Blood stream infections after allogeneic stem cell transplantation: a single-center experience with the use of levofloxacin prophylaxis.

Blood stream infections (BSIs) remain one of the major causes of morbidity and mortality for patients receiving an allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, we evaluated the incidence and characteristics of BSI within 1 year after allogeneic HSCT in 269 consecutive adult patients who received antibacterial prophylaxis with levofloxacin. Cumulative incidence of BSI was 12% (95% confidence interval, 8-16%). Bacteria were responsible for 30 out of the 32 BSI, while fungi were responsible for 2 episodes of BSI. The median onset of BSI was day 8 (range 1-328 days) post transplant, and 66% of BSI occurred before neutrophil recovery. Gram-positive organisms accounted for 60% (n=18) of bacteremia, and gram-negative isolates for 40% (n=12) of the cases. Coagulase-negative staphylococci were the most commonly isolated gram-positive pathogens (53% of the cases), while Escherichia coli was the most commonly isolated gram-negative bacteria (58% of the cases). Candida albicans and Candida guillermondii were isolated from patients with candidemia. Resistance to fluoroquinolones (FQ) was common with 13% of gram-positive isolates being susceptible to FQ, while 50% of the gram-negative rods were susceptible to FQ. Crude mortality and mortality attributable to BSI were both 3% (1 of 32). In conclusion, our data suggest that despite the emergence of antibiotic resistance, FQ prophylaxis may be considered an appealing approach in allogeneic HSCT recipients and is also worth evaluating in randomized studies.

Nucleolar organizer region counts predict complete remission, remission duration, and survival in adult acute myelogenous leukemia patients.

PURPOSE: The analysis of the nucleolar organizer regions (AgNORs) was performed in patients with acute myelogenous leukemia (AML) to verify the role of cell proliferation in predicting complete remission (CR) and survival. MATERIALS AND METHODS: Bone marrow biopsies from 40 adult patients with AML were stained with the argyrophilic method. The mean AgNOR number (AgNOR count) was calculated for each case. After induction therapy, patients who achieved CR received intensive consolidation; two underwent autologous and four allogeneic bone marrow transplantations (BMT). RESULTS: The mean AgNOR count for the whole series was 6.6 (SD = 1.35); it was higher in CR patients than in resistant ones (P = .02). The median duration of CR was 26 months for patients with an AgNOR count greater than 6.6, but only 6 months for those with lower counts (P = .01). Sixteen patients who achieved a CR relapsed and 14 reached a second CR; the median duration of second CR was 16 months for patients with AgNOR count greater than 6.6, but only 5 months for those with lower counts (P = .01). The median survival time for the whole series was 14 months, with 30% of patients alive and in continuous CR at 103 months. Survival was longer for patients with an AgNOR count greater than 6.6 (33 months) than for those with lower counts (6 months; P = .0009). In multivariate analysis, when CR was excluded from the model, AgNOR count appeared as an independent prognostic variable (P = .005). CONCLUSION: AgNOR analysis is a suitable method to assess cell proliferation in bone marrow biopsies and can predict CR, remission duration, and survival in AML patients.

Multiple myeloma: VMCP/VBAP alternating combination chemotherapy is not superior to melphalan and prednisone even in high-risk patients.

The efficacy of alternating vincristine, melphalan (M), cyclophosphamide, prednisone/vincristine, carmustine, doxorubicin, and prednisone (VMCP/VBAP) polychemotherapy was compared with the M and prednisone (MP) regimen as induction treatment in multiple myeloma (MM). Three hundred four MM patients entered this study between March 1983 and July 1986; the analysis was performed in December 1989. The treatment groups did not show significant differences with respect to major prognostic factors. Median overall survival was 33.8 months. In the VMCP/VBAP and MP arms, after 12 induction chemotherapy cycles, 59.0% and 47.3% (P less than .068) of the patients achieved an M component reduction greater than 50%. No significant difference was observed in the two treatment arms in terms of remission duration (21.3 v 19.6 months, P less than .66) and survival (31.6 v 37.0 months, P less than .28). Patients younger than 65 years did not show any advantage from the alternating polychemotherapy. At diagnosis, the plasma cell labeling index (LI) and serum beta-2 microglobulin (beta 2-m) were evaluated in 173 and 183 patients, respectively. A significantly reduced survival was observed for patients with LI greater than or equal to 2% (16.4 months) or beta 2-m greater than or equal to 6 mg/L (20.4 months). Even in these poor-risk subgroups, VMCP/VBAP was not superior to MP.

