FOXO1 has an oncogenic role in adult germinal center-derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1, resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples, respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3, or members of the phosphatidylinositol 3-kinase signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

AimsRecent attempts to study MYC distribution in human samples have been confounded by a lack of agreement in immunohistochemical staining between antibodies targeting the N-terminus and those targeting the C-terminus of the MYC protein. The aim of this study was to use a novel in-situ hybridization (ISH) approach to detect MYCmRNA in clinically relevant samples, and thereby determine the reliability of MYC-targeting antibodies.Methods and resultsWe performed immunohistochemistry on human formalin-fixed paraffin embedded normal colon (n = 15), hyperplastic polyp (n = 4) and neoplastic colon samples (n = 55), using the N-terminally directed antibody Y69, and the C-terminally directed antibody 9E10. The MYC protein distributions were then compared with the location of MYCmRNA, determined by ISH. We found that the localization of MYCmRNA correlated well with the protein distribution determined with the N-terminally directed antibody Y69, and was also associated with expression of the proliferation marker Ki67. The protein distribution determined with the C-terminally directed antibody 9E10 was not always associated with MYCmRNA, Y69, or Ki67, and indeed often showed a reciprocal pattern of expression, with staining being strongest in non-proliferating cells.ConclusionsThe observed discrepancy between the staining patterns suggests that the significance of 9E10 in immunohistochemical staining is currently uncertain, and therefore should be interpreted with caution.

Fine needle aspirates from Burkitt's lymphoma and other tumours transferred directly into ThinPrep® PreservCyt® (Cytyc UK Ltd, Crawley, UK) buffered alcohol fixative retain their cellular and viral antigens and nucleic acids for many months at ambient temperatures. Despite the presence of blood and debris, cells dried onto slides from droplets and post-fixed in formalin, or sections of paraffin-embedded cell blocks from formalin post-fixed pellets, prove adequate for morphology, immunocytochemistry, in-situ hybridization and molecular biological analyses. Where there is lack of expertise in making thin smears or hospitals lack pathology laboratories and services, PreservCyt® provides an excellent medium for transport elsewhere for diagnosis and research.

Chronic myelomonocytic leukaemia (CMML) is a clonal disorder with myelodysplastic/myeloproliferative features. Its diagnosis is based on the presence of peripheral blood monocytosis and bone marrow aspirate findings, according to World Health Organization criteria. However, bone marrow trephine biopsy (BMTB) features characteristic of CMML have not been adequately investigated. We studied BMTB in 20 cases of CMML-1 and three cases of CMML-2 and compared with ten cases of polycythaemia vera, ten cases of chronic myeloid leukaemia (chronic phase) and ten cases of florid myeloid hyperplasia (MH). Furthermore, we evaluated the use of CD34, CD117 and CD68 (PGM-1) antibodies in diagnosis and subtyping of CMML and in differentiating from other categories. Hypercellularity, high myeloid:erythroid ratio, increased proportion of 'myelo/monocytic' cells often seen as clusters and/or nodules, absence of eosinophilia, and presence of abnormal localisation of immature precursors (ALIP) and dysmegakaryopoiesis can aid in the diagnosis of CMML in BMTB. CD68 (PGM-1) positive cells amounted to 20.7 +/- 6.1% cells among CMML trephines. The proportion of CD34(+) cells among cases of CMML-1 was =5% and among CMML-2 was >/=10% cells in two of three cases and 5% in the other case. Morphological and immunohistochemical features of BMTB samples are extremely helpful in the diagnosis of CMML.

An increase in leucocyte apoptosis and impaired clearance of apoptotic cells has been observed in patients with systemic lupus erythematosus (SLE). Apoptotic cells are likely to be a key source of autoantigens in SLE as they express many of the nuclear autoantigens (in surface blebs and apoptotic bodies) that are relevant to this disease. The clearance of apoptotic cells is usually a rapid process, such that few cells are usually seen in the extracellular environment in vivo. We report a case in which multiple apoptotic bodies were observed in the bone marrow of a patient with SLE that was complicated by an immune-mediated pancytopenia. We have subsequently examined the frequency of apoptotic cells, identified morphologically, and by caspase-3 staining in bone-marrow trephine samples taken from patients with SLE over a 10-year period of follow-up. A high proportion of bone marrows contained apoptotic debris. The novel demonstration of apoptotic bodies in vivo in patients with SLE is unusual and supports the notion that the marrow may be a target organ in the disease. Their abundance is also consistent with the hypothesis that normal clearance mechanisms are defective and/or overwhelmed in SLE.

