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P. Towne

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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-046 - ALK IHC is Highly Sensitive to Fixation Parameters (ID 5792)

      14:30 - 15:45  |  Author(s): P. Towne

      • Abstract

      An ALK genetic translocation event occurs in ~2-7% of non-small cell lung carcinoma patients (NSCLC), resulting in the constitutive expression of an active chimeric ALK protein, which leads to tumor proliferation. Ventana has developed a fully automated immunohistochemistry (IHC) assay using the VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody (ALK (D5F3)) to detect the ALK protein in formalin fixed paraffin embedded tissue. ALK IHC testing is becoming more widespread in NSCLC; however, information concerning the impact of pre-analytical conditions on the ALK antigen is limited. We used human cell lines expressing ALK, generated as xenografts to evaluate the effect of Fixation Type, Time, and Delay to Fixation (Ischemia) on the ALK antigen as measured by the ALK (D5F3) antibody staining intensity.

      NCI-H2228 human NSCLC cell lines were used to generate xenograft tumors in SCID mice. In order to assess ischemia (delay to fixation) H2228 xenografts were used to model ischemia in 10% NBF. The impact of fixation on staining performance of the ALK (D5F3) primary antibody was assessed using the H2228 xenografts to model fixation type and fixation time for 10% NBF, Zinc Formalin, 95% Alcohol, Alcoholic Formalin Acid, B5, and Prefer for 1, 6, 12, 24 and 72 hours. Each of the stained slides were evaluated by a pathologist using a qualitative assessment and scored on a 0-3+ intensity scale (0 = no staining detected, 1+= weak staining detected, 2+= moderate staining detected, 3+ = strong staining detected).

      Fixation in all time points in B5, Prefer, and AFA, as well as ethanol, severely compromised staining intensity of ALK (by decreasing staining intensity greater than 0.5 points). Ischemia greater than 6 hours also decreased staining intensity. In contrast, EGFR (5B7) and TTF1 (SP141) antigens were robust across a wide range of fixation times and types, as well as ischemia times.

      The ALK antigen is highly sensitive to fixative time, type and ischemia. The data indicate that the detection of ALK by IHC is impacted by fixation conditions. Strikingly, antigens detected by other lung antibodies (EGFR, TTF1) are more robust in comparison across a range of conditions. Standardized pre-analytical conditions are critical to achieve appropriate staining for ALK IHC and to mitigate the risk of false negative results. The recommendations for pre-analytical conditions for ALK are to fix at least 6 hours in 10% neutral buffered formalin.