Long-Smoldering T-prolymphocytic Leukemia: A Case Report and a Review of the Literature.

T-prolymphocytic leukemia (T-PLL) is a rare malignancy of mature T-cells with distinct clinical, cytomorphological, and molecular genetic features. The disease typically presents at an advanced stage, with marked leukocytosis, B symptoms, hepatosplenomegaly, and bone marrow failure. It usually follows an aggressive course from presentation, and the prognosis is often considered dismal; the median overall survival is less than one year with conventional chemotherapy. This case report describes a patient with T-PLL who, after an unusually protracted inactive phase, ultimately progressed to a highly invasive, organ-involving disease. After initial treatments failed, a novel treatment approach resulted in a significant response.

The cytokine-mediated crosstalk between primary human acute myeloid cells and mesenchymal stem cells alters the local cytokine network and the global gene expression profile of the mesenchymal cells.

Interactions between acute myeloid leukemia (AML) blasts and neighboring stromal cells are important for disease development and chemosensitivity. However, the molecular mechanisms involved in the cytokine-mediated crosstalk between mesenchymal stem cells (MSCs) and AML cells are largely unknown. Leukemic cells derived from 18 unselected AML patients were cultured with bone marrow MSCs derived from healthy donors; the populations then being separated by a semipermeable membrane. Coculture had only minor effects on MSC proliferation. The unique cytokine network in cocultures was determined by high constitutive MSC release of certain cytokines (especially IL-6 and vascular endothelial growth factor) and constitutive release of a wide range of soluble mediators by primary AML cells. However, the AML cell release varied considerably between patients, and these differences between patients were also reflected in the coculture levels even though supra-additive effects were seen for many mediators. These effects on the local cytokine network were dependent on a functional crosstalk between the two cell subsets. The crosstalk altered the global gene expression profile of the MSCs, especially expression of genes encoding proteins involved in downstream signaling from Toll like receptors, NFκB signaling and CCL/CXCL chemokine release. Thus, primary AML cells alter the functional phenotype of normal MSCs.

Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma.

AIMS: We aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL). METHODS: Clonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis. RESULTS: BM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I-III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0-1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival. CONCLUSIONS: Our results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor.

Effects of the Sangvia blood collection system on patients undergoing elective hip surgery.

We have conducted a randomized controlled study where 164 patients were randomized to receive autologous salvaged blood collected by Sangvia™ Blood Salvage System or allogeneic red cell concentrates if transfusion was indicated by clinical judgement. The study was powered to detect if transfusion of autologous blood reduced the occurrence of postoperative infections. We found no statistical significant difference in postoperative infection rate between the groups, but this may be due to the fact that postoperative infections were diagnosed in only five patients. Increased C-reactive protein concentrations slightly above level of significance indicate that autologous blood transfusions stimulate the patient's immune system. However, there was no indication of increased transfusion reaction rate, including febrile reactions, in the autologous group. Transfusion of autologous blood did not reduce the use of allogeneic red cell concentrates. The mean use of allogeneic red cell concentrates was 0.93 units (both groups combined), indicating that the transfusion policy may have been too liberal. There was a highly significant inverse correlation between pre-operative haemoglobin concentration and transfusion of allogeneic blood. In a patient population with a low frequency of postoperative infection, a larger study is needed to clarify if autologous salvaged blood protects against postoperative infections.

Is there a scientific basis for a recommended standardization of collection and cryopreservation of peripheral blood stem cell grafts?

High-dose chemotherapy followed by autologous stem cell transplantation has been used extensively during the last two decades in the treatment of hematologic malignancies. The vast majority of recent transplantations have been performed using mobilized peripheral blood stem cells, because they have become the preferred source of hematopoietic cells rather than bone marrow stem cells. The mobilization is achieved by growth factors, eventually combined with chemotherapy, and the cells are then harvested and cryopreserved until reinfusion. Despite extensive use for many years, few attempts have been made to standardize the various steps in mobilization, harvesting and cryopreservation. Furthermore, the autografts only represent relative stem cell enrichment and contain a wide range of more mature hematopoietic and immunocompetent cells; the potential clinical importance of these normal cells is largely unknown and represents an additional non-standardized factor in this treatment. We have reviewed the various methodologic approaches for stem cell mobilization, collection and cryopreservation of autografts with a special focus on the cryopreservation procedures, immunocompetent cells in the graft, and cytokine content of the graft supernatant. We conclude that the factors/aspects mentioned above should be standardized in future clinical studies of autotransplantation for human hematologic malignancies. Alternatively, detailed methodologic descriptions should be required when the results are published. Standardization of autograft preparation and cryopreservation will be achieved if/when transplantation units assess and adopt new standards based not only on the technology but, more importantly, on the quality of evidence and data related to that technology/methodology.

