T(11;14)(q13;q32) in Mantle Cell Lymphoma Patients Is Present Only on Mature Malignant Cells. Fluorescence-Activated Cell Sorting and Fluorescence in Situ Hybridization Analysis
Authors: Vorobyev Vladimir I. , Gretsov Evgenii’ M , Obukhova Tatyana N , Domracheva Elena V , Lorie Yuri Yu , Kravchenko Sergey K , Vorobjev Ivan A 
Published: October 2019

Abstract
Abstract 4405

Background:
Mantle cell lymphoma (MCL) is a distinct incurable B-cell neoplasm with a median survival of 3 to 5 years. t(11;14)(q13;q32) is the hallmark of the disease leading to overexpression of protooncogene cyclin D1. At least 60% of MCL patients have unmutated VH genes. Chemoresistance to conventional treatment and continuous disease relapses even after high dose therapy and autologous stem cell transplantation define the clinical course of the disease. A possible cause of this frequent failures may be that t(11;14)(q13;q32) occurs in early hematopoietic precursors, capable of multilineage differentiation.

Aim:
evaluation of t(11;14)(q13;q32) in different hematopoietic lineages of MCL patients.

Methods:
Bone marrow mononuclears from 12 MCL patients (including pleomorphic and blastoid variants) were sorted by Fluorescence-Activated Cell Sorting (BD FACSVantage SE) to divide the following cell lineages: CD45+CD34+ (progenitor cells); CD45+CD5+CD19+light chain Ig (mantle cell lymphoma); CD45+CD5-CD19+ (normal B-cells); CD45+CD14+ (monocytes); CD45+CD3+ (T-cells); CD45-GlyA+ (erythrokaryocytes) and granulocytes by light scattering, excluding CD14+CD45+ cells. The purity of sorted cells was checked by flow cytometry (BD FACSCanto II) and correlated to the number of sorted cells – if there were more than 50 thousands cells the purity was more than 92% (usually more 97%), but if there were less than 20 thousands cells the purity was 80% (never less). After sorting cells in the test-tube were washed from the PBS, fixed in methanol and glacial acetic acid mixture and layered onto slides by Cytospin centrifuge (Cellspin II Tharmac). The probes have been denatured at 75°C for 2 minutes, and hybridized with dual fusion LSI IGH-CCND1 probes (Vysis Inc.) at 37°C for 17–20 hours. Signals were visualized with Olympus IX-61 microscope with triple filter cube (DAPI / FITC / TexasRed).

Results:
t(11;14)(q13;q32) was present in 97% of sorted mantle cell lymphoma cells (range 84–100%), whereas in cells of all other lineages including normal B-cells t(11;14)(q13;q32) was not found at all. However, direct assessment of t(11;14)(q13;q32) in CD34+CD45+ cells and in normal B-cells was difficult due to the small number of these cells. In one case (classic variant MCL) we have sorted two “independent” cell populations 1 log different in the level of expression CD5: CD5dimCD19+ and CD5+CD19+. In both subpopulations t(11;14)(q13;32) was detected in 98,5% cell nuclei, indicating no correlation between CD5 level and fusion gene expression. In three cases using together FACS and FISH we were able to detect the population of MCL cells in bone marrow as small as 0,1–0,05%, whereas histology examination and standard FISH analysis failed to detect bone marrow involvement.

Conclusion:
Out data give evidence for the absence of t(11;14)(q13;q32) in early hematopoetic cells. FACS allows increase FISH sensitivity in 100–1000 times.

Disclosures:
No relevant conflicts of interest to declare.