Bassem Sawan

The 3D nuclear organization of telomeres marks the transition from Hodgkin to Reed-Sternberg cells

To get an insight into the transition from mononuclear Hodgkin cells (H cells) to diagnostic multinuclear Reed–Sternberg cells (RS cells), we performed an analysis of the three-dimensional (3D) structure of the telomeres in the nuclei of the Hodgkin cell lines HDLM-2, L-428, L-1236 and lymph node biopsies of patients with Hodgkins disease. Cellular localization of key proteins of the telomere-localized shelterin complex, the mitotic spindle and double-stranded DNA breaks was also analyzed. RS cells show significantly shorter and significantly fewer telomeres in relation to the total nuclear volume when compared with H cells; in particular, telomere-poor ‘ghost’ nuclei are often adjacent to one or two nuclei displaying huge telomeric aggregates. Shelterin proteins are mainly cytoplasmic in both H and RS cells, whereas double-stranded DNA breaks accumulate in the nuclei of RS cells. In RS cells, multipolar spindles prevent proper chromosome segregation. In conclusion, a process of nuclear disorganization seems to initiate in H cells and further progresses when the cells turn into RS cells and become end-stage tumor cells, unable to divide further because of telomere loss, shortening and aggregate formation, extensive DNA damage and aberrant mitotic spindles that may no longer sustain chromosome segregation. Our findings allow a mechanistic 3D understanding of the transition of H to RS cells.

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

In Epstein–Barr virus (EBV) negative Hodgkins cell lines and classical EBV-negative Hodgkins lymphoma (HL), Reed–Sternberg 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 μm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (P<0.0001) and formation of telomere aggregates in RS cells (P<0.0001), 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.

Abstract 4725: The 3D nuclear telomere organization of Hodgkin-cells is different between patients entering rapid remission and patients with refractory or relapsing disease

Our recent study has utilized innovative 3D Q- FISH and three-dimensional (3D) imaging to define the 3D nuclear telomere organization in mono-nucleated Hodgkin (H) and multi-nucleated Reed-Sternberg (RS) cells of Hodgkin9s lymphoma (HL) derived cell lines and diagnostic patient biopsies (Leukemia. 2009). These characteristics were found in both, classical EBV negative and EBV-associated, LMP1 expressing HL (Lab Invest. 2010). However, it is still unknown whether the 3D telomere profile at diagnostic biopsy is different in patients entering rapid remission after initiation of standard chemotherapy (ABVD) compared to patients with refractory or relapsing disease. In this study, we investigated by 3D telomere Q-FISH diagnostic biopsies of HL patients entering rapid complete remission and diagnostic biopsies from patients with refractory or relapsing disease. 8 diagnostic biopsies of 8 patients entering rapid remission (after 1-4 cycles of ABVD) and 8 diagnostic biopsies of 5 patients including 2 with primary refractory disease (progressing after 4-8 cycles of ABVD) and 3 cases relapsing 1-3 years after late remission (post 6 -8 cycles of ABVD) were analyzed the 3D telomeric structure by 3D Q-FISH and TeloView software (Cytometry A. 2005). We found that RS-cells of all patients from both groups showed significant increase in very short telomeres when compared to the mononuclear precursor H-cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2011-4725

Significance of CD23 expression in diffuse areas of follicular lymphoma.

To the Editor: Follicular lymphoma (FL) derives from germinal center B cells and is characterized phenotypically by coexpression of CD20, Bcl2, Bcl6, and CD10. Typically the growth pattern is follicular but diffuse areas can also be found. Lack of CD10 antigen expression and expression of CD23 is well documented particularly in interfollicular area. CD23 expression is often reported to be nonspecific. It is more frequent in grade I follicular lymphoma of the groin region. Recently, in a review about the common problems encountered in hematopathology practice, Isaacson mentioned that follicular lymphoma cases comprise the majority of lymphoproliferative disorders submitted to consultation. According to his experience, immunohistochemical criteria seem to have aggravated the problem of distinguishing between entities. Among others, CD23 expression in follicular lymphoma gives rise to insecurity. We share this experience with the author but we would like to mention to the readers to keep in mind that composite lymphoma, for example, follicular lymphoma and small lymphocytic lymphoma (SLL) may exist, and then CD23 expression must be interpreted carefully when expressed by interfollicular lymphocytes. Composite lymphomas have been well documented. It is defined by the coexistence of 2 morphologically and phenotypically distinct types of lymphoid neoplasm that may occur within the same organ or tissue. However, reported cases of composite lymphoma involving either SLL or FL are very rare and little is known about their pathogenesis. To illustrate this issue, we report an 89-year-old man who presented with cutaneous single nodule of the left forearm. No other involvement was detected. Histologic examination of the surgically resected lesion revealed a lymphoid lesion showing follicular and diffuse areas (Figs. 1A, B). FIGURE 1. A, Nodular pattern is dominant. B, Focally expansive interfollicular areas. C, Nodular and diffuse areas are CD20 +. D, Interfollicular areas are CD10 negative.