Radiological assessment of the PRF/BMSC efficacy in the treatment of aseptic nonunions: A retrospective study on 90 subjects.

BACKGROUND: Nonunion is a major orthopaedic concern because of treatment difficulty, high costs and devastating effects on the patients' life quality. Therefore, there is interest in the use of bone substitutes and cell-based strategies to augment fracture repair. We aimed to verify if Platelet Rich Fibrin (PRF) added with bone marrow stromal cells (BMSC) was able to improve the reparative process in the aseptic nonunion, and to establish whether it was worthwhile with atrophic nonunion. The primary outcome was radiological union. As secondary endpoint, the healing time was assessed, and the radiological consolidation grade at each follow-up. METHODS: We identified 113 subjects with tibia or femur nonunion and retrospectively created two groups. Group A was constituted by 56 subjects who underwent the standard procedure, i.e. Judet decortication with/out internal fixation devices, and opposite cortical homoplastic stick. In 57 patients, the standard procedure was modified by adding PRF and BMSC carried by homologous lyophilised bone chips (group B). The same surgeon performed all the operations. To our knowledge, no data are reported in the literature about such application. Since a "gold standard" for healing quantification does not exist, a new scoring radiological system was applied, at 1.5, 3, 6, 12 and 24 months after treatment. RESULTS: At the final 24-month follow-up, the radiological union percentage was 94,12 in group B and 95,12% in group A. A decreased healing time was demonstrated in the presence of PRF/BMSC in comparison with the standard procedure. When we compared the radiological scores at each follow-up, we found that the PRF/BMSC combination significantly improved the consolidation grade at 1.5-, 3- and 6-month follow-up in femurs and at 1.5-month follow-up in tibiae. Furthermore, an improved consolidation grade was demonstrated in the atrophic subjects treated with adjuvants compared to atrophic patients treated with the standard procedure at 1.5-month follow-up. CONCLUSIONS: This study supports the concept that the use of PRF/BMSC, during the standard procedure, is effective in shortening nonunion healing time. It could allow an early mobilization of patients, minimizing suffering, and could be an effective tool to reduce the health-care costs resulting from this issue. LEVEL OF EVIDENCE: Therapeutic level III.

Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel or platelet gel and bone marrow stromal cells.

BACKGROUND: There is great interest in the use of bone substitutes to improve bone repair. We compared the osteogenic potential of lyophilized bone chips combined with platelet gel, or with platelet gel and bone marrow stromal cells, with that of lyophilized bone chips alone in the healing of a high tibial osteotomy. METHODS: A prospective, randomized, controlled study was performed, and a standardized clinical model was applied. Thirty-three patients undergoing high tibial osteotomy to treat genu varum were enrolled and assigned to three groups. During the osteotomy, lyophilized bone chips with platelet gel were implanted into eleven patients (Group A), lyophilized bone chips with platelet gel and bone marrow stromal cells were implanted in twelve patients (Group B), and lyophilized bone chips without gel were placed in ten patients as controls (Group C). Six weeks after surgery, computed tomography-guided biopsies of the grafted areas were performed and the specimens were analyzed by histomorphometry. Clinical and radiographic evaluation was performed at six weeks, twelve weeks, six months, and one year after surgery. RESULTS: Histomorphometry at six weeks showed significantly increased osteoblasts and osteoid areas in both Group A (p = 0.006 and p = 0.03, respectively) and Group B (p = 0.009 and p = 0.001) in comparison with controls, as well as increased bone apposition on the chips (p = 0.007 and p = 0.001, respectively), which was greater in Group B than in Group A (p < 0.05). Group B showed significantly higher revascularization than the controls (p = 0.004). Radiographs revealed a significantly higher rate of osseointegration in Groups A and B than in the controls at six weeks (p < 0.005 and p < 0.0001, respectively). At the final evaluation at one year, the osseointegration was still better in Groups A and B than in Group C; however, all patients had complete clinical and functional evidence of healing. CONCLUSIONS: Adding a platelet gel or a platelet gel combined with bone marrow stromal cells to lyophilized bone chips increases the osteogenetic potential of the lyophilized bone chips and may be a useful tool in the treatment of patients with massive bone loss.

