A phase I/IIa clinical trial of autologous hematopoietic stem cell transplantation in amyotrophic lateral sclerosis.

OBJECTIVE: To verify the safety and potential effect on ALS progression of a low-intensity immunosuppressive regimen followed by autologous hematopoietic stem cell transplantation (aHSCT) in amyotrophic lateral sclerosis (ALS) patients. METHODS: ALS eligible patients underwent a set of clinical and laboratory evaluations at T-4 (screening), T-1 (pre-treatment visit), and for the 12 consecutive months after treatment (T3, T6, T9, T12). We evaluated the tolerability of the procedure, its efficacy on clinical course and quality of life (QoL). RESULTS: Eight of the 11 ALS patients enrolled received the established immunoablative protocol. The procedure was well tolerated and side effects were those expected. One patient died 4 months after the conditioning regimen and another patient underwent tracheotomy just before T3 for a sudden respiratory failure, but he is still alive 4 years after the procedure without being ventilated any more. A third patient died 10 months after conditioning. In the other cases, there was no statistical difference in all functional measures and QoL pre- and post-treatment; however, a transitory slopes' reduction of ALSFRS-R and seated SVC% after the conditioning procedures was reported. Moreover, although not statistically significant, trends of reduction of CD4 + and increment of CD8 + were found. CONCLUSIONS: aHSCT was overall well tolerated, but it was not followed by any significant modification in disease progression. Considering the negative results of this small trial, further studies aimed to evaluate the possible efficacy of the aHSCT using a higher-intensity regimen should be carefully and with caution evaluated.

BCR/ABL1 fluorescence in situ hybridization fusion signals on both copies of chromosome 22 in a Philadelphia-masked chronic myeloid leukemia case: implication for the therapy.

The cytogenetic hallmark of Chronic Myeloid Leukemia (CML) is the presence of Philadelphia (Ph) chromosome, which results from a reciprocal translocation t(9;22)(q34;q11). In this report, we describe a CML patient with no evidence of Ph chromosome but trisomy of chromosome 8 as single cytogenetic abnormality and a typical e14a2 (b3a2) BCR-ABL1 fusion transcript. Fluorescence In Situ Hybridization (FISH) analysis revealed an uncommon signal pattern: the fusion signals were located on both copies of chromosome 22. During the course of the disease the appearance of the p.(Tyr315Ile) mutation was recorded. To the best of our knowledge this is the first Ph chromosome-negative CML case with e14a2 (b3a2) BCR-ABL1 transcript and p.(Tyr315Ile) mutation.

Tregs and GvHD prevention by extracorporeal photopheresis: observations from a clinical trial.

The aim of the present study was to evaluate the circulating T regulatory cells (Tregs) in patients undergoing extracorporeal photopheresis (ECP) for the prevention of chronic graft-versus-host disease (GvHD) and to search for any correlation between Tregs counts and chronic GvHD occurrence. Among n = 12 patients with complete longitudinal data, the median cumulative values of absolute peripheral Tregs counts were 21.64 and 63.49 cells/µL for patients who developed chronic GvHD and those who did not develop it, respectively (p = 0.05). The analysis of the median absolute counts of peripheral HLA-DR + Tregs provided similar results, showing that 20% (1 out of 5) and 100% (7 out of 7) of patients with HLA-DR + Tregs values of > 5 cells/µL were in the GvHD and non-GvHD groups, respectively (p = 0.01). In conclusion, the present results support the involvement of Tregs in the prevention of chronic GvHD in patients receiving ECP and suggest Tregs count as a potential biomarker of ECP effectiveness. Future strategies are needed to enhance Tregs expansion and/or activity in conjunction with ECP for an effective chronic GvHD prevention.

A double-hit High-grade B-cell lymphoma with three-way translocation t(3;8;14)(q27;q24;q32) involving BCL6, MYC, and IGH.

We describe an High-grade B-cell lymphoma case, in which a complex translocation t(3;8;14) with effects on the genes BCL6, MYC, and IGH, was detected. This case could be the first double-hit lymphoma with a single chromosome rearrangement causing the double effect with three genes involved.

