Long-term prognosis for 1-year relapse-free survivors of CD34+ cell-selected allogeneic hematopoietic stem cell transplantation: a landmark analysis.

CD34+ cell selection significantly improves GvHD-free survival in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, specific information regarding long-term prognosis and risk factors for late mortality after CD34+ cell-selected allo-HSCT is lacking. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT for AML (n=164), ALL (n=33) or myelodysplastic syndrome (n=79). At 5 years' follow-up after the 1-year landmark (range 0.03-13 years), estimated relapse-free survival (RFS) was 73% and overall survival (OS) 76%. The 5-year cumulative incidence of relapse and non-relapse mortality (NRM) were 11% and 16%, respectively. In multivariate analysis, Hematopoietic Cell Transplantation Comorbidity Index score⩾3 correlated with marginally worse RFS (hazard ratio (HR) 1.78, 95% confidence interval (CI) 0.97-3.28, P=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26-5.08, P=0.004). Despite only 24% of patients with acute GvHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GvHD associating with increasingly poorer survival on multivariate analysis (P<0.0001). Of 63 deaths after the landmark, GvHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. Although prognosis is excellent for patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GvHD.

Virus-specific T-cell banks for 'off the shelf' adoptive therapy of refractory infections.

Adoptive immunotherapy with transplant donor-derived virus-specific T cells has emerged as a potentially curative approach for the treatment of drug-refractory EBV+lymphomas as well as CMV and adenovirus infections complicating allogeneic hematopoietic cell transplants. Adoptive transfer of HLA partially matched virus-specific T cells from healthy third party donors has also shown promise in the treatment of these conditions, with disease response rates of 50-76% and strikingly low incidences of toxicity or GVHD recorded in initial trials. In this review, we examine the reported experience with transplant donor and third party donor-derived virus-specific T cells, identifying characteristics of the viral pathogen, the T cells administered and the diseased host that contribute to treatment response or failure. We also describe the characteristics of virus-specific T-cell lines in our center's bank and the frequency with which in vitro culture promotes expansion of immunodominant T cells specific for epitopes that are presented by a limited array of prevalent HLA alleles, which facilitates their broad applicability for treatment.

T-cell depleted allogeneic hematopoietic cell transplants as a platform for adoptive therapy with leukemia selective or virus-specific T-cells.

Allogeneic hematopoietic cell transplants adequately depleted of T-cells can reduce or prevent acute and chronic GVHD in both HLA-matched and haplotype-disparate hosts, without post-transplant prophylaxis with immunosuppressive drugs. Recent trials indicate that high doses of CD34+ progenitors from G-CSF mobilized peripheral blood leukocytes isolated and T-cell depleted by immunoadsorption to paramagnetic beads, when administered after myeloablative conditioning with TBI and chemotherapy or chemotherapy alone can secure consistent engraftment and abrogate GVHD in patients with acute leukemia without incurring an increased risk of a recurrent leukemia. Early clinical trials also indicate that high doses of in vitro generated leukemia-reactive donor T-cells can be adoptively transferred and can induce remissions of leukemia relapse without GVHD. Similarly, virus-specific T-cells generated from the transplant donor or an HLA partially matched third party, have induced remissions of Rituxan-refractory EBV lymphomas and can clear CMV disease or viremia persisting despite antiviral therapy in a high proportion of cases. Analyses of treatment responses and failures illustrate both the advantages and limitations of donor or banked, third party-derived T-cells, but underscore the potential of adoptive T-cell therapy in the absence of ongoing immunosuppression.

Patterns of relapse and progression in multiple myeloma patients after auto-SCT: implications for patients' monitoring after transplantation.

