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The aim of this study was to retrospectively analyse clinical characteristics of chronic GVHD (cGVHD) and requirements for immunosuppressive treatment (IST) in patients receiving reduced-intensity stem cell transplantation (RIST). About 29 patients who underwent RIST between September 1999 and April 2003 were evaluable for cGVHD; they were compared to an age-matched cohort of 29 patients who received conventional stem cell transplantation (CST).A total of 26 patients in the RIST group and 24 in the CST group developed cGVHD, which was graded as limited disease in 15 (52%) and 12 (41%) cases, respectively, and as extensive disease in 11 (38%) and 12 (41%) cases, respectively. Kaplan-Meier estimates of the risk of cGVHD at 1 year after transplant were 96 and 82%, respectively (p = 0.4). The median day of onset of cGVHD was 117 (range 93-220) in RIST group and 112 (range 77-225) in CST group. The skin was the most common target organ, involving 22 (84%) patients in the RIST group and 17 (71%) in the CST group. The probability of withdrawal from systemic IST at 3 years was 63 and 52% in the two groups, respectively, (p = 0.7). By multivariate analysis, RIST was the only, independent, prognostic factor for the development of refractory cGVHD (p = 0.01).In conclusion, we did not find major differences between patients receiving RIST and CST respect to timing, clinical characteristics and incidence of cGVHD. Refractory disease was more frequently observed in patients receiving RIST, although the probability of withdrawal from systemic IST was not significantly different between the two groups.

Twenty-one samples of grapefruit seed extract (GSE) either from marketed products or provided by an apiculturist were analysed to verify their inhibition activity, in particular against Paenibacillus larvae subsp. larvae, responsible for American foulbrood. The bactericide capacity of GSE has been measured in Bacillus subtilis BGA, Bacillus cereus 11778, Bacillus cereus K250 and Micrococcus luteus 9341a; these bacteria are normally used in the laboratory to study inhibitors. The results showed that not all GSE have the same inhibitory activity and two of those analysed do not inhibit the five bacteria used. Considering that 19 samples inhibited American foulbrood bacillus, the authors conclude that the use of a natural product (such as GSE) to control this important disease of bees, can be used as a substitute for chemotherapeutic products, after appropriate expedients.

Several studies have compared bone marrow (BM) and peripheral blood (PB) as stem cell sources in patients receiving allografts, but the cell doses infused have not been considered, especially for BM. Using the ALWP/EBMT registry, we retrospectively studied 881 adult patients with acute myelocytic leukemia (AML), who received a non-T-depleted allogeneic BM (n = 515) or mobilized PB (n = 366) standard transplant, in first remission (CR1), from an HLA-identical sibling, over a 5-year period from January 1994. The BM cell dose ranged from 0.17 to 29 x 10(8)/kg with a median of 2.7 x 10(8)/kg. The PB cell dose ranged from 0.02 to 77 x 10(8)/kg with a median of 9.3 x 10(8)/kg. The median dose for patients receiving BM (2.7 x 10(8)/kg) gave the greatest discrimination. In multivariate analyses, high-dose BM compared to PB was associated with lower transplant-related mortality (RR = 0.61; 95% CI, 0.39-0.98; P =.04), better leukemia-free survival (RR = 0.65; 95% CI, 0.46-0.91; P =.013), and better overall survival (RR = 0.64; 95% CI, 0.44-0.92; P =.016). The present study in patients with AML receiving allografts in first remission indicates a better outcome with BM as compared to PB, when the dose of BM infused is rich.

Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT. The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients. Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/microl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups. Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.

