Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy.

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 bla(OXA-58) alone, 21 of 202 also carried bla(OXA-23) , and 27 of 202 carried bla(OXA-23) alone. No isolates were positive for bla(OXA-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.

Dissemination of an NDM-2-producing Acinetobacter baumannii clone in an Israeli rehabilitation center.

New Delhi metallo-ß-lactamase (NDM-1) was initially identified in various Enterobacteriaceae and recently in Acinetobacter baumannii. This study described the clonal dissemination of an NDM-2-producing A. baumannii isolate in an Israeli rehabilitation ward and the genetic surroundings of the gene. The bla(NDM-2) gene was surrounded by the ble and trpF genes downstream and two copies of the ISAba125 on both sides. These are the first NDM-producing A. baumannii strains in Israel from patients with no previous travel or hospitalization on the Indian subcontinent.

Cytogenetic and fluorescence in situ hybridization monitoring in Ph+ Chronic Myeloid Leukemia patients treated with imatinib mesylate.

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.

Interferon-alpha protects Philadelphia-negative progenitors from exhaustion in chronic myeloid leukemia patients with cytogenetic response.

INTRODUCTION: Normal immature hematopoietic progenitors are relatively well preserved in most patients newly diagnosed with chronic myeloid leukemia, but tend to decline rapidly with time. Such exhaustion could reflect a suppressive effect of the Philadelphia positive clone expansion and/or be induced by Interferon-alpha treatment. MATERIALS AND METHODS: A total of 51 CML patients were classified into three groups. Newly diagnosed untreated patients were group A (n=30). Of the 21 treated individuals with Interferon-alpha, for at least 12 months, 15 showed no cytogenetic response (group B) while six showed persisting major/complete response (group C). Patients belonging to groups A and B were mobilized with chemotherapy plus G-CSF while patients of group C received a short course of G-CSF only. RESULTS: Patients responding to IFN-alpha (group C) showed comparable numbers of bone marrow Ph- long-term culture initiating cells to those of newly diagnosed individuals (group A): 8.5 (<1-65)/10(6) MNC vs 10.5 (<1-30), while non-responders had markedly lower numbers: <1 (<1-5). The amount of Ph- LTC-IC collected was significantly lower in patients of group B 1.8 (0-325)x10(2)/kg than in patients of either group A 31.3 (0-952)x10(2)/kg (P<0.002) or group C 109 (8-259)x10(2)/kg (P<0.01). Interestingly, five patients of group B who had 100% Ph+ metaphases, but Ph- progenitors in their bone marrow, mobilized normal amounts of Ph(-) progenitors. CONCLUSION: These findings suggest that the decline of normal hematopoietic progenitors, currently observed in the majority of CML patients, is not induced by IFN-alpha treatment, but it is likely due to the expanding leukemic clone. They also indicate that normal hematopoietic reservoir is consistently preserved in patients given IFN-alpha early after diagnosis and achieving a stable cytogenetic response.

Reduced intensity thiotepa-cyclophosphamide conditioning for allogeneic haemopoietic stem cell transplants (HSCT) in patients up to 60 years of age.

UNLABELLED: 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 x 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.

Autografting with Ph-negative progenitors in patients at diagnosis of chronic myeloid leukemia induces a prolonged prevalence of Ph-negative hemopoiesis.

OBJECTIVE: In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myeloreductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. MATERIALS AND METHODS: We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%-100% Ph-negative PBPC for a median time of 28 months (range 18-50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). RESULTS: Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%-100% to 0%-25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21-43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term-culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p < 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p < 0.03). CONCLUSION: We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.

Peripheral plasma amino acid abnormalities in rehabilitation patients with severe brain injury.

