Cytochemical characteristics of leukopoietic differentiation in murine erythroleukemic (Friend) cells.

The 3BM-78 murine Friend erythroleukemia cell line was obtained by in vitro transformation of bone marrow cells of DBA/2J mice by the polycythemic Friend virus complex. Thirty-five subclones have been isolated and tested for their ability to express various markers of blood and bone marrow cells. Upon dimethyl sulfoxide treatment, the cells differentiated along the erythroid pathway as shown by morphological evidence and by their increased synthesis of hemoglobin and spectrin. In addition, a high proportion of dimethyl sulfoxide-induced cells stained positive for specific esterase, a marker characteristic of granulocytic cells. Of these cells, about 20% stained positive for both hemoglobin-peroxidase and specific esterase. Analysis of the subclones showed that the expression of these markers for erythroid and leukopoietic differentiation was uncoordinated. Further dissection of expression was obtained by the use of two potent tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-benzoate, which in general inhibited specific esterase more than hemoglobin peroxidase expression. Inhibition was related to the structure of the phorbol diester and was unrelated to toxicity. No evidence was found for other markers characteristic of different pathways of differentiation, such as Fc and C3 receptors, cell surface immunoglobulins, theta antigen, or the capacity to phagocytose inert particles. All cells stained positive for nonspecific esterase activity in both the presence and the absence of dimethyl sulfoxide. This staining was only partially fluoride sensitive. In unstimulated cultures, a few cells also reacted for myeloperoxidase, Sudan black staining and, very rarely, alkaline phosphatase staining. These findings support the view that 3BM-78 cells are leukemic cells which, despite a prevalent commitment to erythroid differentiation, retain the genetic determinants for some traits of leukopoietic differentiation. These traits may be expressed under suitable culture conditions.

Treatment of elderly patients (> or =60 years) with newly diagnosed acute promyelocytic leukemia. Results of the Italian multicenter group GIMEMA with ATRA and idarubicin (AIDA) protocols.

In all, 134 elderly patients (median age 66 years, range 60-75 years) with newly diagnosed acute promyelocytic leukemia (APL) were enrolled in two successive protocols of the Italian multicenter group GIMEMA. All patients received an identical induction with all-trans retinoic acid and idarubicin; 116 (86%) entered complete remission (CR), two (2%) were resistant and 16 (12%) died during induction. After CR, 106 patients received further therapy whereas 10 did not, because of refusal (n=5) or toxicity (n=5). Consolidation consisted of three chemotherapy courses in the AIDA protocol (AIDA, 67 patients) or, since 1997, of an amended protocol including only the first cycle (amended AIDA, aAIDA, 39 patients). In the AIDA group, 43 patients (64%) completed consolidation, while seven (11%) and 17 (25%) patients were withdrawn after first and second courses, respectively; nine patients (13%) died in CR and 12 (18%) relapsed. In the aAIDA group, all patients received the assigned treatment; two patients (5%) died in CR and six (15%) relapsed. In the AIDA and aAIDA series, the 3-year overall and discase-free survival rates were 81 and 83% (P=NS), 73 and 72% (P=NS), respectively. We highlight here the frequency and severity of complications linked to intensive chemotherapy in this clinical setting and suggest that, in APL of the elderly, less intensive postremission therapy allows significant reduction of severe treatment-related toxicity and may be equally effective.

High numbers of mobilized CD34+ cells collected in AML in first remission are associated with high relapse risk irrespective of treatment with autologous peripheral blood SCT or autologous BMT.

The faster hematopoietic recovery after autologous peripheral blood SCT (APBSCT) in patients with AML may be offset by an increased relapse risk as compared with autologous BMT (ABMT). The EORTC and GIMEMA Leukemia Groups conducted a trial (AML-10) in which they compared, as second randomization, APBSCT and ABMT in first CR patients without an HLA compatible donor. A total of 292 patients were randomized. The 5-year DFS rate was 41% in the APBSCT arm and 46% in the ABMT arm with a hazard ratio (HR) of 1.17; 95% confidence interval=0.85-1.59; P=0.34. The 5-year cumulative relapse incidence was 56% vs 49% (P=0.26), and the 5-year OS 50% and 55% (P=0.6) in the APBSCT and ABMT groups, respectively. APBSCT was associated with significantly faster recovery of neutrophils and platelets, shorter duration of hospitalization, reduced need of transfusion packed RBC and less days of intravenous antibiotics. In both treatment groups, higher numbers of mobilized CD34+ cells were associated with a significantly higher relapse risk irrespective of the treatment given after the mobilization. Randomization between APBSCT and ABMT did not result in significantly different outcomes in terms of DFS, OS and relapse incidence.

