Impact of preleukapheresis cell counts on collection results and correlation of progenitor-cell dose with engraftment after high-dose chemotherapy in patients with germ cell cancer.

PURPOSE: To identify predictive factors for a good leukapheresis yield and to determine peripheral-blood progenitor cell (PBPC) dose requirements for rapid hematopoietic engraftment. PATIENTS AND METHODS: Seventy-one patients with germ cell cancer (GCC) underwent PBPC harvest for autologous transplantation following high-dose therapy. Aphereses were performed after chemotherapy during granulocyte colony-stimulating factor (G-CSF) administration. RESULTS: A median of two aphereses (range, two to five) resulted in 4.6 x 10(8) mononuclear cells (MNC)/kg, 15.7 x 10(4) colony-stimulating units granulocyte-macrophage (CFU-GM)/kg, and 6.0 x 10(6) CD34+ cells/kg. Peripheral blood MNC count correlated significantly with number of harvested CD34+ cells per kilogram (r = .49; P < .0001) and with CFU-GM count per kilogram (r = .35; P < .002). Circulating CD34+ cells from peripheral blood gave the best correlations to collected CD34+ cells per kilogram (r = .92; P < .0001), as well as to harvested CFU-GM per kilogram (r = .48; P < .0001). A preleukapheresis number of CD34+ cells greater than 4 x 10(4)/mL was highly predictive for a PBPC collection yield that contained more than 2.5 x 10(6) CD34+ cells/kg harvested by a single leukapheresis. After autologous transplantation, 41 patients were assessable for hematopoietic engraftment. They engrafted in a median time of 9 days (range, 7 to 18) to a WBC count greater than 1.0 x 10(9)/L, 10 days (range, 7 to 18) to an absolute neutrophil count (ANC) greater than 0.5 x 10(9)/L, and 11 days (range, 7 to 62) to a platelet (PLT) count greater than 20 x 10(9)/L. Good correlations were seen between reinfused CD34+ cell count and recovery of WBC count, ANC, and PLT count, with r values of .65 (P < .001), .65 (P < .001), and .45 (P < .03), respectively. Patients reinfused with a PBPC dose greater than 2.5 x 10(6) CD34+ cells/kg recovered hematopoiesis in a significantly shorter time than patients who received less than 2.5 x 10(6) CD34+ cells/kg. CONCLUSION: Rapid hematopoietic engraftment can be achieved by a PBPC dose of greater than 2.5 x 10(6) CD34+ cells/kg. When circulating preleukapheresis CD34+ cell counts are greater than 4 x 10(4)/mL, a PBPC autograft that contains more than 2.5 x 10(6) CD34+ cells/kg can be collected by a single leukapheresis.

Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow: a randomized comparison.

PURPOSE: To compare autologous bone marrow (BM) with peripheral-blood progenitor cells (PBPC) as hematopoietic rescue after high-dose chemotherapy (HDCT). PATIENTS AND METHODS: From January 1991 until April 1993, 47 consecutive patients with relapsed or refractory germ cell tumors were randomized to either BM harvest or collection of PBPC mobilized by chemotherapy plus granulocyte colony-stimulating factor (G-CSF). After additional conventional-dose salvage treatment, all patients received HDCT with carboplatin 1,500 mg/m2, etoposide 2,400 mg/m2, and ifosfamide 10 g/m2 with either BM or PBPC rescue. RESULTS: Forty-six patients were assessable for hematologic reconstitution, and one patient died on day +4 before engraftment. Rescue using PBPC resulted in a significantly shorter recovery time to neutrophil counts more than 500/microL (10.0 v 11.0 days, P < .01), neutrophil counts more than 1,000/microL (10.0 v 12.0 days, P = .001), and platelet counts more than 20,000/microL (10.0 v 17.0 days, P < .01), as well as in fewer days to transfusion independence from RBCs (8.0 v 12.0, P < .05) and platelets (9.0 v 12.0, P < .01) and fewer days of intravenous (IV) antibiotics (9.0 v 11.0, P < .05). However, no statistical differences in transfusion requirements or in other clinical outcome variables were observed. Overall survival and event-free survival also were not different in the two study arms. CONCLUSION: We conclude that the use of PBPC mobilized by chemotherapy plus G-CSF results in sustained trilineage reconstitution after HDCT, which occurs more rapidly as compared with BM. The earlier hematologic reconstitution in patients with PBPC rescue significantly reduces the time to transfusion independence.

In situ immunocytochemical staining method for haemopoietic cells grown in vitro.