Early responder myeloma: kinetic studies identify a patient subgroup characterized by very poor prognosis.

In order to assess the prognostic value of rapid tumor mass reduction in responding multiple myeloma (MM) patients, 100 consecutive patients were analyzed, and bone marrow plasma cell kinetic characteristics were evaluated at diagnosis. Forty-two patients obtained a tumor mass reduction greater than or equal to 50% with three cycles of chemotherapy and within 3 months (early responder myeloma [ERM]), and 23 in greater than 3 months (slow responder myeloma [SRM]). Survival rates in these two groups were not statistically different (P = .07). The labeling index (LI) of bone marrow plasma cells was significantly higher in ERM patients than in SRM patients (1.8 +/- 2.0 v 0.8 +/- 0.7, P = .006). The LI was used to separate the ERM patients into two well-defined subgroups. ERM patients with a LI greater than or equal to 2% showed a median survival of 16.4 months, whereas ERM patients with a LI less than 2% did not reach the median survival at 46.9 months (P less than .0044). Remission duration was also significantly different: 12.2 months in the high LI subgroup and 26.3 months in the low LI subgroup (P less than .0025). Early response itself does not correspond to shorter remission duration and shorter survival, but it is a poor prognostic factor if associated with a high plasma cell proliferative activity.

Intensified and high-dose chemotherapy with granulocyte colony-stimulating factor and autologous stem-cell transplantation support as first-line therapy in high-risk diffuse large-cell lymphoma.

PURPOSE: In our previous study with MACOPB, we identified a high-risk group of patients with a poor 3-year survival rate of 29%. These patients were defined as having at diagnosis advanced-stage disease with high tumor burden (TB) and elevated lactate dehydrogenase (LDH) level or bone marrow (BM) involvement. A novel therapeutic scheme was investigated to improve the outcome of these patients. PATIENTS AND METHODS: Fifty patients with high-risk diffuse large-cell lymphoma (DLCL) were enrolled. The therapeutic scheme includes three phases: induction with 8 weeks of MACOPB; intensification with a 3-day course of mitoxantrone 8 mg/m2 plus high-dose cytarabine (HDARA-C) 2 g/m2 every 12 hours plus dexamethasone 4 mg/m2 every 12 hours (MAD protocol) and granulocyte colony-stimulating factor (G-CSF) 5 microg/kg on days 4 to 17 to harvest peripheral-blood progenitor cells (PBPC); consolidation with carmustine (BCNU), etoposide, ARA-C, and melphalan (BEAM) regimen; plus autologous stem-cell transplantation (ASCT) with PBPC, marrow, or both. RESULTS: Thirty-six patients (72%) achieved a complete response (CR), 11 (22%) showed no response (NR), and three (6%) died of toxicity. Among the 22 PRs or NRs after the induction phase, 56% of patients achieved a CR with subsequent intensified therapy. With a median follow-up duration of 32 months, the overall survival and failure-free survival rates were 56% and 50%, respectively. The disease-free survival rate is 69% at 32 months. Leukapheresis after MAD and G-CSF yielded a median of 32 x 10(6)/kg CD34+ cells and 80 x 10(4)/kg granulocyte-macrophage colony-forming units (CFU-GM). Thirty-nine patients were autografted and 11 did not undergo ASCT: six because of disease progression, four due to toxicity, and one because of patient refusal. The median times to achieve engrafment were 11 days (range, 7 to 19) to a neutrophil count greater than 0.5 x 10(9)/L and 12 days (range, 8 to 60) to a platelet count greater than 50 x 10(9)/L. CONCLUSION: This sequential scheme with intensified and high-dose chemotherapy with ASCT is feasible with moderate toxicity and may improve the outcome in high-risk DLCL.

Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts.

The inv(16)(p13q22) chromosomal rearrangement associated with FAB M4Eo acute myeloid leukemia (AML) subtype is characterized by the presence of the CBFbeta/MYH11 fusion transcript that can be used to detect minimal residual disease (MRD). However, qualitative RT-PCR studies of MRD have so far produced conflicting results and seem of limited prognostic value. We have evaluated retrospectively MRD in a large series of CBFbeta/MYH11-positive patients employing both qualitative and quantitative (real-time PCR) approaches. 186 bone marrow samples from 36 patients were examined with a median follow-up of 27.5 months; 15 patients relapsed during follow-up. In qualitative studies, carried out by 'nested' RT-PCR assay, all patients in complete remission (CR) immediately after induction/consolidation therapy were found to be PCR positive. However, follow-up samples at later time points were persistently negative (except one case) in patients remaining in continuous CR (CCR) for more than 12 months. 16 patients were evaluated by quantitative real-time PCR assay: CBFbeta/MYH11 transcript copy number was normalized for expression of the housekeeping gene ABL, expressed as fusion gene copy number per 10(4) copies of ABL. A 2-3 log decline in leukemic transcript copy number was observed after induction/consolidation therapy. After achieving CR, the mean copy number was significantly higher in patients destined to relapse compared to patients remaining in CCR (151 vs 9, P < 0.0001 by Mann-Whitney test). Moreover, in CCR patients, the copy number dropped below the detection threshold after the treatment protocol was completed and remained undetectable in subsequent MRD analysis in accordance with results obtained by qualitative RT-PCR. On the contrary, in the seven patients who relapsed, the copy number in CR never declined below the detection threshold; thus a cut-off value discriminating these two groups of patients could be established. The findings of our study, if confirmed, might confer an important predictive value to quantitative real-time PCR determinations of MRD in patients with inv(16) leukemia.

Risk groups of myeloma patients by histologic pattern and proliferative activity.

We investigated the histologic pattern and the cell proliferative activity of myeloma cells by the analysis of the nucleolar organizer regions (AgNORs) in bone marrow biopsy specimens from 150 multiple myelomas at diagnosis. The objective was an attempt to define risk groups of myeloma patients. On univariate analysis, the percentage of bone marrow plasma cells (BMPC%), the pattern of infiltration, the degree of plasma cell (PC) atypia, the marrow fibrosis, and the number of AgNOR/PC were correlated with survival time. On multivariate analysis, only AgNOR counts and pattern of infiltration retained independent prognostic significance. At 4-year followup, all patients with BMPC% < or = 20, interstitial pattern of invasion, and well-differentiated (G1) PC plus AgNOR/cell < or = 3.32 were alive, while no patient with BMPC% >50, diffuse pattern of infiltration, and poorly differentiated (G3) PC plus AgNOR/cell >5.15 survived (p < 0.0001). In conclusion, the histologic pattern and proliferative activity of myeloma cells, evaluated by AgNOR counts, are reliable predictors of survival in myeloma. Both parameters can be easily assessed in the same biopsy specimen, are reproducible, and permit identification of a group of patients with favourable outcome at 4-year followup. Thus, bone marrow biopsy should always be included in the diagnostic procedures for myeloma patients.

Cytotoxic T lymphocyte precursor frequency as a predictor of acute graft-versus-host disease in bone marrow transplantation from HLA-identical siblings.

