Analysis of a breakpoint cluster reveals insight into the mechanism of intrachromosomal amplification in a lymphoid malignancy

A distinct sub-group of B-cell precursor acute lymphoblastic leukemia, defined by intrachromosomal amplification of chromosome 21 (iAMP21), is restricted to older children and has been associated with a poor outcome. Accurate diagnosis is important for appropriate risk stratification for treatment. It could be improved by understanding the initiating mechanism. iAMP21 is characterized by amplification of a 5.1-24 Mb region of chromosome 21, which includes the RUNX1 gene. It is thought to arise through a breakage-fusion-bridge (BFB) mechanism. Breakpoints initiating BFB cycles were determined from recent array data from 18 patients. Three occurred within the PDE9A gene. Other patients with breakpoints in PDE9A were identified by fluorescence in situ hybridization and molecular copy number counting. Sequencing defined a 1.7 Kb breakpoint cluster region, positioned 400 bp distal to an extensive region enriched for CA repeats with the potential to form Z-DNA. None of the rearranged sequences showed the inverted repeat structure characteristic of BFB; instead PDE9A was fused to intergenic regions of chromosome 21 or to genes on other chromosomes. These observations indicated that previously unrecognized complex events, involving microhomology-mediated end joining, preceded or accompanied initiation of the BFB cycle. A chi-like heptomer, CCTCAGC, contained four of the breakpoints, two within PDE9A and two within partner Alu-repeat sequences. This heptomer was closely homologous to a breakpoint hotspot within the TCF3 gene, suggesting involvement of a common novel recombinogenic mechanism that might also contribute to the recombinogenic potential of Alu repeats. These findings provide insight into potential mechanisms involved in the formation of iAMP21.     

Focal increases of fetal macrophages in placentas from pregnancies with histological chorioamnionitis: potential role of fibroblast monocyte chemotactic protein-1

Citation Toti P, Arcuri F, Tang Z, Schatz F, Zambrano E, Mor G, Niven‐Fairchild T, Abrahams VM, Krikun G, Lockwood CJ, Guller S. Focal increases of fetal macrophages in placentas from pregnancies with histological chorioamnionitis: potential role of fibroblast monocyte chemotactic protein‐1. Am J Reprod Immunol 2011; 65: 470–479 Problem Histopathological chorioamnionitis (HCA) is caused by microbial‐driven infiltration of leukocytes to the maternal‐fetal interface resulting in adverse neonatal outcomes in a subset of pregnancies. The role of placental villus macrophages (i.e. Hofbauer cells, HBCs) in the pathophysiology of HCA is unelucidated. Method of study The number of HBCs in human term placental villi in HCA and control groups was compared using immunohistochemistry. Levels of monocyte chemotactic protein (MCP‐1) expression were measured in primary cultures of syncytioytrophoblasts (SCTs) and fibroblasts (FIBs) treated with bacterial compounds [lipopolysaccharide (LPS) and peptidoglycan] and pro‐inflammatory cytokines (TNF‐α and IL‐1β) using ELISA and quantitative real‐time PCR. Results Immunohistochemistry revealed a focal increase in HBCs in HCA. Treatment of FIBs with LPS, IL‐1β, and TNF‐α significantly increased MCP‐1 mRNA and protein expression. Conversely, MCP‐1 mRNA and protein levels were virtually undetectable in treated and untreated SCTs. Conclusion These results demonstrate cell‐type‐specific regulation of MCP‐1 expression in human placenta. A model is presented in which bacterial products and inflammatory cytokines initiate a fibroblast‐driven cytokine cascade resulting in recruitment of fetal monocytes to placenta which focally increases levels of HBCs in pregnancies complicated by HCA.     