OBJECTIVE: Acute severe brain injury causes an increased mobilization of amino acids from tissue. The plasma amino acid profile of patients undergoing rehabilitation after brain injury is unknown. This study was aimed at delineating the plasma amino acid profile of rehabilitation patients with brain injury. DESIGN: Peripheral plasma aminogram, lactate, pyruvate, glycerol, ketone body, and carnitine concentrations were determined in 11 patients with brain injury (34.6+/-15 years old, 60+/-16.8 days after injury) and in 8 controls. Resting energy expenditure and nitrogen balance were also determined. RESULTS: (1) All essential amino acids and about 50% of nonessential amino acids were significantly lower in brain injury patients than in controls (p < .05). (2) Plasma amino acids were lower irrespective of either energy and protein intake or nitrogen balance. (3) Total carnitine concentration and esterified/free carnitine ratio were higher in brain injury patients than in controls (p < .05). CONCLUSIONS: Rehabilitation patients with brain injury may have an important reduction of their plasma aminogram. Muscle tissue depletion and the persistence of a hypercatabolic state caused by subclinical infections, pressure sores, and immobility may contribute to this reduction.

Complex chromosome rearrangements may locate the bcr/abl fusion gene sites other than 22q11.

BACKGROUND AND OBJECTIVE: From 5-8% of Philadelphia (Ph) positive patients with chronic myeloid leukemia (CML) show variant translocations in which at least a third chromosome in addition to 9q34 and 22q11 is involved. The formation mechanisms and clinical significance of variant Ph translocations are still unclear. The BCR/ABL chimeric gene encoding for chimeric proteins is always present and maps on the 22q- regardless of the type of translocation. We studied two apparently Ph negative CML patients with unusual karyotypes both showing a typical b3a2 rearrangement. DESIGN AND METHODS: Dual-color fluorescence in situ hybridization (FISH) can visualize BCR and ABL genes and localize the BCR/ABL fusion gene. We used FISH to study the formation mechanisms of variant Ph translocations in two patients. RESULTS: The chimeric BCR/ABL gene was located on a locus other than the expected 22q11 in both patients. In the first case the fusion signal was present on the 9q34 band whereas in the second patient it was detected on chromosome 8, involved in masked Ph formation. INTERPRETATION AND CONCLUSIONS: The location of the hybrid BCR/ABL gene on chromosomes other than 22q- is a rare event which can only be observed using the FISH technique. When these unusual translocations occur the hypothesis most often put forward is that several consecutive cytogenetic events have taken place. The factors which regulate the formation of these breakpoints have yet to be clarified. The FISH technique allows the identification of chromosome rearrangements that could not otherwise be detected by conventional banding procedures. The location of the hybrid BCR/ABL gene on sites other than 22q11 represents a rare type of variant Ph translocation. The real frequency and clinical significance of such rearrangements need to be investigated.

Coexistence of normal and clonal haemopoiesis in aplastic anaemia patients treated with immunosuppressive therapy.

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.

Changes of chromatin condensation in one patient with ataxia telangiectasia disorder: a structural study.

Differential scanning calorimetry and quantitative fluorescence microscopy have been employed to characterize the structure and organization of in situ chromatin in lymphoblastoid cells obtained from one ataxia telangiectasia (A-T) patient and one healthy family member. The proven capability of these biophysical techniques to measure changes of chromatin condensation directly inside the cells makes them very powerful in studying the eventual structural changes associated with the appearance of a pleiotropic genetic disorder such as ataxia telangiectasia. A-T syndrome is genetically heterogeneous and can be induced by different mutations of a single gene. The aim of this work is to determine whether the genetic mutation exhibited by the A-T patient of this study may be associated with modifications of chromatin structure and organization. Both the calorimetric and the fluorescence microscopy results acquired on cells from the A-T patient show that the structure and distribution of nuclear chromatin in situ change considerably with respect to the control. A significant decondensation of the nuclear chromatin is in fact associated with the appearance of the A-T disorder in the A-T patient under analysis, together with a rearrangement of the chromatin domains inside the nucleus.

