Peripheral blood stem cells for allogeneic transplantation.

The use of peripheral blood stem cells (PBSC) for allogeneic transplantation (PBSCT) is increasing steadily so that it cannot be considered an experimental practice any longer. Collection of PBSC requires the treatment of donors with G-CSF. With this drug, 10 to 16 mg/kg/day, side-effects are acceptable, but thrombocytopenia may follow the PBSC harvest. After transplant, allogenic PBSC engraft quickly in comparison with marrow. This has been shown for platelets, and to a lesser extent for granulocytes. Stability of graft has been documented by DNA analysis. With PBSC a high number of T- and NK-cells is infused, with a possible increase of GVL effect. However, we only have experimental evidence in the mouse that this may be the case. Incidence of acute GVHD equals that after BMT, but data on chronic GVHD are controversial, with an increased incidence reported in some studies. There is currently no indication for T-cell depletion of PBSC in HLA-identical sibling pair transplants. Experiments with CD34+ cell selection have sometimes produced a paradoxical increase of acute GVHD. The challenge of allogeneic PBSCT is improvement in survival, but available data only show that results are no worse than BMT. Prospective studies of allogeneic PBSCT versus bone marrow transplantation are in progress in Europe and USA.

Successful engraftment of allogeneic PBSC after conditioning with busulfan alone.

A 44-year-old woman with AML, while receiving a conditioning treatment with BU-CY for an allogeneic sibling transplant, developed septic shock with pulmonary embolism and heart failure. Conditioning was stopped at the end of the busulfan course and cyclophosphamide omitted. After antibiotics, dopamine and steroids the patient was allografted, using the donor's G-CSF-primed PBSC. She recovered her peripheral blood counts promptly and developed an acute GVHD grade II that responded to steroids. The DNA microsatellite analysis showed full donor engraftment up to a year from transplantation. This case suggests that the use of PBSC may facilitate engraftment in the absence of an effective immunosuppression during conditioning.

Three to six year follow-up of normal donors who received recombinant human granulocyte colony-stimulating factor.

One hundred and one donors who had received filgrastim (rhG-CSF) for the purpose of donating either granulocytes or peripheral blood stem cells (PBSC) for their relatives more than 3 years ago were contacted. All donors had received daily rhG-CSF at a median dose of 16 microg/kg/day (range 3-16) for a median of 6 days (range 3-15 days). All collection procedures were completed and short-term side-effects of rhG-CSF were mild in the majority of the donors. At a median time interval of 43.13 months (range 35-73), the donors were contacted to assess whether adverse effects related to rhG-CSF administration had occurred. Prior to rhG-CSF two donors had cancer, one had a myocardial infarction, one was hepatitis C virus positive, one had a history of sinusitis, one had Graves' disease and two had arterial hypertension. None worsened with the rhG-CSF administration but the donor with a history of infarction had an episode of angina following apheresis, and the donor with Graves' disease had a stroke 15 months after rhG-CSF. Two pregnancies occurred after the rhG-CSF administration and one donor was 2-3 weeks pregnant during rhG-CSF treatment. Three pregnancies resulted in two normal births and one in a spontaneous abortion of a pregnancy which occurred more than 2 years following rhG-CSF. In the time following rhG-CSF administration two donors developed cancer (breast and prostate cancer) at a follow-up of 70 and 11 months, respectively. One donor developed lymphadenopathy 38 months after the rhG-CSF, which spontaneously resolved. Blood counts were obtained in 70 donors at a median follow up of 40.4 months (range 16.8-70.8). Hematocrit was 43% (median, range 36.8-48), white blood cells were 5.7 x 109/l (median, range 3-14), granulocytes 3.71 x 109/l (median, range 1. 47-10.36), lymphocytes 1.67 x 109/l (median, range 0.90-3.96), monocytes 0.46 x 109/l (median, range 0.07-0.87) and platelet counts were 193.0 x 109/l (median, range 175.0-240.0). This study indicates that short-term administration of rhG-CSF to normal donors for the purpose of mobilizing the PBSC or granulocytes appears safe and without any obvious adverse effects more than 3 years after the donation. Bone Marrow Transplantation (2000) 25, 85-89.

