Graft-versus-host disease (GVHD) is a major risk factor for secondary malignancy after hematopoietic stem cell transplantation. Squamous cell carcinoma (SCC) of the skin and mucous membranes are especially frequent in this setting where aneuploidy and tetraploidy are associated with aggressive disease. The current study is directed to the mechanism of neoplasia in this setting. Un-manipulated keratinocytes from areas of oral GVHD in 9 patients showed tetraploidy in 10–46% of cells when examined by florescence in situ hybridization (FISH). Keratinocytes isolated from biopsy sites of GVHD but not from normal tissue showed even greater numbers of tetraploid cells (mean 78%, range 15–85%; N=9) after culture. To mimic the inflammatory process in GVHD, allogeneic HLA- mismatched lymphocytes were mixed with normal keratinocytes. After two weeks, substantial numbers of aneuploid and tetraploid cells were evident in cultures with lymphocytes and with purified CD8 but not CD4 cells. Telomere length was substantially decreased in the lymphocyte-treated sample. No mutations were present in p53 gene, although haploinsufficiency for p53 due to loss of chromosome 17 was common in cells exposed to lymphocytes. These findings suggest that in GVHD, inflammation and repeated cell division correlates with the development of karyotypic abnormalities.
Patients with aplastic anemia and other autoimmune diseases such as rheumatoid arthritis are more likely to develop myelodyplasia when compared to healthy controls. Inflamed tissue from Barrett’s esophagus, areas of bronchitis, healing burn tissue, and bowel with ulcerative colitis commonly show increased numbers of tetraploid and aneuploid cells, suggesting that inflammation may have a role in promoting genomic instability. We recently demonstrated the common occurrence of aneuploid cells in the 15 buccal smears of transplant patients with graft versus host disease involving the oral mucosa but not in transplant patients without GVSH when these cells were examined by fluorescence in situ hybridization (FISH). Aneuploidy could be reproduced in 5 normal keratinocyte cultures following incubation with allogeneic HLA-mismatched lymphocytes. Oxidative stress and repeated cell division with telomere shortening may play a role in producing karyotypic abnormalities. Alternatively, in the AA bone marrow factors such as increased cytokine expression and the presence of cytotoxic T cells in marrow may lead to a relatively improved survival of cells with certain chromosomal abnormalities. In this respect, we previously demonstrated survival advantages for trisomy 8 and monosomy 7 relative to diploid cells (Proc Natl Acad Sci2006; 10314483: Blood. 2007;109(6):2399). In order to more thoroughly assess the role played by inflammation in producing aneuploidy, we examined five samples of normal bone marrow co-cultured for two weeks with allogeneic, autologous lymphocytes (effector to target ratio of 2:1), or with interferon alone (1000 u/mL); media was changed after 24 hours and FISH was performed using probes to chromosomes 7,8 and 20 after two weeks. Slides were read by three different individuals blinded to the identity of the slides. All bone marrow samples with allogeneic lymphocyte and with interferon but not those cultured with autologous lymphocytes showed significant numbers of aneuploid cells; Aneuploidy was most prominent in bone marrow treated with allogeneic lymphocytes (monosomy 7 mean control=1%±1%; treated=11±2%; p=0.01; trisomy 8 control=0±0%; treated=6±1%; p=0.06; deletion 20 control=1±1%; treated=14%; p=0.01; N=5). In order to ensure that these changes were not secondary to apoptosis-related DNA disintegration and to determine if aneuploidy occurred in cells capable of self renewal, bone marrow cells were exposed to allogeneic lymphocytes and to interferon g (1000 units/mL) in short-term methylcellulose culture for two weeks; we subsequently picked cells from hematopoietic colonies, pooled these cells, and stained them with annexin-FITC. Cells were sorted by flow cytometry and annexin-negative cells were subjected to FISH using centromeric probes to chromosome 8, 7 and 20. These isolated cells, derived from single progenitors, still demonstrated significant numbers of trisomy 8, monosomy 7, and deletion (20) by FISH (Fig. 1). Spectral karyotyping (SKY) analysis demonstrated chromosomal abnormalities in 13/25 (52%) of cells and prominent telomeric fusion in many metaphase preparations (Fig II). In order to determine whether telomeric shortening could play a role in fusion of chromosomes and subsequently aneuploidy, we measured telomere length by Southern hybridization: bone marrow cells cultured with allogeneic lymphocytes showed a 20% decline in telomere length relative to control populations. These in vitro data suggest that inflammatory changes may be associated with genomic instability, possibly secondary to accelerated telomere attrition in response to regeneration. Fig 1 Fig 1. Fig 2 Fig 2.
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