British Society of Haematology, Slide Session presented at the Annual Scientific Meeting, Brighton 2009.

Seven cases were discussed by an expert panel at the 2009 Annual Scientific Meeting of the British Society of Haematology. These cases are presented in a similar format to that adopted for the meeting. There was an initial discussion of the presenting morphology, generation of differential diagnoses and then, following display of further presenting and diagnostic information, each case was concluded with provision of a final diagnosis.

FIP1L1-PDGFRA positive chronic eosinophilic leukaemia and associated central nervous system involvement.

Interstitial deletion involving chromosome 4q12 generates the novel tyrosine kinase fusion protein encoded by FIP1L1-PDGFRA, which is present in many patients previously labelled as having hypereosinophilic syndrome, initially reported in 2003. Reports in recent literature document excellent clinical and molecular response to the tyrosine kinase inhibitor imatinib (Glivec). This report describes the case of a 58-year-old lady, diagnosed with FIP1L1-PDGFRA positive hypereosinophilic disorder, who subsequently developed symptoms related to an intracranial lesion. Biopsy and molecular genetic studies confirmed a diffuse infiltrative lesion, with evidence of FIP1L1-PDGFRA gene fusion. Initiation of imatinib treatment led to impressive clinical and radiological response.

Post-transplant lymphoproliferative disorders presenting as gingival overgrowth in patients immunosuppressed with ciclosporin. A report of two cases.

BACKGROUND: Post-transplant lymphoproliferative disorder (PTLD) can occur in patients maintained on immunosuppressive therapy following transplantation. This paper describes two cases of PTLD occurring in gingival tissues, in patients receiving ciclosporin following cardiac transplantation. TREATMENT: The lesions were localised to gingival tissues, mimicking ciclosporin-induced gingival overgrowth. They were removed surgically and the ciclosporin dose reduced to help prevent recurrence. CONCLUSION: The importance of histopathological examination of all tissue removed during routine gingivectomy procedures for ciclosporin-induced gingival overgrowth is highlighted.

Unusual splenic sinusoidal iron overload in sickle cell/haemoglobin D-Punjab disease.

Sickle cell/haemoglobin D-Punjab disease is a disorder with similar clinical features to sickle cell anaemia. This report describes the case of an 11 year old boy with this disease who was treated with regular transfusions from infancy. He underwent splenectomy at the age of 10 years for hypersplenism. Histology of the spleen revealed a striking pattern of heavy sinusoidal endothelial iron loading, with only moderate uptake by macrophages. Possible explanations for this unusual distribution of iron include phagocytosis of sickled erythrocytes by sinusoidal endothelial cells or direct endothelial iron uptake via transferrin receptors. Transfusion programmes ameliorate the symptoms of sickle cell disease but the dangers of iron overload should always be remembered.

Loss of heterozygosity at chromosome 9p21 is a frequent finding in enteropathy-type T-cell lymphoma.

Enteropathy-type T-cell lymphoma (ETL) and ulcerative jejunitis (UJ) are rare disorders often occurring in patients with coeliac disease. The genetic events associated with the accumulation of intraepithelial lymphocytes in coeliac disease and tumour development are largely unknown. Deletions at chromosome 9p21, which harbours the tumour suppressor genes p14/ARF, p15/INK4b, and p16/INK4a, and 17p13, where p53 is located, are associated with the development and progression of lymphomas. To examine whether deletions at 9p21 and 17p13 play a role in ETL, 22 cases of ETL and seven cases of UJ were screened for loss of heterozygosity (LOH) by tissue microdissection and polymerase chain reaction (PCR) analysis for microsatellite markers. Furthermore, p53 and p16 protein expression was examined by immunohistochemistry. In addition, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis for detection of mutations in exons 5-8 of the p53 gene was performed in five cases of ETL and three cases of UJ. LOH was found in at least one microsatellite marker at the 9p21 locus in 8 of 22 (36%) ETLs, but not in UJ. Five of nine (56%) tumours composed of large cells showed LOH at 9p21, as opposed to two of eight (25%) tumours with small- or medium-sized cell morphology. The region spanning the p14/p15/p16 gene locus was most frequently affected (five cases); LOH at these markers coincided with loss of p16 protein expression in all of these cases. p53 overexpression was demonstrated in all ETLs examined and in four of seven cases of UJ. However, no alterations of the p53 gene were detected by LOH or PCR-SSCP analysis. The results of this study show that LOH at chromosome 9p21 is frequent in ETL, especially in tumours with large cell morphology; this finding suggests that gene loss at this locus may play a role in the development of ETL.

Quantitation of cyclin D1 over-expression using competitive fluorescent reverse transcription polymerase chain reaction: a tool for the differential diagnosis of mantle cell lymphoma.