The measurement of precursor frequencies of donor anti-recipient cytotoxic T lymphocytes (CTL-p) has been shown to predict the incidence and the severity of acute graft-versus-host disease (aGVHD) in unrelated donor bone marrow transplantation (BMT). In HLA-identical sibling BMT, where aGVHD is most likely caused by minor histocompatibility antigen mismatches, this assay did not appear to be sensitive enough to provide similar predictive information. In this study, the CTL-p frequencies and the incidence and severity of aGVHD in 51 onco-hematological patients transplanted from HLA-identical siblings were compared. Sibling donors were selected on the basis of HLA identity using serological typing for HLA-A, B, C antigens, whereas HLA-DRB was tested by molecular analysis. Sibling identity was also confirmed by DNA heteroduplex analyses. Fifteen out of 21 (71%) patients with high precursor frequency (>1:100 x 10(3)) and 12 out of 30 (40%) with low precursor frequency (<1:100 x 10(3)) experienced clinically significant (II-IV) aGVHD. A significant correlation (P = 0.04) between CTL-p frequency and severe aGVHD was demonstrated. Moreover there was a positive trend for a high frequency response according to an increasing grade of aGVHD, which was statistically significant (P = 0.04). In our experience the CTL-p assay is a helpful predictive test for aGVHD in HLA-identical sibling BMT, indicating high risk patients possibly requiring additional prophylaxis.

AgNORs and myeloma prognosis.

The argyrophilic nucleolar organizer regions (AgNORs) were analysed in bone marrow biopsies from 80 patients with multiple myeloma (MM) at presentation. The mean AgNOR number per MM cell (AgNOR counts) and their distribution within the nucleus (configuration) were assessed. AgNOR counts were significantly associated with several recognized prognostic factors: Durie and Salmon clinical staging system (p = 0.02), percentage of plasma cells (PCs) in aspirates (p = 0.01) and in bone marrow biopsies (p = 0.0000), pattern of bone marrow involvement (p = 0.0003), calcaemia (p = 0.0005) and creatininaemia (p = 0.0003). AgNOR counts were also associated with the degree of PC differentiation (p = 0.0000). A single central cluster of 2-3 large-sized AgNORs (configuration A) was evident in most G1 MM; one cluster of 4-5 medium-sized dots or two clusters of 2-4 dots (configuration B) were seen in most G2 MM; many small-sized, scattered dots were present in G3 MM (configuration C). AgNOR counts and configuration were related to the prognosis: in the univariate analysis, the 5 year survival rate was 7% for cases with > 4.5 AgNORs/cell and 46% for cases with < or = 4.5 AgNORs/cell (p = 0.01), 53% for configuration A, 12% for configuration B and 0% for configuration C (p = 0.0000). AgNOR counts (p = 0.02) and configuration (p = 0.000) were independent prognostic variables in the multivariate analysis. The AgNOR counts were significantly higher in "fulminant myeloma" than in less aggressive cases (p = 0.002). The plasma cell labelling index (LI%), evaluated in 44 MM patients, showed significant correlation with prognosis: the 5 year survival rate was 51% for LI% < or = 1 and 17% for LI% > 1 (p = 0.02). More than 70% of patients with low LI% had low AgNOR counts and more than 70% of patients with high LI% had high AgNOR counts (p = 0.007). AgNOR counts and configuration reflect the myeloma cell mass, the degree of differentiation and the kinetics of the myeloma cells. They offer an exact evaluation of the tumour characteristics and can be useful additional parameters for MM prognosis.

[Adenopathies in chronic myeloid leukemia (CML). Study of 161 cases]. / Adenopatie nella leucemia mieloide cronica (LMC). Studio su 161 casi.

161 cases of CML have been studied. Clinically significant adenopathies were present in 3,2% of the patients at the moment of diagnosis, and in the subsequent course they appeared in 7% of them. The behaviour of adenopathies showed to be unrelated to: --splenomegaly; --blastic metamorphosis in the peripheral blood or in the marrow (which they often preceded from 3 to 26 months); --hematological sensibility to cytostatic therapy; and furthermore they often acted as the most important clinical and therapeutic problem. From the cyto-histological point of view three features have been observed: 1) blastic metamorphosis in a lymphnode showing features of myeloid metaplasia; 2) blastic invasion in a lymphnode without any sign of myeloid metaplasia; 3) malignant lymphoma. Cytological examination of imprints and ultrastructural studies, besides the usual histological investigations, proved to be useful for the definition of the above mentioned features. In lymphomatous forms, together with the study of the cariotype and the research of the Ph' chromosome, the performance of immunocytological investigations is also necessary.

