Prevention, monitoring and treatment of cardiovascular adverse events in myeloma patients receiving carfilzomib A consensus paper by the European Myeloma Network and the Italian Society of Arterial Hypertension.

BACKGROUND: The novel proteasome inhibitor carfilzomib alone or in combination with other agents is already one of the standard therapies for relapsed and/or refractory multiple myeloma (MM) patients and produces impressive response rates in newly diagnosed MM as well. However, carfilzomib-related cardiovascular adverse events (CVAEs) - including hypertension (all grades: 12.2%; grade ≥3: 4.3%), heart failure (all grades: 4.1%; grade ≥3: 2.5%) and ischemic heart disease (all grades: 1.8%; grade ≥3: 0.8%) - may lead to treatment suspensions. At present, there are neither prospective studies nor expert consensus on the prevention, monitoring and treatment of CVAEs in myeloma patients treated with carfilzomib. METHODS: An expert panel of the European Myeloma Network in collaboration with the Italian Society of Arterial Hypertension and with the endorsement of the European Hematology Association aimed to provide recommendations to support health professionals in selecting the best management strategies for patients, considering the impact on outcome and the risk-benefit ratio of diagnostic and therapeutic tools, thereby achieving myeloma response with novel combination approaches whilst preventing CVAEs. RESULTS: Patients scheduled to receive carfilzomib need a careful cardiovascular evaluation before treatment and an accurate follow-up during treatment. CONCLUSIONS: A detailed clinical assessment before starting carfilzomib treatment is essential to identify patients at risk for CVAEs, and accurate monitoring of blood pressure and of early signs and symptoms suggestive of cardiac dysfunction remains pivotal to safely administer carfilzomib without treatment interruptions or dose reductions.

CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells.

OBJECTIVES: In this study, we compared a classic single-platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. BACKGROUND: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the 'gold standard' for such determinations. METHODS/MATERIALS: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen-thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen-thawed samples for viability by a bead-based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. RESULTS: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL(-1) , the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen-thawed samples, the analysis of viability was comparable for both techniques. CONCLUSIONS: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL(-1) or CD45+ cells µL(-1) is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.

Impact of patient: donor HLA disparity on reduced-intensity-conditioned allogeneic stem cell transplants from HLA mismatched unrelated donors for AML: from the ALWP of the EBMT.

Patients with acute myeloid leukaemia (AML) who lack a matched sibling or unrelated donor commonly undergo transplantation from a donor matched at 9/10 HLA-A, -B, -C, -DRB1, -DQB1 alleles, and it is unclear if a specific locus mismatch is preferable to any other. We therefore studied 937 patients with AML in complete remission transplanted using a reduced intensity conditioning regimen from an unrelated donor mismatched at a single allele. In a multivariate analysis, patient age, adverse karyotype and patient cytomegalovirus (CMV) seropositivity were correlated with decreased leukaemia free survival (LFS) and overall survival (OS). There was no significant difference in LFS or OS between patients transplanted from donors mismatched at HLA-A, -B, -C or -DRB1 in comparison to a HLA-DQB1 mismatched transplant. In a multivariate analysis, patients transplanted with a HLA-A mismatched donor had higher rates of acute graft-versus-host disease (GVHD) and non-relapse mortality (NRM) than patients transplanted with a HLA-DQB1 mismatched donor. Patient CMV seropositivity was associated with an increase in NRM and acute GVHD and reduced LFS and OS, regardless of donor CMV status. For CMV seropositive patients lacking a fully matched donor, alternative GVHD and CMV prophylaxis strategies should be considered.

Palifermin reduces incidence and severity of oral mucositis in allogeneic stem-cell transplant recipients.

In this multicenter study, 30 patients undergoing matched related or unrelated allogeneic stem-cell transplantation for leukemia were treated with palifermin, and retrospectively compared to a matched control group. Palifermin recipients transplanted with an unrelated donor showed a significant reduction of severity, incidence and duration of oral mucositis WHO grades 2-4. In addition, in the palifermin group the use of opioid analgesics and the duration of total parenteral nutrition decreased, whether stem cells were used from matched related or unrelated donors. No beneficial influence of palifermin on the incidence and severity of acute GVHD (aGVHD) was apparent. The incidence and duration of febrile neutropenia, documented infections, hematopoietic recovery or overall survival remained unchanged. The most common adverse effects included rash or erythema, generally mild and transient in appearance. Thus, the administration of palifermin was generally well tolerated and safe, and significantly reduced oral mucositis whereas--regardless of donor status--no effect on the incidence and severity of aGVHD was seen.

