Flow Cytometry and Cytogenetics of Fine Needle Aspiration Biopsy Samples Is a Reliable Method for Diagnosing Burkitt Lymphoma. Evaluation of 78 Cases from a Single-Institution
Authors: Grzegorz Rymkiewicz , Blachnio Katarzyna , Grygalewicz Beata , Bystydzienski Zbigniew , Woroniecka Renata , Sledz-Gawronska Beata , Romejko-Jarosinska Joanna , Domanska-Czyz Katarzyna , Zajdel Michalina , Sikorska-Mali Kinga , Siwicki Jan Konrad , Pienkowska-Grela Barbara , Prochorec-Sobieszek Monika , Walewski Jan 
Published: October 2019

Abstract
Background: The diagnosis of Burkitt lymphoma (BL) is usually based on histopathology (HP), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) of lymph node or extranodal tissue excisional biopsy and occasionally, on flow cytometry (FCM) of cell suspensions. Accuracy of classical HP/IHC method is highly dependent on the quality sample fixation and takes substantial processing time. In addition, a range of antibodies used in IHC may be insufficient because CD20+/BCL6+/CD10+/BCL2- is not specific for BL. Furthermore, a need for surgery in cases of abdominal presentation may cause delay in initiating curative systemic treatment. Gene expression profiling studies demonstrated that HP/IHC criteria for distinguishing BL from diffuse large B-cell lymphoma (DLBCL) are often inadequate, and 20-30% of BL and DLBCL cases are misdiagnosed and thus inadequately treated. In addition, 5-10% of BL cases have no chromosomal translocation involving the MYC gene (MYC¯) detectable by FISH. We have recently described a BL MYC¯ with recurrent chromosome 11q aberrations.
Methods: We evaluated 78 cases of BL, initially diagnosed with HP/IHC, by FCM, classical cytogenetics (CC), and FISH, using cell suspension obtained with fine needle aspiration biopsy (FNAB) of peripheral lymph nodes (n=39), abdominal mass (n=31), cerebrospinal fluid (n=1), pleural effusion (n=4), and bone marrow (n=3). The samples containing at least 70% BL cells were evaluated. 68 cases were MYC+(male/female 52/16, median age/range 34/3-64/, HIV+: 9) and 10 were MYC¯ (male/ female 9/1, median age/range 25/18-62/, all HIV-). Antigen expression on BL cells was compared with normal lymphocytes as well as isotype controls. Antigen expression level was categorized into: [–] - no expression (<20% cells positive), [+/–] - expression on ≥20%, <100% cells, and [+] - expression on 100% of cells. Expression of CD (19, 20, 22, 23, 52, 79β), FMC7, HLADR, and CD (5, 25, 38, 43, 44, 45, 16&56, 56, 52, 62L, 71), BCL2 on BL cells was described as mean fluorescence intensity (MFI) value in comparison to MFI of these antigens on B - and T - lymphocytes, respectively. This procedure allows quantitative and comparative evaluation of pan-B antigens (i.e.: CD19 vs CD20 vs CD22 on BLs). Dim or bright expression was also defined on dot plots. All 78 cases showed cytological features of classical or non-classical BL on smears and all were confirmed by cytogenetic analysis (CC - 56/78pts, FISH - 78/78) with simple karyotype and set of FISH probes including MYC, BCL2 and BCL6. In addition, CCND1, ATM, MLL and telomeric 11q probe were used to evaluate BL MYC¯ cases.
Results: We identified characteristic qualitative and quantitative immunophenotypic pattern in both MYC+ and MYC¯ BL subgroups. All MYC+ and MYC¯ BL cases were CD45/CD19/CD20/CD22 - positive (MFI CD20>MFI CD19>MFI CD22), majority were CD10/CD43/CD52/CD56/CD79/HLA-DR - positive, and often FMC7 and CD138 positive while they were usually negative for CD5/CD11c/CD23/CD25/CD62L/BCL-2. Expression of CD62L was mostly seen on the small subpopulation of cells. We found a heterogenic type of CD44 and surface light/heavy chains (IgH) of immunoglobulin expression in both types of BL. The moderate intensity expression of clonal sIg and no κ/λ expression was found in 82% and 18% of MYC+, and in 70% and 30% MYC¯ BL cases, respectively.The most frequent pattern of IgH expression was IgM+ (48% and 33,5% in MYC+ and MYC¯, respectively) as well both IgD+/IgM+ (40% and 33,5% in MYC+ and MYC¯, respectively). In addition, proliferative activity measured by CD71 expression was detected in all BL samples on 100% of cells. MYC+ cases demonstrated significantly higher CD38 expression level and absence of CD16&CD56 expression compared to MYC¯ cases.
Conclusions: Combined use of FNAB/FCM/CC/FISH is a reliable method for diagnosing BL. BL has a characteristic immunophenotype on FCM examination. MYC+ and MYC¯ cases differ in the level of CD38 and CD16&CD56 expression. CD38 overexpression correlates with MYC rearrangement in MYC+ cases. FCM of a cellular suspension obtained by the FNAB is a safe, relatively non-traumatic, rapid (2 hours), and cost-effective procedure at our institution. Newly diagnosed BL is an oncologic emergency and a quick diagnostic decision is of critical importance in clinical practice. Therefore, we believe that FNAB/FCM/CC/FISH should be the optimal method for diagnosing BL in reference centers.

Disclosures
No relevant conflicts of interest to declare.