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.

Evaluation of bone healing enhancement by lyophilized bone grafts supplemented with platelet gel: a standardized methodology in patients with tibial osteotomy for genu varus.

Orthopedic practice may be adversely affected by an inadequate bone repair that might compromise the success of surgery. In recent years, new approaches have been sought to improve bone healing by accelerating the rate of new bone formation and the maturation of the matrix. There is currently great interest in procedures involving the use of platelet gel (PG) to improve tissue healing, with satisfactory results both in vitro and in maxillofacial surgery. Otherwise, to our knowledge, only a preliminary clinical study was undertaken in the orthopedic field [Kitoh et al., Bone 2004;35:892-898] and the efficacy of PG is still controversial. Our paper focuses on the effect on bone regeneration by adding PG to lyophilized bone chips used for orthopedic applications. The clinical model and the laboratory methodology were standardized. As a clinical model, we employed the first series of patients of a randomized case-control study undergoing high tibial osteotomy (HTO) for genu varus. Ten subjects were enrolled: in 5 patients lyophilized bone chips supplemented with PG were inserted during tibial osteotomy (group A); 5 patients were used as a control (group B) and lyophilized bone chips without gel were applied. Forty-five days after surgery, computed tomography scan guided biopsies of grafted areas were obtained and the bone maturation was evaluated by a standardized methodology: the osteogenic and angiogenic processes were semi-quantitatively characterized by using histomorphometry, and the mineral component of the lyophilized and host bone was analyzed by using X-ray diffraction technique with sample microfocusing and microradiography. Lyophilized bone with PG seems to accelerate the healing process, as shown by new vessel formation and deposition of newly formed bone, with no evidence of inflammatory cell infiltrate, when compared with lyophilized bone without gel. On the contrary, lyophilized bone undergo a resorption process, and a fibrous tissue often fills the spaces between chips. A histiocytic/giant-cell reaction is sometimes present. Otherwise, no differences have been found concerning microstructure. Our findings show the reliability of the methodology used to monitor early bone repair. The completion of the study and the evaluation of the ultimate clinical outcome are necessary in order to verify PG in vivo effects in orthopedic surgery.

Infections caused by filamentous fungi in patients with hematologic malignancies. A report of 391 cases by GIMEMA Infection Program.

BACKGROUND AND OBJECTIVES: To evaluate the clinical characteristics of patients with hematologic malignancies developing a filamentous fungi infection (FFI) and to define the prognostic factors for their outcome. DESIGN AND METHODS: A retrospective study, conducted on patients admitted to 14 Hematology divisions of tertiary care or university hospitals, participating in the GIMEMA Infection Program, over a ten-year period (1988-1997). The study included patients with hematological malignancies and a histologically and/or microbiologically proven or probable FFI. RESULTS: We included 391 patients (male/female: 262/129, median age 49 years) with hematologic malignancies (225 acute myeloid leukemia, 67 acute lymphocytic leukemia, 30 chronic myeloid leukemia, 22 non-Hodgkin's lymphoma, 12 myelodysplastic syndrome, 10 aplastic anemia, 7 Hodgkin's disease, 8 chronic lymphocytic leukemia, 5 multiple myeloma, and 5 hairy cell leukemia) who developed a proven FFI. Eighty percent of the patients had been neutropenic for an average of 14 days before the infection, and 71% had an absolute neutrophil count lower than 0.5 x 10(9)/L at the time of FFI diagnosis. The primary sites of infection were: lungs (85%), nose and paranasal sinus (10%), and other sites (5%). The diagnosis was made while still alive in 310 patients (79%), and at autopsy in the remaining 81 patients (21%). Chest X-ray was diagnostic in 77% of patients with pulmonary FFI, while computed tomography (CT) scan of the thorax was positive in 95% of cases. A significant diagnostic advantage for CT scan was observed in 145 patients who had both a chest X-ray and CT scan. Aspergillus was identified as the cause of FFI in 296 patients, Mucorales in 45 patients, Fusarium in 6 patients and other filamentous fungi species in 4 patients, while in a further 40 patients no agent was identifiable. The overall mortality rate three months after the diagnosis of FFI was 74%, and fungal infection had been the cause of death in 51% of patients. INTERPRETATION AND CONCLUSIONS: Our retrospective study shows that FFI still remains a life-threatening complication in neutropenic patients. Despite appropriate treatment, half of the patients die due to this complication. The use of glucocorticoids and recovery from neutropenia are the most important prognostic factors. Mucorales infections are associated with a significantly poorer prognosis than those due to Aspergillus spp.