Triple antifungal therapy for severe systemic candidiasis allowed performance of allogeneic stem cell transplantation.

Systemic candidiasis is a rare but life threatening complication in immunosuppressed patients undergoing allogeneic SCT. Combination of new antifungal agents may improve outcome in this patient population. Here, triple anti-mycotic therapy is described in an relapsed ALL patient in urgent need of allogeneic bone marrow transplantation. The patient with T-cell acute lymphoblastic leukemia of thymic differentiation achieved remission after treatment according to the German ALL protocol 07/03. Two months after the consolidation therapy relapse occurred requiring high dose chemotherapy with allogeneic stem cell transplantation. One day after start of the conditioning regimen the patient showed skin manifestations typical for septic mycosis and blood cultures became positive for Candida krusei while on fluconazole prophylaxis. Because of the limited sensibility of fluconazole resistant candida species to liposomal amphotericin B and the high mortality rate in patients with systemic candidiasis, voriconazole was added immediately to liposomal amphotericin B. Since fever did not resolve and the conditioning therapy for allogeneic transplantation was not yet completed caspofungin was added. Skin manifestation responded to this triple anti-mycotic combination and peripheral blood stem cells from an unrelated donor were transplanted. With the triple antifungal therapy the patient finally became afebrile, skin manifestations showed complete resolution and blood cultures became negative. Three months after the onset of systemic candidiasis the patient was fully active with no signs of fungal infection and in haematological and molecular remission. Mycotic sepsis at the start of myeloablative conditioning therapy in heavily pretreated acute leukemia patients is usually considered as not allowing successful allogeneic transplantation. Thus this case demonstrates, that allogeneic stem cell transplantation is feasible in patients presenting with systemic candidiasis if combined antifungal therapy with liposomal amphotericin B, caspofungin and voriconazole is given.

Risk factors for treatment failure after allogeneic transplantation of patients with CLL: a report from the European Society for Blood and Marrow Transplantation.

For young patients with high-risk CLL, BTK-/PI3K-inhibitors or allogeneic stem cell transplantation (alloHCT) are considered. Patients with a low risk of non-relapse mortality (NRM) but a high risk of failure of targeted therapy may benefit most from alloHCT. We performed Cox regression analyses to identify risk factors for 2-year NRM and 5-year event-free survival (using EFS as a surrogate for long-term disease control) in a large, updated EBMT registry cohort (n= 694). For the whole cohort, 2-year NRM was 28% and 5-year EFS 37%. Higher age, lower performance status, unrelated donor type and unfavorable sex-mismatch had a significant adverse impact on 2-year NRM. Two-year NRM was calculated for good- and poor-risk reference patients. Predicted 2-year-NRM was 11 and 12% for male and female good-risk patients compared with 42 and 33% for male and female poor-risk patients. For 5-year EFS, age, performance status, prior autologous HCT, remission status and sex-mismatch had a significant impact, whereas del(17p) did not. The model-based prediction of 5-year EFS was 55% and 64%, respectively, for male and female good-risk patients. Good-risk transplant candidates with high-risk CLL and limited prognosis either on or after failure of targeted therapy should still be considered for alloHCT.

Long-term survival of patients with CLL after allogeneic transplantation: a report from the European Society for Blood and Marrow Transplantation.

Even with the availability of targeted drugs, allogeneic hematopoietic cell transplantation (allo-HCT) is the only therapy with curative potential for patients with CLL. Cure can be assessed by comparing long-term survival of patients to the matched general population. Using data from 2589 patients who received allo-HCT between 2000 and 2010, we used landmark analyses and methods from relative survival analysis to calculate excess mortality compared with an age-, sex- and calendar year-matched general population. Estimated event-free survival, overall survival and non-relapse mortality (NRM) 10 years after allo-HCT were 28% (95% confidence interval (CI), 25-31), 35% (95% CI, 32-38) and 40% (95% CI, 37-42), respectively. Patients who passed the 5-year landmark event-free survival (N=394) had a 79% probability (95% CI, 73-85) of surviving the subsequent 5 years without an event. Relapse and NRM contributed equally to treatment failure. Five-year mortality for 45- and 65-year-old reference patients who were event-free at the 5-year landmark was 8% and 47% compared with 3% and 14% in the matched general population, respectively. The prospect of long-term disease-free survival remains an argument to consider allo-HCT for young patients with high-risk CLL, and programs to understand and prevent late causes of failure for long-term survivors are warranted, especially for older patients.

