Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling

Abstract

Objectives: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. Material and Methods: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the “dot-type” (representing late G₂ phase) and “mitotic” immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. Results: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G₂ phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G₂ phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G₂ staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G₂/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). Conclusions: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G₂ phase B cells in reactive folliculi directs to physiological G₂/M checkpoint blockade. In contrast, accelerated G₂/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

RETRACTED: Impact of Histone Modifications and Their Therapeutic Targeting in Hematological Malignancies

“…Additionally, the phosphorylation of histone 3 serine 10 (H3S10) is responsible for the immediate condensation of chromatin in the late G2 phase of the cell cycle and, thus, can function as a marker that is present only in dividing cells [ 119 ]. Indeed, the levels of phosphorylated H3 have been used to determine mitotic activity and to experimentally, as well as clinically, measure cellular proliferation in different cancer types, including lymphomas [ 120 , 121 , 122 ].…”

Histone Modifications and Their Targeting in Lymphoid Malignancies

“…Thus, the discovery of compounds that target histone phosphorylation is an interesting approach for the treatment of oncological patients. In their study, Méhes et al [215] showed an enhanced G2 phase transition due to the abrupt chromatin phosphorylation in neoplastic B-cells. Moreover, during the Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 November 2021 G2 phase, phospho-H3 co-localizes with the Aurora B kinase which is known to be frequently overrepresented in NHL patients [216,217].…”

Histone Modifications and Their Targeting in Lymphoid Malignancies

“…Phosphorylation of H3 at serine 10 (phospho-H3) results in immediate condensation of the chromatin in the late G2 phase of the cell cycle, and thus, is a marker present in dividing cells [ 207 ]. Indeed, mitotic activity determined by phospho-H3 counting has been experimentally and clinically used to measure cell proliferation in different types of cancer, including lymphoma [ 207 , 208 , 209 , 210 ]. Thus, the discovery of compounds that target histone phosphorylation is an interesting approach for the treatment of oncological patients.…”