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C. McCulloch

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    P1.03 - Poster Session 1 - Technology and Novel Development (ID 150)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.03-001 - Multiplexing technology for in situ biomarker profiling of Non-Small Cell Lung Cancer (NSCLC) (ID 1467)

      09:30 - 16:30  |  Author(s): C. McCulloch

      • Abstract

      NSCLC is a heterogeneous neoplasm comprising several histologic types, etiology, genetics, survival and response to therapy. Accurate analysis of these subtypes has increased sample requirements, which is challenging in the era of minimally invasive procedures. A recent survey of 90 US pathologists presented at ASCO 2013 meeting, concluded insufficient sample availability in 6% of all NSCLC samples recently handled by these pathologists. Moreover, subcellular localization of marker expression linked to tumor pathobiology necessitates methodological advancement. With the development of a new platform that allows in situ, multiplexed sub-cellular analysis of over 60 proteins, this project aims to demonstrate the feasibility of detailed in situ molecular profiling and perform comparative analysis of known cancer pathways and prognostic markers on the same serial section.

      Multiplex immunofluorescence staining and imaging of over 30 biomarkers, including several RTKs, cell adhesion molecules, select members of PI3K, MAPK/ERK, JAK/STAT pathways, angiogenesis, hypoxia, proliferation and chemotherapy resistance markers were performed on replicate FFPE tissue microarrays (TMA) from 382 samples. Cell-level and subcellular-level marker expressions were quantified using image analysis algorithms and compared between serial sections. Associations between marker expressions and histological subtypes and survival were investigated in European male smokers. Multivariate analysis was performed using logistic regression and Cox proportional hazard models on over 300 quantitated features of marker expression. All models controlled for age. Serial sections were modeled separately and combined to improve confidence in associations. EGFR and cMET positivity was evaluated using whole cohort median expression values to define positive cells and the summary statistics are reported using 10% positive cells as cutoff for characterizing positive samples.

      In concordance with previous reports, differential expression of RRM1, CK5 and CK7 was observed in SCC vs AD in the high grade, early stage male smokers (N=86). With a 10% cell positivity threshold, 72.7% (76.3%, serial section (SS)) of all male smokers (N=183 (190, SS)) were positive for EGFR. EGFR positivity was higher in SCC, 83.9% (86.0%, SS) compared to AD 54.9% (61.8%, SS). Opposite was observed for cMET with 81.7% (78.9%, SS) of AD characterized as positive compared to only 58.0% (57.0%, SS) SCC. Among the several previously reported prognostic markers evaluated in this study, only CA9 expression was associated with overall patient survival with a hazard ratio of 1.47, p-value 0.0005 (N=278). Again, analysis of serial section produced a similar result confirming the robustness of the platform.

      The study demonstrates the capabilities of multiplexing technology (MultiOmyx[TM]) for assessment of limited lung samples, encompassing topographic expression features and the ability to observe relationships between markers through in situ pathway profiling. Additionally, by evaluating markers on exactly the same sample set (same section), a direct comparison of their relative significance in predicting course of disease is now feasible.