Proliferation-based T-cell selection for immunotherapy and graft-versus-host-disease prophylaxis in the context of bone marrow transplantation.

Graft-versus-host disease (GvHD) caused by alloreactive T cells within the graft is a major drawback of allogeneic BMT, but depletion of T cells leads to higher rates of relapse, opportunistic infections and graft failure. Therefore, selective removal of GvHD-inducing alloreactive T cells might be beneficial. We describe here the separation of alloresponsive T cells, based on carboxyfluorescein succimidyl ester labeling, in vitro allostimulation and FACS-sorting. In vivo effects of the separated cell populations were investigated in the context of allogeneic BMT in murine models: in vitro resting T cells were shown to survive in the allogeneic host and retain immunoreactivity against 'third-party' antigens. As demonstrated in two different transplantation models, elimination of proliferating cells significantly reduces GvHD but offers no advantages to using T-cell-depleted bone marrow alone concerning engraftment and tumor control. Transplanting T cells that proliferate in response to tumor antigens in vitro may narrow down the spectrum of antigens recognized by T cells and therefore reduce GvHD while maintaining graft-facilitating function and tumor control. Therefore, selecting tumor-reactive T cells on the basis of their proliferative response in vitro may be beneficial for the recipient, less time consuming than T-cell cloning and still reduce the extent of GvHD.

Successful autologous stem cell transplantation in a severely immunocompromised patient with relapsed AIDS-related B-cell lymphoma.

There is now evidence that the tolerability and response to systemic chemotherapy in HIV-infected patients with AIDS-related lymphoma (ARL) is significantly improved by highly active antiretroviral therapy. Here we report an severely immunocompromised AIDS patient with recurrent ARL who was successfully treated with autologous stem cell transplantation (ASCT). We also review the current literature of ASCT in HIV-infected patients.

67Ga citrate versus 99mTc-labeled LL2-Fab' (anti-CD22) fragments in the staging of B-cell non-Hodgkin's lymphoma.

The value of 67Ga citrate scanning as a transferrin receptor agent was compared in this study with a 99mTc-labeled anti-CD22 (B-cell) Fab' fragment (LL2) in patients with low- and high-grade B-cell non-Hodgkin's lymphoma (NHL). Thirteen patients with histologically confirmed NHL were examined prospectively with both radiopharmaceuticals within 8 days. The results of immunoscintigraphy were compared with those of 67Ga scanning and the clinical and radiological workup (computed tomography, ultrasound, and magnetic resonance imaging) of the patients. The overall sensitivity of 67Ga citrate and 99mTc-labeled LL2 fragment was each 80% in a total of 43 lesions. Low-grade lymphoma patients had a higher sensitivity in LL2 imaging (82% versus 71%), and high-grade lymphoma patients in 67Ga citrate scanning (100% versus 75%). The target:background ratio in low-grade NHL for LL2 was 1.43 +/- 0.3:1 versus 1.8 +/- 0.5:1 in 67Ga scans; in high-grade NHL, 1.49 +/- 0.35:1 versus 2.2 +/- 0.8:1, respectively. Single-photon emission computed tomography imaging was necessary in 21.7% of the patients 4 h after injection to localize the lesions. In conclusion, the overall sensitivity of 99mTc-labeled LL2 is comparable to 67Ga citrate scanning in patients with B-cell NHL. 99mTc-labeled LL2 antibodies are rapid to use, are safe, and need a shorter imaging time (24 h versus 72 h). Because of these advantages, 99mTc-labeled LL2 may be superior to 67Ga scanning for the staging of lymphoma patients.

Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer.