Thromboembolic complications and pharmacological prophylaxis in orthopaedic surgery.

Two thousand, three hundred and three patients who had undergone major orthopaedic surgery were statistically analysed for the incidence of complications comparing three regimens of prophylaxis and coexisting diseases; 2090 patients did not present postoperative complications. PTE occurred in 0.65% (one fatal). The mortality rate was 0.34% and the incidence of haemorrhage (haematoma and one gastric haemorrhage) was 3.8%. Patients treated with indobufen had a shorter hospital stay and the need for homologous blood transfusions was lower than for patients treated with calcium heparin. The rate of PTE was notably different in the three groups, being lower in the group treated with enoxaparin, although this result was not found to be statistically significant.

Contemporaneous acute myeloid leukaemia and sarcoidosis. Report of three cases.

Three cases of contemporaneous acute myeloid leukaemia (AML) and sarcoidosis are described. The possible pathogenic mechanisms concerning their concurrent appearance are discussed: if sarcoidosis impaired T-cell response, it could perhaps predispose the development of AML; alternatively, the development of sarcoidosis during AML may be due to a reaction linked to a diffuse release of tumour antigens with a subsequent formation of a non-caseating granulomata.

Mucormycosis in patients with haematological malignancies: a retrospective clinical study of 37 cases. GIMEMA Infection Program (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto).

A retrospective study of 37 patients with haematological malignancy (21 acute myeloid leukaemia, 11 acute lymphoid leukaemia, two lymphoma, two hairy cell leukaemia, one Hodgkin's disease) and histologically documented mucormycosis was conducted to evaluate the clinical characteristics and ascertain the factors which influenced the outcome from mycotic infection. Patients were admitted to 18 haematology divisions in tertiary care or university hospitals in Italy between 1987 and 1995. Fever, thoracic pain, dyspnoea and cough were the most frequent presenting symptoms. At the onset, 89% patients were neutropenic (neutrophil counts < 0.5 x 10(9)/l) with a median duration of previous neutropenia of 14 d (range 6-60). The most frequent sites of infection were lungs (81%), CNS (27%), sinus (16%), liver (16%) and orbital space (10%). Only three patients were asymptomatic. A correct in vivo diagnosis was made in only 13 (35%) patients. When performed, thoracic and cranial CT scan were the most useful diagnostic investigations. Despite the fact that 26 febrile patients were treated with empirical antifungal treatment, 28 of the 37 patients (76%) died from fungal infection at a median time of 17 d from the onset of clinical symptoms. Nine patients were cured by antifungal therapy plus, in five cases, radical surgery procedures. An analysis of factors influencing outcome demonstrated that the resolution of chemotherapy-induced neutropenia and prolonged treatment with amphotericin B and, if feasible, radical surgical debridement treatment, were significantly correlated with recovery from infection. Mucormycosis, a rare filamentous fungal infection that occurs most frequently in neutropenic acute leukaemia patients, is characterized by a high mortality rate. Extensive and aggressive diagnostic and therapeutic procedures are essential to improve the prognosis in these patients.

