentation
      • Slides

      Background:
      Precision therapy with tyrosine kinase inhibitor (TKI) crizotinib and ceritinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) has advanced rapidly in recent years as a new paradigm in personalized cancer therapy. However, acquired drug resistance despite initial response still remains the rule, necessitating further investigations into mechanisms of resistance and novel therapies to overcome progressive resistant disease. Liquid-biopsy using peripheral blood circulating tumor cells (CTCs) as a minimally-invasive tool to determine patient’s disease status, tumor cells molecular-genomic make-up and evolution during therapies, is highly desirable. There is still an unmet need to develop affordable and robust technology platforms to empower such liquid-biopsy assay of CTC. There are relative advantages and pitfalls with various CTC platforms and a method to capture CTC in an unbiased fashion without pre-definition would be beneficial.

      Methods:
      We conducted pilot studies with adoption of two different CTC detection and enrichment platforms. First, we used the CellSearch[®] “positive-selection” platform through EpCAM immunomagnetic separation to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative-selection” CTCs capture-isolation platform, based on unbiased immunomagnetic removal of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to enable CTC isolation without predefined CTC criteria. Pilot studies utilizing ALK+ H3122 cell line and ALK+ patients’ blood samples were performed for assay optimization and comparison. Whole genome sequencing using Illumina HiSeq x TEN was performed after whole genome amplification of the CTC tumor gDNA with paired-normal germline DNA for genomic interrogation.

      Results:
      Using ALK+ NSCLC patients’ peripheral blood samples, we demonstrated the presence of the EML4-ALK fusion (variant 1) in QPCR assay from the enriched CTC isolated using the CellSearch® platform. Also, CellSearch[®] enumeration in our pilot ALK+ cohort revealed a trend of correlation between the CTC numbers and disease status. The spike-in experiment in “negative selection” CTC platform enriched the spiked H3122 cells by 10,000 fold from the nucleated blood cells. Applying our “negative-selection” CTC assay to a patient with known EML4-ALK variant 1 (EML4-ex13/ALK-ex20) fusion lung cancer during disease progression on crizotinib, we detected the specific EML4-ALK variant 1 fusion in QPCR assay from the CTC enriched “eluate” fraction, but not in the “feed” fraction, whether the CellSearch® platform revealed any CTCs or not. In our index case of ALK-rearrangement NSCLC, we successfully performed whole genome sequencing analysis on the pretreatment negative-selection CTCs in comparison with the germline DNA and pretreatment lymph node tumor biopsied tissue tumor DNA. Our preliminary WGS results revealed similar genomic landscapes between the CTC and the biopsied tumor tissues.

      Conclusion:
      Taken together, our pilot CTC study results support the high sensitivity of the unbiased “negative selection” enrichment platform and its potential to empower molecular and genomic determinations in lung cancer. We also demonstrated the feasibility of the negative-selection CTC liquid-biopsy platform to achieve whole genome sequencing analysis of the captured CTCs.

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