Objective: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described., Methods: Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity., Results: Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity., Conclusion: We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation., (Copyright 2003 International Society for Experimental Hematology)

High-dose chemotherapy followed by allogenic stem cell transplantation has been extensively used for the treatment of patients with hematological malignancies. Unfortunately, this life saving procedure is limited to a subset of patients who are in good medical condition due to the increased risk of regimen-related toxicity and graft-versus-host disease that occur with increasing age and poor performance status. On the other hand, it became apparent that the curative potential of transplantation was not solely due to the conditioning regimen but also to the graft-versus-leukemia effect mediated by alloreactive donor T cells. These observations led to the development of new transplant strategies using less intensive preparative regimens that would allow donor cell engraftment without the toxicity of myeloablative conditioning as a method of exploiting a graft-versus-malignancy effect in patients ineligible for conventional marrow grafts. Although follow-up is relatively short, preliminary results are encouraging and demonstrate the feasibility of non-myeloablative transplants in patients with heterogeneous diseases and disease status who Current challenges include defining the optimal regimen to promote full donor engraftment and the malignancies susceptible to this approach. The present review summarizes the most recent results obtained in this attractive field.

A reduced-intensity conditioning regimen was investigated in 45 patients with hematologic malignancies who were considered poor candidates for conventional myeloablative regimens. Median patient age was 49 years. Twenty-six patients previously failed autologous transplantation, and 18 patients had a refractory disease at the time of transplantation. In order to decrease nonrelapse mortality, and enhance the graft-versus-tumor effect, a program was designed in which a reduced conditioning with thiotepa, fludarabine, and cyclophosphamide was associated with programmed reinfusions of donor lymphocytes for patients without graft-versus-host disease (GVHD), not achieving clinical and molecular remission after transplantation. GVHD prophylaxis consisted of cyclosporine A and methotrexate. Seventeen patients received marrow cells and 28 received mobilized hematopoietic cells. All patients engrafted. The probability of grades II-IV and III-IV acute GVHD were 47% and 13%, respectively. The probability of nonrelapse mortality, progression-free survival, and overall survival were 13%, 57%, and 53%, respectively. Thirteen patients in complete remission had a polymerase chain reaction marker for minimal disease monitoring; 10 achieved molecular remission after transplantation. Nine patients received donor lymphocytes: one patient with mantle cell lymphoma had a minimal response, one patient with refractory anemia with excess of blasts in transformation achieved complete remission, and 7 patients did not respond. At a median follow-up of 385 days (range, 24 to 820 days), 25 patients (55%) were alive in complete remission. Although longer follow-up is needed to evaluate the long-term outcome, the study shows that this regimen is associated with a durable engraftment, has a low nonrelapse mortality rate, and can induce clinical and molecular remissions.

Unrelated donor bone marrow transplant (UD-BMT) has become an attractive alternative source of hematopoietic cells for patients lacking a matched sibling. The aim of this paper was to report on results of the 696 UD BMTs performed in 31 Italian institutions during the first 10 years of activity of the Italian Bone Marrow Donor Registry (IBMDR). In 1989 the Italian Bone Marrow Transplant Group (GITMO) established the IBMDR to facilitate donor search and marrow procurement for patients lacking an HLA identical sibling. By end of December 1999, 260,000 HLA-A, B typed volunteer donors had been cumulatively registered and 2,620 searches had been activated for Italian patients. At least one HLA-A, B, DRB1 matched donor was found for 54% of the patients and 696 UD BMTs were performed. In 50% of cases the donor was found in the IBMDR and in 50% in 15 other Registries. The average time from search activation to transplant was 6 months for disease other than CML. For CML it was 14 months. Actuarial 12-month transplant-related mortality (TRM) was 68% in patients grafted between 1979 and 1992 and 44% for patients grafted after 1993. Twenty-eight per cent of patients developed grade III or IV acute GvHD and 24% developed extensive chronic GvHD. The rate of disease free survival at three years was 57% for patients with 1st chronic phase CML, 37% for patients with 1st or 2nd CR ALL, 31% for AML or MDS patients < or = 18 years of age and 54% for patients with inborn errors. We conclude that the IBMDR has benefited a substantial number of patients lacking a matched sibling and has facilitated the recruitment of UDs into the international donor pool. The long time required for the search is the major obstacle to the success of this programme. This suggests that early transplant and a decrease in TRM could further improve these encouraging results.

