M. A. Boogaerts

Clinical and molecular features of FIP1L1-PDFGRA (+) chronic eosinophilic leukemias.

Detection of the FIP1L1-PDGFRA fusion gene or the corresponding cryptic 4q12 deletion supports the diagnosis of chronic eosinophilic leukemia (CEL) in patients with chronic hypereosinophilia. We retrospectively characterized 17 patients fulfilling WHO criteria for idiopathic hypereosinophilic syndrome (IHES) or CEL, using nested RT-PCR and interphase fluorescence in situ hybridization (FISH). Eight had FIP1L1-PDGFRA (+) CEL, three had FIP1L1-PDGFRA (−) CEL and six had IHES. FIP1L1-PDGFRA (+) CEL responded poorly to steroids, hydroxyurea or interferon-α, and had a high probability of eosinophilic endomyocarditis (n=4) and disease-related death (n=4). In FIP1L1-PDGFRA (+) CEL, palpable splenomegaly was present in 5/8 cases, serum vitamin B12 was always markedly increased, and marrow biopsies revealed a distinctively myeloproliferative aspect. Imatinib induced rapid complete hematological responses in 4/4 treated FIP1L1-PDGFRA (+) cases, including one female, and complete molecular remission in 2/3 evaluable cases. In the female patient, 1 log reduction of FIP1L1-PDGFRA copy number was reached as by real-time quantitative PCR (RQ-PCR). Thus, correlating IHES/CEL genotype with phenotype, FIP1L1-PDGFRA (+) CEL emerges as a homogeneous clinicobiological entity, where imatinib can induce molecular remission. While RT-PCR and interphase FISH are equally valid diagnostic tools, the role of marrow biopsy in diagnosis and of RQ-PCR in disease and therapy monitoring needs further evaluation.

Trisomy 3 in marginal zone B-cell lymphoma: A study based on cytogenetic analysis and fluorescence in situ hybridization

Trisomy 3 represents the most frequent and consistent chromosomal abnormality characterizing the recently defined entity marginal zone B‐cell lymphoma (MZBCL). By cytogenetic analysis and/or fluorescence in situ hybridization (FISH) on interphase nuclei we found an increased copy number of chromosome 3 in 22/36 (61%) successfully analysed cases, including 8/12 cases with extranodal MZBCL, 8/13 cases with nodal MZBCL, and 6/11 patients with splenic MZBCL. Sensitivity of interphase cytogenetics was somewhat higher than that of conventional cytogenetic investigation. Structural chromosomal changes involving at least one chromosome 3 were seen in 11/20 cases with an increased copy number of chromosome 3: +del(3)(p13) was demonstrated in three cases, and was the sole chromosomal abnormality in one of them; +i(3)(q10) was seen in two other patients; and rearrangements involving various breakpoints on the long arm of chromosome 3 were found in the remaining cases. FISH on metaphase spreads confirmed these structural abnormalities and additionally showed two unexpected translocations involving chromosome 3. We conclude that: (1) trisomy 3 occurs in a high proportion of extranodal, nodal and splenic MZBCL; (2) FISH on interphase nuclei is an additional and sensitive tool in detecting an increased copy number of chromosome 3 in MZBCL; (3) additional structural abnormalities involving the long arm of chromosome 3 are frequent but non‐recurrent and are perhaps secondary changes; and (4) abnormalities such as +del(3)(p13) and +i(3)(q10) suggest that genes located on the long arm of chromosome 3 are of particular importance in the pathogenesis of MZBCL.

Morphologic, immunologic and cytogenetic studies in erythroleukaemia : evidence for multilineage involvement and identification of two distinct cytogenetic-clinicopathological types

Summary. Clinical features, as well as morphology, immunophenotype and cytogenetics were retrospectively studied in 20 patients with an original diagnosis of erythroleukaemia (EL) reclassified according to the FAB criteria. Fifteen patients had de novo EL, five patients had therapy‐related EL. Myelodysplasia preceded the onset of EL in eight cases and myelodysplastic features involving multiple haemopoietic lineages were observed at leukaemia presentation in all cases. Immunologic findings confirmed multilineage involvement, showing sub‐populations of cells expressing platelet‐associated markers in more than 50% of cases tested and the presence of a myelomonocytic component, besides glycophorin A‐positive cells. Cytogenetically, major karyotype aberrations (MAKA), defined by the presence of three or more aberrant events in the same clone, were observed in 14 cases, minor karyotype aberrations (MIKA) were observed in four cases and normal karyotype in two cases. No differences in the cytological‐cytogenetic picture of our patients with de novo EL and with therapy‐related EL were found suggesting that aetiological factors and/or pathogenetic mechanisms common to EL and secondary leukaemia may exist. All patients with MAKA had leftward shift of erythropoiesis with proerythroblasts and basophilic erythroblasts usually representing more than 50% of all erythroid cells. In patients with MIKA or normal karyotype, maturation of erythroid cells, though morphologically abnormal, was quantitatively preserved and early erythroblasts never exceeded 25% of erythroid cells. Clinically, the haemoglobin level at presentation, as well as in the proportion of patients achieving complete remission after chemotherapy, appeared to be lower in the maturation arrest‐MAKA group as compared to the preserved maturation‐MIKA/normal karyotype group. Median survival was shorter in the former group (3.5 months) than in the latter (median 13 months). Morphologic‐immunologic‐cytogenetic studies thus allow for the identification of two distinct cytogenetic‐clinicopathological types of EL.