Intensive chemotherapy for acute myeloid leukemia differentially affects circulating TC1, TH1, TH17 and TREG cells.

BACKGROUND: Several observations suggest that immunological events early after chemotherapy, possibly during the period of severe treatment-induced cytopenia, are important for antileukemic immune reactivity in acute myeloid leukemia (AML). We therefore investigated the frequencies of various T cell subsets (TC1, TH1, TH17) and CD25+ FoxP3+ TREG cells in AML patients with untreated disease and following intensive chemotherapy. RESULTS: Relative levels of circulating TC1 and TH1 cells were decreased in patients with severe chemotherapy-induced cytopenia, whereas TH17 levels did not differ from healthy controls. Increased levels of regulatory CD25+ FoxP3+ T cells were detected in AML patients with untreated disease, during chemotherapy-induced cytopenia and during regeneration after treatment. TH17 and TH1 levels were significantly higher in healthy males than females, but this gender difference was not detected during chemotherapy-induced cytopenia. Finally, exogenous IL17-A usually had no or only minor effects on proliferation of primary human AML cells. CONCLUSIONS: We conclude that the effect of intensive AML chemotherapy differ between circulating T cell subsets, relative frequencies of TH17 cells are not affected by chemotherapy and this subset may affect AML cells indirectly through their immunoregulatory effects but probably not through direct effects of IL17-A.

Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: the hematological experience.

In vitro studies have demonstrated that cancer-specific T cell cytotoxicity can be induced both ex vivo and in vivo, but this therapeutic strategy should probably be used as an integrated part of a cancer treatment regimen. Initial chemotherapy should be administered to reduce the cancer cell burden and disease-induced immune defects. This could be followed by autologous stem cell transplantation that is a safe procedure including both high-dose disease-directed chemotherapy and the possibility for ex vivo enrichment of the immunocompetent graft cells. The most intensive conventional chemotherapy and stem cell transplantation are used especially in the treatment of aggressive hematologic malignancies; both strategies induce T cell defects that may last for several months but cancer-specific T cell reactivity is maintained after both procedures. Enhancement of anticancer T cell cytotoxicity is possible but posttransplant vaccination therapy should probably be combined with optimalisation of immunoregulatory networks. Such combinatory regimens should be suitable for patients with aggressive hematological malignancies and probably also for other cancer patients.

Early pre-engraftment, functional, in vitro responsiveness of T lymphocytes in allotransplanted, acute leukemia patients: proliferation and release of a broad profile of cytokines, possibly predictive of graft-versus-host disease.

Previous studies of T cell reconstitution following allogeneic stem cell transplantation have described long-lasting T cell defects, including decreased levels of autocrine proliferating CD4+ T cells. However, T cell functions during the early phase of conditioning-induced, pre-engraftment pancytopenia have not been characterized previously. We used a whole blood assay to investigate T cell proliferation and cytokine release during the period of pre-engraftment cytopenia. The study included 13 acute leukemia patients receiving myeloablative conditioning followed by transplantation of G-CSF-mobilised peripheral blood stem cells derived from HLA-matched family donors. Maximal proliferation and cytokine release could not be reached by anti-CD3 stimulation alone, but was dependent on the presence of additional costimulation with anti-CD28. Circulating T cells showed a broad cytokine release profile after activation, and the highest levels were detected for IFNgamma, GM-CSF and IL-6. Correlation analyses showed that TNFalpha/IL-4/IL-5/IL-13 in particular were released as a separate cluster, IFNgamma and GM-CSF correlated strongly, whereas IL-17 showed a weak correlation to IL-6 only. The capacity of circulating T cells derived during pre-engraftment cytopenia to release high levels of IFNgamma, IL-6 and IL-17 in response to in vitro activation with anti-CD3+anti-CD28 showed statistically significant correlations with later acute GVHD. We conclude that allotransplanted patients have a functional T cell system even during the pre-engraftment period of severe pancytopenia.

Use of different DMSO concentrations for cryopreservation of autologous peripheral blood stem cell grafts does not have any major impact on levels of leukocyte- and platelet-derived soluble mediators.