Immunotherapies directed to the proto-oncogene product HER-2/neu, which is overexpressed on a subset of breast and other carcinomas, currently receive considerable attention. We have investigated cell-mediated effector mechanisms of HER-2/neu antibodies against breast cancer cell lines. Compared to unfractionated control blood, whole blood from patients during granulocyte colony-stimulating factor (G-CSF) treatment exhibits significantly enhanced lysis (P < 0.001) of SK-BR-3 cells in the presence of HER-2/neu antibody 520C9. The extent of tumor cell killing correlated positively (r = 0.74) to polymorphonuclear neutrophil (PMN) blood counts. Fractionation of whole blood into plasma, mononuclear cells, and PMNs showed major killing capacity to reside in the granulocyte fraction. PMNs were efficiently cytolytic with a panel of HER-2/neu antibodies and against various breast cancer cell lines. Experiments with blocking antibodies to Fc(gamma)R documented Fc(gamma)RII (CD32) as the major trigger molecule for monoclonal antibody 502C9-mediated cytotoxicity. Killing via 520C9 was significantly influenced by an allotypic polymorphism of Fc(gamma)RIIa, the CD32 molecule expressed on PMNs. In reverse antibody-dependent cell-mediated cytotoxicity experiments with a panel of HER-2/neu-directed bispecific antibodies, Fc(gamma)RIII (CD16) proved to be an efficient trigger molecule in blood from healthy volunteers. During G-CSF treatment, however, Fc(gamma)RI (CD64)-expressed on monocytes and G-CSF primed, but not on healthy donor PMNs-became the predominant cytotoxic trigger molecule. Thus, G-CSF application increased effector cell numbers for HER-2/neu-directed immunotherapy, and G-CSF primed PMNs proved particularly effective with a [HER-2/neu x Fc(gamma)RI] bispecific antibody. These findings support clinical trials with HER-2/neu-directed antibodies in combination with G-CSF in breast cancer patients overexpressing HER-2/neu.

Evaluating antibodies for their capacity to induce cell-mediated lysis of malignant B cells.

Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on the antibody isotype. We then compared killing mediated by chimeric IgG1 antibodies with that from Fc gammaRI-directed bispecific antibodies, targeting classical HLA class II, or the Lym-1 and Lym-2 antigens. The latter two are variant forms of HLA class II, which are highly expressed on the surface of malignant B cells but which are found only at low levels in normal cells. With blood from G-CSF-treated donors, bispecific antibodies showed enhanced killing compared to their chimeric IgG1 derivatives, because they were more effective in recruiting Fc gammaRI-expressing PMN cells. G-CSF- and Fc gammaRI-directed bispecific antibodies to HLA class II, therefore, seem to be an attractive combination for lymphoma therapy.

Therapy with OKT3 monoclonal antibody in refractory T cell acute lymphoblastic leukemia induces interleukin-2 responsiveness.

Administration of cytokines to patients with leukemia or lymphoma may recruit dormant malignant cells into cell cycle and thus make them more susceptible to chemotherapy. We treated a patient with refractory T cell acute lymphoblastic leukemia (ALL) with OKT3 monoclonal antibody and observed a dramatic but transient decrease of lymphoblasts. The T ALL cells were rather mature by morphology and immunophenotyping, expressing CD7, CD4, CD8 and CD3 surface antigens and nuclear TdT. Cytogenetic analysis revealed inversion of chromosome 14(q11q32.1). A total of 500 mg OKT3 (maximum dose 50 mg/day) was given. A decrease of lymphoblasts in the blood and a reduction of spleen size was observed. Complement levels dropped remarkably. Despite increasing serum levels of tumor necrosis factor, treatment was well tolerated overall. CD3 therapy induced strong IL-2 responsiveness of the lymphoblasts. Thus, OKT3 antibody treatment not only significantly decreased CD3-positive tumor cells, but also induced IL-2-mediated proliferation. This may also allow sequential application of CD3 and IL-2 to render certain T cell tumors more susceptible to chemotherapy.

Low- versus high-dose radioimmunotherapy with humanized anti-CD22 or chimeric anti-CD20 antibodies in a broad spectrum of B cell-associated malignancies.