Characterization and enumeration of haemopoietic cells grown in vitro is routinely performed either on unstained cultures or on cultures stained by cytochemical agents. This report describes a novel method for the immunocytochemical analysis of cells grown in plasma clots. The stain is performed in situ by subjecting the whole plasma clot in the culture dish to the staining procedure. The growth of early haemopoietic progenitor cells was monitored in cultures from normal human peripheral blood and proliferating progenitor cells were identified with an anti-human transferrin receptor monoclonal antibody followed by a two-layer immunoperoxidase stain. The number of transferrin receptor positive clusters demonstrable after 5 days of culture was similar to the number of haemopoietic colonies detectable after 12 days of culture.

An immunocytochemical method for the detection of fluorochrome-labelled DNA probes hybridized in situ with cellular RNA.

Various detection systems for in situ hybridization of nucleic acids are currently used. We report here an immunocytochemical detection system which is based on the detection of FITC-labelled DNA/mRNA hybrids and takes advantage of FITC molecules attached covalently to the DNA probes prior to hybridization. In situ hybridization on cytocentrifuge spots is followed by the application of an anti-fluorescein antibody thus permitting detection of mRNA/DNA-FITC hybrids. The anti-FITC antibody reaction is demonstrated by an indirect immunocytochemical peroxidase-staining method. The T lymphoblast cell line Jurkat and the cDNA for the TCR-beta chain were chosen to establish the technique.

Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen. Identification of proliferating cells by monoclonal antibodies.

Using a two-colour immunofluorescence technique, we have investigated the mitogenic effects of phytohaemagglutinin-M (PHA-M) and of pokeweed nitrogen (PWM) on human lymphocyte subsets. These were identified by CD1, CD3, CD4, CD8, and CD16 monoclonal antibodies, and proliferation was demonstrated by a polyclonal anti-transferrin antibody. Evidence has been obtained for the generation of a population expressing both the CD4 and CD8 antigens simultaneously, in short-term cultures of peripheral blood mononuclear cells in the presence of PWM and of PHA-M.

Binding of mitogenic plant lectins to human lymphocytes. Flow cytometric analysis.

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.

Influenza vaccination in liver transplant recipients.

BACKGROUND: The immunogenicity of the trivalent inactivated influenza split virus vaccine (Infusplit SSW 97/98) containing A/Bayern/07/95 (H1N1)-like (A/Johannesburg/82/96 [NIB-39]), A/Wuhan/359/95 (H3N2)-like (A/Nanchang/933/95 [Resvir-0]), and B/Beijing/184/93-like (B/Harbin/7/94) hemagglutinin antigens was tested in liver transplant recipients (TXL-R). SUBJECTS AND METHODS: Serum antibody titers were determined 21+/-2 days after a single vaccination in 62 adult TXL-R and 59 adult volunteers. RESULTS: Protective postimmunization antibody titers for the three antigens were similar in TXL-R (protection rates 92%, 92%, and 95%) and the comparison group (97%, 100%, and 100%). Adverse reactions were mild and less frequent in TXL-R. A significant decrease of CD8+CD38+ lymphocytes after vaccination was found in TXL-R. No association between antibody response and age, gender, time interval since transplantation, anti-hepatitis B surface antigen immunoprophylaxis, or immunosuppressive medication was detected. CONCLUSION: Our results show that the vaccine is safe and effective and should be recommended to TXL-R.

Overexpression of the Raf-1 proto-oncogene in human myeloid leukemia.

The Raf-1 protein, a cytoplasmic serine/threonine kinase, plays an important role in signal transduction pathways. In order to examine the role of Raf-1 in human myeloid leukemia, we determined raf-1 mRNA expression by Northern blot analysis in blast cell samples from 27 acute myeloid leukemia (AML) cases and peripheral blood mononuclear cells from six healthy donors. A normal raf-1 transcript size was detected in all cases investigated. However, overexpression of raf-1 mRNA was found in 2 of 27 AML cases, both of which were erythroleukemias (AML, FAB M6).

A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts.

Recently, the regulatory authorities have begun to show interest in haematopoietic stem cell products. On a professional rather than a regulatory basis, the International Society for Hematotherapy and Graft Engineering (ISHAGE) has established the Foundation for the Accreditation of Haematopoietic Cell Therapy (FACHT), which has drawn up guidelines for standards and accreditation of such activity. In Europe, the regulatory environment with regard to haematopoietic stem cell grafts, processing and storage are currently less stringent. However, in 1998 the European Joint Accreditation Committee Euro-ISHAGE/EBMT (JACIE) prepared a regulatory document 'Standards for Blood and Marrow Progenitor Cell Collection, Processing and Transplantation' which was approved by the EBMT General Assembly. The major objectives were to promote quality of medical and laboratory practice in haematopoietic progenitor cell transplantation. The standards extend and detail the pre-existing activity of EBMT centres including all phases of collection, processing and administration of these cells. This is the platform for the proposed reference protocol for CD34(+) cell enumeration and clinical validation of quality assessment to ensure that appropriate standards of work and product quality are established and will be maintained.