High incidence of chronic GVHD after primary allogeneic peripheral blood stem cell transplantation in patients with hematologic malignancies.

To assess feasibility and potential advantages of PBSC allograft, we transplanted nine patients (age 20-47 years) with advanced or poor-risk hematologic malignancies. These included eight HLA-identical sibling transplants and one partially matched. Cells were collected from donors by apheresis after rh-G-CSF 10-16 micrograms/kg/day for 4-5 days, and stored at 4 degrees C until infusion. Patients were conditioned with busulfan 16 mg/kg and cyclophosphamide 200 mg/kg, and received GVHD prophylaxis with CSA-MTX. The graft consisted of PBSC alone, with a median of 101.2 (range 28-254.2) x 10(4)/kg CFU-GM, 6.84 (range 4.57-15.9) x 10(6)/kg CD34+ cells and 2.5 (range 1.2-6) x 10(8)/kg CD3+ cells. An ANC > 0.5 x 10(9)/1 occurred on (median) day 13 range 11-17), and a platelet count > 50 x 10(9)/l on (median) day 15 (range 12-29) post graft. One patient died of ARDS on day 13, the others are alive 96-485 (median 245) days from the graft. Two patients have relapsed, one of them with isolated CNS involvement. Acute GVHD (grade I-II) occurred in three patients and severe chronic GVHD in six patients, with no relationship to CSA withdrawal. This unexpected incidence of chronic GVHD might be linked to the high number of CD3+ cells in the graft, contributing to a favourable GVL effect.

Allogeneic transplantation of unmanipulated peripheral blood stem cells in patients with multiple myeloma.

In multiple myeloma (MM), allogeneic bone marrow transplantation may produce complete and durable responses, but is accompanied by significant transplant-related mortality (TRM). To assess feasibility and possible advantages offered by the use of allogeneic, growth factor-primed PBSC instead of marrow, we analyzed the data of 10 patients with MM (IgG = 6, IgA = 1, BJ = 2, non-secreting = 1; stage II = 1, stage III = 8, plasma-cell leukemia = 1) who received an allogeneic transplant with PBSC. Their age ranged between 35 and 53 years (median 45). All were HLA-identical to their sibling donors. Prior to allograft, six patients received standard-dose chemotherapy (DAV or CY-Dexa) and four a sequential intensified scheme with autologous PBSC support. At the time of transplantation, three patients were in CR, three in PR, three had refractory disease, one progressive disease. Patients were conditioned with busulfan-melphalan (n = 9) or busulfan-cyclophosphamide (n = 1), and were allografted with unmanipulated PBSC obtained by apheresis after treatment with G-CSF alone (n = 6) or GM-CSF followed by G-CSF (n = 4). All patients engrafted, with 0.5 x 10(9)/l PMN and 50 x 10(9)/l platelets on (median) day 13. Four patients had > or =grade II acute GVHD (grade II in 3, grade III in 1). Following allograft, CR was achieved in 71% patients. Eight are currently alive, with six in CR at a median of 18.5 months (range 7-28) from the transplant. Two patients died, 1 and 4 months from the allograft, respectively, and one is alive with progression. A PCR analysis of IgH rearrangement showed that residual disease was no more molecularly detectable in four out of seven evaluated patients following allograft. The results suggest that PBSC may improve the therapeutic efficacy of allogeneic transplant in MM, not only by a reduction of TRM but also by an improvement of rate and quality of response.

Chromosomal abnormalities in Waldenström's macroglobulinemia.

We report the results of cytogenetic studies of direct bone marrow (BM) preparations and of short-term BM and peripheral blood (PB) cultures from 17 patients with Waldenström's macroglobulinemia. We noted clonal chromosome changes in 10 patients. Abnormalities affected chromosomes X, Y, 2, 4, 5, 15, 16, 18, 19, 20, 21, and 22; in particular, chromosomes 2, 4, and 5 were involved in structural changes: a homogeneously staining region [hsr(2)], a der(4)t(4;?)(q32;?), and a 5q+. The other chromosomes were involved in numerical abnormalities, such as pseudodiploidy (a 46,X, -X, + 15 clone), loss of chromosome Y, and monosomy of chromosomes 16, 18, 19, 20, 21, and 22. Nonclonal chromosome rearrangements were also observed. The results are discussed in comparison with the few data reported in the literature, and the finding of an hsr in the long arm of chromosome 2 is emphasized; indeed, this is the first report of hsr in WM.

