3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost' nuclei and very short telomeres

Abstract

In Epstein-Barr virus (EBV) negative Hodgkin's cell lines and classical EBV-negative Hodgkin's lymphoma (HL), ReedSternberg cells (RS cells) represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. However, the three-dimensional (3D) telomere organization in latent membrane protein 1 (LMP1)-expressing RS cells of EBV-associated HL is not known. We performed a 3D telomere analysis after quantitative fluorescent in situ hybridization on 5 mm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (Po0.0001) and formation of telomere aggregates in RS cells (Po0.0001), when compared with the mononuclear precursor Hodgkin cells (H cells). Telomere-poor or telomere-free 'ghost' nuclei were a regular finding in these RS cells. These nuclei and their telomere content strongly contrasted with the corona of surrounding lymphocytes showing numerous midsized telomere hybridization signals. Both H cells and RS cells of two EBV-negative HL cases analyzed in parallel showed 3D telomere patterns identical to those of LMP1-expressing cases. As a major advance, our 3D nuclear imaging approach allows the visualization of hitherto unknown profound changes in the 3D nuclear telomere organization associated with the transition from LMP1-positive H cells to LMP1-positive RS cells. We conclude that RS cells irrespective of LMP1 expression are end-stage tumor cells in which the extent of their inability to divide further is proportional to the increase of very short telomeres, telomere loss, aggregate formation and the generation of 'ghost' nuclei. Mononuclear H cells are the precursors of multinuclear RS cells, 6-9 and endomitotic multinucleation is associated with disturbed cytokinesis and jumping translocations 10,11 pointing to a severe telomere dysfunction. 12,13 Telomeres are the nucleoprotein complexes at the ends of chromosomes in which a number of specific proteins, either binding telomere proteins directly or a protein complex, termed 'shelterin,' is directly associated with telomeric DNA. [14][15][16] Profound changes in the three-dimensional (3D) nuclear organization of telomeres are the hallmark of the transition from mononuclear H cells to multinuclear RS cells in EBVnegative Hodgkin cell lines and in classical EBV-negative HL. We recently showed that EBV-negative RS cells represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. 1 However, nothing is known about the 3D telomere organization in LMP1-expressing H and RS cells of EBV-associated Hodgkin's disease (HD). In this study, we document 3D telomere dynamics in LMP1-expressing H and RS cells and show conformity with those observed in EBV-negative HD analyzed in parallel.

Dynamic chromosomal rearrangements in Hodgkin's lymphoma are due to ongoing three-dimensional nuclear remodeling and breakage-bridge-fusion cycles

“…We conclude that: (i) chromosomes 9 and 22 are not observed in their regular territories in Hodgkin cells irrespective of their mono-or multi-nucleated nature; (ii) the spatial distribution and number of these two chromosomes is different from nucleus to nucleus; (iii) some nuclei of RS cells are chromosome-poor containing no or very little chromosome 9 or 22 material; (iv) such nuclei can be considered 'ghost' cell nuclei; 1,9 and (v) the number of overlaps between the two chromosomes seen in interphase nuclei may vary between H and RS cells.…”

“…8 Different pathways lead to such instability, but, in the context of HL, telomere dysfunction is a prime mechanism leading to the transition of H to RS cells. 1,9,10 As first shown by Müller 11 and McClintock, 12,13 chromosomal endto-end fusions create dicentric chromosomes that form anaphase bridges and break upon completion of a cell division cycle. After completion of cell division, both daughter cells remain with two 'broken' chromosome ends that represent a double-strand break.…”

“…This refers to both telomeres 1,9 and chromosomes (this study). Chromosomes 9 and 22 are indicators of this situation.…”

“…10,38 Taken together, we propose that H cells are tumor cells with altered nuclear architecture and resulting chromosomal rearrangements at a level that still supports tumor growth. In contrast, RS cells 'struggle' to keep dividing; not only is their nuclear architecture further disrupted, not only do they contain 'ghost cell nuclei' with little telomeric material 1,9 and low levels of chromosomal material, they also have an uneven distribution of genetic material within each of their sub-nuclei ( Figure 2B). Chromosomal territories are disrupted, chromosomal rearrangements continue to occur, aneuploidy increases and genetic diversity within a single RS cell (referring to each of its sub-nuclei) is enhanced with each attempt to divide.…”

Novel automated three‐dimensional genome scanning based on the nuclear architecture of telomeres

“…chromosome number, and 3D nuclear architecture of telomeres, have been used to define the transition of mononucleated H-cells to multi-nucleated Reed-Sternberg cells in Hodgkin's Lymphoma (14)(15)(16)(17), to classify subgroups of glioblastoma patients (11) to detect premalignant cells in mouse plasmacytoma and early lesions in cervical cancer (18).…”