Mobilization of peripheral blood progenitor cells by disease-specific chemotherapy in patients with soft tissue sarcoma.

We investigated peripheral blood progenitor cell (PBPC) mobilization by disease-specific chemotherapy in patients with metastatic soft tissue sarcoma (STS). Nine patients, five females and four males, aged 12-51 years, pretreated by one to nine courses of cytotoxic chemotherapy, underwent STS-specific mobilization followed by G-CSF at 5 microg/kg/day. PBPC were collected by 19 conventional-volume aphereses (8-12 l) with one to four procedures in individual patients. Leukaphereses started on median day 15 (range 13-18) from the first day of mobilization chemotherapy at medians of 25.8 x 10(3) WBC/microl (6.8-46.9), 3.5 x 10(3) MNC/microl (1.1-8.8), 122 x 10(3) platelets/microl (72-293) and 30.7 CD34+ cells/microl (6.7-207.8). Cumulative harvests resulted in medians of 4.6 x 10(8) MNC/kg (3.0-6.4), 2.9 x 10(6) CD34+ cells/kg (1.1-11.1) and 12.0 x 10(4) CFU-GM/kg (2.0-37.8). Eight patients underwent high-dose chemotherapy (HDCT) followed by PBPC rescue. Seven patients recovered hematopoiesis at medians of 12 days (8-15) for ANC >0.5 x 10(3)/microl and 14 days (8-27) for platelets >20 x 10(3)/microl. One patient, who received 1.6 x 10(6) CD34+ cells/kg, exhibited delayed ANC recovery on day +37 and failed to recover platelets until hospital discharge on day +55. We conclude that in patients with metastatic STS, who are pretreated by standard chemotherapy, PBPC can be mobilized by a further course of STS-specific chemotherapy plus G-CSF. One to four conventional-volume aphereses result in PBPC autografts that can serve as hematopoietic rescue for patients scheduled for HDCT.

Fractionated total body irradiation and high-dose VP-16 with purged autologous bone marrow rescue for children with high risk relapsed acute lymphoblastic leukemia.

Twenty-two children with ALL in high risk second (n = 13), third or subsequent complete remission (n = 9) were treated with high-dose VP-16 60 mg/kg and fractionated total body irradiation (fTBI) 12 Gy, 2 x 2 Gy daily followed by autologous BM rescue. Prior to transplantation all patients had been treated according to intensive German BFM front-line or BFM relapse protocols. In all cases the marrow was purged using monoclonal antibodies attached to magnetic microspheres. All patients engrafted. There was no severe toxicity related to the pre-transplant high-dose chemoradiotherapy. Two patients died in the early course of transplantation from infections (Legionella and Aspergillus). Sixteen patients relapsed within 259 days (median 109 days); 13 died from leukemia. Four patients are alive in CR at a median of 1328 days with a Karnofsky score of 100%. The Kaplan-Meier estimation shows a probability of event-free survival (EFS) of 18% and a probability of relapse of 80%. Considering the otherwise poor prognosis of these children the results are acceptable although the high relapse rate is still disappointing. We conclude that high-dose VP-16 and fTBI combined with ABMT is a curative treatment for some children and should therefore be considered for those who lack an HLA-identical sibling donor. In future better therapy concepts are needed either in pre-transplant conditioning regimens or in post-transplant treatment schedules.

Imprecision of counting CFU-GM colonies and CD34-expressing cells.

Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unit-granulocyte/macrophage) and of CD34-expression haemopoietic cells as assessed by multiparameter flow cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively assesses the short- and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold doses have been established. Above these thresholds rapid engraftment is almost invariable but below these thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data suggest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose.

HIV-Related Lymphoblastic Lymphoma.

The clinical records of 17 patients with HIV-associated lymphoblastic mostly Burkitt-type lymphomas, are reviewed (54% of a total of 31 patients with HIV-associated malignant lymphomas, treated between 1/85-1/90). The lymphomas were diagnosed histologically with additional immuno-histochemical analyses, or cytologically, with subsequent immunocyto-chemistry. All patients were homosexual and HIV antibody positive, and the average age was 39 y. At initial staging evaluation an Ann Arbor stage III or IV was present in 15 patients (88%); a CDC-stage II of HIV-infection was present in 10 patients, CDC-stage III in 5 patients, and CDC-stage IV in 2 patients. An extranodal or mixed nodal/extranodal pattern of organ involvement was seen in 14 cases, with predominance of the gastrointestinal tract (30%) and the bone marrow (30%). The response rate to chemotherapy (CR + PR) was 81%, a CR was achieved in 53% of the patients, and relapses within a few months after CR were common. Survival following relapses in the CR- and PR group was similar, namely 5.2 and 4.9 months respectively. 2 patients in the CR-group and 1 patient in the PR group have been alive for 13, 19 and 30 months. An optimal therapeutic regimen for this disorder does not seem to have been found yet.