BACKGROUND AIMS: Infusion of stem cell autografts can be associated with adverse effects. Necrotic normal leukocytes, cytokines or intracellular mediators released from leukocytes and platelets or the cryo-protectant dimethyl sulfoxide (DMSO) may contribute to this. Cryopreservation using 5% instead of 10% DMSO improves CD34(+) cell viability and therefore we investigated whether using less DMSO had favorable outcomes on leukocyte viability and levels of various soluble mediators in the graft supernatant. METHODS: Peripheral blood autografts were harvested by 20 apheresis procedures in 16 cancer patients, and autograft samples were cryopreserved with 2%, 4%, 5% and 10% DMSO and stored for 5-6 years. After thawing, the viability of neutrophils and lymphocytes was analyzed by flow cytometry and supernatant levels of soluble mediators were determined by enzyme-linked immunosorbent assay (ELISA) analyzes. RESULTS: The highest viability of both neutrophils and lymphocytes was detected with 4% and 5% DMSO, whereas decreased viability was observed with 2% and 10% DMSO. Low or undetectable levels of leukocyte-derived interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha and CXCL8, high levels of platelet-derived CCL5 and CXCL4, and high levels of monocyte-derived soluble CD14 were measured independent of the DMSO concentration, except for slightly increased CXCL8 and decreased CXCL4 levels with 2% DMSO. Perforin levels showed a significant inverse correlation with the DMSO concentration. CONCLUSIONS: The use of different DMSO concentrations affects the viability of normal leukocytes in autologous peripheral blood stem cell grafts, but has only minor effects on supernatant levels of leukocyte- and platelet-derived soluble mediators.

Long-term cryopreservation of autologous stem cell grafts: a clinical and experimental study of hematopoietic and immunocompetent cells.

BACKGROUND: Autologous stem cell transplantation(ASCT) is used in the treatment of several malignancies.Harvesting sufficient peripheral blood progenitor cells (PBPCs) for a potential second autotransplantation at the time of relapse several years after diagnosis is becoming an increasingly common practice. STUDY DESIGN AND METHODS: Cryopreserved PBPCs were prepared with different concentrations of dimethyl sulfoxide (DMSO; 2, 4, 5, and 10%) and stored for at least 5 years before the recovery of CD34+ cells and various T- and natural killer (NK)-cell subsets were analyzed by flow cytometry. Furthermore,clinical variables for myeloma patients having a second autotransplantation with long-term-stored autografts were evaluated. RESULTS: The number of viable CD34+ cells in longterm-stored grafts was higher when autografts were cryopreserved with 4 or 5% than with 2 and 10%DMSO. The number of viable CD34+ cells was reduced by 13.9% after 5 years of cryostorage in 5% DMSO.Lymphocyte viability was also higher with 4 or 5%DMSO. However, the frequencies of several T-cell subsets showed DMSO-dependent differences,whereas NK-cell subsets did not. Furthermore, after a second autotransplantation with long-term-stored PBPC grafts at the time of myeloma relapse (median storage time, 42 months) all 17 patients reached neutrophil counts exceeding 0.5 x 109/L and platelet counts exceeding 20 x 109/L within 15 days. There was no difference in engraftment between patients receiving autografts preserved with 5 and 10% DMSO. CONCLUSION: PBPC autografts can safely be stored for at least 5 years in 5% DMSO and used for ASCT.

Thrombelastography.

BACKGROUND: Thromboelastography (TEG) records the continuous profiles of whole blood coagulation by measurement of the viscoelastic changes associated with fibrin polymerization, and thereby provides a global assessment of haemostatic function. In the past decades there has been an increasing interest for TEG in clinical practice. In this paper we present the rationale for the method and a discussion of the possible application of TEG. MATERIAL AND METHODS: This review is based on personal experience and literature retrieved from searches in PubMed. RESULTS AND INTERPRETATION: Currently TEG is used with standard coagulation tests to decrease the risk for bleeding and reduce the homologous blood transfusion in cardiac surgery with cardiopulmonary bypass and in liver surgery. Other applications are severe trauma, obstetric medicine, haemophilia and hypercoagulable conditions. Development of a modified TEG, using heparin in combination with reptilase and factor XIIIa, has the potential to monitor the effects of platelet inhibiting drugs. It should be kept in mind that the TEG is a global test of coagulation and therefore the need for additional haemostatic tests should be evaluated when applicable. The main advantage for TEG is an inexpensive patient near method for quick evaluation of the patient's global haemostatic system. Used by experienced hands, TEG is a valuable haemostatic test, the future of which is already present.

[Granulocyte transfusion]. / Granulocyttransfusjon.

BACKGROUND: Granulocyte transfusion is used in the treatment of severe, sustained or complicated infection and neutropenia. In recent years, the method's efficacy has improved and its availability increased. After the introduction of granulocyte colony-stimulating factor (G-CSF) there has been a growing interest for granulocyte transfusion, and effective methods for collection and transfusion of granulocytes are now in clinical use. This paper presents clinical, immunological and ethical challenges, our own experience with granulocyte harvesting and documentation of efficacy. MATERIAL AND METHODS: The paper is based on our own experience with granulocyte transfusion and literature retrieved though a non-systemic search. RESULTS: The efficacy of granulocyte transfusion with respect to morbidity and mortality is still debated, and the method currently has no place in routine treatment of documented infection and neutropenia. However, the treatment could be an alternative for patients with inadequate response to conventional treatment and for whom sustained neutropenia is expected. The combined use of G-CSF, hydroxyethyl starch and corticosteroids considerably increases the yield of granulocytes collected for transfusion. INTERPRETATION: Granulocyte transfusion is clinically feasible, but more research is needed to define clinical indications and to document the procedure's efficacy. Larger randomized controlled efficacy trials are needed.

