Clara Cesana

Prognostic Factors for Malignant Transformation in Monoclonal Gammopathy of Undetermined Significance and Smoldering Multiple Myeloma

PURPOSE To evaluate the natural history of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), identify early predictors of evolution, and assess whether associated conditions correlate with disease progression. PATIENTS AND METHODS A total of 1,231 consecutive patients with either MGUS (n = 1,104) or SMM (n = 127) diagnosed from July 1975 to March 1998 were included in the study. Cumulative survival probability and cumulative probability of transformation into lymphoproliferative disease were calculated by means of the Kaplan-Meier estimator. Univariate and multivariate Cox models were used to identify possible predictors of malignant evolution. RESULTS Cumulative transformation probability at 10 and 15 years was 14% and 30%, respectively. At a median follow-up of 65 months (range, 12 to 239 months), 64 MGUS cases (5.8%) evolved to multiple myeloma (MM) (n = 43), extramedullary plasmacytoma (n = 1), primary amyloidosis (n = 1), Waldenströms macroglobulinemia (n = 12), non-Hodgkins lymphoma (n = 6), and B-chronic lymphocytic leukemia (n = 1). At a median follow-up of 72 months (range, 12 to 247 months), 25 SMMs (19.7%) evolved to overt MM. A lower evolution risk was observed in MGUS than in SMM (P <.0001). Greater than 5% marrow plasmacytosis, detectable Bence Jones proteinuria, polyclonal serum immunoglobulin reduction, and high erythrocyte sedimentation rate (ESR) were independent factors influencing MGUS transformation. SMM progression correlated with greater than 10% marrow plasma cells, detectable Bence Jones proteinuria, and immunoglobulin (Ig) A isotype. Neither concomitant diseases nor immunosuppression correlated with progression. CONCLUSION Careful evaluation of marrow plasmacytosis, urinary paraprotein, background immunoglobulins, ESR, and paraprotein isotype might help identify at presentation patients with benign monoclonal gammopathies requiring stricter monitoring.

CD34 + cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) are functionally different from CD34 + cells mobilized by G-CSF

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34+ cells, we analyzed nucleated cells (NC), CD34+ cells, committed progenitor cells and long-term culture initiating-cells (LTC-IC) in 52 leukaphereses from 26 patients with lymphoid malignancies, mobilized either by CY+G-CSF (n = 16) or G-CSF alone (n = 10). Thirty-four aphereses from the CY+G-CSF group and 18 aphereses from the G-CSF group were investigated. According to the study design, leukaphereses were carried out until an average number of 7 × 106 CD34+ cells/kg body weight were collected. The mean (± s.e.m.) numbers of CD34+ cells mobilized per apheresis by CY+G-CSF and G-CSF were not significantly different (2.76 ± 0.6 × 108 vs 2.53 ± 0.4 × 108, P ⩽ 0.7). This resulted from a mean number of NC that was significantly lower in the CY+G-CSF products than in the G-CSF products (12.4 ± 1.7 × 109 vs 32 ± 5.4 × 109, P ⩽ 0.0001) and a mean incidence of CD34+ cells that was significantly higher in the CY+G-CSF products than in the G-CSF products (2.9 ± 0.6% vs 0.9 ± 0.2%, P ⩽ 0.0018). The mean (± s.e.m.) number of CFU-GM collected per apheresis was significantly higher in the CY+G-CSF group than in the G-CSF group (37 ± 7 × 106 vs 14 ± 2 × 106, P ⩽ 0.03). Interestingly, CY+G-CSF-mobilized CD34+ cells had a significantly higher plating efficiency than G-CSF-mobilized CD34+ cells (25.5 ± 2.9% vs 10.8 ± 1.9%, P ⩽ 0.0006). In addition, the mean number of LTC-IC was significantly higher in the CY+G-CSF products than in the G-CSF products (6.3 ± 1 × 106 vs 3.3 ± 0.3 × 106, P ⩽ 0.05). In conclusion, our data provide evidence that CY+G-CSF and G-CSF induce the mobilization of CD34+ cells with different clonogenic potential. As mobilized PBPC containing large numbers of progenitors lead to safer transplantation, this issue may have implications for planning mobilization strategies.

Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6–14 months). Viability was always >97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80–87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 ± 7% vs 57 ± 4%, P ⩽ 0.4) or in purity (89 ± 2% vs 81 ± 6%, ⩽ 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.

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.

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

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 × 106 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.

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.

Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor

OBJECTIVE We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). MATERIALS AND METHODS Healthy donors (n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 microg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. RESULTS Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34+ cells (r = 0.6, p = 0.004), CFU-GM (r = 0.79, p = 0.0005) and CFC (r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells (r = 0.68, p = 0.001), numbers of day 4 CD341 cells (r = 0.52, p = 0.005), CFU-GM (r = 0.63, p = 0.002), and CFC (r = 0.61, p = 0.003). CONCLUSION Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.

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

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.