Acute myeloid leukaemia (AML) with multilineage dysplasia (MD) is one of the four main categories of AML in the World Health Organization (WHO) classification. The role of bone marrow trephine biopsy (BMTB) histology and immunohistochemistry in the diagnosis of AML-MD is currently unclear. BMTBs were studied in 11 cases of AML-MD and two cases of myelodysplasia that subsequently transformed to AML. Among them, six cases showed trilineage dysplasia and seven showed bilineage dysplasia. With respect to conforming to the WHO definition of AML, documentation of an increased proportion of immature myeloid cells was possible on morphology and counting of immature cells following immunostaining with CD34, CD117 or HLA-DR antibodies. Recognition and quantification of dysplastic features in the haemopoietic lineages was made easier by immunohistochemistry with antibodies to ret40f (glycophorin C), myeloperoxidase, CD61 and/or CD42b, CD34, CD117 and HLA-DR. Based on this relatively small series of cases we show the utility of BMTB and immunohistochemistry as an aid to the diagnosis of AML-MD. This has to be seen not just in light of its utility at diagnosis, but also the role the diagnostic BMTB would play for purposes of comparison when follow-up BMTBs are submitted in this group of patients.

Monoclonal Gammopathy of Unknown Significance (MGUS) is a non-malignant condition characterized by a serum paraprotein, low numbers of plasma cells on bone marrow examination(<10%) and the absence of end organ damage. The transformation rate of MGUS to multiple myeloma is frequently quoted as about 1% per year. IgG, IgM and IgA paraproteins account for virtually all cases of MGUS with only the rare literature report of IgD or IgE isotypes. To our knowledge there is only one published case of IgE MGUS (Ludwig and Vormittag 1980; BMJ , 281:6239, 539–540). IgE Myeloma is an extremely rare entity and reported to have a comparatively poorer prognosis, but the incidence of pre-existing IgE MGUS is unknown. Here we report the case of a 74 year old gentleman with a stable low level IgE paraprotein. The patient presented in March 2003 for investigation of a persistent borderline normocytic anaemia recorded as 13.3g/dl at initial review (lower range of normal 13.5g/dl) with an ESR of 27mm/h. Co-morbidity included hypertension, ischaemic heart disease and hypercholesterolemia but general examination was unremarkable. Screening investigations revealed normal haematinics, renal function and corrected calcium. Analysis of immunoglobulins revealed mild immunoparesis of his IgM titre only (0.33g/l; Reference range 0.5–2.0g/l) and the presence of both a blood and urinary paraprotein. The blood paraprotein was quantified as 3g/l with immunofixation identifying kappa light chain restriction without the presence of one of the commonly encountered heavy chain isotypes (IgG, IgM or IgA). Further probing of the abnormal gamma globulin region revealed the paraprotein to be IgE in isotype. Immunofixation of a spot urine sample revealed kappa light chain restriction with a protein concentration of 0.03g/l. Bone marrow examination demonstrated 1% plasma cells on the aspirate with mild dysplastic change within the erythroid series. Cytogenetics were not performed. The trephine specimen revealed 10% plasma cells with strong immunostaining for IgE, dysplastic features within the megakaryocytic and erythroid lineages together with confirmation of kappa light chain restriction. Interestingly, recent further immunostaining of the original specimen revealed the plasma cells to be CD56-ve but cyclin D1 positive. Skeletal survey was normal. His investigations fulfil the WHO diagnostic criteria for MGUS, although it could be argued that with 10% plasma cells in the trephine biopsy, he could be classified as a borderline smouldering myeloma. Four years later, however, this gentleman remains well on routine follow-up with no evidence of myeloma related organ or tissue impairment. His most recent paraprotein from August 2007 is stable at 4g/l. The reporting of plasma cell dyscrasias with rare immunoglobulin isotypes should be encouraged to contribute to the bank of knowledge of these infrequently encountered entities.