Autologous peripheral blood progenitor cells cryopreserved with 5 and 10 percent dimethyl sulfoxide alone give comparable hematopoietic reconstitution after transplantation.

BACKGROUND: Previous in vitro studies have demonstrated decreased apoptosis and necrosis in peripheral blood progenitor cells (PBPCs) cryopreserved with 5 percent instead of 10 percent dimethyl sulfoxide (DMSO). This study was carried out to investigate whether these in vitro findings were supported by clinical data concerning hematopoietic engraftment after autologous stem cell transplantations with PBPCs cryopreserved with 5 and 10 percent DMSO. STUDY DESIGN AND METHODS: During a 6-year period, 103 consecutive patients with newly diagnosed multiple myeloma (MM; n = 58) and lymphoma (n = 45) were transplanted with autologous PBPCs. Throughout the first part of the period cells were cryopreserved with 10 percent DMSO and later with 5 percent. A retrospective comparison was carried out of the clinical results for these two groups. RESULTS: No significant difference in median time to neutrophil and platelet (PLT) engraftment was demonstrated for MM and lymphoma patients transplanted with PBPCs cryopreserved with 5 or 10 percent DMSO. Time until neutrophil counts of more than 0.5 x 10(9) per L was 10 days both for the 5 and 10 percent MM groups and 12 days for both the 5 and the 10 percent lymphoma patients. Median time until stable PLT counts of more than 20 x 10(9) per L was 11 days in all four groups. In addition, transfusion requirements and duration of days admitted to hospital did not differ between the groups. CONCLUSION: The routines for cryopreservation of autografts vary considerably between transplantation centers, and this makes it difficult to compare different clinical studies. Our results suggest that cryopreservation with 5 percent DMSO alone followed by storage in nitrogen is a simple, highly standardized, and safe procedure for cryopreservation of autologous stem cell graft.

Reduced exercise capacity in genetic haemochromatosis.

BACKGROUND: Many patients with genetic haemochromatosis complain about fatigue and reduced physical capacity. Exercise capacity, however, has not been evaluated in larger series of haemochromatosis patients treated with repeated phlebotomy. DESIGN AND METHODS: We performed exercise echocardiography in 152 treated haemochromatosis patients (48+/-13 years, 26% women) and 50 healthy blood donors (49+/-13 years, 30% women), who served as controls. Echocardiography was performed at rest and during exercise in a semiupright position on a chair bicycle, starting from 20 W, increasing by 20 W/min. Transmitral early and atrial velocity and isovolumic relaxation time were measured at each step. Ventilatory gas exchange was measured by the breath-to-breath-technique. RESULTS: Compared with healthy controls, haemochromatosis patients were more obese and less trained. More of them smoked, and 17% had a history of cardiovascular or pulmonary disease. Adjusted for training, the left ventricular function and dimensions at rest did not differ between the groups. During exercise the haemochromatosis patients obtained a significantly lower peak oxygen (O2) uptake (28.1 vs. 34.4 ml/kg per min, P<0.001). In a multiple regression analysis haemochromatosis predicted lower peak O2 uptake independently of significant contributions of sex, age, and height, as well as of systolic blood pressure and log-transformed isovolumic relaxation time at peak exercise, whereas no independent association was found with weight or physical activity (multiple R=0.74, P<0.001). Adding genotype, s-ferritin, prevalence of smoking, or history of cardiopulmonary disease among the covariates in subsequent models did not change the results. CONCLUSION: Genetic haemochromatosis, even when treated with regular phlebotomy, is associated with lower exercise capacity independently of other covariates of exercise capacity.

[Survival after high-dose therapy with autologous stem cell support]. / Overlevelse etter høydosebehandling med autolog stamcellestøtte.

BACKGROUND: High-dose therapy with autologous stem cell support has been carried out in our hospital since 1996. We have recently collected survival data on patients who have undergone this procedure. MATERIAL AND METHODS: The study population comprised 111 patients, 58 of whom had been diagnosed with multiple myeloma, 38 with various forms of malignant lymphoma, 11 with sarcoma and 4 with testicular cancer. RESULTS: Median survival from reinfusion of stem cells was 74.8 months for patients with myeloma, 47.8 months for patients with malignant lymphoma and 11.7 months for patients with sarcoma. Three-year survival was 72.2 % for myeloma patients and 54.8 % for lymphoma patients. While the survival slope decreased steadily throughout the study period for patients with multiple myeloma, it seemed to level out after approximately 18 months for patients with malignant lymphomas. INTERPRETATION: Our data on myeloma patients show survival comparable to previously published data. For malignant lymphoma patients, our data support the assumption that high-dose therapy has the potential for cure.