Abstract 2716: A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma

Abstract

Abstract Background: Classical Hodgkin Lymphoma (CHL) ranks among the most frequently occurring lymphomas and is marked by Hodgkin and Reed-Sternberg (HRS) cells, frequently multi-nucleated neoplastic cells that are derived from germinal center B cells. The genetic alterations involved in the pathogenesis of this lymphoma have been difficult to study because HRS cells are rare and are surrounded by robust, non-neoplastic infiltrate, therefore they frequently represent less than 0.1% of cells within involved lymph nodes. Consequently, methods that isolate these rare cells are required to understand pathobiology of this lymphoma. We used the AccuCyte® - CyteFinder® system (RareCyte) to develop a method for detection and isolation of HRS cells. Methods: A 6-parameter immunofluorescence assay was designed to identify HRS cells that included HRS markers (CD40, CD30, CD15), CD3, combined CD20/CD14 and a cell membrane permeability viability dye. The assay was tested using the KMH2 Hodgkin lymphoma cell line spiked into PBMC and transferred to well slides for imaging by CyteFinder. This instrument utilizes 6-color scanning microscopy coupled with predefined algorithms to identify rare cell populations. The assay was then applied to a frozen fine needle aspirate (FNA) sample taken from the lymph node of a patient with histologically confirmed CHL. The FNA sample was thawed, prepared into a suspension, stained and analyzed as above. Cells identified with HRS phenotype were individually retrieved using the integrated CytePicker module. PCR of framework regions of the immunoglobulin heavy chain gene (IGH) was performed on single HRS cells (InvivoScribe) to assess clonality. Results: Of ~200 KMH2 cells spiked into blood, the assay identified 180 cells (90%) with the correct HRS phenotype. HRS phenotype cells were identified in the patient lymph node FNA sample. Two individual cells analyzed by IGH framework region PCR demonstrated identical size PCR products, indicating clonality of the B cell receptor rearrangement. Conclusion: A multi-parameter assay was successfully developed for identifying rare HRS cells with the CyteFinder system. Cells with HRS phenotype in a lymph node FNA sample were demonstrated to have clonal IGH rearrangements, demonstrating that actual HRS cells were identified. This method may be a useful tool for the diagnosis of CHL by identification of rare HRS cells from FNA specimens, as well as to investigate the molecular genomics of CHL. Citation Format: Lance U'Ren, David Wu, Jonathan R. Fromm, Jackie Stilwell, Eric Kaldjian. A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2017-2716