Mantle cell lymphoma is characterized by the presence of the t(11;14)(q13;q32) translocation that causes over-expression of the BCL-1 gene and consequent overproduction of its gene product cyclin D1. We have developed a competitive fluorescent reverse transcription polymerase chain reaction assay for the detection and semiquantitation of cyclin D1 over-expression. Using this assay a definitive ratio of the expression of cyclin D1 to cyclins D2 and D3 can be determined, provided good quality RNA is available. A single upstream primer derived from a consensus sequence found in cyclins D1, D2, and D3 was labeled at the 5' end using a fluorescent dye. Downstream primers specific to cyclins D1 and D2 were designed and used in conjunction with a previously published D3 specific primer. The fluorescently labeled PCR products were separated by electrophoresis using an ABI 377 DNA sequencer. Fluorescence emitted from each product was used to determine the ratio of expression of cyclin D1 to D2 and D3 by assigning a dosage quotient [D1/(D2+D3)]. The mean dosage quotient recorded from samples representing 29 non-MCL patients was 0.03 (SD +/- 0.03), the maximum value being 0.11. Samples from eight patients with a diagnosis of MCL generated values greater than 2. Calculation of a dosage quotient using this competitive fluorescent reverse transcription polymerase chain reaction assay allows unequivocal identification of patients with over-expression of cyclin D1, providing a new tool for the differential diagnosis of MCL.

Splenic marginal zone atrophy and progressive CD8+ T-cell lymphocytosis in HIV infection: a study of adult post-mortem spleens from Côte d'Ivoire.

AIMS: Progressive changes have been reported in lymph nodes in HIV infection, but few accounts describe altered splenic histology at different stages of the disease. Investigation of splenic changes accompanying the progressive CD4+ T-cell depletion that occurs in HIV infection could shed light on normal immunological interactions in this organ. Therefore, we assessed the amount and distribution of lymphoid tissue in spleens from adults with documented early or advanced HIV disease. METHODS AND RESULTS: Immunohistochemistry was used to study splenic tissue collected in an extensive autopsy survey of HIV+ adults in West Africa. Compared with post-mortem spleens from HIV- West African adults and control UK spleens, those from HIV-infected patients showed severe atrophy of white pulp B- and T-cell compartments. In early and advanced HIV disease, marginal zone atrophy was significant. Peri-arteriolar lymphoid sheaths contained increased numbers of CD8+/CD45RO+ T-cells in advanced HIV disease. In red pulp, early and advanced cases showed a lymphocytosis of CD8+/CD45RO- T-lymphocytes. CONCLUSIONS: Atrophic changes were more extreme in advanced than early HIV infection. Reduced marginal zone function possibly explains the known predisposition of HIV+ patients to infection by encapsulated bacteria. Possible immunological consequences of these CD8+/CD45RO+ (peri-arteriolar lymphoid sheaths) and CD8+/CD45RO- (red pulp) responses deserve further study. Comparison of West African and UK control spleens indicated that there were no major ethnic differences in spleen structure to prevent extrapolation of our results to European adults.

Identification of four new translocations involving FGFR1 in myeloid disorders.

The 8p11 myeloproliferative syndrome (EMS) is associated with three translocations, t(8;13)(p11;q12), t(8;9)(p11;q33), and t(6;8)(q27;p11), that fuse unrelated genes (ZNF198, CEP110, and FOP, respectively) to the entire tyrosine kinase domain of FGFR1. In all cases thus far examined (n = 10), the t(8;13) results in an identical mRNA fusion between ZNF198 exon 17 and FGFR1 exon 9. To determine if consistent fusions are also seen in the variant translocations, we performed RT-PCR on four cases and sequenced the products. For two patients with a t(8;9), we found that CEP110 exon 15 was fused to FGFR1 exon 9. For two patients with a t(6;8), we found that FOP exon 5 (n = 1) or exon 7 (n = 1) was fused to FGFR1 exon 9. To determine if FGFR1 might be involved in other myeloid disorders with translocations of 8p, we developed a two-color FISH assay using two differentially labeled PAC clones that flank FGFR1. Disruption of this gene was indicated in a patient with a t(8;17)(p11;q25) and Ph-negative chronic myeloid leukemia in association with systemic malignant mast cell disease, a patient with acute myeloid leukemia with a t(8;11)(p11;p15), and two cases with T-cell lymphoma, myeloproliferative disorder, and marrow eosinophilia with a t(8;12)(p11;q15) and ins(12;8)(p11;p11p21), respectively. For the patient with the t(8;11), the chromosome 11 breakpoint was determined to be in the vicinity of NUP98. We conclude that 1) all mRNA fusions in EMS result in splicing to FGFR1 exon 9 but breakpoints in FOP are variable, 2) two-color FISH can identify patients with EMS, and 3) the t(8;17)(p11;q25), t(8;11)(p11;p15), t(8;12)(p11;q15), and ins(12;8)(p11;p11p21) are novel karyotypic changes that most likely involve FGFR1.