JAK2V617F activating mutation is associated with the myeloproliferative type of chronic myelomonocytic leukaemia.

BACKGROUND: Chronic myelomonocytic leukaemia (CMML) is a haematopoietic malignancy with heterogeneous clinical and morphological features. It is classified in the World Health Organization myeloproliferative-myelodysplastic overlap category. JAK2(V617F) mutation can be found in a large percentage of patients with myeloproliferative neoplasms. AIMS: To investigate the association between JAK2(V617F) mutation and clinical, haematological and bone marrow histological features in CMML and to verify whether the mutation is associated with the myeloproliferative type of the disease. METHODS: 78 consecutive patients with newly diagnosed CMML from 2004 to 2008 were included in the study. JAK2(V617F) mutation was assessed using direct sequencing of exon 14 or by allele-specific PCR from total peripheral blood or bone marrow samples. RESULTS: JAK2(V617F) mutation was identified in eight cases (10.2%). All patients with the mutation presented with splenomegaly and had a significantly higher haemoglobin level and neutrophil count than patients without the mutation. All bone marrow biopsies of JAK2(V617F)-mutated CMML showed increased erythropoiesis, a marked myeloid and megakaryocytic hyperplasia with occasionally clustered megakaryocytes, and a mild or moderate (grade 1 or 2) fibrosis; six cases showed an increased number of dilated sinusoids and reactive lymphoid nodules. CONCLUSIONS: The results indicate that JAK2(V617F) mutation is associated with clinical and morphological features of the myeloproliferative type of CMML. Therefore, JAK2 mutation analysis together with bone marrow morphology could help in a more appropriate classification of the disease.

Lack of correlation between objective response and death rate in multiple myeloma patients treated with oral melphalan and prednisone.

An analysis of survival by response category was performed on 76 patients with stage II and III multiple myeloma, who were treated with oral melphalan and prednisone. The analysis demonstrated a survival advantage for responders over non-responders only in stage III patients (32.3 months for responders vs. 15.6 months for non-responders, p = 0.03). However, two possible sources of error must be considered: a) the poor prognosis of early responders that may adversely affect the survival of all responders, and b) the bias introduced by the 'guarantee time' of responders (i.e., the time on-study required to detect the response). Exclusion from the analysis of the unfavourable subgroup of 'early' responders (median survival 14.7 mos.) provided an improvement of the difference in survival between the remaining 'slow' responders and non-responders in stage III (p = 0.005) as well as in the series as a whole (p = 0.025). Because of the consistent 'guarantee time' of slow responders, the Mantel-Byar test (which credits survival to responders only after the response has been obtained) was then applied. The survival advantage of slow responders over non-responders, previously observed in all patients, particularly those with stage III, was not confirmed by the Mantel-Byar test (chi-square 0.831 and 1.457, respectively), thus supporting the hypothesis of an equal death rate over time in each response category. It therefore appears that the usual response criteria (which require at least a 50% reduction of the myeloma protein) should perhaps be reassessed, as they seem to be an inadequate parameter for evaluation of treatment effectiveness in multiple myeloma.

Quantitation of melphalan in plasma of patients by reversed-phase high-performance liquid chromatography with electrochemical detection.

An analytical procedure for the separation and determination of melphalan in human plasma was carried out. A simple high-performance liquid chromatographic method with electrochemical detection was developed taking advantage of the high sensitivity of the electrode redox reaction. The sample pretreatment consisted of a direct extraction of the interferents rather than of melphalan, owing to the difficulty of extraction of the drug, and was very simple, rapid and reproducible.

Prognostic significance of serum albumin in chronic lymphocytic leukemia.

