Meinolf Suttorp

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: safety, kinetics of mobilization, and composition of the graft.

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) markes the general anaesthesia of the donor unnecessary and may result in more rapid engraftment and faster recovery of the immune system. We have studied G‐CSF‐mediated PBPC mobilization in healthy donors and analysed the cellular composition of the resulting PBPC grafts. PBPC grafts were obtained from nine healthy donors (18‐67 years old) for allogeneic or syngeneic transplantation. Six donors received 10 μg/kg G‐CSF per day, the others 5‐6 μg/kg. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in all donors primed with 10 μg/kg. With 10 μg/kg, a 31‐fold (9‐62) enrichment of circulating CD34+ cells was observed with peak values constantly occurring on day 5 after the start of G‐CSF administration. Starting harvest on day 5, one to three collections on consecutive days yielded 5.5 x 106/kg (0.9‐10.7) CD34+ cells, 219 x 106/kg (106–314) T cells, and 34 x 106/kg (23–67) NK cells per 10 litres leukapheresis volume. Altogether, PBPC grafts contained 3 times more CD34+ cells, 7 times more T cells, and 20 times more NK cells than five allogeneic marrow grafts that were analysed for comparison. The yield of CD34+ cells per 10 litres apheresis volume as well as the height of the CD34+ peak in peripheral blood were inversely correlated to the age of the donor. In the donors primed with 5–6μg/kg G‐CSF the increase of circulating CD34+ cells (4–7‐fold enrichment) and the CD34+ cell yield per 10 litres leukapheresis volume (1 x 106/kg [0.8‐2‐2]) was much smaller compared with the 10μg/kg group. In conclusion, sufficient amounts of PBPC capable of restoring haemopoiesis in allogeneic recipients can be mobilized safely by administration of G‐CSF (10 μg/kg s.c. for 5 d) in healthy donors, and harvested with one or two leukapheresis procedures. Whether the large numbers of T‐cells and NK cells that are contained in the collection products may influence graft‐versus‐host and graft‐versus‐leukaemia reactivities of PBPC grafts remains to be determined.

Autografting of highly purified peripheral blood progenitor cells following myeloablative therapy in patients with lymphoma: a prospective study of the long-term effects on tumor eradication, reconstitution of hematopoiesis and immune recovery.

In a prospective study, we have investigated CD34+ selection of peripheral blood progenitor cells (PBPC) for autotransplantation in patients with lymphoma. Twenty-six consecutive patients (10 follicular lymphomas, seven mantle cell lymphomas, seven B-CLL, two immunocytomas) were mobilized using chemotherapy plus G-CSF. Sufficient numbers of PBPC could be collected from 24 patients and were immunoselected with the semiautomated Isolex 300 (n = 17) or the fully integrated Isolex 300i (n = 7) devices. The selection products were assayed by PCR amplification of clonal CDRIII or t(14;18) rearrangements for residual tumor cell content. Residual disease and long-term hematopoietic and immune recovery were studied by assessing the following parameters at 3, 6, and 12 months post-transplant: CDRIII or t(14;18) PCR, platelet count, lymphocyte subsets, serum IgG, serum IgA, and measles titer. With the Isolex 300 device 26% (10–65) of input CD34+ cells were recovered with a median purity of 89.2% (49.4–98.9) after CD34+ selection. The Isolex 300i device allowed significantly better recoveries (46% (22–86)) and purities of CD34+ cells (98.8% (92.2–99.2)). The overall purging efficacy was 3.2 (0.6–5.1) log. Twenty patients have been reinfused with CD34+ selected grafts after myeloablative preparation. Rapid engraftment occurred in all patients. With a median follow-up of 28 (19–42) months, 14 patients are alive without clinical or molecular evidence of disease recurrence, whereas five have relapsed and one additional patient shows persistent presence of the disease-specific molecular marker without clinical progression. Cellular and serological parameters of hematopoietic and immune functions were largely normal at 12 months post-transplant including the measles titer which was present in all patients. Kinetics of immunohematopoietic recovery were similar to those of 12 control patients who had received unmanipulated PBPC during the same time period except for the recovery of CD4+ CD45RA+ T cells which was significantly delayed in the CD34+ group. During the first year post-transplant, transient monoclonal or oligoclonal gammopathies were observed in seven of 16 study patients. We conclude that CD34+ selection with the Isolex system allows preparation of highly purified CD34+ fractions and effective tumor cell depletion. The CD34+ products can be reinfused safely after myeloablative treatment and result in sustained hematopoietic and immune recovery. The fact that all patients retained their specific measles immunity suggests that myeloablative treatment with reinfusion of highly purified CD34+ PBPC is not immunoablative.