A phase II trial on alpha-interferon (alpha IFN) effect in patients with monoclonal IgM gammopathy.

Waldenström's macroglobulinemia (WM) is an incurable disorder of B cells. Following occasional reports of response to alpha interferon (IFN) and in view of its effectiveness in hairy cell leukemia, we tested this agent in a relatively large group (n = 88) of patients who had an IgM monoclonal component (MC) greater than 10 g/l. Thirty eight patients had a MC > 30 g/l and were classified as Waldenström's macroglobulinemia (WM), while fifty had either WM in an early stage or an IgM monoclonal gammopathy of undeterminated significance (all of them operationally classified as IgM-MGUS). All patients received IFN 3 MU/day for one month and then 3 times/week. Response to treatment was mainly based on MC reduction in two consecutive determinations (> 50%: major response; 25-50%: minor response). Of 36 evaluable WM patients, 12 had a major and 6 a minor response; of 41 evaluable IgM-MGUS patients, 2 had a major and 6 a minor response. In WM patients with a major response, MC reduction was associated with disappearance of hyperviscosity symptoms, raised Hb level and reduced bone marrow lymphoplasmacytosis. At the dose used, tolerance was excellent in the majority of patients; only 15% withdrew from the study due to side effects. Although single cases and very small series have already been reported, no large study collecting quantitative data on the effects of alpha IFN in WM has been published so far. Our results suggest that IFN treatment is not indicated for patients with a low monoclonal component, while it is of clinical benefit in about 50% of patients with IgM > 30 g/l.

Transplantation of unmanipulated allogeneic PBSC: preliminary report on 24 patients.

To explore the feasibility and potential advantages of PBSC in allogeneic transplantation, we grafted 24 patients (age 16-57, median 37) with different hematologic diseases (ALL = 10, AML = 5, MM = 4, NHL = 2, CML = 1, MDS = 1, AA = 1), 23 HLA-identical to their siblings and 1 partially matched. Cells were collected from donors by apheresis after G-CSF 10 to 16 mg/kg/day for 4 to 5 days, and stored at 4 degrees C until infusion. The patients were conditioned with chemotherapy regimens including busulfan and cyclophosphamide in the majority of cases and received GVHD prophylaxis with CSA-MTX in all but two. The graft consisted of PBSC alone, with a median of 143.5 (range 18.1-358.9) x 10(4)/kg CFU-GM, 9.0 (range 3.3-18.0) x 10(6)/kg CD34+ cells and 2.8 (range 1.2 to 8.6) x 10(8)/kg CD3+ and cells. An ANC >0.0.5 x 10(9)/L was recovered on (median) day 13 (range 11-17), and a platelet count >50 x 10(9)/L on (median) day 13 (range 12-55) post graft. There was no correlation between CD34+ cells or CFU-GM number in the inoculum and time to hematologic reconstitution. Acute GVHD (grade II-IV) occurred in 10 out of 22 (45%), chronic GVHD in 10 out of 18 evaluable (55%) patients. We found no relationship between occurrence of acute or chronic GVHD and number of CD3+ cells in the graft. Four patients relapsed and 7 died after transplantation. Fifteen patients are currently alive and disease-free 67 to 710 (median 286) days from the graft. Allogeneic transplantation with unmanipulated PBSC ensures a fast and stable engraftment. Acute GVHD incidence and severity seems comparable to that of bone marrow transplantation, but there may be an increase in chronic GVHD, mainly of the extensive form.

[Repetitive movement of the upper limbs: results of a current exposure evaluation and a clinical investigation in workers employed in the preparation of pork meat in the province of Modena]. / Movimenti ripetitivi degli arti superiori: risultati della valutazione dell'esposizione attuale e dell'indagine clinica negli addetti ad un reparto di lavorazione di carni suine del modenese.