Allogeneic bone marrow transplantation for Fanconi anemia.

Five patients (age range 7-14 years) received allogeneic bone marrow transplantation (BMT) for Fanconi anemia (FA). All patients showed progressive pancytopenia associated with congenital malformations. Diagnosis was confirmed by studies of cellular hypersensitivity to the clastogenic effect of the DNA crosslinking agent diepoxybutane. The conditioning regimen consisted of low dose cyclophosphamide (5 mg/kg x 4) and fractionated total body irradiation (167 cGy x 3). For graft-versus-host disease prophylaxis one patient was given cyclosporin alone while the remaining four patients received a combination of cyclosporin and two doses of methotrexate. Marrow was given unmanipulated from HLA-identical siblings. All patients are alive 18-67 months after grafting with Karnofsky scores of 100% and normal hemopoiesis of donor origin. Modifications in transplant protocols such as those here described have resulted in a decreased risk of severe transplant-related complications. These results confirm that BMT is a curative therapy in FA patients and should be considered as a first choice treatment if an HLA-identical donor is available.

Meningitis due to penicillin-resistant Streptococcus pneumoniae in patients with chronic graft-versus-host disease.

Two episodes of meningitis due to penicillin-resistant Streptococcus pneumoniae occurring in two patients with chronic graft-versus-host disease (GVHD) are reported. Both patients were treated with ceftazidime. The first patient died, unresponsive to therapy. The second patient showed clinical improvement, reverting to her baseline mental status. This report draws attention to the fact that in chronic GVHD patients: (1) bacterial prophylaxis does not ensure protection against encapsulated bacteria; (2) rapid microbiological investigation is recommended with any upper respiratory tract infections.

Cytogenetic survey of 80 patients with acute nonlymphocytic leukemia.

The authors report a cytogenetic survey of 80 patients with acute nonlymphocytic leukemia. The prognostic value of chromosome aberrations has been evaluated with three methods. The first one showed that patients with NN or AN bone marrow cellularity have a significantly better prognosis than those with AA cellularity; the second method confirmed the relatively good prognosis for patients with t(8;21) and abnormal 16 and a poor one for those with rearrangements of chromosomes 5 and/or 7. The authors also noted, surprisingly, that patients with hyperdiploidy had a significantly poorer prognosis than those with hypodiploidy and especially pseudodiploidy. The third method showed that patients with very complex karyotypes and a worse outcome than those with simple changes. Finally, they discuss the prognostic value of unusual and/or undeciphered chromosome changes detected in 18 patients, with a mean survival of 9.6 months, showing that these changes have a negative prognostic significance.

Chromosome changes in 19 patients with Waldenström's macroglobulinemia.

We report on 19 patients with Waldenström's macroglobulinemia (WM) who were studied cytogenetically at the onset and during progression of the disease. We found a high frequency of chromosome changes confirming the claim of other authors that, during progression of the disease, a large number of residual neoplastic cells, insensitive to conventional chemotherapy, persist. In turn, this may be the cause of the difficulty of inducing remission (21% of cases) and of the short survival (mean, 35 months). In our experience it is difficult to identify the primary chromosome abnormalities because of the late clinical stage at which the chromosomes were examined. However, changes involving chromosomes #10, #11, and #12 may be unfavorable events in patients with WM.

Complex chromosome translocations of standard t(8;21) and t(15;17) arise from a two-step mechanism as evidenced by fluorescence in situ hybridization analysis.

