Correlation of CD30 Expression on Neoplastic Mast Cells in Systemic Mastocytosis Assessed By Immunohistochemistry Versus Multiparameter Flow Cytometry and Correlation to Clinical Parameters
Authors: Bellos Frauke , Sotlar Karl , Schnittger Susanne , Haferlach Claudia , Haferlach Torsten , Kern Wolfgang 
Published: October 2019

Abstract
Background: Systemic mastocytosis (SM) is rarely diagnosed and presents with highly variable clinical manifestation from taking rather indolent to very aggressive courses, mast cell leukemia being the most aggressive variant. Expression of CD30 (Ki-1 antigen) has been detected on some neoplastic mast cells (MC) by immunohistochemical staining (IHC) in bone marrow (BM) biopsies and presence of CD30 has been reported to be correlated to more aggressive variants of SM. Assessmentof CD30 expression by multiparameter flow cytometry (MFC) might not only contribute to improved diagnostic accuracy but correlation of the hereby detected CD30 expression with clinical SM parameters might also give further insights into disease biology.
Aims: Comparison of CD30 expression detected by either MFC or IHC on MC of patients with SM and correlation of results with patient and disease characteristics. Correlation of MFC detected CD30 expression with cytogenetics (CG) determined by chromosome banding analysis and molecular genetics (MG).
Methods: For this study, CD30 expression was analyzed in BM samples from 93 patients with SM by MFC using a five color staining assay with monoclonal antibodies against CD30, CD45, CD117, CD2 and CD25. We identified MC based on CD45 positivity and bright expression of CD117. Based on aberrant coexpression of CD2 and/or CD25 neoplastic MC were identified. On those MC, mean and median fluorescence intensities (MFI, medFI) of CD30 were determined and related to CD30 MFI and medFI in lymphocytes to derive CD30 index. Moreover, data on MC infiltration and CD30 expression by IHC was assessed in 22 patients and examination of CG and MG was done in 44 and 80 patients, respectively. KIT D816V mutation was analyzed using melting curve-based DNA mutation analysis applying PNA-mediated PCR clamping according to Sotlar et al. [Am J Pathol. 162: 737-746, 2003]. Results of MFC detected CD30 expression was correlated to those of CD30 expression examined with IHC and to the results of CG and MG.
Results: 42 patients were female and 51 male. Median age was 59 years (20-87 years). While we found normal karyotypes in 41 patients, 3 patients showed aberrant karyotypes. KIT D816V mutation was seen in 74/80 patients (93%). Diagnosis of concurrent hematological non-mast cell disease (AHNMD) was made in 14/93 patients (15%). Mean (±SD) MC infiltration was 20%±26% (range, 1.5%-85%) by IHC and 0.4%±1.8% (range, 0.01%-17%) by MFC. Mean (±SD) CD30 index was 19±20 (range, 3-154), mean (±SD) CD30 expression by IHC was 9%±17% (range, 0%-70%). Percentages of MC infiltration detected by IHC and MFC correlated significantly (p=0.002, r=0.819). No correlation of MFC CD30 index (MFI and medFI) with age, sex, concomitant AHNMD, grade of MC infiltration or percentage of CD30 positive MC by IHC was found. Interestingly, a significantly higher medFI CD30 index and a trend to higher MFI CD30 index were seen in patients with normal karyotype (12.2±6.2 vs 6.3±2.7, p=0.037 and 15.1±9.4 vs. 11.6±10.3, n.s., respectively) versus those with an aberrant karyotype. We also detected a trend to higher MFI and medFI CD30 index in patients with KIT D816V mutation (19.8±21.8 vs. 10.2±7.4, n.s. and 24.0±89.0 vs 10.0±5.8, n.s., respectively).
Conclusions: Assessment of CD30 expression as a dynamic parameter on neoplastic MC in patients with SM can be reliably performed by MFC. A stronger expression of CD30 expression on neoplastic MC harbouring an aberrant karyotype was found. CD30 expression seems also stronger on MC from patients harbouring KIT D816V mutations compared to those who do not. CD30 expression on neoplastic MC in patients with SM should be further analyzed combining analyses by MFC and IHC to substantiate the present findings.

Disclosures
Bellos: MLL Munich Leukemia Laboratory: Employment. Sotlar:Ludwig-Maximilians-University: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.