Comparison of different strategies of molecular genetic monitoring following autologous stem cell transplantation in patients with follicular lymphoma.

Two different molecular genetic methods were compared for their suitability for monitoring minimal residual disease in patients with follicular lymphoma (FL) treated with high-dose therapy and autologous stem cell transplantation. Fifteen patients were selected because of a specific PCR-amplifiable t(14;18) mbr translocation. PCR amplification of rearrangements of the complementary region III (CDRIII) of the immunoglobulin heavy chain gene was also carried out. After autologous stem cell transplantation, patients were prospectively monitored with both molecular genetic methods. Seven of the 15 patients with detectable t(14;18) prior to transplantation were persistently negative during follow-up to 32 months post transplant. None of these patients relapsed, whereas four of eight patients with positive PCR signals post transplant relapsed. Comparing t(14;18) and PAGE results, we observed six patients showing clonal signals in CDRIII PAGE in spite of persistent negativity of t(14;18) PCR. We concluded that in patients with FL, t(14;18) PCR is superior to CDRIII PCR in terms of sensitivity and specificity. A positive t(14;18) PCR during the first year post transplant is highly predictive for disease recurrence. CDRIII PCR may be used for monitoring in t(14;18) negative lymphomas. However, due to the poor specificity of conventional gel electrophoresis PCR, the use of clone-specific probes is highly desirable.

Reduced-intensity allogeneic stem cell transplantation as salvage treatment for patients with indolent lymphoma or CLL after failure of autologous SCT

Reduced-intensity allogeneic stem cell transplantation as salvage treatment for patients with indolent lymphoma or CLL after failure of autologous SCT

THE ROLE OF THE SPLEEN AFTER BONE MARROW TRANSPLANTATION FOR PRIMARY MYELOFIBROSIS

central component, unlike that observed in normal individuals. These patterns were not due to proteolysis. since there was no difference when blood samples were collected with inhibitors of calcium-activated proteases (Gralnick et al, 1985). The patients platelet vWf contents and multimer patterns were normal. Infusion of DDAVP normalized the factor VIII/vWf complex. The mean peaks of vWERCoF (78.5 u/dl) and vWEAg (71.8 u/dl) were observed at 30 min; at 60 min that of factor V1II:C (1 78 u/dl). 120 min after DDAVP infusion, FVIII/vWf complex reached levels almost similar to pre-DDAVP conditions. In contrast, in 10 normal subjects, the factor VIII/vWf complex after DDAVP was characterized by a constant peak at 120 min, in agreement with that observed in a group of patients with inherited classic type I vWd. The significant shortening in the half-life of the patients vWf supports the hypothesis that the decrease in plasma levels of vWf may be due to instability of the molecule in the circulation rather than, or in addition to, a simple decrease in synthesis. Post-DDAVP plasma showed enhanced amounts of all multimers and an early appearance of unusual large vWf components, as in the normals (Fig 1) . The doublet morphology was more evident than in the pre-DDAVP samples. The staining intensity of all multimers became weaker earlier than in normal subjects and in classic type I vWd. Multimer British journal of Haematology, 1992, 81

Granulocyte-colony-stimulating factor induces increased serum levels of soluble interleukin 2 receptors preceding engraftment in autologous bone marrow transplantation

Summary The serum levels of soluble interleukin 2 receptors (sIL‐2R) were determined in 19 patients who received highdose chemotherapy and an autologous or syngeneic bone marrow transplant (BMT) for treatment of Hodgkins disease (n= 18) or non‐Hodgkins lymphoma (n= 1). Twelve patients received granulocyte colony‐stimulating factor (G‐CSF) from day 0 or day +1 after autologous BMT until the white blood cell count had been stable for 9 d above 1 × 109/1, the remaining seven patients did not receive growth factors, In all G‐CSF‐treated patients the sIL‐2R levels increased steadily in the early post‐transplant course, even in the absence of infection. This increase was statistically significant 2‐4 d prior to the appearance of leucocytes in the peripheral blood (median 340 pm versus median 256 pm immediately after BMT, P<0.025) and peaked with the appearance of first peripheral blood leucocytes (median 536 pm, P<0.001). Cessation of G‐CSF administration resulted in a decline of sIL‐2R levels. In contrast, five of seven patients without G‐CSF treatment did not exhibit an sIL‐2R increase before or at the time of engraftment. Infection was associated with a rise of sIL‐2R levels. A correlation between sIL‐2R levels and total leucocyte count, lymphocyte count, or CD25 + lymphocyte count was not evident.