Localization of aspergillosis to the central nervous system among patients with acute leukemia: report of 14 cases. Gruppo Italiano Malattie Ematologiche dell'Adulto Infection Program.

We retrospectively studied a consecutive series of 100 patients with acute leukemia and aspergillosis to evaluate the clinical findings and risk factors for colonization of the central nervous system (CNS) by Aspergillus species. The diagnosis of CNS aspergillosis was made in 14 patients on the basis of the following criteria: neurological signs of CNS involvement (13 of 14 patients); cerebral CT scan findings (9 of 12); microbiological findings (6 of 12); and histological findings at autopsy (11 of 11). The majority of patients had severe neurological complications (i.e., hemiparesis or seizures), due mainly to brain abscesses or mycetomas. Autopsies were performed on 11 of 14 patients and provided evidence that CNS localization was secondary to invasive aspergillosis; in each case, the most likely primary focus of infection was the lung. Although all patients had received oral antimycotic prophylaxis and had received timely empirical antifungal treatment, they all died within a median time of 5 days from the onset of neurological symptoms. Analysis of the characteristics of patients with invasive aspergillosis did not reveal any difference between those with CNS localization and those without CNS localization.

FLAG (fludarabine, cytarabine, G-CSF) as a second line therapy for acute lymphoblastic leukemia with myeloid antigen expression: in vitro and in vivo effects.

Thirteen consecutive adult patients with primary refractory (n = 5) or relapsed (n = 8) acute lymphoblastic leukemia (ALL) were treated by an induction schedule (FLAG) consisting of Fludarabine (30 mg/sqm/d) plus high dose Cytarabine (HD-ara-C: 2 g/sqm/d) (d 1-5) and G-CSF (from d O to polymorphonuclear recovery). Patients achieving complete remission (CR) were administered a second FLAG course as consolidation and were then submitted to an individualized program of post-remission therapy, depending on the patient's age and performance status. CR was achieved in 8/12 evaluable cases (67%). The median CR duration was 22.5 w. CR attainment was significantly related to the co-expression of lymphoid and myeloid antigens. ALL/My+ patients achieved CR in 6/6 evaluable cases vs. 2/6 for ALL/My+. In vitro 3H ara-C incorporation into cellular DNA resulted significantly increased by Fludarabine (in 7/9 tested cases) and, furthermore, by the association of Fludarabine G-CSF in 5 evaluable ALL/My+ cases; in contrast, no effect of G-CSF addition to Fludarabine was observed in 4 ALL/My. Myelosuppression was observed in all patients: the median time to neutrophils > 0.5 x 10(9)/1 was 16.3 d (range 13-22) and 16.2 d (range 9-29) to platelets > 20 x 10(9)/1. Nonhematological toxicity was minimal. In conclusion, FLAG is an active and tolerable combination in refractory ALL, particularly in cases with myeloid antigen expression where G-CSF appears to improve efficacy, probably increasing ara-C incorporation into the DNA of leukemic cells.

MEC (mitoxantrone, etoposide and intermediate dose cytarabine): an effective induction regimen for previously untreated acute non-lymphocytic leukemia.

Twenty-three patients with acute non lymphocytic leukemia (ANLL), were treated with a single-6 day course of Mitoxantrone 6mg/m2/day, Etoposide 80mg/m2/day and intermediate dose Cytarabine (ara-C) 1g/m2/day (MEC). Patients who achieved complete remission (CR) were submitted to a 4-day-course of MEC as consolidation. Seventeen patients (73.9%) obtained CR, five patients (22.7%) were resistant to the treatment and one patient died during induction. Median remission duration was 11 months; overall median survival was 16 months. Relapses occurred in 11 patients; eight patients are still alive: 6 in 1st, 2 in 2nd CR (mean survival 20.1 months, range 17-26). All patients experienced severe myelosuppression comparable to that observed after classical induction cycles including ara-C in continuous intravenous infusion; none, however, died of infection. Non-hematologic toxicity was minimal; in particular, neurotoxicity was not observed. According to our results, the MEC regimen, which was previously demonstrated to be active in refractory patients, represents an effective induction treatment in ANLL, with an acceptable toxicity.

