This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and ...multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried blaOXA-58 alone, 21 of 202 also carried blaOXA-23, and 27 of 202 carried blaOXA-23 alone. No isolates were positive for blaOXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.
Transplant‐related mortality (TRM) remains a major problem in older patients undergoing allogeneic haemopoietic stem cell transplants (HSCTs). We have therefore explored a less intensive conditioning ...in 33 patients with a median age of 52 years (range 43–60) transplanted from human leucocyte antigen (HLA)‐identical siblings. The underlying disease was chronic myeloid leukaemia (n = 15), acute myeloid leukaemia (n = 6), myelodysplasia (n = 7) or a chronic lymphoproliferative disorder (n = 5); 15 patients (45%) had advanced disease. The regimen consisted of thiotepa (THIO; 10 mg/kg) on day −5 and cyclophosphamide (CY; 50 mg/kg) on days −3 and −2 (total dose 100 mg/kg). The source was bone marrow (BM) (n = 17) or granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood (PB) (n = 16), which were infused without manipulation. Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and a short course of methotrexate. Mean time to achieve a neutrophil count of 0·5 × 109/l was 17 d (range 11–23) and full donor chimaerism was detected in 79% of patients by day 100. Acute GVHD grade III or IV occurred in 3% of patients. Chronic GVHD was seen in 45% of patients, with a significant difference for PB (69%) compared with BM transplants (23%) (P = 0·009). For BM grafts, the actuarial 2‐year TRM was 6%, the relapse 56% and survival 87%; for PB grafts, these figures were, respectively, 27%, 33% and 68%. Twenty‐five patients are alive at a median follow‐up of 762 d (range 216–1615) and 20 patients (60%) remain free of disease. Thirteen patients (39%) received donor lymphocyte infusion (DLI) either for persisting or relapsing disease and six patients had complete remission. In conclusion: (i) patients up to the age of 60 years can be allografted with reduced intensity conditioning; (ii) the procedure was associated with a low transplant‐related mortality, particularly for bone marrow grafts, because of a lower risk of chronic GVHD; and (iii) DLI were required after transplant in half the patients for persisting disease or relapse.
Donor lymphocyte infusions (DLI) were given between June 1990 and March 1996 to 18 patients with chronic myeloid leukemia (CML) for the treatment of cytogenetic (n = 6) or hematologic relapse (n = ...12) following an allogeneic bone marrow transplant (BMT). Patients were divided in two groups: patients in group A (n = 8) received a large dose of donor lymphocytes (> or = 1 x 10(8)/kg), whereas patients in group B (n = 10) received escalating numbers of cells (2 x 10(5) up to 2 x 10(8)/kg). The median number of DLI in group A was 2 (range 1-3); the median number of infusions in group B was 7 (range 3-9). Acute GVHD occurred in 12 patients (grades I-III) and was a major cause of death in two. The risk of developing GVHD correlated with the number of cells infused: 37%, 14%, 5% and 0% for DLI with cells > or = 1 x 10(8), 2 x 10(7)/kg, 2 x 10(6)/kg, and 2 x 10(5)/kg, respectively (P = 0.01). Median transaminase levels were found to be significantly increased in patients with, as compared to patients without, acute GVHD (GPT 412 vs 28 IU/l; P = 0.03). Severe aplasia occurred in four and was a contributing cause of death in two patients. Overall, four patients died as a consequence of DLI and all received > 1 x 10(8)/kg cells: the actuarial risk was 38% in group A and 14% in group B (P = 0.1). There were 10 complete and three partial cytogenetic responses: the actuarial probability at 5 years of being Ph negative was 69%: it was 46% for group A and 85% for group B (P = 0.1). The longest patient is now 6 years post-DLI, Ph negative, BCR-ABL negative. The actuarial 3 year survival is 38% in group A and 86% in group B (P = 0.06). The study confirms that DLI post-BMT is not innocuous and that there is a definite long-lasting antileukemic effect in patients with CML. It also suggests that: (1) the risk of developing GVHD correlates with the number of infused cells; (2) that significant elevations of serum GPT levels are associated with GVHD; and (3) that the use of escalating doses of cells may allow the identification of side-effects and discontinuation of infusions before life-threatening GVHD has developed.
Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we ...investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow‐up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI‐AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG‐AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG‐AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively).
This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow‐up of 4 years.
Imatinib mesylate determines a favorable clinical course in most Ph positive Chronic Myeloid Leukemia (CML) patients in the chronic phase. Cytogenetic response is usually evaluated by analyzing 20-25 ...bone marrow metaphases using standard banding techniques. Since this methodology has very low sensitivity, we compared the results obtained by standard banding techniques to the ones obtained by fluorescent in situ hybridization (FISH). This was also done to identify any possible discrepancies between the two techniques. We analyzed 40 Ph+ CML patients in the chronic phase who had previously been treated with interferon alpha (IFNalpha) and who were receiving imatinib. The studies were performed by utilizing the same BM cell samples fixed in acetic acid/methanol, before imatinib therapy and then quarterly. Comparison of cytogenetic results to FISH results at 3 and 6 months of imatinib treatment showed that some patients who had achieved major cytogenetic response (i.e.<35% of examined metaphases showing Ph), showed retention of a higher number of persisting Ph+ cells when examined by FISH, and they did not achieve major FISH response (i.e. <35% of examined interphase cells show the BCR-ABL fusion signal). The discrepancy we found between the results that were obtained by analyzing metaphases and interphase cells disappeared in the subsequent examinations. Moreover, we found that 4 patients (10%) were still Ph+ in all the metaphases we examined even though they achieved excellent clinical response. On the basis of this small series of patients, we suggest that cytogenetic evaluation of patients on imatinib therapy should be performed by utilizing the classic banding technique (metaphase examination), but also by using the FISH technique (interphase examination), since the two methodologies may provide different results.
Myelodysplastic syndromes (MDS) are stem cell diseases but it is still controversial whether chromosomal abnormalities occurring in these disorders affect a multipotent stem cell or a committed ...progenitor. We studied a case of refractory anemia with ringed sideroblasts (RARS) and monosomy 7 in 100% of examined metaphases. Using the fluorescence in situ hybridization (FISH) technique with a probe specific for the centromeric region of chromosome 7, we demonstrated that 15% of BM cells fixed in acetic acid/methanol exhibited a normal diploid karyotype. Applying the FISH technique on PB cells smeared onto a slide, we observed that lymphocytes maintain two chromosomes 7, whereas other leukocytes exhibited monosomy 7. Our study confirms that chromosomal abnormalities found in MDS can occur in cells capable of differentiation along granulocytic and monocytic lineages, but not along the lymphocytic lineage.
'.the leukemic stem line is a small minority within the total cell mass;. when the leukemic stem line is not exceeding the normal stem cell numbers, its proliferation may still be under partial ...control.' LG Lajtha, Blood Cells 1981; 7: 45-62 We performed cytogenetic analysis on fresh bone marrow cells and on progenitor cell colonies in a patient who relapsed after allogeneic BMT for CML and was subsequently treated with donor lymphocyte infusions (DLI). Two Philadelphia-positive clones were identified at relapse. One clone displayed an additional chromosomal abnormality most likely induced by radio-chemotherapy and therefore arising in a single cell. This cell displays the characteristics of a stem cell, since it was able to support 20% of Ph-positive hemopoiesis for 5 months. If the progeny of a single Ph-positive stem cell account for 20% of hemopoiesis, a very low number of leukemic stem cells may sustain relapse after allogeneic BMT. This is in keeping with two observations: (1) at relapse, long-term culture initiating cells (LTC-IC) were all donor-derived and Ph-negative; (2) on average, the pace of the disease is very slow after relapse following allogeneic-BMT. Therefore, we hypothesize that a small number of leukemic stem cells may be involved in the initial events of relapse following BMT for CML.
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