Both CD22 and CD20 have been used successfully as target molecules for radioimmunotherapy (RAIT) of low-grade B cell non-Hodgkin's lymphoma. Because both CD20 and CD22 are highly expressed relatively early in the course of B cell maturation, and because its expression is maintained up to relatively mature stages, we studied the potential of the humanized anti-CD22 antibody, hLL2, as well as of the chimeric anti-CD20 (chCD20) antibody, rituximab (IDEC-C2B8), for low- or high-dose (myeloablative) RAIT of a broad range of B cell-associated hematological malignancies. A total of 10 patients with chemorefractory malignant neoplasms of B cell origin were studied with diagnostic (n = 5) and/or potentially therapeutic doses (n = 9) of hLL2 (n = 4; 0.5 mg/kg, 8-315 mCi of 131I) or chCD20 (n = 5; 2.5 mg/kg, 15-495 mCi of 131I). The diagnostic doses were given to establish the patients' eligibility for RAIT and to estimate the individual radiation dosimetry. One patient suffered of Waldenström's macroglobulinemia, eight patients had low- (n = 4), intermediate- (n = 2) or high- (n = 2) grade non-Hodgkin's lymphoma, and one patient had a chemorefractory acute lymphatic leukemia, after having failed five heterologous bone marrow or stem cell transplantations. Three of these 10 patients were scheduled for treatment with conventional (30-63 mCi, cumulated doses of up to 90 mCi of 131I) and 7 with potentially myeloablative (225-495 mCi of 131I) activities of 131I-labeled hLL2 or chCD20 (0.5 and 2.5 mg/kg, respectively); homologous (n = 6) or heterologous (n = 1) stem cell support was provided in these cases. Good tumor targeting was observed in all diagnostic as well as posttherapeutic scans of all patients. In myeloablative therapies, the therapeutic activities were calculated based on the diagnostic radiation dosimetry, aiming at lung and kidney doses < or = 20 Gy. Stem cells were reinfused when the whole-body activity retention fell below 20 mCi. In eight assessable patients, five had complete remissions, two experienced partial remissions (corresponding to an overall response rate of 87%), and one (low-dose) patient had progressive disease despite therapy. In the five assessable, actually stem-cell grafted patients, the complete response rate was 100%. Both CD20 and CD22 seem to be suitable target molecules for high-dose RAIT in a broad spectrum of hematological malignancies of B cell origin with a broad range of maturation stages (from acute lymphatic leukemia to Waldenström's macroglobulinemia). The better therapeutic outcome of patients undergoing high-dose, myeloablative RAIT favors this treatment concept over conventional, low-dose regimens.

Antibodies TC-12 ("unique") and TH-111 (CD96) characterize T-cell acute lymphoblastic leukemia and a subgroup of acute myeloid leukemia.

To extend the panel of monoclonal antibodies useful for immunophenotyping of acute leukemias, two new reagents, TC-12 and TH-111, were developed. TC-12 was found "unique," and TH-111 was assigned to the recently defined CD96 cluster. Both reagents show little reactivity with blood and bone marrow nucleated cells but define a major (TH-111: 78.3%) or an important (TC-12: 45.6%) subset of T-cell acute lymphoblastic leukemia (ALL). In addition, in acute myeloid leukemia (AML), the expression of TC-12 was found in 64 (20.2%) of 317 and TH-111 in 97 (29.1%) of 333 of these patients. TC-12 positivity in AML was virtually restricted to the Fab subtypes M0, M1, M2, and M6. In the group of immature AML characterized by the coexpression of CD7 as well as CD117 and CD34 positivity, leukemic blasts frequently disclosed the TC-12 and TH-111 antigen. Although the TC-12 antigen could not be determined, TH-111 immunoprecipitated the TACTILE (CD96) antigen and, when expressed, was found to be associated with the transferrin receptor. These reagents may help not only to define and dissect T-cell ALL, but also to characterize a subgroup of immature AML at the divergence of T-cell and myeloid lineage.

Cryopreserved human cultured T cells as responder cells for the quantitative measurement of interleukin-2: improvement of the assay.

Large batches of human cultured T cells (CTC) were cryopreserved for later use as responder cells in a proliferation assay for measurement of interleukin (IL)-2 activity. Cryopreservation of CTC could be carried out without considerable loss in viability and cryopreserved and fresh cells showed equally good responses to IL-2. The conditions of IL-2-dependent CTC growth were analyzed, which led to a better evaluation of test results, and had important implications for the calculation of relative IL-2 activity. The repeated use of the same batch of cryopreserved CTC reduced test variability and provided an assay system that allows reliable and reproducible measurement of human IL-2 activity.

Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies.

Studies with gene-modified mice have recently reinforced the importance of Fc receptor-mediated effector mechanisms for the therapeutic efficacy of rituxan and herceptin - two clinically approved antibodies for the treatment of tumor patients. We investigated Fc receptor-dependent tumor cell killing by mononuclear and granulocytic effector cells - comparing human IgG1 antibodies against CD20 or HER-2/neu with their respective FcgammaRI (CD64)-, FcgammaRIII (CD16)-, or FcalphaRI (CD89)-directed bispecific derivatives. With blood from healthy donors as effector source, human IgG1 and FcgammaRIII (CD16)-directed bispecific antibodies proved most effective in recruiting mononuclear effector cells, whereas tumor cell killing by granulocytes was most potently triggered by FcalphaRI-directed bispecific constructs. Granulocyte-mediated tumor cell lysis was significantly enhanced when blood from G-CSF- or GM-CSF-treated patients was investigated. Interestingly, however, both myeloid growth factors improved effector cell recruitment by different mechanisms, which were furthermore dependent on the tumor target antigen, and on the selected cytotoxic Fc receptor.

Detection of soft-tissue infections and osteomyelitis using a technetium-99m-labeled anti-granulocyte monoclonal antibody fragment.

UNLABELLED: Imaging of osteomyelitis and soft-tissue infections can be problematic with currently available agents; bone scans are often false-positive. Indium-111-oxine and 99mTc-HMPAO white blood cell (WBC) scans require ex vivo handling of blood with potential exposure to infectious agents, and 99mTc-antigranulocyte (IgG1) antibodies need 24 hr for final diagnosis. METHODS: We investigated the use of 99mTc-murine anti-granulocyte monoclonal Fab' fragment in 20 patients with suspected osteomyelitis of soft-tissue infections. All patients also had 99mTc bone scans and 111In-oxine or 99mTc-HMPAO white blood cell scans. The final diagnosis was confirmed by culture, biopsy, surgery, follow-up, x-rays, CT or MRI. In vitro studies performed on granulocytes demonstrated no effect on their function when the anti-granulocyte monoclonal antibody fragment was added. RESULTS: Sensitivity, specificity and diagnostic accuracy to detect infection was 88%, 75% and 80%, respectively. All lesions could be detected as early as 1 hr after injection of the antibody fragment. In comparison, WBC scanning had values of 86%, 78% and 81%, respectively. Some lesions could only be detected 24 hr following the injection of labeled WBCs. LeukoScan had three false-positives and WBC scanning had two false-positives. CONCLUSIONS: Immunoscintigraphy with 99mTc-NCA-90 Fab' fragments offers rapid localization of foci, rapid and simple use, a negligible HAMA response rate, no effect on granulocyte function and an accuracy comparable to WBC scanning.

Heterogeneity of T-acute lymphoblastic leukemia (T-ALL) cell lines: suggestion for classification by immunophenotype and T-cell receptor studies.

Hematopoietic cell lines are often used as representatives for a certain cell differentiation lineage and stage, particularly in immunological and hematological studies. Acute lymphoblastic leukemia (ALL) of T-cell type is a rather heterogeneous group of ALL at least by immunophenotyping. Our aim was to present a comprehensive characterization of frequently used T-cell leukemia cell lines and to suggest a correlation with the normal differentiation pattern. A total of 16 T-ALL cell lines were analyzed for their immunophenotype and for T-cell receptor (TCR) rearrangement and expression. The panel of 20 cell surface markers included two new monoclonal antibodies (MoAb), TC-12 and TH-111, which were raised in our laboratory and detect subpopulations of T-cell ALL. TC-12 was typed 'unique', TH-111 was assigned to the CD96 cluster at the Vth Conference on human leucocyte differentiation antigens (HLDA). We categorized the 16 cell lines into the four groups pro-T, pre-T, cortical T and mature T differentiation stage according to the recent proposal of the European Group for the Immunological Characterization of Leukemias (EGIL). Interestingly, none of the T-cell lines were found to be alike. In conclusion, it appears necessary to consider the particular differentiation stage of each individual cell line when using T-cell leukemia lines as models for malignant or normal T cells.