Jordi Juncà

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)]

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ABLx104 also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with ⩽0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels ⩽10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Hepatosplenic T-Gammadelta Lymphoma in a Patient with Crohn's Disease Treated with Azathioprine

Hepatosplenic γ δ T-cell lymphoma (HS γ δ TCL) is an uncommon type of peripheral T-cell lymphoma, which has been associated in some cases with immunosuppression, mainly after solid organ transplants. We describe a case of HS γ δ TCL with a leukaemic course in a patient with Crohns disease who had received azathioprine during the previous 55 years. Sinusoidal infiltration by atypical lymphocytes was observed in the liver, spleen and bone marrow and the typical cytogenetic abnormalities (isochromosome 7 and trisomy 8) were found. The patient did not respond to intensive chemotherapy. This case shows the importance of ruling out HS γ δ TCL in patients with hepatosplenomegaly, B-symptoms and any immunosuppressive condition.

CXCR4 and SDF-1 expression in B-cell chronic lymphocytic leukemia and stage of the disease

The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked to an overexpression of the chemokine receptor CXCR4 and increased in vitro functional response to its natural ligand CXCL12 (SDF-1). The CXCR4/SDF-1 system appears to be important for tissue localization and increased survival of B-CLL cells. The aim of our study was to examine if CXCR4 expression and SDF-1 blood levels were correlated to clinical and pathological stage of B-CLL. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) techniques were used to determine CXCR4 expression and SDF-1 plasma levels, respectively, in a cohort of 51 patients diagnosed with B-CLL to correlate these measurements with several parameters that define the clinical stage of the disease. We confirmed that CXCR4 was consistently expressed on circulating B-CLL cells with a fluorescence intensity that was five-fold greater than in cells from healthy volunteers. There was a correlation between CXCR4 expression and leukocyte count (r: 0.55, p<0.01), and CD19+/CD5+ cells (r: 0.63, p<0.01). Interestingly, the group of B-CLL patients showed lower SDF-1 plasma levels compared to the control group. However, there was no correlation between CXCR4 or SDF-1 expression and the clinical stage of disease or the pattern of bone marrow infiltration. The results obtained suggest that other factors, and not only alteration in the SDF-1/CXCR4 chemokine system, must account for marrow infiltration of neoplastic cells observed in B-CLL and that CXCR4 could be involved in other features that exhibit malignant B cells, such as increased survival, rather than in their homing or migration to the bone marrow.

Predictive factors for poor peripheral blood stem cell mobilization and peak CD34+cell count to guide pre-emptive or immediate rescue mobilization

BACKGROUND AIMS Failure in mobilization of peripheral blood (PB) stem cells is a frequent reason for not performing hematopoietic stem cell transplantation (HSCT). Early identification of poor mobilizers could avoid repeated attempts at mobilization, with the administration of pre-emptive rescue mobilization. METHODS Data from the first mobilization schedule of 397 patients referred consecutively for autologous HSCT between 2000 and 2010 were collected. Poor mobilization was defined as the collection of < 2 × 10(6) CD34(+)cells/kg body weight (BW). RESULTS The median age was 53 years (range 4-70) and 228 (57%) were males. Diagnoses were multiple myeloma in 133 cases, non-Hodgkins lymphoma in 114, acute myeloid leukemia or myelodysplastic syndrome in 81, Hodgkins lymphoma in 42, solid tumors in 17 and acute lymphoblastic leukemia in 10. The mobilization regimen consisted of recombinant human granulocyte-colony-stimulating factor (G-CSF) in 346 patients (87%) and chemotherapy followed by G-CSF (C + G-CSF) in 51 (13%). Poor mobilization occurred in 105 patients (29%), without differences according to mobilization schedule. Diagnosis, previous therapy with purine analogs and three or more previous chemotherapy lines were predictive factors for poor mobilization. A CD34(+)cell count in PB > 13.8/μL was enough to ensure ≥ 2 × 10(6) CD34(+)cells/kg, with high sensitivity (90%) and specificity (91%). CONCLUSIONS The prevalence of poor mobilization was high, being associated with disease type, therapy with purine analogs and multiple chemotherapy regimens. The threshold of CD34(+) cell count in PB identified poor mobilizers, in whom the administration of immediate or pre-emptive plerixafor could be useful to avoid a second mobilization.

Immunophenotype of acute myeloid leukemia with NPM mutations: Prognostic impact of the leukemic compartment size

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Translocation (3;8)(q27;q24) in two cases of triple hit lymphoma.

Unclassifiable lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma is a new category of B-cell lymphoma appearing in the new World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. This lymphoma usually shows MYC rearrangements with non-IGH genes in the setting of a complex karyotype possibly involving BCL2 and, less frequently, BCL6 rearrangements. According to the presence of two or three rearrangements, these lymphomas are called double-hit lymphomas or triple-hit lymphomas (THL), respectively. Here we report two cases of THL with MYC, BCL2, and BCL6 rearrangements and t(3;8)(q27;q24) diagnosed in one center in the last two years.

Electron Microscopic Identification of Parvovirus Virions in Erythroid and Granulocytic-Line Cells in a Patient with Human Parvovirus B19 Induced Pancytopenia

In this short report we describe a patient with human parvovirus B19 (HPV B19)-induced transient pancytopenia. Parvovirus virions were seen by electron microscopy in both erythroid and granulocytic precursors. Erythroid cells are not the only targets in these cases. We draw attention to this disorder so that physicians involved with hematological disorders and transplantation be more aware of this infection.

Usefulness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

In up to 5–15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B‐cell lymphoma and 3/10 with T‐cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T‐cell clonality should be based on all the available clinical and analytical data.

Expression of CD43 in chronic lymphoproliferative leukemias

CD43 has been used on histological samples for the differential diagnosis of lymphoproliferative disorders but there is scarce data on its use by flow cytometry (FC). We set out to characterize the expression of CD43 by FC in B‐cell lymphoproliferative disorders and to determine its possible role in the differential diagnosis of these malignancies.

Erythema nodosum as a presenting feature of T-cell-rich B-cell lymphoma

Erythema nodosum (EN) consists in the appearance of erythematous and tender red nodules on the pretibial surface of both legs and, less frequently, on the thighs and forearms. Histopathologically, it corresponds to septal panniculitis without vasculitis. EN has been most commonly associated with sarcoidosis, drug intake, streptococcal infections, and tuberculosis; however, association with lymphoproliferative disorders is uncommon. The term «T-cell-rich B-cell lymphoma» was introduced by Ramsay et al. to describe a pathological sub-type of non-Hodgkins lymphoma (NHL) in which neoplastic B cells are mixed with a predominant population of non-neoplastic T cells [4]. We report herein a patient with T-cell-rich B-cell lymphoma preceded by erhythema nodosum within the 3 months prior to diagnosis, a previously unobserved association (MEDLINE, 1982-1994)