Auto-SCT (ASCT) is widely used in first-line treatment of multiple myeloma (MM). However, most patients eventually relapse or have progression of disease (R/POD). Although precise knowledge of R/POD patterns would be important to generate evidence-based surveillance recommendations after ASCT, such data is limited in the literature, especially after introduction of the free light chain assay (FLCA). This retrospective study examined the patterns of R/POD after first-line ASCT in 273 patients, using established criteria. At the time of R/POD, only 2% of patients had no associated serological evidence of R/POD. A total of 85% had asymptomatic R/POD, first detected by serological testing, whereas 15% had symptomatic R/POD with aggressive disease, early R/POD and short survival, with poor cytogenetics and younger age identified as risk factors. Although occult skeletal lesions were found in 40% of asymptomatic patients tested following serological R/POD, yearly skeletal surveys and urine testing were poor at heralding R/POD. We found a consistent association between paraprotein types at diagnosis and R/POD, allowing informed recommendations for appropriate serological monitoring and propose a new needed criterion using FLCA for patients relapsing by FLC only. Our findings provide important evidence-based recommendations that strengthen current monitoring guidelines after first-line ASCT in MM.

Cancer-testis antigen expression and immunogenicity in AL amyloidosis.

Light-chain amyloidosis (AL) is a plasma cell dyscrasia closely related to multiple myeloma. In multiple myeloma, the cancer-testis antigens (CTAs) CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A CTAs are expressed in up to 80% of cases. In this study, we investigated the expression and immunogenicity of several CTAs in patients with AL amyloidosis in a total of 38 bone marrow specimens by employing standard immunohistochemistry techniques on paraffin-embedded archival tissues. Plasma samples from 35 patients (27 with matched bone marrow samples) were also analyzed by ELISA for sero reactivity to a group of full-length CTA proteins. CT7 was present in 25/38 (66%) while CT10 was demonstrated in 3/38 and GAGE in 1/38 AL amyloid cases. The expression pattern was mostly focal. There were no significant differences with regard to organ involvement, response to treatment, or prognosis in CTA positive compared to negative cases. None of the specimens showed spontaneous humoral immunity to CT7, but sero reactivity was observed in individual patients to other CTAs. This study identifies CT7 as the prevalent CTA in plasma cells of patients with AL amyloidosis. Further analyses determining the biology of CTAs in AL amyloidosis and their value as potential targets for immunotherapy are warranted.

Rapid selection of antigen-specific T lymphocytes by retroviral transduction.

Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT(+) T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT(+) fraction relative to the untransduced T-cell population. NIT(+) fractions transduced on day 8 possessed more CD4(+) T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

PNH cells are as sensitive to T-cell-mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria.

The mechanism responsible for the bone marrow failure that is almost invariable in paroxysmal nocturnal haemoglobinuria (PNH) is unknown. Based on the close association between PNH and idiopathic aplastic anaemia, a plausible pathogenetic model predicts that, in PNH, autoreactive T cells specific for haemopoietic stem cells (HSCs) cause depletion of normal HSCs, whereas PNH HSCs escape this T-cell-mediated attack. In this study, we addressed the hypothesis that PNH HSCs are resistant to the cytotoxic effect of T cells because they lack surface expression of one or more glycosylphosphatidylinositol (GPI)-linked molecules. We tested the sensitivity of normal and PNH Epstein-Barr virus (EBV)-transformed B-cell lymphoblastoid cell lines (BLCLs) to the cytotoxic effect of autologous EBV-specific T-cell lines and clones from a patient with PNH in an in vitro experimental system. We found that the PNH BLCLs were no less sensitive to T-cell-mediated cytotoxicity than non-PNH isogenic BLCLs, indicating that GPI-linked molecules on the surface of target cells are not required for killing by T cells. This suggests that the mechanism whereby PNH HSCs survive T-cell attack is not because of the lack of surface expression of one or more GPI-linked molecules. By implication, other mechanisms become more probable.

Phenotype of lymphocyte subsets after autologous peripheral blood stem cell transplantation.