All-trans retinoic acid in the treatment of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by uni- or multilineage maturation defects of the bone marrow. Controversial therapeutic results have been obtained using growth factors or differentiating agents such as 13-cis retinoic acid. In this pilot study we evaluated the effects of all-trans retinoic acid (ATRA) in 10 MDS patients (5 male, 5 female). Six patients had refractory anemia (RA), 1 had refractory anemia with excess of blasts (RAEB), and 3 had refractory anemia with excess of blasts in transformation (RAEB-t). All patients received the same dose of ATRA (45 mg/sqm/day) orally for 6 weeks. A rise in hemoglobin concentration > 1g/dl was observed in 3/10 patients, while 5/10 patients showed an increase in granulocyte count > 0.5 x 10(9)/l without concomitant increase in the percentage of blast cells in the bone marrow. A rise in the platelet count > 50 x 10(9)/l was observed in 1/10 patients. All the effects were transient and maximal responses were obtained by the fourth week of treatment. Thereafter, the peripheral blood counts started to drop again, reaching pre-therapy values by the end of the treatment. This phenomenon could be attributed either to the exhaustion of an ATRA-responding cell pool, the development of cellular resistance to ATRA or to a reduction of plasma ATRA levels after prolonged treatment. According to our results, it seems that ATRA might have therapeutic efficacy in MDS, particularly if its effect could be improved by combinations with other differentiating agents or growth factors.

Molecular analysis of PML-RAR alpha fusion mRNA detected by reverse transcription-polymerase chain reaction assay in long-term disease-free acute promyelocytic leukaemia patients.

By use of RT-PCR of PML/RAR alpha, we evaluated bone marrow aspirates in 10 patients with APL in long-term disease-free status after induction chemotherapy and consolidation (median 54 months: range 33-101 months from complete remission). All patients were in clinical and cytogenetic remission at the time of molecular evaluation (range 32-96 months from CR). All patients but one were found to be RT-PCR negative at the molecular level for the expression of PML-RAR alpha transcript, confirming that the majority of the patients with long-term survival of APL are characterized by the eradication of the neoplastic clone.

Fatal haemoptysis in pulmonary filamentous mycosis: an underevaluated cause of death in patients with acute leukaemia in haematological complete remission. A retrospective study and review of the literature. Gimema Infection Program (Gruppo Italiano Malattie Ematologiche dell'Adulto)

A retrospective study on a consecutive series of 116 patients affected by acute leukaemia with documented pulmonary filamentous mycosis (FM) admitted between 1987 and 1992 to 14 tertiary-care hospitals in Italy was made in order to evaluate the characteristics of those patients who developed fatal massive haemoptysis. In 59/116 cases of pulmonary FM the infection was the principal cause of death and in 12 of these patients a massive haemoptysis was responsible for death. The diagnosis of FM infection was made ante-mortem in only four out of these 12 patients. The autopsy was performed in 11/12 patients and documented a FM infection. The mycetes isolated were: Hyphomycetes spp. (three patients), Mucorales spp. (two patients), Aspergillus spp. (seven patients). At the time of the massive haemoptysis the mean neutrophil count was 7.2 x 10(9)/l, and no patient had relevant thrombocytopenia (mean 184 x 10(9)/l, range 28-350) or coagulative abnormalities. The mean time which elapsed between resolution of chemotherapy-induced neutropenia (WBC < 10(9)/l) and occurrence of haemoptysis was 7 d. No signs or symptoms predictive of this fatal complication were identified. Massive haemoptysis can be the cause of death in patients with acute leukaemia and pulmonary FM which in the majority of patients was not diagnosed in vivo. This complication occurs most frequently shortly after the recovery from chemotherapy-induced aplasia. The mechanism of lesion is unknown, but it may involve the vascular tropism of FM and the release of leucocyte enzymes. Better preventive and therapeutic antifungal treatments are needed to avoid this serious, albeit rare, complication.