A Case of Blastic Plasmacytoid Dendritic Cell Neoplasm Extensively Studied by Flow Cytometry and Immunohistochemistry.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with aggressive clinical course and poor prognosis. Diagnosis is based on detection of CD4+ CD56+, CD123high, TCL-1+, and blood dendritic cell antigen-2/CD303+ blasts, together with the absence of lineage specific antigens on tumour cells. In this report we present a case of BPDCN presenting with extramedullary and bone marrow involvement, extensively studied by flow cytometry and immunohistochemistry, who achieved complete remission after acute lymphoblastic leukemia like chemotherapy and allogeneic hematopoietic stem cell transplantation.

Rapid detection of t(15;17)(q24;q21) in acute promyelocytic leukaemia by microwave-assisted fluorescence in situ hybridization.

Acute promyelocytic leukaemia (APL) is a hematologic malignancy characterized by the rearrangement of the PML and RARα genes, mostly due to a reciprocal chromosomal translocation t(15;17)(q24;q21). A quick APL diagnosis is essential for starting a prompt suitable therapy. We describe a new rapid diagnostic laboratory approach to detect the PML-RARα rearrangement, which gives clear genetic results within 30 min of hybridization. It combines quick cell harvesting, fluorescence in situ hybridization performed with commercial DNA probe and microwave beams supplied by a domestic microwave oven. Copyright © 2015 John Wiley & Sons, Ltd.

Response of steroid-refractory chronic graft-versus-host disease to extracorporeal photopheresis correlates with the dose of CD3+ lymphocytes harvested during early treatment cycles.

BACKGROUND: Extracorporeal photopheresis (ECP) is a recognized second-line treatment for steroid-refractory chronic graft-versus-host disease (cGVHD). Treatment course is usually long, expensive, and demanding for patients, so predictors for response are needed. We carried out a retrospective study on cGVHD patients treated at our institution with the aim to identify a possible correlation between apheretic yields composition and probability of response. STUDY DESIGN: Patients treated for at least 6 months were eligible for the study. Flow cytometry data, including absolute counts of lymphocytes and their subpopulations in ECP products from cGVHD patients, were collected. For each cell population 1) the median dose per procedure harvested during the first 3 months of treatment and 2) the cumulative dose collected in the same period were compared with clinical response. RESULTS: A total of 726 ECP procedures were performed in 15 patients. Overall response, defined as either a complete response (CR) or a partial response according to National Institutes of Health criteria, was obtained in 10 of 15 patients (66.7%), and CR, in eight of 15 (53.3%). According to Cox regression analysis, the probability of achieving an overall response is significantly correlated with the median number of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes collected during the early treatment phase (first 3 months). CONCLUSION: Our data suggest that CD3+ cell evaluation in ECP during the early phase of treatment course could predict response and help identify patients who deserve further treatment.

Prognostic relevance of the flow cytometric count of medullar blasts in myelodysplastic syndromes.

OBJECTIVE: The medullar blast count is a milestone in the prognostic assessment in myelodysplastic syndromes (MDS). The optical microscopy (OM) may sometimes be inaccurate in this disease. The aim of this work is to test the flow immunocytometric (FCM) determinations of medullar immature cells (CD45(±) ) and the expression, among them, of CD33, CD34, and CD117 markers, for their prognostic relevance. METHODS: In a retrospective analysis of 98 patients affected by MDS, the IPSS was re-calculated by means of the FCM determination of blasts. Survival of patients at low or intermediate-1 IPSS risk was compared with the survival of patients at intermediate-2 or high IPSS risk. In the 64 cases with OM blast count lower than 5%, the survival of patients with the FCM count of medullar blasts ≤2% was compared with that of patients with FCM count >2%. RESULTS: Each single marker had a prognostic weight comparable to the optical blast count. The FCM blast count was particularly efficient in distinguishing the risk of having up to 2% or more than 2% of blasts in patients without OM excess of blasts. CONCLUSION: This method is interesting as prognostic tool, especially in patients without excess of blast.

Identification of hematopoietic progenitor cell donor characteristics predicting successful mobilization: results of an Italian multicenter study.