Recent progress on the role of Axl, a receptor tyrosine kinase, in malignant transformation of myeloid leukemias.

Receptor tyrosine kinases (RTK) play an important role in the signal transduction of normal and malignant cells. There are different families of RTKs which are mainly characterized by differences in the ligang-binding extracellular domains. Axl (or UFO/Ark) is the first member of a new class of RTK with two fibronectin type III domains and two immunoglobulin-like domains present at the extracellular domain. The axl-gene has been isolated by means of gene transfection studies using DNA of patients with chronic myelogeneous leukemia. For a previous and the present study, we used a sensitive reverse-transcriptase polymerase chain reaction assay to detect axl's mRNA in cells from normal and malignant hematopoietic tissue. Axl's mRNA expression was mainly detected in myelo-monocytic cells, whereas much weaker transcription was seen in lymphatic cells and in lymphatic leukemias. In normal bone marrow, axl was heavily transcribed in marrow stromal cells. Further, we analysed Axl protein expression using monoclonal antibody M50 in peripheral stem cell harvests; in most harvests, no co-expression of CD34 and Axl was detected. However, in one patient with AML in complete remission, Axl was co-expressed on 80% of the CD34-positive population. These data show that axl is preferentially expressed in monocytes and stromal cells. Furthermore, a fraction of CD34-positive progenitor cells may express Axl. The exact mechanism for transformation of myeloid progenitor cells through Axl, however, remains to be determined.

Immune parameters in patients with anxiety or depression during psychotherapy.

BACKGROUND: Numerous studies have described distinctive immunological findings in patients with depression. In contrast, only very little is known about the possible influence of anxiety disorders on the immune system. It is also unknown whether treatment with psychotherapy alone has any influence on immunological variations in patients with psychiatric disorders. METHODS: We measured immunological and psychological parameters in patients with minor depression (N=10) or anxiety disorder (N=13) over an 8-week course of inpatient psychotherapy. Data for patients and a group of healthy controls (N=11) were recorded three times in 4-week intervals. A FACS analysis revealed the composition of lymphocyte subpopulations. The production of reactive oxygen species (ROS) by phagocytes was analyzed using lucigenin-enhanced chemiluminescence. RESULTS: On admission, patients with anxiety disorder showed a markedly elevated ratio of CD4(+) (T helper) versus CD8(+) (T suppressor/cytotoxic) lymphocytes compared to healthy controls (P<0.001) and minor depressives (P<0.01). The increased ratio in patients with anxiety disorder could mainly be attributed to a reduced count in CD8(+) T cells compared to healthy controls (P<0.01) and depressives (P<0.05). There were no differences between patients with depression and healthy controls with respect to the CD4(+)/CD8(+) ratio. We did not observe any differences in the production of ROS by phagocytes in patients compared to healthy controls. The CD4(+)/CD8(+) ratio remained elevated in patients with anxiety disorders during the following 8 weeks. There were no significant changes in this parameter over the course of the inpatient treatment. LIMITATIONS: As a pilot study on the immune status in patients with anxiety disorders, the study's main limitation is the relatively low number of patients observed. CONCLUSIONS: In this study we demonstrated for the first time marked immunological changes in patients with anxiety disorders. In addition, our results provide preliminary evidence that these immunological variations are not reversible by an 8-week course of inpatient psychotherapy alone.

[Lymphocytic infiltrations of the orbit in MRT and CT. Lymphoma, pseudolymphoma and inflammatory pseudotumor]. / Lymphozytäre Infiltrationen der Orbita in MRT und CT. Lymphom, Pseudolymphom und entzündlicher Pseudotumor.

MR and CT examinations of 16 patients with lymphoma, pseudolymphoma and inflammatory pseudotumours were analysed to describe morphologic features of lymphocytic infiltration. Density and signal did not allow for differentiation, but localisation was the most important criterion: lymphoma and pseudolymphoma were located in the anterior superior orbit, inflammatory pseudotumours being retrobulbar lesions. Differentiation is of clinical importance, since both lymphoma and pseudolymphoma are accompanied by generalised malignant lymphoma while inflammatory pseudotumours are localised. Imaging of topographic relations of lens, optic nerve and lesion on sagittal MR images was found helpful for radiation therapy planning.