The authors report the results of cytogenetic and fluorescence in situ hybridization (FISH) analysis performed on complex chromosome translocations (CCTs) of t(8;21) and t(15;17) standard translocations associated with two M2 subtypes of acute myeloid leukemia (AML-M2) and four acute promyelocytic leukemia (APL), respectively. In one of two AML-M2 patients FISH analysis showed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) and on chromosome 1 at band p32, suggesting that the t(8;21) occurred as the primary step. In the second AML-M2 patient. FISH displayed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) but not on the third rearranged chromosome. Therefore, it is unclear whether chromosome 2 was rearranged secondary to the standard t(8;21). In four APL patients, FISH analysis showed material derived from chromosome 17 on the der(15). Moreover, in two patients with an i(17q) FISH disclosed material from chromosome 15 at the ends of both arms of the i(17q), suggesting that it occurred after the standard t(15;17). In the remaining two APL patients, FISH showed material from chromosome 15 on the der(17) and on chromosome 21 at band q22 in one case, and material of the p arm of chromosome 17 on chromosome 4 at band q11 in the other, demonstrating that in these two cases the first mutation also had been the t(15;17). Therefore, FISH analysis revealed that CCTs in five patients were secondary changes which occurred after standard t(8;21) and t(15;17), thus clarifying the hierarchy of the cytogenetic events, their role in the pathogenesis of the disease, and the associated clinic-hematologic findings.

Autologous blood stem cell transplantation in malignant lymphomas: an Italian Cooperative Study.

Twenty-three patients with malignant lymphoma, (7 Hodgkin's, and 16 non-Hodgkin's) in different phases of disease were autografted in 4 Italian Haematology institutions using only chemotherapy-mobilized blood stem cells (BSC) collected by apheresis. Clinical and laboratory data were analysed centrally and showed mean collection yields of 8.1 x 10(8) kg mononuclear cells (MNC) (SE 0.5; range 2.6-13.8) and 24.1 x 10(4) kg CFU-GM (SE 7.4; range 1.4-162.9). The mean times required to attain 0.5 x 10(9)/l neutrophils and 50 x 10(9)/l platelets after marrow-ablative high-dose chemo+radiotherapy and BSC reinfusion were 14.9 days (SE 1.5; range 7-38) and 18.6 days (SE 2.6; range 6-49) respectively. The incidence of early deaths was < 5% and the requirement for support with blood product transfusion was moderate. The progression free survival (PFS) is > 50% at 3 years with a median follow-up of 17.3 months. Results were significantly better for patients autografted in remission. These results suggest that autologous blood stem cell transplantation (ABSCT) may be proposed for the primary treatment of poor prognosis malignant lymphomas. However, ABSCT needs to be compared with autologous bone marrow transplantation (ABMT) followed by infusion of growth factors to accelerate recovery.

Superficial inflammatory and primary neoplastic lymphadenopathy: diagnostic accuracy of power-doppler sonography.

OBJECTIVE: To evaluate the sensitivity, specificity and diagnostic accuracy of a cut-off of the resistive index of 0.5 for the differentiation between inflammatory and neoplastic primary lymphadenopathies. SUBJECTS AND METHODS: We measured the resistive index of superficial enlarged lymph nodes in a total of 50 patients (29 males and 21 females; age range 12-72 years, mean age 41.6 year) using an ATL 5000 HDI. A resistive index greater than or equal to 0.5 indicated an inflammatory lymph node and a resistive index <0.5 was consistent with neoplastic primary lymphadenopathies. The gold standard was either surgical biopsy or lymph-node reduction seen with ultrasound examination after antibiotic therapy. RESULTS: The sensitivity of the resistive index for distinguishing inflammatory from neoplastic lymphadenopathy was 84.6%, the specificity 100% and the diagnostic accuracy 95.7% (P < 0.001, statistically significant). CONCLUSION: The results of this study indicate that power-Doppler using a resistive index cut-off of 0.5 was a valid technique for distinguishing between inflammatory and primary neoplastic lymph nodes in patients with superficial lymphadenopathies.

Patterns of cytomegalovirus retinitis in immunocompromised patients treated with 9-(2-hydroxy-1-(hydroxymethyl)ethoxymethyl) guanine (ganciclovir).

Five immunocompromised patients, four with AIDS and one who had undergone bone marrow transplantation, showing ocular signs of cytomegalovirus retinitis, were treated with 9-(2-hydroxy-1-(hydroxymethyl)ethoxymethyl) guanine (Ganciclovir), given intravenously at the dose of 5 mg/kg twice daily for a period ranging from 10 to 20 days. At the end of the treatment, in 4 of 5 patients, the ophthalmoscopic picture had improved, with reduced exudation and an arrest in the progression of retinal necrosis, the pattern clearly indicating a trend towards organization and scarring. Complete resolution of the retinitis without subsequent relapse was observed only in the bone marrow transplant patient, who recovered immunologically, whereas improvement of the eye involvement was only transient in the three AIDS patients.

