Distribution of the c-MYC gene product in colorectal neoplasia.

AIMS: Recent 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 MYC mRNA in clinically relevant samples, and thereby determine the reliability of MYC-targeting antibodies. METHODS AND RESULTS: We 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 MYC mRNA, determined by ISH. We found that the localization of MYC mRNA 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 MYC mRNA, Y69, or Ki67, and indeed often showed a reciprocal pattern of expression, with staining being strongest in non-proliferating cells. CONCLUSIONS: The 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.

Bone marrow trephine morphology and immunohistochemical findings in chronic myelomonocytic leukaemia.

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 /=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.

Bone marrow trephine findings in acute myeloid leukaemia with multilineage dysplasia.

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.

Burkitt's lymphoma: maximising the use of fine needle aspirates by long-term preservation for diagnosis and research.

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.

In vivo evidence for apoptosis in the bone marrow in systemic lupus erythematosus.

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.

The expression of minichromosome maintenance protein-2 in normal and abnormal megakaryocytes and comparison with the proliferative marker Ki-67.

The minichromosome maintenance (Mcm) and Cdc6 proteins are important regulators of eucaryotic DNA replication. In most normal tissues, a similar proportion of cells express Mcm-2 and Ki-67. The present study showed that in both normal and abnormal states, the proportion of megakaryocytes expressing Mcm-2 is roughly seven times as many as those that express Ki-67. This is likely to be related to the process of endomitosis and endoreduplication. We also demonstrated that a significantly lower proportion of megakaryocytes in myelodysplastic syndrome express Mcm-2. These findings provide new insights into megakaryocyte biology.

Promiscuous expression of Epstein-Barr virus genes in Burkitt's lymphoma from the central African country Malawi.

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.