Relapse after allogeneic BMT for chronic myeloid leukemia (CML) may be sustained by a small number of leukemic 'stem cells': a hypothesis.

'.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.

Normal primitive haemopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time.

We carried out studies to quantify Ph-negative progenitors both in steady state and during regeneration after chemotherapy and G-CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady-state bone marrow, Ph-negative long-term culture initiating cells (LTC-IC) and Ph-negative colony-forming-cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P<0.001 and P<0.02, respectively). The absolute number of mobilized Ph-negative progenitors was markedly higher in group A versus group B (P<0.02 for LTC-IC, P<0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph-negative LTC-IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph-negative LTC-IC in the bone marrow predicted the yield of Ph-negative LTC-IC mobilized into peripheral blood (P<0.001). The bone marrow frequency of Ph-positive LTC-IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph-negative circulating progenitors would appear to be soon after diagnosis.

A unique carboxy-terminus truncation mutant of the retinoic acid receptor alpha gene associated with a variant marker chromosome in a retinoic acid resistant HL-60 subline.

In order to contribute to the study of the molecular basis of leukemic cellular resistance to the induction of differentiation by all-trans retinoic acid (RA) we have generated and analyzed a mutant, RA-resistant HL-60 cell line. Molecular analysis of the retinoic acid receptor alpha (RARalpha) cDNA disclosed, in one of the two alleles, a novel mutation consisting of a 7-base deletion in the ligand binding domain that includes part of a FokI restriction endonuclease site previously described. As a consequence of this deletion and translational frame-shift, a stop signal is created that truncates the protein at codon 421, disrupting an essential functional component of the receptor. Transducing an epitope tagged RARalpha into the mutant is sufficient to inhibit clonal growth in the presence of RA. Standard cytogenetic analysis, fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis revealed the presence of two RARalpha loci, and showed a composite karyotype with additional abnormalities with respect to the parental line, including a chromosome 8 insertion in a chromosome previously known as marker three.

Complex structural involvement of chromosome 7 in primary myelodysplastic syndromes determined by fluorescence in situ hybridization.

Cytogenetic analysis of 72 consecutive de novo myelodysplastic syndrome patients revealed monosomy 7 in 12 cases. In 4 of these cases, the -7 was the only abnormality, whereas the remaining 8 cases showed additional chromosomal aberrations. Fluorescence in situ hybridization (FISH) utilizing chromosome 7 alpha-satellite and painting probes and other specific probes, when necessary, provided evidence of unusual and unsuspected structural rearrangements involving chromosome 7. FISH analysis showed that the small fragment found in one patient and the ring found in each of two other patients were chromosome 7-derived rings. FISH also revealed the insertion of chromosome 7 sequences into autosomes in three other patients and unusual translocations in the remaining two patients. By comparing the results obtained by using banding techniques to those obtained by using the FISH technique, we deduced the involvement of chromosome 7 with partial deletion of the short arm in all eight examined patients. Our study confirms the ability of FISH to detect chromosomal aberrations that would otherwise not be identified and the tendency of chromosome 7 to be involved in many different rearrangements. From a clinical point of view, we confirm that patients affected by myelodysplastic syndromes with complex karyotypes involving chromosome 7 do not respond to treatment and have a poor prognosis.

Allogeneic hemopoietic stem cell transplantation for patients with high risk acute lymphoblastic leukemia: favorable impact of chronic graft-versus-host disease on survival and relapse.