Exposure assessment tests were undertaken to measure the biomechanical overload factors affecting the upper limbs. The tests were carried out on a group of 86 workers employed on the cutting, boning and trimming line of a pork meat processing plant. Anamnestic screening and clinical tests targeted at correlated disorders were also performed and were followed by instrumental tests. The results are reported with respect to frequency of repetitive technical actions, degree of muscular involvement, postural risk, several complementary factors and distribution of recovery periods. The clinical investigation showed a high prevalence of carpal tunnel syndrome, tendon disorders of the hand and epicondylosis of the elbow, in addition to other disorders. The report confirms the presence of additional risks for the workers, both in the past and under present circumstances, and suitable preventive measures are formulated.

Testicular relapse of AML during chronic graft-versus-host disease induced by donor leukocyte infusion.

Treatment options for acute leukemia relapsing after allogeneic BMT include conventional chemotherapy or a second transplant; however, results are rather discouraging, the first option being associated with poor survival and the second with a high mortality rate. More recently, donor leukocyte infusion (DLI) from the original donor has been used for relapsed patients in an attempt to induce a graft-versus-leukemia (GVL) effect. This procedure is partially devoid of the toxicity inherent to a second BMT. At our Institution, a 36-year-old patient with biphenotypic AML in second complete remission after relapse following allogeneic BMT was treated with peripheral blood stem cell (PBSC)-enriched donor leukocytes, obtained after in vivo priming with rhG-CSF. The patient experienced extensive cGVHD but developed a testicular relapse while in full hematologic remission. After irradiation of the sanctuary site he remains free of disease, still with chronic GVHD, 21 months after bone marrow relapse. This case suggests that immunologically privileged sites are inaccessible to GVHD/GVL effect. This should be considered when planning salvage transplants procedures in patients at risk for extramedullary involvement.

Assessment of blocking activity in patients with at least two consecutive spontaneous abortions.

The activity of the blocking factor (BF) was studied in 88 patients affected by recurrent abortion syndrome (RAS) by means of the determination of lymphocyte proliferation rate (LPR) in one-way mixed maternal-paternal lymphocyte cultures (MLC). Forty patients (45.5%) showed inadequate blocking activity (LPR greater than 80%). Pearson's correlation did not reveal any link between patient age and LPR (m2 = 0.031), or between LPR and number of aborted pregnancy (m20.058). Fifteen of the 88 patients had had only two consecutive abortions, but Pearson's correlation did not result in any particular differences in the link between LPR and number of abortions in this group; we therefore felt perfectly justified in extending the study to these subjects.

Mobilization and collection of PBSC in healthy donors: comparison between two schemes of rhG-CSF administration.

Procurement of a high number of progenitor cells is of primary interest in allogeneic PBSC transplantation. We have retrospectively compared toxicity, mobilization effect and progenitor cell yields of two different rhG-CSF schedules in 11 consecutive healthy individuals donating their PBSC. Five of them received rhG-CSF 16 micrograms/kg/d for 4 subsequent d in 2 divided subcutaneous injections (group A); similarly, 6 donors received rhG-CSF 10 micrograms/kg/d for 5 d (group B). The aphereses were started the last day of rhG-CSF treatment; 9 donors underwent 2 aphereses, one underwent 1 and another 3 procedures, always on subsequent days. Toxicity was mild, but moderate thrombocytopenia developed following apheretic collections, irrespective of rhG-CSF schedule. In all the donors WBC, as well as circulating CD34+ cells, CFU-GM, CFU-GEMM and BFU-E dramatically increased over the baseline values, peaking on d 5 or 6, with no statistical difference between the 2 groups for the height of the cell peaks. Also the peripheral lymphoid cell populations (CD3+, CD19+ and CD56+/CD3-) increased following the rhG-CSF administration. The number of MNC, CFU-GM, BFU-E, CFU-GEMM, as well as CD34+, CD3+, CD19+ and CD56+/CD3- cells collected by apheresis showed no statistical difference in the 2 groups. Overall, 8 of the 11 donors collected the target number of CD34+ cells > 4 x 10(6)/kg ideal recipient body weight with the first apheresis, with no difference between the 2 groups. Mobilization with rhG-CSF in healthy donors enables the collection of large number of progenitor cells with modest side effects. A schedule of 10 micrograms/kg for 5 d is as effective as 16 micrograms/kg for 4 d. A single apheresis would be enough in 80% of cases.