Tryptase-positive mast cells accompany lymphocytic as well as lymphoplasmacytic lymphoma infiltrates in bone marrow trephine biopsies.

AIMS: To investigate the specificity of increased bone marrow mast cell numbers in lymphoplasmacytic lymphoma (LPL) and to evaluate the relationship between mast cell number and the immunoglobulin phenotype of neoplastic lymphoid cells. METHODS AND RESULTS: Retrospective study of bone marrow trephine biopsy specimens from patients with LPL, compared with selected cases representing chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM) of known immunoglobulin light and heavy chain phenotype. Bone marrow mast cells were counted following immunohistochemical staining of sections for mast cell tryptase. We have confirmed previous observations that mast cell numbers are increased in bone marrow infiltrates of LPL. However, we found similarly high mast cell numbers in CLL. High mast cell numbers were associated with neoplastic lymphoid cells expressing an IgM kappa phenotype. Mast cell numbers were low in all cases of MM studied and in controls with no lymphoma present. We observed an apparent bias towards kappa light chain expression in our cases of LPL. CONCLUSIONS: Mast cell number should not be considered a reliable factor in the differential diagnosis of LPL and CLL when assessing bone marrow histology. Possible bias towards kappa light chain expression in LPL requires further study, as do the mechanism and functions of mast cell recruitment by neoplastic lymphoid cells.

Molecular analysis reveals somatically mutated and unmutated clonal and oligoclonal B cells in T-cell-rich B-cell lymphoma.

This paper describes the immunohistology and molecular genetics of 18 cases of T-cell-rich B-cell lymphoma (TCRBL). In all cases, the large B cells stained strongly for CD20, with more variable expression of CD79a, and were negative for CD30 and CD15. The majority of T cells were predominantly positive for TIA-1 and negative for CD57; a large population of histiocytes was present in all cases. Epstein-Barr virus (EBV)-coded RNA (EBER) was found in B blasts from four cases and in one case was present among the background lymphoid cells. IgH PCR products were generated in 16/18 cases and revealed clonal, oligoclonal and polyclonal PCR products in 12, two and two cases, respectively. In addition, TCRG clonal gene rearrangements were identified in two cases. TCRB gene rearrangements were polyclonal. Sequence analysis of seven cases with clonal/oligoclonal IgH gene rearrangements revealed functional sequences with predominant V(H)3 gene usage associated with various D genes and J(H)4 or J(H)6 gene segments. Four cases displayed varying degrees of replacement and silent mutations (1.8-21%), with one case exhibiting intraclonal heterogeneity; the distribution of mutations was indicative of antigen selection in three cases. The remaining three cases, including two cases with functional oligoclonal IgH rearrangements, harboured unmutated V region genes. The EBV-positive cases were associated with clonal, oligoclonal and polyclonal PCR products and with mutated and germline clonal sequences. These data indicate that TCRBL may be a heterogeneous entity associated with clonal and oligoclonal B cells derived from both germinal centre and naïve B cells.

Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies.

AIMS: To establish a robust method of extracting DNA from paraffin wax embedded bone marrow trephine (PBMT) biopsies for the amplification of relatively long polymerase chain reaction (PCR) products. METHOD: Xylene and ethanol were used to remove paraffin wax from eight formalin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine different PCR primers sets, including those used to detect chromosomal translocations (t(11;14) and t(14;18), and clonal B cell populations. A t(11;14) PCR product of approximately 600 base pairs (bp) was sequenced using dye terminator cycle sequencing. RESULTS: All eight DNA samples extracted from PBMT biopsies were amplified successfully to generate DNA fragments up to 643 bp in length. Chromosomal translocations and immunoglobulin gene rearrangements were detected by PCR in some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunoglobulin heavy chain (IgH) gene sequences, consistent with the presence of this translocation. CONCLUSIONS: This method enables PCR analyses of PBMT biopsies that were not previously possible, offering the prospect of improved accuracy of diagnosis and the monitoring of patients with bone marrow disease.

Selective genetic analysis of p53 immunostain positive cells.

