Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia. Correlation between VEGF expression and the FAB category

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-American-British (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (> 90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category.     

Expression of the monoclonal antibody HECA-452 defined E-selectin ligands in Langerhans cell histiocytosis

The cutaneous lymphocyte associated antigen (CLA) recognized by the monoclonal antibody (moAb) HECA-452 plays a major role in the homing of lymphocyte subpopulations to the skin by binding to E-selectin on dermal microvessels. The factors responsible for the immigration of Langerhans cells (LC) and their precursors into the skin are still unknown, but because normal resting LC are also capable of expressing CLA, the antigen was proposed as a candidate LC-homing structure. To gain insight into these mechanisms, the expression of HECA-452 on neoplastic LC within and outside the skin was investigated in paraffin-embedded sections from 44 patients with localized and disseminated forms of Langerhans cell histiocytosis (LCH) presenting with proliferating cells positive for CD45, CD1a, S100 and HLADR. Irrespective of the clinical presentation or the type of organ involved, HECA-452-positive LC were detected in all biopsies tested (range 5->90%). The most prominent HECA-452 reactivity was observed in skin lesions and in areas with accumulations of eosinophilic granulocytes. Our data provide evidence for a heterogeneous expression of sLe^x/sLe^a structures in various stages of activated and/or differentiated LCH cells. Remarkably, CLA-antigen expression on LCH-cells was not restricted to cutaneous sites. In view of recent findings on the expression of HECA-452 on resting epidermal LC, our data are compatible with the view that local cytokine production by keratinocytes or cells from the surrounding infiltrate induce and/or modulate CLA expression on LC in both cutaneous and extra-cutaneous sites.This work is dedicated to Professor Dr. Thaddäus Radaszkiewicz, who died in September 1995     

Immunophenotypic characterization of human bone marrow endosteal cells

In order to determine the relationship between bone marrow (bm) endosteal cells (EDC) and hemopoietic progenitors, we have analyzed the immunophenotype of EDC using various antibodies (Ab) against mesenchymal antigens. The Ab were applied on paraffin sections of normal bm (iliac crest, n=17; talus, n=1; phalanx, n=1), myeloregenerative bm (after chemotherapy), and hematologic disorders (acute myeloid leukemia (AML), n=8; chronic myeloid leukemia (CML), n=6; myelodysplastic syndromes (MDS), n=14; severe aplastic anemia (SAA), n=4; essential thrombocythemia (ET), n=2; idiopathic (primary) osteomyelo-fibrosis (IMF), n=1; polycythemia vera (PV), n=1). In normal bm, EDC were found to react with Ab against vimentin, tenascin, alpha-smooth muscle actin, osteocalcin, CD51, and CD56, but did not react with Ab against CD3, CD15, CD20, CD34, CD45, CD68, or CD117. An identical phenotype of EDC was found in AML, MDS, SAA, ET, IMF, PV, myeloregenerative bm, and peripheral bones lacking active hemopoiesis (talus, phalanx). In patients with CML, EDC reacted with Ab to CD51, but did not react with Ab to CD56. Based on their unique antigen profile, EDC were enriched from normal bm by enzyme digestion and cell sorting. However, these enriched cells (CD56+, CD45-, CD34-) did not give rise to hemopoietic cells under the culture conditions used, i.e. in the presence of the growth factors IGF-1, bFGF, SCF, IL-3, and GM-CSF Together, our data do not support the hypothesis that EDC are totipotent mesenchymal progenitors giving rise to hemopoietic cells.     

CD25 indicates the neoplastic phenotype of mast cells: a novel immunohistochemical marker for the diagnosis of systemic mastocytosis (SM) in routinely processed bone marrow biopsy specimens

The diagnosis of systemic mastocytosis (SM) is based primarily on the histologic and immunohistochemical evaluation of a bone marrow trephine biopsy specimen. Although mast cell (MC) specific antigens like tryptase and chymase are detectable in routinely processed tissue, no immunohistochemical markers that can be used to discriminate between normal and neoplastic MCs are yet available. We have investigated the diagnostic value of an antibody against CD25 for the immunohistochemical detection of MCs in bone marrow sections in 73 patients with SM and 75 control cases (reactive marrow, n = 54; myelogenous neoplasms, n = 21) and correlated the results with the presence of c-kit mutations. While MCs in almost all patients with SM (72 of 73) expressed CD25, none of the control samples contained CD25-positive MCs. Irrespective of the SM subtype, most of neoplastic MCs expressed CD25. In 3 patients with advanced MC disease, pure populations of neoplastic MCs were obtained and found to express CD25 mRNA by RT-PCR analysis. In addition, all patients with CD25-positive MCs contained c-kit mutations, while all control cases exhibited wild type c-kit. CD25 therefore appears to be a reliable immunohistochemical marker for the discrimination of neoplastic from normal/reactive MCs, with potential as a diagnostic tool in SM.     