Effective treatment of autoimmune hemolytic anemia and hairy cell leukemia with interferon-α

To the Editor: Hairy cell (HC) leukemia (HCL) is characterized by splenomegaly, pancytopenia and typical HCs in peripheral blood (PB), bone marrow (BM), spleen and liver (1.). HCL-related autoimmune hemolytic anemia (AIHA) is extremely unusual (2–5.). Although interferon-a is effective in HCL (6.), its use is controversial in HCL-related autoreactive disorders due to its well known capacity to enhance autoimmunity (7–9.). In September 1998, a 57-yr-old female presented weight loss, jaundice and hepatosplenomegaly. PB showed hemoglobin 6.8 g/dL, white blood cells 4.8r10/L (normal differential count), platelets 153r10/L, MCV 104 fL, aniso-poikilocytosis with microspherocytes, and reticulocytes 660r 10/mL. Lactate dehydrogenase, total/unconjugated bilirubin and b2-microglobulin were 537 U/L, 2.45/1.87 mg/dL, and 3.1 mg/mL (ranges 10–500 U/L, 0.25–1/0.2–0.7 mg/dL, and 0.6–2.6 mg/mL), respectively. Haptoglobin was 8 mg/dL (range 32–205 mg/dL). The direct antiglobulin test was positive for IgG and C3d. An eluate showed a 1 : 4 autoantibody titer, but was unable to identify any specificity. Anti-DNA/antinuclear antibodies, rheumatoid factor, and circulating immune complexes were unremarkable. Serologic and liver function tests suggested a hepatitis B virus (HBV) surface antigen carrier status. BM showed a hypercellularity with erythroblastosis, and no lymphoid infiltration. An idiopathic AIHA was diagnosed. Oral prednisone 1 mg/kg/d was started .(Fig. 1.). After hemoglobin and haptoglobin normalization, the drug was tapered (10 mg/wk). During prednisone dose reduction, periodical hepatitis reactivations were recorded. Seven months after diagnosis, chronic compensated hemolysis recurred, and maintenance prednisone (7.5 mg/d) was continued. b2-microglobulin progressively increased until August 1999, when progressive pancytopenia, further spleen enlargement and PB HCs positive for the tartrate-resistant acid phosphatase (TRAP) reaction and CD19, CD20, CD22, CD11c, and CD25 appeared. BM showed an interstitial infiltration with lymphoid cells TRAP, CD20, DBA44, and a moderate increase in reticulin. Liver biopsy demonstrated focal infiltration with lymphoid cells CD20, DBA44, and cirrhosis. HCL was diagnosed. In January 2000, interferon-a 1 MU s.c. three times per week was started and increased by 0.5 MU/wk every 2 wk up to 3 MU three times per week. Prednisone (7.5 mg d) was continued. Shortly after interferon-a initiation, hemoglobin increase, haptoglobin normalization, PB HC disappearance and b2-microglobulin/spleen size reduction were recorded. In June 2000, a onemonth interferon-a withdrawal [from lamivudine (100 mg/d p.o.) initiation until HBV-DNA negativization and prednisone suspension] induced transient hemolysis recurrence. Nine months after interferon-a initiation, HCL partial remission was associated with stable normal haptoglobin, despite no change in direct antiglobulin test results. Immunosuppression is frequently unsuccessful in HCL-related AIHA (3–5.), and not recommended for HCL. The effectiveness of drugs specific for HCL on an associated AIHA, however, is not well recognized. In our case, splenectomy could not be performed nor could cytotoxic drugs be administered because of liver cirrhosis, and interferon-a was the only feasible therapeutic option for the HCL. In keeping with previously reported interferoninduced improvement of AIHA in primary cold agglutinin disease (10, 11.) and with the incidentally observed reduction/disappearance of autoantibody titers during interferon treatment for HCL (7.), our autoimmune hemolysis recovered in parallel with HCL clinical response to interferon-a and relapsed during temporary interferon-a withdrawal. AIHA pathogenetic mechanisms in HCL are unknown. In our case, AIHA precedence to the leukemia diagnosis might suggest that the Eur J Haematol 2002: 68: 120–121 Printed in UK. All rights reserved Copyright # Blackwell Munksgaard 2002

In vitro growth of mobilized peripheral blood progenitor cells is significantly enhanced by stem cell factor.

The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony‐forming units (CFU‐Mix), erythroid burst‐forming units (BFU‐E) and granulocyte‐macrophage colony‐forming units (CFU‐GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self‐renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long‐term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long‐term culture (LTC‐IC). Addition of SCF (50 ng/ml) to methylcellulose cultures stimulated with maximal concentrations of G‐CSF, GM‐CSF, interleukin 3 and erythropoietin significantly increased the growth (mean ± SE) of CFU‐Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p ≤ 0.0001), BFU‐E (47 ± 10 versus 32 ± 6, p ≤ 0.002) and CFU‐GM (173 ± 31 versus 112 ± 20, p ≤ 0.0001). Mean (± SE) percentages of SCF‐dependent CFU‐Mix, BFU‐E and CFU‐GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. Mean (± SE) LTC‐IC growth per 2 × 106 nucleated cells was 221 ± 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r =.87; p ≤ 0.0001) between LTC‐IC and SCF‐dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF‐dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.