Multiplexed IHC analysis to enable Hodgkin lymphoma differential diagnosis on a single slide.

Abstract

e19536 Background: Routine diagnosis of classical Hodgkin lymphoma is performed with a panel of immunohistochemistry markers to evaluate the biomarker expression profile of the relatively rare Hodgkin cells. One of the key challenges of this technique is that serial immunostains are used and hence it can be difficult or impossible to locate the same Hodgkin cell on adjacent slides. Given the rarity of the Hodgkin cells coupled with the number of markers that are needed for a definitive diagnosis, we developed a new technique in which a single patient slide is multiplexed with nine different antibodies . Methods: One FFPE tissue section from 11 cases was probed for the following nine biomarkers: CD30, CD15, CD45, Pax5, CD20, CD79a, OCT2, Bob1, and CD3. An initial 10x whole slide fluorescent image of CD30 was acquired and presented to the pathologist who based on this staining selected regions of interest for higher magnification (40x) imaging of the CD30 and the other antibodies. The fluorescent images acquired were processed for interpretation using an in-house developed viewing tool. The pathologist was able to view each biomarker as a standard grayscale, monochromatic image, an overlay of two or more biomarkers, or as a virtually created molecular DAB image. Results: A correct diagnosis of classical Hodgkin lymphoma vs. other was able to be made using the MultiOmyx platform in all cases. Subjectively, the pathologist noted that the novel methodology allowed for a significantly more confident assessment of marker expression on the Hodgkin cells in the seven cases of classical Hodgkin lymphoma, eliminating many issues of staining ambiguity and allowing recognition of subtle nuances of staining intensity in the Hodgkin cells. The CD30+ cells in the four other cases, three cases of B-cell lymphoma and one case of lymphocyte predominance Hodgkin lymphoma, showed a B-cell profile that was distinguishable from the classical Hodgkin cell phenotype. Conclusions: This new method of fluorescent multiplexing on a single tissue section allows more accurate interpretation of the biomarker expression profile on the same Hodgkin cell. It is likely that this paradigm can be expanded to a greater range of challenging cases in hematopathology.