Splenic mastocytosis: report of two cases and detection of the transforming somatic C-KIT mutation D816V

In the vast majority of patients with systemic mastocytosis (SM), the bone marrow is the primary extracutaneous site of disease. In addition to bone marrow involvement, other visceral organs such as the spleen, liver or the gastrointestinal tract, may also be affected. However, isolated involvement of a single extramedullary organ is rarely seen in SM. We report on two patients with SM with splenic involvement, lack of 'diagnostic' mast cell (MC) infiltrates in the bone marrow, and absence of skin lesions. In one patient, a myelodysplastic syndrome was diagnosed prior to the detection of SM. Both patients presented with massive splenomegaly and multifocal MC infiltrates in splenic tissues. These MCs also expressed CD25 as well as the C-KIT mutation D816V. In consecutive examinations, the mutation was also detected in the bone marrow in both patients suggesting diffuse infiltration with neoplastic cells. In summary, our data show that the spleen can be a primary site of disease in rare cases of SM. Mastocytosis should therefore be considered as a (rare) differential diagnosis in patients with splenomegaly of unknown etiology.     

Post-transplant acute myeloid leukemia (PT-AML).

Acute myeloid leukemia following organ transplantation (PT-AML) is a rare event with only a few published cases in the literature. We present three patients who developed AML (FAB M1, M5, M4) after renal, double lung or liver transplantation. Molecular analysis detected a t(9;11) in one patient and documented the recipient origin of AML in a second patient. All patients were treated with chemotherapy. Immunosuppression was reduced to cyclosporin A (CsA) and prednisone in two patients and to prednisone alone in one patient. Two patients achieved a complete remission (CR), with a remission duration of 4.6 months in one patient, the other patient died from septicemia after 15.2 months in CR. One patient was refractory to chemotherapy and died from septicemia. This report together with the documented cases in the literature suggests that PT-AML (1) develops after a median interval of 5 years after transplantation with variable latency (range, <1-17 years); (2) is heterogeneous with respect to FAB classification; (3) shows chromosomal and molecular changes typical of therapy-related AML (t-AML: -7, +8, 11q23, inv16, t(15;17)); (4) standard chemotherapy is feasible after reduction of immunosuppression and produces a CR rate of 56% with a median remission duration of 4.6 months and an overall survival of 2.6 months; (5) the major complications are early death (25%), gram-negative septicemia, progressive disease or relapse. This review provides diagnostic and therapeutic experiences and guidelines for the management of this increasing group of post-transplant patients.     

The proportion of reticulated platelets is higher in bone marrow than in peripheral blood in haematological patients.

Since the detection that platelets originate from megakaryocytes (MK), the site of megakaryocyte fragmentation has been disputed. Some authors have even postulated that platelets are solely produced in the lungs. Thus, we have directly measured platelet generation in the bone marrow (BM) by comparing the relative number of young RNA-containing, so-called reticulated platelets (%RP) in the BM and in the peripheral blood (PB). Two separate prospective, cross sectional trials have been conducted in patients routinely undergoing BM biopsies for diagnostic purposes. In the first part of the study 30 patients with stem cell or bone marrow transplantation were examined. The second part of the study was performed in 62 haematological patients visiting the outpatient's clinic. Median %RP were higher in BM than in PB (p <0.001). In the second part of the study the difference averaged 133% (interquartile range: 30-383%). There was a moderate correlation between %RP in BM and in PB (r = 0.67; p <0.001). The absolute number of RP in PB correlated weakly with the number of megakaryocytes (0.42; p = 0.001), which was due to a correlation between the platelet counts and the megakaryocyte counts (r = 0.55; p <0.001 in biopsies). Two patients with autoimmune antibodies against GPIIb/IIIa exhibited 10% and 16% RP in PB, and had 29% and 59% RP in BM, respectively. It is concluded that the relative number of RP is significantly higher in BM than in blood. This supports the notion that platelets are at least in part released from MK in the bone marrow, particularly in patients suffering from immune thrombocytopenia.     

Oncogenic role of miR‐155 in anaplastic large cell lymphoma lacking the t(2;5) translocation

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non‐Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin‐anaplastic lymphoma tyrosine kinase (NPM–ALK) fusion protein (ALCL ALK⁺). However, little is known about the molecular features and tumour drivers in ALK‐negative ALCL (ALCL ALK^?), which is characterized by a worse prognosis. We found that ALCL ALK^?, in contrast to ALCL ALK⁺, lymphomas display high miR‐155 expression. Consistent with this, we observed an inverse correlation between miR‐155 promoter methylation and miR‐155 expression in ALCL. However, no direct effect of the ALK kinase on miR‐155 levels was observed. Ago2 immunoprecipitation revealed miR‐155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPβ and SOCS1. To investigate its function, we over‐expressed miR‐155 in ALCL ALK⁺ cell lines and demonstrated reduced levels of C/EBPβ and SOCS1. In murine engraftment models of ALCL ALK^?, we showed that anti‐miR‐155 mimics are able to reduce tumour growth. This goes hand‐in‐hand with increased levels of cleaved caspase‐3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR‐155 induces IL‐22 expression and suppresses the C/EBPβ target IL‐8. These data suggest that miR‐155 can act as a tumour driver in ALCL ALK^? and blocking miR‐155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1). © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Effective treatment of primary plasma cell leukemia with thalidomide and dexamethasone - a case report

Plasma cell leukemia (PCL) is an aggressive disease with poor prognosis and a median survival of only 2-6 months. Currently, no standard therapy is available, but intensive polychemotherapy appears to be more effective than the conventional melphalan plus prednisone. However, the efficacy of thalidomide in PCL has not yet been widely evaluated. Recently, treatment with thalidomide has been reported to yield promising results in refractory multiple myeloma. Here, we report on a patient with primary PCL in whom first-line treatment with thalidomide/dexamethasone resulted in a rapid response and achievement of a very good partial remission.