2007 Winter Meeting - London - The Pathological Society of Great ...

O5Immunohistochemical Study of Plasmacytoid Dendritic Cellsin Lymph Nodes Draining Breast Cancer by BDCA2 andCD123 Antibodies{P} R Ahmad 1 , H El-Hassi 1 ,MBurke 2 , H Singhal 2 ,SC Knight 1 ,NM Aqel 21 Antigen Presentation Research Group, Faculty of Medicine, ImperialCollege London, Northwick Park and St Marks Campus, London, UnitedKingdom, 2 Northwick Park Hospital, London, United KingdomBackground. Plasmacytoid dendritic cells (PDC) are a distinct subset of antigenpresenting cells which are major producers of Interferon type-1 and contributeto development of immune response. These cells are known as “plasmacytoidmonocytes, PM” in tissue sections. They are found in groups in paracoretex ofreactive lymph nodes (LN), close to high endothelial venules (HEV). PM arecharacterised by their bright expression of CD123 (IL-3 alpha-chain receptor);an important cytokine for PM survival, proliferation and differentiation) and bytheir specific expression, in frozen section, of an antibody reactive with blooddendritic cells (BDCA2).Methods. We examined PM in frozen sections of 40 axillary LN draining breastcarcinoma by using two antibodies, namely BDCA2 and CD123. The antigenlocalisation was performed using biotinylated detection system.Results. PM stained strongly for BDCA-2 and CD123 in frozen sections of allLN; they were found scattered discretely in LN paracortex and in groups, closeto HEV. A few PM were found in subcapsular LN sinus.Conclusions. 1. Identification of PM in LN paracortex, discretely and in groupsclose to HEV, confirms previous data on the localisation of these cells inreactive lymph nodes. 2. PM were observed in LN subcapsular sinus; this maysuggest that some PM migrate to LNs through the afferent lymphatics. 3.Consistent demonstration of PM in LN draining breast cancer suggests apossible role for PM in mounting an immune response towards cancer cells 4.BDCA-2 was found to be highly specific to PM; this can facilitate the use ofthis antibody to isolate a pure population of PM in flowcytometric studies.O6Richter’s Transformation of Chronic Lymphocytic Leukaemia{P} MA Catherwood 1 , L Venkatraman 2 ,MEl-Agnaf 3 ,TCM Morris 11 Belfast City Hospital, Belfast Northern Ireland, United Kingdom, 2 RoyalVictoria Hospital, Belfast Northern Ireland, United Kingdom, 3 UlsterHospital, Dundonald Northern Ireland, United KingdomBackground: Richter’s transformation of CLL into high grade lymphomaoccurs in up to 10% of cases. In CLL, unmutated IgVH status implies a poorprognosis in comparison to mutated IgVH genes. The mutational status andcytogenetic aberrations of CLL’s that transform to Richter’s syndrome are notwell understood.Purpose: To determine the mutational status and cytogenetic abnormalities inCLL patients with Richter’s transformation.Methods: BIOMED-2 protocol and primers were employed for determiningIgVH mutational status and gene usage. Mutations were identified bycomparison with the germline sequence using IMGT and Ig Blast databases.Chromosomal abnormalities were determined using interphase fluorescent insitu hybridisation (FISH) and commercial CLL set FISH probes.Results: Of the 270 CLL patients studied, 6 patients transformed to high gradeB-cell lymphoma (3 diffuse large B-cell lymphoma, 2 prolymphocyticleukaemia and 1 classical Hodgkin’s lymphoma). Two of these patients hadmutated VH genes, while the remaining 4 possessed unmutated IgVH genes.On FISH analysis the 2 mutated cases showed no detectable abnormality (n=2).The unmutated sub-group consisted of: biallelic del 13q14 (n=1), del 11q23(n=1), trisomy 12 (n=1) and del 17p13 (n=1).Conclusions: These results suggest that Richter’s transformation may occurirrespective of mutational status and chromosomal aberrations and may be dueto other factors including treatment related immunosupression.O7Expression and Regulation of Hypoxia-Inducible Factor (HIF)in Osteoclasts and GCTB{P} HJ Knowles, NA AthanasouUniversity of Oxford, Oxford, United KingdomThe alpha subunit of the transcription factor Hypoxia-Inducible Factor (HIF) isover-expressed in many human cancers where it is commonly associated withpoor prognosis and resistance to therapy. Giant cell tumour of bone (GCTB)contains numerous osteoclast-like multinucleated giant cells and a mononuclearcomponent predominantly comprising macrophages and stromal osteoblast-likecells. We have analysed expression of HIF-1α, HIF-2α and the HIF downstreamtarget gene BNIP3 by immunohistochemistry in 12 GCTB. Giant cells wereimmunoreactive for HIF-2 > HIF-1 with cytoplasmic > nuclear staining. BNIP3expression generally correlated with that of HIF-1α. A variable proportion ofmononuclear stromal cells and macrophages were also positive for all factors.HIF expression was further analysed by Western blot in normal humanmonocyte-derived osteoclasts and the osteoblastic cell line MG-63. In both celltypes the osteoclastogenic factors M-CSF and HGF induced HIF-1α and HIF-2α expression for the period 2-16h post-stimulation. This is the firstdemonstration of HIF expression in either GCTB or human osteoclasts.Induction of HIF by growth factors that promote osteoclast differentiation /activity and its expression in osteoclasts, osteoblasts and macrophages in vivosuggests a potentially important role for the HIF pathway in osteoclastdifferentiation and the pathogenesis of GCTB.O8Ossification During Distraction Osteogenesis in Patients Fittedwith Ilizarov Frames{P} E Byrne, C Evans, C HutchinsonUniversity of Manchester, Manchester, United KingdomThis study aims to find a pattern of ossification in the fracture gap of patientsfitted with tibial Ilizarov frames and assess how this pattern varies with timefrom the operation.A series of 55 digitised anterior-posterior plain x-ray films from 17 tibias fittedwith Ilizarov frames were analysed. The pixel densities from set sections ofeach fracture gap were obtained using Image J. An increase in pixel densitywas assumed to be ossification. The results were standardised using a sectionof tibial cortical bone distant from the fracture gap.The results show pixel density is greater at the proximal and distal ends of thefracture gap than the centre, this becomes less apparent with time. Pixel densityis greater at the medial aspect of the fracture gap than the lateral aspect, thisbecomes more apparent with time.We conclude that ossification starts at the proximal and distal ends of thefracture gap progressing toward the centre with time. The medial aspect of thefracture gap ossifies more readily than the lateral aspect, this is seen throughoutthe process. These findings correlate well with previous animal studies,validating this method as a potentially useful tool in research and clinicalpractice.Winter Meeting (191 st ) 3–5 January 2007 Scientific Programme51