Evaluation of azlocillin-amikacin combination for empirical therapy of infection in febrile neutropenic patients.

We have evaluated the azlocillin-amikacin combination, given at a daily dose of 200 mg/kg and 15 mg/kg respectively, in the treatment of 62 consecutive febrile granulocytopenic patients (less than 500 PMN/microliters) affected by hematological disease. The effectiveness of the treatment was assessed in 60 patients, 44 (73%) of whom responded within 96 hours from the beginning. 36 of the responders showed microbiological and clinical infections, 2 had clinically documented pneumonia and 6 a possible infection. No improvement was obtained in 16 patients; 7 of whom suffered from clinical and microbiological infection, 2 from pulmonary mycosis, 4 from possible infection and 3 from doubtful infection. Seven of these patients subsequently responded to a proven antibiotic treatment, while only one of the remaining responded to a second-line empirical antibiotic schedule. These results suggest that the combination of azlocillin-amikacin was able to overcome about two-thirds of the infections, representing an effective remedy for the empiric treatment of febrile neutropenic patients.

Clinical efficacy and in vitro activity of the ciprofloxacin-azlocillin combination (ratio 1:10) against gram-negative bacteria from non-neutropenic haematologic patients.

The authors report treatment of eighteen haematologic patients (twelve male, six female, age between 21 and 78 years), suffering from upper respiratory tract (ten patients) and/or lower urinary tract (eight patients) infections caused by Gram-negative germs, with a combination of ciprofloxacin-azlocillin in the ratio 1:10. Before treatment, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Fractional Inhibitory Concentration (FIC) index of Gram-negative isolates from patients for the ciprofloxacin-azlocillin combination were evaluated. The in vitro experiments revealed a synergistic activity of the combination for 85% of isolates, while at the same concentration ciprofloxacin alone was 100% effective, and azlocillin alone was 50% effective. The combination was administered to patients as follows: ciprofloxacin: 750 mg "per os" every 12 h; azlocillin 5 g intravenously every 8 h for a therapeutic cycle of 8 days. Seventeen of the eighteen patients that were treated with the combination showed complete eradication of the causative pathogen, sixteen of the eighteen patients recovered fully, whereas the other two showed significant improvements. The tolerability of the combination was excellent in seventeen patients and only one patient developed symptoms of mild gastric intolerance. The results presented here warrant further interest in studies of this antibiotic combination.

Comparison of norfloxacin and pefloxacin in the prophylaxis of bacterial infection in neutropenic cancer patients.

In this study the efficacy of norfloxacin and pefloxacin for the antibacterial prophylaxis of granulocytopenia was compared in cancer patients following cytostatic treatment. A total of 136 patients was randomly selected to receive either norfloxacin or pefloxacin. Nineteen patients remained afebrile in the norfloxacin group compared with thirty one in the pefloxacin group (p = 0.045). Twenty four microbiologically documented infections (twelve with and twelve without bacteraemia) occurred in sixty seven patients taking norfloxacin, and twelve in sixty nine patients taking pefloxacin (five with and seven without bacteraemia) (p = 0.015). Only one infection caused by Gram-negative bacilli was observed in the pefloxacin group compared with seven in the norfloxacin group (p = 0.019). In conclusion, both microbiological and clinical results showed pefloxacin to be a better antibacterial agent than norfloxacin for these patients.

Early hematopoietic reconstitution after autologous transplantation with blood-derived stem cells in a patient with advanced lymphoma.

A 30-year-old man with advanced non-Hodgkin lymphoma underwent repeated leukaphereses for harvesting blood-derived hemopoietic stem cells. Collection was started 8-10 days after the end of L-VAMP therapy (3 cycles). Nine procedures were performed and a total of 65.4 x 10(9) mononuclear cells (0.87 x 10(9)/Kg) were collected, processed, cryopreserved and stored in liquid nitrogen. The yields of CFU-GM, BFU-E and CFU-GEMM were respectively 964 x 10(4) (12.4 x 10(4)/Kg), 249 x 10(4) (3.2 x 10(4)/Kg) and 798 x 10(4) (10.4 x 10(4]. The patient received a myeloblative regimen consisting of fractionated total body irradiation (1200 cGy) and cyclophosphamide (120 mg/kg) followed by infusion of his own thawed cells. Early trilineage hematopoietic recovery was first observed on day +8; 1 x 10(9)/l WBC were reached on day +11, 0.5 x 10(9)/l PMN on day +13 and 50 x 10(9)/l platelets on day +11. Course was uneventful and the patient was discharged from hospital on day +21. Eight months after transplant the patient is in continuous unmaintained complete remission with normal blood cell counts. This reports suggests that complete and sustained engraftment can be achieved with peripheral stem cells recruited after "soft" chemotherapy.