BACKGROUND: Low levels of serum albumin have been reported to be associated with a poor prognosis in lymphoproliferative disorders. METHODS AND RESULTS: Clinical and laboratory data were retrospectively evaluated in a series of 342 patients with chronic lymphocytic leukemia (CLL). In univariate analysis, survival was significantly influenced (p less than 0.01) by traditional prognostic factors: number of lymphoid areas involved, volume of adenopathies, presence and degree of hepatomegaly and splenomegaly, anemia, thrombocytopenia, peripheral blood lymphocytosis (greater than 60 x 10(9)/l), percentage of bone marrow lymphocytes (greater than 50%). Among variables not included in the traditional staging systems, age over 70, hypoproteinemia (less than 6 gr/dl) and hypoalbuminemia (less than 3.5 gr/dl) adversely affected prognosis. All the most widely adopted staging systems recognize no more than three groups of patients with statistically different outcomes. In multivariate analysis, the prognostic value of serum albumin was independent of both age and the group of variables included in each staging system considered. CONCLUSIONS: We suggest that evaluation of the serum albumin level could be useful, in future multicenter studies, for further implementation of the staging of CLL.

Correlation between argyrophilic nucleolar organizer region counts and labelling index in multiple myeloma.

The correlation between the bromodeoxiuridine (BrdU)-labelling index (LI) of plasma cells and a new proliferation marker, the Argyrophilic Nucleolar Organizer Regions (AgNORs), was investigated in 44 myeloma patients at diagnosis. A preliminary analysis was made to verify the reproducibility of the assessment of plasma cell infiltration (PC%) in bone marrow aspirates, used to collect cells for LI determination, and in bone marrow biopsies, used for AgNORs evaluation. Although an overall good correlation was observed between PC% in biopsies and aspirates (r=0.58, p=0.001), the ratio between PC% in biopsies and in aspirates ranged form 0.35 to 7.5. Only 17 patients (38.6%) were within the 0.5-1.5 range. A positive correlation between LI and AgNORs was observed in these patients (r=0.68, p=0.003), whereas the correlation was lost in patients with higher ratio between PC% in biopsies and in aspirates (r=0.08, p=0.69). The prognostic significance of AgNORs was confirmed by survival analysis, showing a reduced survival for patients with high (>4.4) AgNOR counts (14 months vs 35 months, p=0.004). The AgNORs analysis therefore allows the simultaneous evaluation of myeloma cell infiltration, degree of differentiation and kinetics of growth in bone marrow biopsies. AgNOR counts deserve to be included in the procedures for diagnosis and prognostic evaluation of myeloma patients.

Changes in SRBC binding capacity and T-surface antigen expression on human peripheral blood lymphocytes stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA).

The expression of surface markers was analyzed on human peripheral blood lymphocytes after exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter which is known to have a mitogenic effect on human T-cells. A significant decrease in the proportion of cells capable of binding to SRBC under different experimental conditions (active (EA), high affinity (EH), total (ET) rosettes) was observed after 20 hr exposure to TPA. After 4 days incubation, the ability to form EH and ET rosettes was recovered, while a significant increase in the proportion of EA and temperature stable (ES) rosettes compared with control cultures was observed. Using monoclonal antibodies directed against different T-lymphocyte subpopulations, a depression in the proportion of cells reacting with OKT3 (pan T) and OKT4 (helper/inducer) antibodies was observed, after both early and late exposure to TPA. On the contrary, the positivity with OKT8 (suppressor/cytotoxic) antibody was not affected at any time of culture. These findings suggest that, in normal blood, TPA can modulate the expression of T-cell receptors, and that T-lymphocytes with helper/inducer phenotype appear to be the target of the TPA-induced membrane changes.

[Expression of HLA-DR structure and a membrane-specific antigen by a granulocytopoietic line of acute leukemic blast cells]. / Espressione della struttura HLA-DR e di un antigene di membrana specifico per la linea granulocitopoietica sui blasti di leucemia acuta.

Blast cells from peripheral blood of 32 patients with acute leukemia were tested for their ability to react with a monoclonal antibody (D1.B6) specific for HLA-DR surface antigen. In order to evaluate the degree of leukemic cell differentiation a monoclonal antibody (R1.B19) specific for the granulocytopoietic lineage was also employed. The results demonstrated that a considerable proportion of blast cell populations expressed the HLA-DR antigen, while only a small fraction of cells expressed the myeloid antigen.