The isolation of p53 immunostain positive cells from histological sections for molecular genetic studies is a difficult task, especially if there are few positive cells. To eliminate contaminating DNA from p53 negative cells, which can obscure the results of molecular assays, a variation on the technique of immunohistoselective sequencing was developed. This is a highly selective approach, whereby immunostained sections of formalin fixed, paraffin wax embedded tissue are exposed to ultraviolet irradiation to damage the DNA in p53 negative cells. The DNA in positive cells remains unaffected because the dark immunostain protects their nuclei from ultraviolet light. Polymerase chain reaction single strand conformation polymorphism of samples enriched with p53 immunostain positive cells has shown that this method can produce pure samples of mutated DNA. The isolation of DNA from minority immunostain positive cells allows a wide range of molecular analyses to be carried out on these samples, which would otherwise be hampered by the problem of contaminating background cells.

Behavioural and physiological responses of sheep to 16 h transport and a novel environment post-transport.

The effect of a novel lairage environment on the ability of sheep to recover from 16 h of transport was investigated. Sheep were transported from grass paddocks to either novel outside paddocks or inside pens, and housed groups were transported to either familiar or novel inside pens. During transport, sheep from outside paddocks lay down less than those from inside pens. In sheep transported to inside pens, those from outside paddocks spent more time lying and spent less time eating; hay and water intakes during the first 12 h post-transport were lower than those previously kept inside. There was no obvious effect of a novel environment post-transport on blood biochemistry, suggesting that the lower post-transport feed and water intakes in a novel environment did not have a significant effect on the ability of the sheep to recover from the feed and water deprivation associated with transport.

The clinical utility of the Revised European-American Lymphoma (R.E.A.L.) Classification: preliminary results of a prospective study in patients with non-Hodgkin's lymphoma from a single centre.

BACKGROUND: The clinical applicability of the Revised European-American Lymphoma (R.E.A.L.) Classification has been demonstrated in several retrospective studies. The present, ongoing study was initiated to evaluate the clinical and pathological utility of the R.E.A.L. Classification compared with the Working Formulation (WF) in a prospective fashion, in an unselected patient population treated at a single institution. PATIENTS AND METHODS: Prospective data were collected on 596 biopsies from 557 patients referred with an initial diagnosis of lymphoma. After initial histologic review, 465 biopsies from 441 patients were confirmed as non-Hodgkin's lyphoma (NHL), 412 of which could be classified in R.E.A.L. and WF. RESULTS: According to WF criteria, 25% were low grade, 58% intermediate grade and 2% high grade, 14% could not be allocated to a WF subtype. According to R.E.A.L., 46% were diffuse large B cell, 19% follicle centre lymphoma, 6% marginal zone, 6% small lymphocytic, 4% mantle cell, and 3% T-cell anaplastic large cell. For those with B-cell NHL, 7% were unclassifiable in WF compared with 1% in R.E.A.L. Corresponding figures for T-cell NHL were 68% and 3%, respectively. CONCLUSIONS: Preliminary results confirm the clinical utility of the R.E.A.L. Classification in a single institution setting, demonstrating that cases were more readily sub-typed in R.E.A.L. compared with WF. Frequencies are comparable with I.L.S.G. data. Further follow up with large patient numbers is on-going to analyse survival data with reference to clinical prognostic factors.

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples.

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.

Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.

Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VH genes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 "clonal signature" was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.

Contribution of monocyte/macrophage differentiation to the stromal layer in human long-term bone marrow cultures.

The stroma of bone marrow is a poorly understood tissue which is believed to have important roles in haemopoietic stem cell maintenance and differentiation. We have undertaken immunohistochemical studies of bone marrow stroma in human long-term bone marrow cultures (hLTBMC) and biopsy specimens in order to characterise the cell and matrix components present. We have found two morphological variants of macrophages to be present consistently in hLTBMC stroma, adherent to a substratum of myofibroblastic cells. Large round macrophages appear to actively phagocytic and are formed in hLTBMC regardless of successful establishment of a myofibroblastic cell layer. Elongated macrophages with dendritic processes appear to be non-phagocytic and form only in the presence of a well-established layer of myofibroblasts. Although the functions of these macrophages are not yet known, they have counterparts within intact human bone marrow and their presence in hLTBMC shows some association with the haemopoietic capacity of the cultures.

Heterogeneity of expression of CD68 and other macrophage-associated antigens in human long-term bone marrow culture.

The authors have performed an immunohistochemical study of intact human long-term bone marrow cultures (hLTBMC) grown directly onto glass slides. Between 4 and 12 weeks of growth, such cultures consist of a complex stromal layer supporting foci of haemopoietic cells which undergo granulocytic and monocytic differentiation. As part of a large panel of antibodies employed to characterize monocytes and macrophages within hLTBMC, we included six different anti-CD68 reagents and three antibodies representing a putative new CD group recognizing a macrophage-associated antigen of 130 kDa molecular weight. These gave heterogeneous immunostaining patterns with macrophages and stromal myofibroblastic cells.