Background and Objective: Peripheral blood stem cells (PBSC) are being increasingly used as an alternative to conventional allogeneic bone marrow (BM) transplantation. This has prompted the Working Group on CD34-Positive Hematopoietic Cells to evaluate the current utilization of allogeneic PBSC in clinical hematology., Evidence and Information Sources: The method employed for preparing this review was that of informal consensus development. Members of the Working Group met three times, and the participants at these meetings examined a list of problems previously prepared by the chairman. They discussed the single points in order to reach an agreement on different opinions and eventually approved the final manuscript. Some of the authors of the present review have been working in the field of stem cell transplantation and have contributed original papers in peer-reviewed journals. In addition, the material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline., State of the Art: Review of the current literature shows that unmanipulated allogeneic PBSC give prompt and stable engraftment in HLA-identical sibling recipients. Despite the much higher number of T-cells infused, the incidence and severity of acute GVHD after PBSC transplant seems comparable to that observed with bone marrow (BM) cells. In comparison to the latter, PBSC probably ensure faster immunologic reconstitution in the early post-transplant period. Controversial results on the incidence and severity of acute-GVHD have been reported when CD34+ selection methods are used. Prospective randomized trials are underway to compare the results of PBSC and BM allogeneic transplantation. In mismatched family donor transplants, T-cell depleted PBSC successfully engraft immune-myeloablated recipients through a megacell-dose effect able to overcome the HLA barrier. Experience with PBSC in the context of unrelated donor transplants is currently anecdotal and prospective trials should be completed before that practice becomes routine. Finally, there is also limited evidence that, following induction chemotherapy, the addition of PBSC to donor lymphocyte infusion (DLI) for treatment of leukemia relapse after BMT may improve the safety and effectiveness of DLI itself. Concerning cord blood (CB) transplants, the most interesting aspects are the ease of CB collection and storage, the low risk of viral contamination and the low immune reactivity of CB cells. This last property has its clinical counterpart in an apparently reduced incidence and severity of acute GVHD both in sibling and unrelated CB transplants, probably making the level of donor/recipient HLA disparity acceptable a greater degree with respect to what is required for transplants from other sources.

Two hundred fifty-three patients with newly diagnosed acute promyelocytic leukemia (APL) were eligible to enter the multicentric GIMEMA-AIEOP "AIDA" trial during the period July 1993 to February 1996. As a mandatory prerequisite for eligibility, all patients had genetic evidence of the specific t(15;17) lesion in their leukemic cells confirmed by karyotyping or by reverse transcription-polymerase chain reaction (RT-PCR) of the PML/RAR alpha fusion gene (the latter available in 247 cases). Median age was 37.8 years (range, 2.2 to 73.9). Induction treatment consisted of oral all-trans retinoic acid (ATRA), 45 mg/m2/d until complete remission (CR), given with intravenous Idarubicin, 12 mg/m2/d on days 2, 4, 6, and 8. Three polychemotherapy cycles were given as consolidation. Hematologic and molecular response by RT-PCR was assessed after induction and after consolidation. At the time of analysis, 240 of the 253 eligible patients were evaluable for induction. Of these, 11 (5%) died of early complications and 229 (95%) achieved hematologic remission. No cases of resistant leukemia were observed. Of 139 cases studied by RT-PCR after induction, 84 (60.5%) were PCR-negative and 55 (39.5%) PCR-positive. One hundred sixty-two patients were evaluable by RT-PCR at the end of consolidation. Of these, 159 (98%) tested PCR-negative and 3 (2%), PCR-positive. After a median follow up of 12 months (range, 0 to 33), the estimated actuarial event-free survival for the whole series of 253 eligible patients was 83% +/- 2.6% and 79% +/- 3.2% at 1 and 2 years, respectively. This study indicates that the AIDA protocol is a well-tolerated regimen that induces molecular remission in almost all patients with PML/RAR alpha-positive APL. Preliminary survival data suggest that a remarkable cure rate can be obtained with this treatment.

