Novel mRNA Detection Assay for Quantifying CD30 Expression in Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma
Authors: Wong Jerry Thwin , Ippolito Thomas , Schlosser Andrea , Hernandez-Ilizaliturri Francisco J. , Neppalli Vishala T. 
Published: October 2019

Abstract
Introduction:
Innovations in detecting and measuring antigen expression is paramount in the current era of targeted therapy and check-point inhibitors. For instance, Brentuximab Vedotin (BV), one of two FDA-approved antibody-drug conjugates, employs an anti-CD30-monomethyl auristatin E (MMAE) immunoconjugate for the treatment of refractory Hodgkin lymphoma and anaplastic large cell lymphoma with positive CD30 expression. However, a lack of correlation between objective response rate and range of CD30 expression has been previously demonstrated, and the limitations of immunohistochemistry in detecting low levels of CD30 has been implicated. Moreover, there is interest in the study of the topology of checkpoint proteins such as programmed cell death-1 ligands (PD-L1) malignant cells and surrounding PD-1 expressing immune cells, which requires assay with high sensitivity, specificity, and accurate spatial localization.
In this study, we implemented a novel CD30 mRNA detection method using the BaseScopeTM assay from Advanced Cell Diagnostics to enable detection of specific CD30mRNA isoforms that correspond the different CD30 protein segments within the neoplastic cells and tumor microenvironment immune cells. Using this method, the CD30 mRNA expression patterns in classical Hodgkin lymphoma (CHL) and anaplastic large cell lymphoma (ALCL) were evaluated.
Methods:
Nineteen CHL and 11 ALCL cases were selected from the Roswell Park Comprehensive Cancer Institute department of pathology archives. CD30 expression by immunohistochemistry (IHC) and CD30 mRNA isoform expression by BaseScope™ assay were measured on formalin fixed paraffin embedded ( FFPE ) tissue specimens. Four probe sets spanning the exon junctions for increased specificity were used to detect domains on the protein corresponding to signal peptide (SP), transmembrane portion (TMP), cytoplasmic portion (CP), and transmembrane and cytoplasmic portion (TMP &CP) of full length CD30 protein. The hybridization, amplification, and staining of probes are based on the manufacturer's protocol. The BaseScope™ assay probe signal is detected in the form of red dots, which are localized to the neoplastic cells and tumor microenvironment immune cells. Semi-quantitative analysis of both positive and negative signals using 100X magnification was performed on 100 neoplastic cells for each probe for each case, by two hematopathologists (VN, JW). For tumor microenvironment immune cells, an average number of positive and negative cells were obtained in five 100X magnification fields.
Results:
In both CHL and ALCL, 100% of the neoplastic cells were positive for CD30 protein by immunohistochemistry. The table below illustrates relative expression of CD30 mRNA isoforms in CHL and ALCL neoplastic cells. Expression of CD30 mRNA in CHL the tumor microenvironment cells is also tabulated.
Conclusions:
Detection of CD30 mRNA isoforms using BaseScope™ assay offers a reliable and reproducible signal detection platform, as an alternative to CD30 protein expression by immunohistochemistry. The BaseScope™ assay provides a high-contrast low noise signal for easy visualization, accurate quantification, and precise positive signal localization. Different CD30 isoform patterns and signal frequency were observed within the neoplastic cells and immune cells in the tumor microenvironment. The CD30 mRNA isoform corresponding to transmembrane domain was a dominant expresser in both CHL and ALCL neoplastic cells and bystander cells in CHL. This novel mRNA-based method can potentially provide a more sensitive method for antigen expression detection and be used in new research studies.
Table. Table.

Disclosures
No relevant conflicts of interest to declare.