BACKGROUND: Peripheral blood (PB) hematopoietic progenitor cells (HPC) collected by apheresis are the first-choice source for allogeneic stem cell transplantation. The target HPC dose is usually considered to be 4 × 10(6) CD34+ cells/kg of the recipient, but higher doses are required in reduced-intensity conditioning and haploidentical transplants. Thus, prolonged stimulation and repeated collections or failure to reach HPC target may occur, increasing risks for donors and recipients. We carried out a retrospective multicenter study on healthy donors, to identify donor variables which may correlate with HPC mobilization. STUDY DESIGN AND METHODS: HPC allogeneic donations from sibling and unrelated donors performed in two centers from 1995 to 2012 were analyzed. We defined a mobilization cutoff of 50 × 10(6) CD34+ cells/L and tested somatic variables, blood counts, and granulocyte-colony-stimulating factor (G-CSF) dose and molecular form. RESULTS: A total of 360 donors were analyzed (male, 201; female, 159; sibling, 348; unrelated, 12; median [range] age, 44.8 [13-80] years). Median peak CD34+ in PB was 54.4 × 10(6) /L (range, 5 × 10(6) -299 × 10(6) ). By multivariate analysis, we identified the following variables to correlate with good mobilization: 1) male sex (p<0.0005); 2) younger age (p=0.007); 3) higher baseline (premobilization) white blood cell (WBC) count (p<0.0005); 4) higher G-CSF dosage (p<0.0005); and 5) use of lenograstim rather than filgrastim (p<0.002). CONCLUSION: In healthy donors it is possible to predict successful HPC mobilization by donor sex, age, WBC count, and G-CSF form and dose. Furthermore, based on these data, it may be possible, at least in parental setting, to modulate G-CSF dosage on the basis of donor characteristics.

Regeneration-associated WNT signaling is activated in long-term reconstituting AC133bright acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/ß-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated ß-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Flow cytometry and cytomorphology evaluation of hematologic malignancy in cerebrospinal fluids: comparison with retrospective clinical outcome.

An independent clinical assessment was compared with flow cytometry (FCM) and cytomorphology results obtained on 227 cerebrospinal fluids investigated for hematologic malignancy, in a retrospective longitudinal study with a median observation time of 11 months. A combined method assessment (CMA), defining "positive" a sample if at least one method gave "positive" results, was also tested. Eleven out of 55 screening samples and 53 out of 166 follow-up samples resulted positive at clinical evaluation. FCM and CM were concordant with positive clinical assessment in 68.5% and 51.5% of cases, respectively. According to CMA, 10.5% of samples (resulting false negative by either FCM or cytomorphology) were rescued as true positive. FCM retained significantly higher accuracy than cytomorphology (p=0.0065) and 100% sensitivity when at least 220 leukocytes were acquired. CMA accuracy was higher than FCM accuracy and significantly higher than cytomorphology accuracy in the analysis of all samples (p<0.0001), samples from mature B/T cell neoplasms (p=0.0021), and samples drawn after intrathecal treatment (p=0.0001). When acquiring ≤220 leukocytes, FCM accuracy was poor, and combining cytomorphology added statistically significant diagnostic advantage (p=0.0043). Although FCM is the best diagnostic tool for evaluating CSF, morphology seems helpful especially when clinically positive follow-up samples are nearly acellular.

Flow cytometry vs cytomorphology for the detection of hematologic malignancy in body cavity fluids.

Flow cytometry and cytomorphology results on 92 body cavity fluids [61 effusions and 31 bronchoalveolar lavage fluids (BALF)] from hematologic malignancy were compared with retrospective clinical outcome. We observed double true positive/negative results in 67 cases (73%), and double false negative results in 2 cases (2%). Immunophenotyping accounted for true positive/negative results in 22 out of 23 mismatched cases (25%), and retained significantly higher accuracy than that of cytomorphology especially in effusions and differentiated lymphoma. In BALF analysis, immunophenotyping and cytomorphology sensitivity was 75% and 0%, respectively. Flow cytometry retains the highest accuracy in detecting neoplastic cells in body cavity fluids.

Prognostic value of circulating CD34+ cells in myelodysplastic syndromes.