FLAG (fludarabine + high-dose cytarabine + G-CSF): an effective and tolerable protocol for the treatment of 'poor risk' acute myeloid leukemias.

Twenty-eight patients with poor prognosis acute myeloid leukemia (AML) received therapy with two courses of fludarabine 30 mg/m2/day + ara-C 2 g/m2/day (days 1-5) and G-CSF 5 mg/kg/day (FLAG) (from day 0 to polymorphonuclear recovery). Eighteen patients were considered 'refractory' (eight primarily resistant, five relapsing within 6 months of initial remission, or at a second relapse; five relapsing after an autologous bone marrow transplantation procedure. Ten cases were defined 'secondary' AML (diagnosis of AML made after a preexisting diagnosis of: myelodysplastic syndrome: five cases; myelodysplastic syndrome after therapy for breast cancer: one case; previously untreated, and concomitant, non-Hodgkin's lymphoma: two cases; Hodgkin's disease treated with chemoradiotherapy: one case). Overall, 15 patients (58%) achieved a complete remission (CR). Two patients died of infection during induction, and 11 had resistant disease. Analyzing the data in relation to selected host and disease characteristics, the response varied widely. The highest CR rates (89%) were obtained in secondary AML; in particular, two cases of 'second-primary' (concomitant with low-grade non-Hodgkin's lymphoma) AML obtained CR for both diseases. Refractory AML differed widely for response: high CR rate (75%), although with short mean CR duration for primary resistance AML, and very poor response (11% CR) for relapsed (early, second, after ABMT) cases. Interestingly, a slow kinetic of leukemic growth in vivo before FLAG administration was significantly related to the response and outcome (p = 0.0002). Hematological and nonhematological toxicities were acceptable. In conclusion, the FLAG regimen has significant antileukemic activity and acceptable toxicity especially in secondary AML, both with and without coexisting lymphoid malignancy.

Pre-treatment with all-trans retinoic acid accelerates polymorphonuclear recovery after chemotherapy in patients with acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) is currently being used as remission induction treatment for acute promyelocytic leukemia (APL). Conventional chemotherapy is added both during and after ATRA treatment, in order to avoid the occurrence of hyperleukocytosis, and to improve the duration of complete remission. In this study we analysed the hematopoietic recovery of 18 consecutive APL patients after standard Idarubicin or Daunorubicin +/- Cytosine-Arabinoside regimens. 9 of the patients were at the onset of the disease, (CHT group) while 9 had been pre-treated with ATRA 45 mg/sqm/day for at least 3 months (ATRA group). 500 PMN/mmc were reached after 20.8 day from the end of treatment in CHT group and after 12.0 days in ATRA group (p = 0.007). Platelets recovery was faster, even though not significantly in ATRA group. Interestingly, PMN recovery in ATRA group was even shorter (p = 0.004) than that obtained in CHT group, after the first course of chemotherapy (treatment in CR vs treatment in CR). If these results are confirmed in a larger study, a protective effect of ATRA on normal residual hemopoiesis should be postulated.

All-trans retinoic acid and induction of apoptosis in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) represents a highly effective treatment for acute promyelocytic leukemia (M3-ANLL). This compound induces the leukemic promyelocytes to differentiate into morphologically and phenotypically mature myeloid cells. The mechanism of action of ATRA is far from fully understood. It has recently been reported that, along with its differentiation activity, ATRA causes apoptosis in the acute promyelocytic leukemia cell line HL-60. In this study we attempted to test whether ATRA is also able to induce apoptosis in fresh leukemic cells from M3-ANLL patients. Our results indicated that although morphological differentiation was detectable in 9/9 M3-ANLL samples after in vitro exposure to ATRA 10(-6) M, the percentage of apoptotic cells in the treated samples did not significantly differ from that obtained in controls (13.1% vs 9.4% respectively, after 8 days exposure). These data suggest that apoptosis does not seem to be the key mechanism by which ATRA exerts its action in M3-ANLL, at least at the blast cell level.