The expression of CD45RA+ and CD45RO+ isoforms on T cells and the recovery of B lymphocytes and NK cells after autologous peripheral blood stem cell transplantation (PBSCT) were studied during the early period following transplantation. The same panel of monoclonal antibodies was used to analyze the lymphocyte subsets after allogeneic bone marrow transplantation (allo-BMT) and in cord blood. The CD4+ subsets regenerated differently from the CD8+ isoforms and the CD4+CD45RA+ subsets appears to be the only thymus-dependent regenerating population post-transplantation. Since the CD8+ CD45RA+ and CD8+ CD45RO+ isoforms recovered to normal levels within a month after PBSCT and within 3 months after allo-BMT, there seems to be no thymic influence on the regeneration of the immature CD8+CD45RA+ subset. The regeneration of the CD4+ cells was markedly delayed, but was faster in the PBSCT recipients, mainly because of the faster recovery of the mature CD4+ CD45RO+ subset. The pattern of surface antigens on T lymphocytes after transplantation did not resemble the antigen pattern on cord blood cells. The CD19+ CD20+ cells recovered earlier in the PBSCT group and remained compromised after allo-BMT during the time studied. The faster B lymphocyte regeneration correlates with the faster reconstitution of the mature CD4+CD45RO+ cells. The pattern of antigens CD38+, HLA-DR+, CD10+ on B lymphocytes of the recovery phase resembled the pattern on B cells of cord blood lymphocytes. The NK cells were not deficient at any time post-transplant in both groups.

Increase of mobilized CD34-positive peripheral blood progenitor cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis.

G-CSF (filgrastim) can effectively mobilize peripheral blood progenitor cells (PBPC) when administered during steady-state hematopoiesis. In this single center study, we compared the effectiveness of two different doses of G-CSF on the mobilization of peripheral blood stem cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis. A first group including 33 patients received 10 micrograms G-CSF/kg BW per day (group A), whereas a second group comprising 34 patients was treated with 24 (2 x 12) micrograms G-CSF/kg body weight (BW) per day (group B) prior to the leukapheresis. A significant difference (P = 0.015) in the total number of CD34+ cells between group A: 11.32 x 10(7) (range 0.34-110.2) and group B: 48.25 x 10(7) (range 1.33-447.4) has been observed in the first leukapheresis product. Moreover, the total number of CFU-GM increased significantly from 34.79 x 10(4) (range 1.07-300.9) to 147.69 x 10(4) (range 1.03- 1204.0) (P < 0.005), and the number of MNC increased from 1.35 x 10(10) (range 0.41-3.09) group A) to 2.93 x 10(10) (range 0.66-9.7) (group B) (P < 0.001). Comparable results were obtained in the second leukapheresis. Our data indicate, that the application of higher doses of G-CSF can significantly improve the effectiveness of mobilizing PBPC during steady-state conditions, and thereby considerably contribute to a safe and fast engraftment as well as a reduced number of leukapheresis procedures to achieve sufficient number of PBPC.

Identification of Staphylococcus hyicus with the API staph strip.

The API Staph Strip system (API System S.A., Montalïeu-Vercieu, France) was compared with conventional methods for identification of Staphylococcus hyicus isolated from cases of exudative epidermitis in swine. The API Staph Strip was found to provide unique profile numbers, namely, 6-514-151, 6-514-153, and 6-516-153. These profile numbers are not listed in the API Staph Strip data base. It was found that the use of this miniaturized system is preferable to conventional methods for the identification of the causal agent of swine exudative epidermitis.

Lancefield group R streptococci associated with pneumonia in swine.

During a 12-month survey of streptococcal infections in swine, 14 of 98 isolates were found to belong to Lancefield group R. Most of these isolates were from pigs with pneumonia. Approximately half the group R isolates were associated with concurrent Pasteurella pneumonia.

Physical aspects of wet granulations II: factors involved in prolonged and excessive mixing.

Evidence is presented that excessive blending in a wet granulation process shifts the packing arrangement of the wet granule, causing it to become dense and nonporous. With prolonged kneading, a large amount of the water-soluble excipients dissolves in the granulating fluid, and these two factors make the drying slower. This result, coupled with the previous finding that a certain time is required to attain an equilibrium size granule, explains why there exists an optimum kneading time for a wet granulation from a mechanical performance point of view.