We studied circulating (C)CD34(+) cells by flow cytometry in 96 patients with myelodysplastic syndromes (MDS) at diagnosis, and in a subset of 35 cases during follow-up. CCD34(+) counts were stratified within both International Prognostic Scoring System (IPSS) and World Health Organization (WHO) categories. Counts >10/microl were associated with poorer leukemia-free survival, a prognostic value for evolution independent from that of WHO, and a higher progression probability within intermediate-risk IPSS and WHO classes. When serial measurements were performed, counts >10/microl more frequently correlated to evolution. Separating newly diagnosed patients on the basis of 10/microl cut-off of circulating CD34(+) cells retains prognostic utility, especially in intermediate-risk MDS.

Clinical characteristics and outcome of immunoglobulin M-related disorders.

We analyzed the clinical features and prognostic factors for transformation of immunoglobulin Mrelated disorders (IgM-RDs) to malignant lymphoproliferative disease (MLD) in 83 patients with IgM-RDs. We studied 19 patients with type I cryoglobulinemias, 56 patients with type II cryoglobulinemias, 5 patients with peripheral neuropathies (PNs), and 3 patients with idiopathic thrombocytopenic purpuras. Fourteen patients with cryoglobulinemias presented with mild to moderate hepatomegaly with or without splenomegaly. Fourteen patients with type II cryoglobulinemias had arthralgias and/or vascular purpura (12 receiving corticosteroids), and 7 presented with PN. These latter patients and those with PNs without cryoglobulinemia were treated with steroids, cyclophosphamide, or polychemotherapy with/without plasma-exchange. Cumulative probability of evolution to MLD at 5 years was 15% (95% CI; 5%-25%). At a median of 62 months (12-195 months), 8 cases of IgM-RDs (8.4%) evolved to overt Waldenstrom's macroglobulinemia (n = 6), 1 case to non-Hodgkin's lymphoma, and 1 case to B-cell chronic lymphocytic leukemia. At univariate analysis, male sex (P = 0.02), IgM level > or = 3 g/dL (P < 0.0001), detectable Bence Jones proteinuria (P = 0.0005), lymphocytosis (P = 0.049), and high erythrocyte sedimentation rate (P = 0.003) significantly correlated with the evolution risk. Age, blood cell counts, b2-microglobulin level, degree of marrow lymphoplasmacytic infiltration, type of cryoglobulinemia, and hepatitis C virus positivity did not correlate with transformation. Although IgM-RDs represent a distinct clinical entity frequently requiring treatment in view of the IgM-related symptoms, their evolution probability and prognostic factors for malignant transformation seem to widely overlap those described for asymptomatic IgM monoclonal gammopathies.

Prognostic factors for transformation in asymptomatic immunoglobulin m monoclonal gammopathies.

We evaluated the risk of transformation of asymptomatic immunoglobulin (Ig) M monoclonal gammopathy (aIgM-MG) into symptomatic lymphoproliferative disease in 384 patients, in subgroups of patients with IgM MG of undetermined significance (MGUS) and smoldering Waldenstrom's macroglobulinemia (sWM). One hundred seventy-two patients with aIgM-MG with bone marrow (BM) histopathology and immunophenotyping were available for analysis. After a median of 45 months (range, 12-233 months), 45 cases of aIgM-MG (11.7%) evolved into lymphoproliferative disease, as follows: symptomatic WM (n = 41), non-Hodgkin's lymphoma (NHL; n = 2), IgM multiple myeloma (IgM-MM; n = 1), and primary amyloidosis (n = 1). Cumulative transformation probability at 5 and 10 years was 8% and 29%, respectively. The parameters significantly correlated with evolution were, at univariate analysis, BM lymphoplasmacytic infiltration, high erythrocyte sedimentation rate, hemoglobin (Hb) level, IgM size, and peripheral lymphocytosis; at multivariate analysis, the parameters were IgM size and peripheral lymphocytosis, with Hb level associated with a trend toward higher progression risk. Of the 138 cases of IgM-MGUS, 14 (10.1%) evolved (13 WM, 1 IgM-MM) after a median of 75 months (range, 12-117 months); of the 34 cases of sWM, 13 (38.2%) progressed to WM after 55 months (range, 13-154 months). In patients with IgM-MGUS, event-free survival at 5 and 10 years was 95% and 83%, respectively, and in patients with sWM, 77% and 42%, respectively (P = 0.0001). Among patients with aIgM-MG, those at high risk of evolution are patients with sWM, a distinct entity with clear BM evidence of NHL.