American Journal of HematologyVolume 81, Issue 1 p. 79-79 Letters and CorrespondenceFree Access CD117 Expression as an aid to identify immature myeloid cells and foci of ALIP in bone marrow trephines Kikkeri N. Naresh, Kikkeri N. Naresh Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this authorIrvin A. Lampert, Irvin A. Lampert Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this author Kikkeri N. Naresh, Kikkeri N. Naresh Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this authorIrvin A. Lampert, Irvin A. Lampert Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this author First published: 20 December 2005 https://doi.org/10.1002/ajh.20452Citations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Valverde LR, Matutes E, Farahat N, Heffernan A, Owusu-Ankomah K, Morilla R, Catovsky D. C-kit receptor (CD117) expression in acute leukemia. Ann Hematol 1996; 72: 11– 15. 2Wells SJ, Bray RA, Stempora LL, Farhi DC. CD117/CD34 expression in leukemic blasts. Am J Clin Pathol 1996; 106: 192– 195. 3Siitonen T, Savolainen ER, Koistinen P. Expression of the c-kit proto-oncogene in myeloproliferative disorders and myelodysplastic syndromes. Leukemia 1994; 8: 631– 637. 4Bene MC, Bernier M, Casasnovas RO, Castoldi G, Knapp W, Lanza F, Ludwig WD, Matutes E, Orfao A, Sperling C, van't Veer MB. The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL). Blood 1998; 92: 596– 599. Citing Literature Volume81, Issue1January 2006Pages 79-79 ReferencesRelatedInformation

We studied expression of CD34 positive cells in the bone marrow trephines of two groups of patients with CML treated with imatinib. In the first group (n=17), patients went on to achieve sustained complete cytogenetic response during their disease course. In the second group (n =33) complete cytogenetic response was not achieved and patients later developed either accelerated or blastic phase disease. We studied paraffin embedded bone marrow trephines stained with the monoclonal antibody QBEND10 during an eighteen month period following initiation of imatinib therapy and recorded the highest CD34 positive cell counts and aspirate blast counts in three six-month intervals. CD34 positive cells were evaluated using three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65 and the third method utilised the highest number of CD34 positive cells expressed in a single high power field (CD34+/hpf), the value being taken from the previous CD34+/10 hpf count. We hypothesised that the overall CD34 positive cell burden and absolute CD34 positive numbers would be more important in predicting the likelihood of complete cytogenetic response and this would be better assessed by volumetric evaluation than percentage calculation relative to other nucleated cells. CD34 expression in endothelial cells was taken as a positive internal control. We aimed to establish which variable: % blast count, % CD34+/500 cells, CD34+/10hpf and CD34+/hpf provided better correlation with subsequent cytogenetic response to treatment. Values for each variable were dichotomised at the following levels; aspirate blast count 5, % CD34+/500 cells 2%, CD34+/10hpf 60 cells and CD34+/hpf 10 cells. Univariate analysis was performed on samples within the first six months, seven to twelve months and thirteen to eighteen months. Only CD34+/10hpf showed significant discriminatory ability throughout the entire eighteen month period (Pearson’s Chi-square 2-sided sig: 0.031, 0.003 and 0.007 in the first six months, seven to twelve months and thirteen to eighteen months samples respectively). By logistic regression, we confirmed that CD34+/10hpf had the best correlation with cytogenetic response during the first year following initiation of imatinib therapy. Larger studies are required to confirm these initial findings and to establish volumetric assessment of CD34 positive cells as a predictor for cytogenetic response.

This course is designed for haematologists and histopathologists with a special interest in haematopathology.The course focuses on the cellular and molecular pathology of the haemopoietic and lymphoid system and has a strong emphasis on microscopy and interpretation.The Special Guest Speaker is Daniel Catovsky (London).

Primary BL in Malawian children has a very high frequency association, approaching 100%, with the human herpesvirus EBV. A detailed study carried out on viral gene expression in these tumours, using both fresh material and methanol-fixed FNAs, showed, contrary to prediction, that most belong to a variant "class II" latency category, with lytic cycle-related genes also expressed. That is, in addition to EBNA1 expression, membrane proteins (LMP1/2A), immediate early (BZLF1) and early (IR2 and IR4) genes, a putative viral oncogene (BARF1), CST (BART) antisense transcripts and the viral bcl-2 homologue are expressed in a high proportion of the BLs. Most, but not all, express the small viral (EBER) RNAs. Two other significant observations were made: (i) in addition to expression of cellular cytokine (IL-10) transcripts in all tumours investigated, the normally silent viral IL-10 homologue was expressed in some tumours; (ii) whereas EBNA1 expression from its restricted Qp promoter was generally observed, the nonrestricted Cp/Wp promoter was also active in some tumours. Viral gene expression in the Malawian [endemic (e)] BLs appears to be more promiscuous than predicted from other studies, but expression accords with the cytopathologic picture of eBLs as a rapidly proliferating cell population accompanied by considerable necrosis, and a clinically diverse disease. A small-scale study of relapse Malawian BLs revealed a different picture of viral association, more akin to systemic BL than eBL, where EBV appears to be absent or present only at very low levels. The significance of these findings is considered.