The use of three different therapeutic protocols according to the immunologic pathology present in three women with recurrent spontaneous abortion and fetal death for immunologic reasons: flucortolone and salicylates; high-dose intravenous gammaglobulins; subcutaneous heparin.

The authors report three cases of immunologic spontaneous abortions and fetal death. In the first patient there was lupus-like anticoagulant activity with a diagnosis of sub-clinical autoimmune disease; the second showed inadequate blocking factor activity, while the third subject presented excessive lymphocytotoxicity. In these cases three different therapeutic protocols were successfully used: flucortolone and salicylates, high-dose intravenous gammaglobulins and subcutaneous heparin.

New perspectives for the therapeutic management of recurrent immunological abortion.

Up to now the only effective therapy for recurrent abortion syndrome due to the absence of the so-called blocking-factor has been active immunotherapy with partner or third-party donor mononucleates. The Authors report their in vivo and in vitro experience with high-dose intravenous gammaglobulin (i IV Ig) in order to treat women with recurrent abortion syndrome. In the Authors opinion there are sufficient experimental reasons for continuing this research with IV Ig obtained from multiparous women plasma pools.

Mobilization and collection of PBSC in healthy donors: a retrospective analysis of the Italian Bone Marrow Transplantation Group (GITMO).

BACKGROUND AND OBJECTIVE: The number of allogeneic transplants of peripheral blood stem cells (PBSC) is rapidly increasing. Collection of PBSC in healthy subjects currently implies the administration of G-CSF or GM-CSF and, of course, the use of apheretic devices. These procedures involve potential risks, in particular the risk of leukemia secondary to growth-factor treatment. To evaluate the current practice of PBSC mobilization and collection, and initially assess the short-term side effects and efficiency of procedures, the GITMO (Gruppo Italiano Trapianti di Midollo Osseo) promoted a retrospective cooperative study among the Italian centers. METHODS: Seventy-six healthy individuals donating to their HLA-identical or partially matched sibling recipients in seven Italian centers form the basis of the present analysis. The data were retrospectively collected by proper forms, pooled and analyzed by means of a commercially available statistical soft package. RESULTS: All donors received G-CSF as mobilizing agent with different schedules according to each single center policy. A median of 2.5 (range 1-4) aphereses per donor were run. The most frequent side effect was bone pain. In no case did the medium term follow-up reveal subjective complaints or laboratory modifications. After G-CSF mobilization, WBC and lymphocytes counts increased to a maximum of (mean +/- SD) 48.1 +/- 15.6 x 10(9)/L and 4.2 +/- 1.5 x 10(9)/L, respectively. The peak was reached on day 5 in both cases. Platelets decreased after the apheretic procedures, reaching a minimum of (mean +/- SD) 77 +/- 26 x 10(9)/L on day 8 and returning to normal values on day 11. Overall, the apheretic collection yielded (mean +/- SD) 18.6 +/- 19.2 x 10(8)/kg donor body weight MNC; 10.4 +/- 5.7 x 10(6)/kg CD34+ cells; 90.6 +/- 75.9 x 10(4)/kg CFU-GM and 4.3 +/- 1.8 x 10(8)/kg CD3+ cells. The target dose of 4 x 10(6)/kg CD34+ cells was harvested in 51.3% donors after a single apheresis, in 85.5% after the second, and in nearly 100% after a maximum of 3 aphereses. INTERPRETATION AND CONCLUSIONS: These data demonstrate that collection of adequate numbers of circulating progenitors is feasible and well tolerated in healthy donors. However, only careful monitoring of donors and international cooperation will help to definitively assess the long-term safety of G-CSF for mobilization of PBSC.