Differences between graft product and donor side effects following bone marrow or stem cell donation

Summary:We report graft product stem cell yields and donor safety results of a randomized multicenter study comparing allogeneic peripheral blood stem cell (PBSC) PBSC transplantation with BM transplantation. Matched HLA-identical sibling donors (n=329) were randomized to filgrastim-mobilized PBSC or bone marrow (BM) donation groups. Median yields per kg recipient weight of CD34+ cells, T cells, and natural killer (NK) cells, respectively, were approximately two-fold, eight-fold, and greater than eight-fold in the PBSC group than in the BM group (CD34+ cells, 5.8 × 106/kg vs 2.7 × 106/kg; T cells, 300.1 × 106/kg vs 35.7 × 106/kg; NK cells, 28.2 × 106/kg vs 3.6 × 106/kg; P<0.001 for each). In connection with the cell collection procedures, PBSC donors spent a shorter median time in hospital than BM donors (0 vs 2 days; median difference −2 days, 95% CI −2 to 2) and had fewer median days of restricted activity (2 vs 6 days; median difference −3 days, 95% CI −4 to 2). Overall, 65% of PBSC donors and 57% of BM donors reported at least one adverse event (AE), most of which were transient, mild–moderate in severity, and without clinical sequelae. PBSC donors experienced predominantly filgrastim-related AEs, while BM donors experienced predominantly harvest-related AEs.

Peripheral blood stem cell transplantation as an alternative to autologous marrow transplantation in the treatment of acute myeloid leukemia

The clinical use of autologous marrow transplantation in acute myeloid leukemia (AML) has been hampered by the inability to collect adequate numbers of cells after remission induction chemotherapy and the notably delayed hematopoietic regeneration following autograft reinfusion. Here we present a study in which the feasibility of mobilizing stem cells was investigated in newly diagnosed AML. Among 96 AML patients, 76 patients (79%) entered complete remission. Mobilization was undertaken with low dose and high dose schedules of G-CSF in 63 patients, and 54 patients (87%) were leukapheresed. A median of 2.0u2009×u2009106 CD34+cells/kg (range 0.1–72.0) was obtained in a median of three leukaphereses following a low dose G-CSF schedule (150u2009μg/m2) during an average of 20 days. Higher dose regimens of G-CSF (450u2009μg/m2 and 600u2009μg/m2) given during an average of 11 days resulted in 28 patients in a yield of 3.6u2009×u2009106 CD34+ cells/kg (range 0–60.3) also obtained following three leukaphereses. The low dose and high dose schedules of G-CSF permitted the collection of 2u2009×u2009106 CD34-positive cells in 46% and 79% of cases respectively (Pu2009=u20090.01). Twenty-eight patients were transplanted with a peripheral blood stem cell (PBSC) graft and hemopoietic repopulation was compared with the results of a previous study with autologous bone marrow. Recovery of granulocytes (>0.5u2009×u2009109/l, 17 vs 37 days) and platelets (>20u2009×u2009109/l; 26 vs 96 days) was significantly faster after peripheral stem cell transplantation compared to autologous bone marrow transplantation. These results demonstrate the feasibility of PBSCT in the majority of cases with AML and the potential advantage of this approach with respect to hemopoietic recovery.

Myelodysplastic Syndromes With Bone-marrow Fibrosis - a Myelodysplastic Disorder With Proliferative Features

SummaryWe report on 22 patients with myelodysplastic syndrome (MDS), all of whom showed striking marrow fibrosis. Variable blood counts, often with teardrop poikilocytosis and a leukoerythroblastic picture, were present at diagnosis. Visceral enlargement was detected in 17 patients with a distinct splenomegaly in seven cases. All cases demonstrated dysplasia in at least two cell lineages. No specific cytogenetic abnormality seems to characterize this group of patients. Southern blot analysis showed no breakpoint cluster region rearrangement as observed in classical chronic myeloid leukemia. Ferrokinetic studies revealed quantitatively deficient erythropoiesis in all except two cases and an abnormally high fraction of ineffective erythropoiesis in all. Splenic erythropoiesis was present in eight patients. The median survival was 18 months. At the time of this report, 12 patients had died. The causes of death were disease progression (7 patients) and infection (5 patients). One might speculate that the present series of cases represents a transition between MDS and myeloproliferative disease, thereby dysplaying characteristics of both groups of diseases.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28− cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients’ T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor’s major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients’ CD57+/CD28− T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-γ, and TNF-α than their CD57−/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28− subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients’ CD57+/CD28− T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28− T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