The effect of cyclosporin A (CyA) immunosuppression on the murine thymic microenvironment and T lymphocyte development has been analysed using monoclonal antibodies to epithelial and lymphocyte subpopulations, macrophages and major histocompatibility complex (MHC) class II antigens in immunohistochemistry and flow cytometry. The major microenvironmental target for CyA-induced damage was the thymic medulla, where a reduction in all epithelial cell subsets, dendritic cells and macrophages was observed. In contrast, the thymic cortex appeared essentially normal. CyA had no detectable effect on the intensity of microenvironmental expression of MHC class II molecules in either cortex or medulla, although the number of MHC class II positive medullary cells was reduced after CyA treatment. CyA also had a differential effect on the thymic lymphocyte populations where there was little change in the Thy-1 bright, CD5 dull, CD4+, CD8+ cortical thymocytes but a depletion of the Thy-1 dull, CD5 bright, CD4 or CD8 single-positive medullary cells. This lymphocyte loss may be due partly to increased migration from thymus to spleen and other peripheral lymphoid organs, and partly to a block in the differentiation stage from cortical to medullary lymphocyte. The thymic microenvironment and lymphocyte subpopulations recover rapidly after cessation of CyA treatment, although there may be longer term functional defects resulting from the CyA-induced injury.

Lipofuscin granules were found in an unusual distribution in an atrophic human spleen. The pigment was confined to the sinus littoral cells and was absent from the splenic histiocytes. The lipofuscin differed from the ceroid-type of lipofuscin found in the various conditions associated with the ‘syndrome of the sea-blue histiocyte’ in its distribution, ultrastructure and its association with splenic atrophy rather than splenomegaly.

Various T cell subsets were characterized by double immunofluorescent staining using monoclonal antibodies (MoAb) in blood, bone marrow (BM) and tissues of 29 patients after allogeneic BM transplantation (BMT). In an attempt to prevent graft versus host disease (GvHD), 15 patients received cyclosporin A (Cy A). In the remaining 14 patients the BM was pre-incubated with a MoAb, OKT3. The regeneration of T4+ subset was delayed and the level of T8+ cells was abnormally high even 1 year after engraftment. This did not have any predictive value for the appearance of complications such as GvHD or severe viral infections. The number of T8+ cells was lower in the group of patients who received Cy A than in the OKT3 group (0.7 +/- 0.2 vs 1.5 +/- 0.3 X 10(9)/1 at day 90). In contrast to normal individuals, the T4/T8 ratio in both blood and regenerating BM of BMT patients was less than 1. A sizeable subset of circulating T cells expressed the phenotype T8+, T10+, HNK-1+, DR+. Circulating cells of this phenotype were transiently very high (up to 50%) in patients with active GvHD or suffering from severe viral infection. This subpopulation of lymphocytes was not found in the epidermal infiltrate that accompanied GvHD where the predominant phenotype was T8+, T1-, T10-, HNK-1-, DR-. We conclude therefore that after BMT the number and phenotype of circulating T cells reflects the T cell distribution seen in the regenerating BM.

The authors review a large body of contemporary immunohistologic findings on the tissue distribution of T lymphocytes in normal and pathological conditions. The suggestions for technological advances in this field are: signal amplification using mixtures of monoclonal antibodies directed against different epitopes on the same antigen (e.g. OKT4A+B+D), triple layer amplification systems using hapten-labelled antibodies, and informative double staining methods with combinations of antibodies labelled with different fluorochromes or enzymes. Review of histological observations in a series of human diseases suggests that imbalances of OKT4+ and OKT8+ subsets of T lymphocytes may represent different types of immunoregulatory disorders. Rheumatoid arthritis and sarcoidosis appear to involve a high level of OKT4+ subpopulation response coupled with an associated appearance of a special type of HLA-DR+ macrophages. It remains to be seen whether normal or self-limited immunological responses (early stages of bacterial infection or delayed-type hypersensitivity reactions) produce OKT4+ and macrophage responses that are characteristically different. Meanwhile, excessive levels of OKT8+ cells have been found in a wide range of recognized or presumed immunoregulatory disorders including: graft-vs.-host reaction and viral infections. These disorders, as well as primary biliary cirrhosis and lichen planus, appear to possess both overlapping and disparate clinical characteristics, and the immunohistological observations may reflect the functional heterogeneity of OKT8+ populations in these diseases. These studies show that histologically meaningful heterogeneity can already be demonstrated for the OKT8+ lymphocyte group.