Lymphohaematopoietic chimaerism after bone marrow transplantation for chronic myeloid leukaemia: results of simultaneous cytogenetic analyses on T-cell colonies, myeloid, and erythroid progenitor cells

Summary. Various lymphohaematopoietic compartments represented by cells from T‐cell colonies, myeloid progenitor cells (CFU‐GM), erythroid progenitor cells (BFU‐E), and bone marrow after short‐term culture (BM) have simultaneously been analysed in 15 patients receiving 17 bone marrow transplants for Philadelphia chromosome (Ph) positive chronic myeloid leukaemia (CML) or acute lymphoblastic leukaemia (ALL). The marrow grafts were not T‐cell depleted. Ten patients without relapse did not show any myeloid cells of host origin until their last follow‐up or until death. However, in four of these patients single lymphoid host cells not carrying the Ph chromosome were found after BMT without clinical consequences. In patients with cytogenetic or haematological relapse Ph positive metaphases were first detected in any of the progenitor cell compartments along with residual donor cells in two of three patients. BM became Ph positive after various time intervals. Another patient with CML became Ph positive in all compartments investigated at the same time. The only patient with Ph positive ALL remained completely Ph negative also when haematological and clinical relapse was evident. All patients with relapse exhibited complex clonal and non‐clonal chromosomal aberrations at the time of recurrence of the Ph chromosome. Such abnormalities not identical to those usually found with evolution of the disease and preferentially occurring in progenitor cells preceded the reappearance of Ph positive metaphases in one of our patients.

Semiquantitative reverse transcription polymerase chain reaction analysis for detection of bcr/abl rearrangement using RNA extracts from bone marrow aspirates compared with glass slide smears after 0, 2 and 4 d of storage

The polymerase chain reaction (PCR) is an established tool for the detection of specific chromosomal aberrations in different haematological malignancies. Owing to fast degradation of RNA, the immediate processing of samples is thought to have a major influence on the reliability of results. Any delay caused by transport may be an obstacle to reverse transcription PCR (RT–PCR)‐based methods in multicentre studies. However, as air‐dried bone marrow smears are usually available, we have improved a method to use smears as a source for routine RT–PCR investigations. We studied whether this source of RNA could overcome problems caused by delayed transport of samples. The aim of the present study was (i) to investigate the influence of a storage period of up to 4u2003d before processing of a specimen by nested bcr/abl RT–PCR, and (ii) to compare bone marrow aspirates with bone marrow smears stored at room temperature in parallel. Bone marrow aspirates and smears were taken from 11 patients with Ph‐positive chronic myeloid leukaemia (CML). PCR results were semiquantified using a limiting dilution assay. We observed a loss of sensitivity <u200a1 log in stored bone marrow aspirates, even after 96u2003h. Results obtained from air‐dried unstained glass slide smears were similar to investigations performed on approximately 1u2003×u2003105 cells of a bone marrow aspirate. We conclude that a storage period of up to 96u2003h has little influence on the detection of a bcr/abl fusion transcript in CML at diagnosis. Glass slide smears were equivalent to bone marrow aspirates in 8 out of 11 cases as a source for RT–PCR analysis when nested PCR was performed.

Acute Megakaryoblastic Leukemia (FAB-M7) in an Infant Presenting with Orbital Chloroma and Meningeal Involvement

Although described as early as 50 years ago [20], acute leukemia of megakaryocytic lineage (AMegL) was only recently added to the French-American-British (FAB) classification as FAB-M7 [2, 3]. To establish the diagnosis for this category, sophisticated techniques like ultrastructural cytochemistry or immunological techniques are required. The limited data available suggest a frequency of 3%-12% in adult acute nonlymphoblastic leukemia (ANLL) [15]. In childhood only 20 cases with a definitive diagnosis of AMegL hade been reported up to the year 1986 [5]. To the best of our knowledge the present case is the first in which cranial chloroma was associated with AMegL.