Seven-day storage of single donor platelets in polyolefin bags: clinical, biochemical, morphological and microbiological evaluation.

We compared the in vitro and in vivo function of fresh and stored platelet concentrates (PCs) collected by an automated continuous-flow blood cell separator (CS 3000 Fenwal) in a closed-system apheresis kit in order to evaluate the possibility of extending the storage time to seven days with the polyolefin container (PL-732). The initial 220 ml platelet volume (5.14 +/- 1.23 x 10(11) was divided into two parts. Half was transfused and the other half was stored for 7 days. All cultured units were negative for bacterial contamination. Mean counts for fresh and stored platelets were respectively 2.34 +/- 0.59 and 2.17 +/- 0.50 X 10(11)/100 ml of PCs (mean recovery 88.7 +/- 11.9%). The pO2 levels were maintained during storage (179.9 +/- 30.5 mmHg) but pCO2, pH, LDH, osmolality, glucose consumption, bicarbonates, ATP, and osmotic stress values changed significantly after 7 days storage. From a clinical point of view, in 14 patients receiving a total of 38 PC transfusions no statistically significant change in corrected post-transfusional levels was observed between fresh and stored PC. Biochemical and morphological data and clinical results suggest that PCs collected with CS-3000 blood cell separator in a closed system and stored for 7 days in polyolefin bags (PL-732) can be satisfactorily employed in clinical practice.

Autologous blood stem cell collection after chemotherapy in patients with sensitive and refractory malignancies: a multicenter retrospective study.

A retrospective study was undertaken to assess the factors affecting the yield of peripheral blood stem cell (PBSC) collections after chemotherapy. Fifty-five patients with malignancies, observed in 4 Italian Institutions from January 1987 to June 1991 were eligible for evaluation. This series included 19 non-Hodgkin lymphoma, 11 multiple myeloma, 9 ovarian cancer, 7 Hodgkin disease, 7 acute non-lymphocytic leukemia, 1 acute lymphoblastic leukemia, 1 neuroblastoma. Five hundred and twenty two PBSC collections were performed on 55 patients after a median of 18 days after the start of chemotherapy. The yields of PBSC collections were related to the dose of cytoreductive chemotherapy exploited for PBSC mobilization and to the number of circulating white blood cells, colony forming unit granulocyte/macrophage (CFU-GM) and the percentage of monocytes at the time of collection. Forty-eight patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant related complications.

Survival after PBSC transplantation and comparison of engraftment speed with autologous and allogeneic marrow transplantation: results of a multicenter study.

We have analyzed the results of a multicenter study on peripheral blood stem cell transplantation (PBSCT) performed on 55 patients suffering from various neoplastic diseases. After myeloablative therapy, they received a median of 6.8 x 10(8)/kg MNC and 11.4 x 10(4)/kg CFU-GM harvested by a median of 9 apheresis after mobilization with chemotherapy alone. As of date, 34 of the 55 patients are alive and 28 of them are in continuous complete remission after a follow-up of 30 months. The probability of survival was related to the disease status at transplant, CR/PR vs. PD (p = 0.0001) and the bone marrow involvement, BM-vs. BM+ (P = 0.009). Furthermore, a comparative study on speed of engraftment and clinical management was conducted on the 55 PBSCT patients as well as on 41 autoBMT and 52 alloBMT patients. Days to reach WBC > 1.0 x 10(9)/L, PMN > 0.5 x 10(9)/L and PLT > 50 x 10(9)/L was 12/14/33 for PBSCT, 17/20/23 for ABMT and 15/16.5/18 for BMT, respectively. Days with fever > 38 degrees C, systemic antibiotic therapy and length of hospitalization was 3/12/36 for PBSCT, 5/18.5/42 for ABMT and 9/25/46 for BMT respectively.