Interaction of CTLA‐4 (CD152) with CD80 or CD86 inhibits human T‐cell activation

Occupancy of CTLA‐4 (cytotoxic T‐lymphocyte antigen‐4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA‐4‐deficient mice, by the impact of anti‐CTLA‐4 monoclonal antibodies (mAbs) on mouse T‐cell activation in vitro and by the impact of CTLA‐4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA‐4, however, remains less clear. The expression and function of human CTLA‐4 were further explored. CTLA‐4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin‐2. Memory T cells expressed CTLA‐4 with faster kinetics than naive T cells. The functional role of human CTLA‐4 was assessed utilizing a panel of four anti‐CTLA‐4 mAbs that blocked the interaction between CTLA‐4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti‐CD3 mAb in the absence of anti‐CD28 mAb, but co‐stimulated T‐cell activation in the presence of anti‐CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA‐4 with its ligands using soluble anti‐CTLA‐4 mAbs, in intact form or as Fab fragments, enhanced T‐cell activation in several polyclonal or alloantigen‐specific CD80‐ or CD80/CD86‐dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA‐4 with its functional ligands, CD80 or CD86, can down‐regulate human T‐cell responses, probably by intracellular signalling events and independent of CD28 occupancy.

Additional Prognostic Value of Bone Marrow Histology in Patients Subclassified According to the International Prognostic Scoring System for Myelodysplastic Syndromes

PURPOSEnThe most recent and powerful prognostic instrument established for myelodysplastic syndromes (MDS) is the International Prognostic Scoring System (IPSS), which is primarily based on medullary blast cell count, number of cytopenias, and cytogenetics. Although this prognostic system has substantial predictive power in MDS, further refinement is necessary, especially as far as lower-risk patients are concerned. Histologic parameters, which have long proved to be associated with outcome, are promising candidates to improve the prognostic accuracy of the IPSS. Therefore, we assessed the additional predictive power of the presence of abnormally localized immature precursors (ALIPs) and CD34 immunoreactivity in bone marrow (BM) biopsies of MDS patients.nnnPATIENTS AND METHODSnCytogenetic, morphologic, and clinical data of 184 MDS patients, all from a single institution, were collected, with special emphasis on the determinants of the IPSS score. BM biopsies of 173 patients were analyzed for the presence of ALIP, and CD34 immunoreactivity was assessable in 119 patients. Forty-nine patients received intensive therapy.nnnRESULTSnThe presence of ALIP and CD34 immunoreactivity significantly improved the prognostic value of the IPSS, with respect to overall as well as leukemia-free survival, in particular within the lower-risk categories. In contrast to the IPSS, both histologic parameters also were predictive of outcome within the group of intensively treated MDS patients.nnnCONCLUSIONnOur data confirm the importance of histopathologic evaluation in MDS and indicate that determining the presence of ALIP and an increase in CD34 immunostaining in addition to the IPSS score could lead to an improved prognostic subcategorization of MDS patients.

A new disease categorization of low-grade myelodysplastic syndromes based on the expression of cytopenia and dysplasia in one versus more than one lineage improves on the WHO classification.

Multilineage dysplasia was advanced by the World Health Organization to increase prognostic accuracy in myelodysplastic syndromes (MDS) classification. We performed a structured cytomorphological examination of bone marrow (BM) in 221 low-grade MDS patients, this in conjunction with strict guidelines for cytopenias. A dysplasia scoring system was developed utilizing dysplastic changes, which were associated with worse outcome on univariate and multivariate analysis corrected for the International Prognostic Scoring System (IPSS). Dysplasia ⩾10% in one BM lineage and one cytopenia constituted the low-risk category UCUD or Unilineage Cytopenia and Unilineage Dysplasia. The high-risk category comprised patients with cytopenia in ⩾2 lineages and dysplasia in ⩾2u2009BM lineages, namely MCMD or Multilineage Cytopenia and Multilineage Dysplasia. Intermediate-risk patients had one cytopenia and multilineage dysplasia, or cytopenia in ⩾2 lineages and unilineage BM dysplasia, designated UCMD/MCUD or Unilineage Cytopenia and Multilineage Dysplasia/Multilineage Cytopenia and Unilineage Dysplasia. This system utilizing cytopenia–dysplasia scoring at diagnosis enabled comprehensive categorization of low-grade MDS cases that predicted for overall as well as leukemia-free survival. Cytopenia–dysplasia categorization added additional prognostic values to the lower risk IPSS categories. This suggests that a standardized dysplasia scoring system, used in conjunction with cytopenia, could improve diagnostic and prognostic sub-categorization of MDS patients.