BACKGROUND AND OBJECTIVE: The best post-remission therapy for patients with acute lymphoblastic leukemia (ALL) is controversial, and hemopoietic stem cell transplantation (HSCT) is one therapeutic option. The goal of this study is to describe long term results of HSCT in high risk ALL patients. DESIGN AND METHODS: Between 1978 and 1996, 170 patient with ALL and a median age of 22 years (1-49), underwent an allogeneic HSCT from HLA-identical siblings (n = 149), family mismatched donors (n = 18) or unrelated HLA matched donors (n = 3); 92% of patients had at least one adverse prognostic factor for high risk ALL at diagnosis; one third (33%) were in first remission (CR1) and the majority (85%) received an unmanipulated HSCT with cyclosporin-methotrexate prophylaxis of graft-versus-host disease (GvHD). RESULTS: After a median follow-up of over 6 years, 59 patients are alive and 111 patients have died of leukemia (46%) or transplant related complications (54%). The actuarial 10 year survival is 53%, 38% and 20%, for patients in CR1, CR2 or advanced phase, respectively. The actuarial survival of patients with (n = 24) of without (n = 46) cytogenetic abnormalities, grafted in CR1/CR2 was respectively 45% and 48% (p = 0.5). The year of transplant had a significant impact in multivariate analysis on transplant related mortality (TRM) (p = 0.0009) but not on relapse (p = 0.3). Chronic GvHD was the most important favorable prognostic factor for survival (p = 0.0014) and relapse (p = 0.0019). INTERPRETATION AND CONCLUSIONS: This study confirms that long term survival can be achieved with HSCT in ALL patients, even those with cytogenetic abnormalities. Transplant mortality has been significantly reduced in recent years, whereas leukemia rate relapse has remained unchanged: the latter is influenced by the occurrence of chronic GvHD. Immune intervention post-HSCT may be considered to address this problem.

High frequency of trisomy 8 in acute promyelocytic leukemia: a fluorescence in situ hybridization study.

Correct diagnosis of acute promyelocytic leukemia (APL) requires proof of the translocation (15;17)(q24;q11), which appears to be absolutely specific for this particular type of myeloid disorder. We studied the karyotypes of 29 consecutive APL patients at diagnosis: in 5 of them banding techniques failed to detect the t(15;17). In these seemingly cytogenetically negative cases, fluorescence in situ hybridization (FISH) with a chromosome 17 painting probe detected a high percentage of mitoses with 3 hybridization signals: one derived from the intact chromosome 17, and 2 from the rearranged chromosomes 15 and 17. Trisomy 8 (+8) as a secondary chromosomal abnormality was observed in 8 cases (27.5%), confirming that the t(15;17) favors the acquisition of an extra chromosome 8. One of these 8 cases showed a marker that was interpreted by FISH analysis as der(8) with duplication of a segment of the long arm carrying the c-MYC allele. Clinical features of patients with t(15;17) and +8 were no different from patients with t(15;17) alone. The usefulness of FISH to standard banding techniques in the detection of specific structural and/or numerical chromosomal abnormalities is confirmed in this report.

Amplified c-MYC sequences localized by fluorescence in-situ hybridization on double minute chromosomes in acute myeloid leukemias.

Double minute chromosomes (dmin) are small acentric fragments frequently observed when karyotyping human tumor cells. They are considered the cytogenetic manifestation of gene amplification. The finding of dmin in leukemia is a rare event usually associated with progression of the disease and unfavorable prognosis. We present four patients affected by myeloid disorders with an abnormal karyotype and a variable number of dmin. In an attempt to clarify the origin of the dmin and the amplified gene, we utilized a fluorescent in-situ hybridization (FISH) technique and a panel of specific probes. The results of the analysis indicate that, although chromosomes 8 are apparently uninvolved, dmin retained c-MYC sequencs in three cases. By observing previously reported cases, we found that the majority of patients with myeloid disorders and dmin showed an amplified c-MYC gene, regardless of the chromosomal abnormalities. The FISH technique proved to be informative in demonstrating gene amplification in both metaphase and interphase cells. Finally, in the one patient carrying a 20q deletion, FISH allowed the detection of a previously unreported translocation between a 16p and the 20q-, confirming the ability of the technique to understand complex karyotypes.

Donor lymphocyte infusions (DLI) in patients with chronic myeloid leukemia following allogeneic bone marrow transplantation.

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.