Background: Reduced expression of HLA class I molecules has been demonstrated for many human neoplasias and is correlated with increased malignancy., Methods: We investigated the correlation between HLA expression on blasts and the number of peripheral blood leukocytes (WBC) at onset in 34 patients with acute non lymphoblastic (ANLL) leukemia. Leukemic blasts at onset were serologically typed for HLA-A,B specificities, and the patients were classified in three groups according to the number of detectable surface HLA antigens., Results: The amount of WBC at onset in patients lacking HLA antigens was significantly higher than in patients with no antigenic loss., Conclusions: A decrease in HLA class I molecules on the cell surface is correlated with a greater expansion of the leukemic clone.

Twenty-one patients with high risk non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) underwent autologous bone marrow transplantation (ABMT). Nine out of 21 patients received in addition to bone marrow (BM) cells also peripheral blood (PB) cells collected by leucapheresis performed in the recovery phase after high-doses of either cyclophosphamide or etoposide (7 patients), or after ara-C (2 patient). All patients receiving BM + PB cells had ABMT as salvage therapy following extensive relapse or progression of their disease. The addition of PB cells to BM cells allowed a significantly faster recovery after ABMT. Median time to reach WBC greater than 1,000/microL was 14 days versus 20.5 days for patients receiving BM only. Furthermore, a reduced requirement of supportive care and a shorter period of isolation was observed. These results confirm that PB cells combined with BM cells allow a prompt hematopoietic reconstitution after myeloablation. In addition, the data demonstrate the efficacy of PB cells collected after various cytotoxic drugs, even in patients previously exposed to cytoreductive regimens.

Blood or bone marrow samples from 15 patients with newly diagnosed acute myeloblastic leukemia undergoing remission induction treatment with daunorubicin, cytosine-arabinoside and 6-thioguanine were tested in vitro. Leukemic cells were incubated for 24 h at 37 degrees C with or without the drugs alone or in combination. A 3-h pulse with labelled precursors of DNA synthesis (3H-thymidine) or protein synthesis (3H-leucine) was then given separately. In vitro growth, expressed as the percentage ratio between labeled precursor uptake in treated cells and in control cells, was compared with the clinical results obtained. Three patients were not considered evaluable (death occurred too early), 8 had a complete response (CR), and 4 were disease resistant to chemotherapy. Leukemic cells of resistant-disease patients showed a significantly lower growth inhibition than cells taken from CR patients, with each drug alone or in combination, when measured with thymidine. Inhibition of leucine uptake was not related to the clinical outcome.

The translocation t(8;16) (p11;p13) was found as the sole deviation from the normal karyotype in three patients with acute monocytic leukaemia. The bone marrow morphology was strikingly similar in the two cases where smears were available for re-evaluation: the leukaemic cells showed signs of differentiation, and active erythrophagocytosis was a particularly conspicuous feature. We suggest that t(8;16) (p11;p13) represents a new consistent abnormality in acute monocytic leukaemia, specifically associated with the differentiated subtype (M5b) and with pronounced phagocytic activity by the leukaemic monocytes.

Diabetes and obesity were noted in 21.3% and 42.3% respectively of 94 patients with adenocarcinoma corporis uteri. Hypertension and ovarian or mammary neoplasia were also common. Obese and diabetic subjects proved more sensitive to treatment with high doses of medroxyprogesterone acetate. Screening for precancerous states or carcinoma of the endometrium in obese and diabetic women is suggested.

The presence of mutations activating the N-ras gene was investigated by the polymerase chain reaction technique in twenty patients with acute myeloblastic leukemia (AML) at onset and in four patients with Ph1 positive chronic myelogeneous leukemia (CML) either in chronic phase or in blast crisis. Four remission samples and four relapses from the AML cases were also studied. Mutations were found in five out of twenty (25%) untreated AML cases at onset. No mutations were detected in the complete remission samples, two of them with N-ras mutations during the leukemic phase. Two out of the four leukemia relapses were positive for the same N-ras mutation shown at presentation, whereas no new mutations were found in the other two initially negative cases. An N-ras mutation appeared during the blast crisis of one of the four CML, which were all negative during the chronic phase. In conclusion, whereas some data appear to be consistent with a role of the N-ras mutations as initiating events in myeloid leukemias, in other cases N-ras activation seems to represent a factor involved in progression. These data suggest that a partial overlapping between initiation and progression factors could exist in naturally occurring tumors.