Acute myeloid leukemia from diagnosis to bone marrow transplantation: experience from a single centre.

We analysed the use of allogeneic bone marrow transplantation (BMT) in the treatment of acute myelogenous leukemia (AML). We evaluated 271 adult patients with newly diagnosed AML treated here between 1983 and 1992; 113 patients (42%) were eligible for BMT because of their age (< 45 years until 1986 and < 50 years later). Of these, HLA typing was performed on 81 patients (72%); 32 patients were not typed (19 had no sibling, 8 had a primary refractory leukemia, 3 died during induction, 1 had important previous toxicity and for one patient there was no recorded reason). Of the 81 typed, 36 patients (44.4%) were found to have an HLA-matched sibling donor and 21 (25%) underwent BMT (8% of the total population); 15 patients did not undergo BMT (6 relapsed before transplantation and did not obtain a second remission, 3 declined the procedure, 1 died during induction, 1 had positive MLR, 1 had positive MLR and HCV hepatitis, 1 was a drug addict with HCV hepatitis, 1 had previous organ toxicity, 1 was psychotic). These data show that only a small fraction of unselected patients with AML can undergo BMT. Such findings make the comparison of BMT with other types of post-remission therapy more complex.

Synergistic cytotoxicity of AZT plus alpha and gamma interferon in chronic myeloid leukemia cell line K562.

We have previously reported that the antineoplastic activity of 3'-azido 3' deoxythymidine (AZT) can be increased by drugs that inhibit "de novo" thymidylate synthesis, such as 5-fluorouracil, methotrexate and hydroxyurea. In the present study we tested the combinations AZT+alpha interferon (IFN) and AZT+gamma IFN on in vitro growth of the human acute-phase chronic myeloid leukemia (CML) cell line K562. After 72 hours incubation, not only AZT+alpha-IFN but also AZT+gamma-IFN were synergistic in inhibiting K562 growth, as demonstrated by isobologram analysis of the data. This enhanced cytotoxicity was confirmed by the evaluation of [3H]AZT incorporation into cellular DNA, that was increased by 50% and 222% in the presence of alpha- and gamma-IFN, respectively. The addition of 50 mumol/l thymidine to the culture medium was able to reduce the cytotoxicity of the drug combinations to the degree observed with each compound alone; furthermore, the increased incorporation of AZT into DNA was completely reversed. These data indicate the existence of a biochemical interaction between AZT and IFNs that results in an increased cytotoxic effect. While the combination AZT+alpha-IFN is currently being tested in HIV-related malignancies, AZT+gamma-IFN is new and deserves further study in human CML acute and chronic phase models, in view of possible clinical applications.

Impairment of GM-CSF production in myelodysplastic syndromes.

In this study we evaluated the production of granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) by enriched bone marrow (BM) macrophages in 15 patients affected by myelodysplasia and 20 normal BM donors. The presence of GM-CSF, IL-6 and TNF-alpha in the culture supernatants of BM macrophages was detectable only after stimulation with lipopolysaccharide (LPS), whereas no differences were present in the amount of IL-6 and TNF-alpha between myelodysplastic patients and normal controls, GM-CSF production appeared eight-fold reduced in BM macrophage culture supernatants from myelodysplastic patients with respect to normal controls. After further experiments, we concluded that the impaired release of GM-CSF by BM macrophages could not be due to a different production kinetic in myelodysplastic patients. Moreover, the number of multipotent (CFU-GEMM), granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) progenitors was significantly impaired in myelodysplastic patients. In conclusion, we demonstrated that the production of GM-CSF by purified adherent cells from MDS patients is markedly impaired in spite of the peripheral blood cytopenia. This selective defect in GM-CSF production, along with an intrinsic defect of haematopoietic progenitor cells, might contribute to the impairment of haematopoiesis always observed in myelodysplastic patients.