Age-related increases in DNA repair and antioxidant protection: a comparison of the Boyd Orr Cohort of elderly subjects with a younger population sample

BACKGROUND: One commonly held theory of ageing is that it is caused by oxidative damage to critical molecules in the body, including proteins, lipids and nucleic acids. Accumulation of oxidative DNA damage with age will occur if there is an increase in reactive oxygen species in the body, or a decline in antioxidant defences, or a reduced efficiency of DNA repair. SUBJECTS AND METHODS: Using the comet assay, we have measured DNA breaks and oxidised purines in lymphocytes from subjects of different age groups: 20-35 (n = 40), 63-70 (n = 35), and 75-82 (n = 22). We also measured the resistance of lymphocyte DNA to H(2)O(2)-induced oxidative damage, and the repair activity of cell-free lymphocyte extracts on a substrate containing 8-oxoguanine. RESULTS: We found an increase in oxidative base damage in old age, but this apparently does not result from deterioration of either antioxidant defence or DNA repair. In fact, both of these tend to increase with age. There were few age-related differences in plasma levels of dietary antioxidants: tocopherols and retinol were higher in the older subjects, while lycopene was highest in the youngest age group. CONCLUSIONS: It is possible, that in old age, antioxidant defences and DNA repair are induced, in response to a higher level of oxidative damage, as mitochondria become more leaky and release more reactive oxygen. It is equally possible that older people, as survivors, had relatively high levels of antioxidant defences and DNA repair earlier in their lives, compared with those who did not survive to such an age.     

The simultaneous detection of trivalent & hexavalent chromium in exhaled breath condensate: A feasibility study comparing workers and controls

The analytical method outlined in this feasibility study has been used to show that trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) can be detected and measured in exhaled breath condensate (EBC) samples. EBC samples and urine samples were collected from a cohort of 58 workers occupationally exposed to hexavalent chromium compounds and 22 unexposed volunteers (control group). Levels of Cr(III) and Cr(VI) were determined in EBC samples and total chromium levels were determined in urine samples. Pre and post working week samples for both EBC and urine were collected in tandem. Total chromium in urine samples was analysed by inductively coupled plasma mass spectrometry (ICP-MS). Analysis of Cr(III) and Cr(VI) in EBC samples used a hyphenated micro liquid chromatography (μLC) system coupled to an ICP-MS. Separation was achieved using an anion exchange micro-sized column. The results showed that the occupationally exposed workers had significantly higher levels of Cr(III) and Cr(VI) in their EBC samples than the control group, as well as higher levels of total chromium in their urine samples. However, for the exposed workers no significant difference was found between pre and post working week EBC samples for either Cr(III) or Cr(VI). This study has established that Cr(III) and Cr(VI) can simultaneously be detected and measured in ‘real’ EBC samples and will help in understanding inhalation exposure.     

Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma

Pediatric ependymomas are enigmatic tumors, and their clinical management remains one of the more difficult in pediatric oncology. The identification of biological correlates of outcome and therapeutic targets remains a significant challenge in this disease. We therefore analyzed a panel of potential biological markers to determine optimal prognostic markers. We constructed a tissue microarray from 97 intracranial tumors from 74 patients (WHO grade II-III) and analyzed the candidate markers nucleolin, telomerase catalytic subunit (hTERT; antibody clone 44F12), survivin, Ki-67, and members of the receptor tyrosine kinase I (RTK-I) family by immunohistochemistry. Telomerase activity was determined using the in vitro-based telomere repeat amplification protocol assay, and telomere length was measured using the telomere restriction fragment assay. Primary tumors with low versus high nucleolin protein expression had a 5-year event-free survival of 74%+/-13% and 31%+/-7%, respectively. Multivariate analysis identified low nucleolin expression to be independently associated with a more favorable prognosis (hazard ratio=6.25; 95% confidence interval, 1.6-24.2; p=0.008). Ki-67 and survivin correlated with histological grade but not with outcome. Immunohistochemical detection of the RTK-I family did not correlate with grade or outcome. Telomerase activity was evident in 19 of 22 primary tumors, with telomere lengthening and/or maintenance occurring in five of seven recurrent cases. Low nucleolin expression was the single most important biological predictor of outcome in pediatric intracranial ependymoma. Furthermore, telomerase reactivation and maintenance of telomeric repeats appear necessary for childhood ependymoma progression. These findings require corroboration in a clinical trial setting.