Long-term benefits and risks of frontline nilotinib vs imatinib for chronic myeloid leukemia in chronic phase: 5-year update of the randomized ENESTnd trial.

In the phase 3 Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients (ENESTnd) study, nilotinib resulted in earlier and higher response rates and a lower risk of progression to accelerated phase/blast crisis (AP/BC) than imatinib in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP). Here, patients' long-term outcomes in ENESTnd are evaluated after a minimum follow-up of 5 years. By 5 years, more than half of all patients in each nilotinib arm (300 mg twice daily, 54%; 400 mg twice daily, 52%) achieved a molecular response 4.5 (MR(4.5); BCR-ABL⩽0.0032% on the International Scale) compared with 31% of patients in the imatinib arm. A benefit of nilotinib was observed across all Sokal risk groups. Overall, safety results remained consistent with those from previous reports. Numerically more cardiovascular events (CVEs) occurred in patients receiving nilotinib vs imatinib, and elevations in blood cholesterol and glucose levels were also more frequent with nilotinib. In contrast to the high mortality rate associated with CML progression, few deaths in any arm were associated with CVEs, infections or pulmonary diseases. These long-term results support the positive benefit-risk profile of frontline nilotinib 300 mg twice daily in patients with CML-CP.

Possible benefit of consolidation therapy with high-dose cytarabine on overall survival of adults with non-promyelocytic acute myeloid leukemia

In adults with non-promyelocytic acute myeloid leukemia (AML), high-dose cytarabine consolidation therapy has been shown to influence survival in selected patients, although the appropriate doses and schemes have not been defined. We evaluated survival after calculating the actual dose of cytarabine that patients received for consolidation therapy and divided them into 3 groups according to dose. We conducted a single-center, retrospective study involving 311 non-promyelocytic AML patients with a median age of 36 years (16-79 years) who received curative treatment between 1978 and 2007. The 131 patients who received cytarabine consolidation were assigned to study groups by their cytarabine dose protocol. Group 1 (n=69) received <1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles. The remaining patients received high-dose cytarabine (≥1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles). The actual dose received during the entire consolidation period in these patients was calculated, allowing us to divide these patients into 2 additional groups. Group 2 (n=27) received an intermediate-high-dose (<27 g/m2), and group 3 (n=35) received a very-high-dose (≥27 g/m2). Among the 311 patients receiving curative treatment, the 5-year survival rate was 20.2% (63 patients). The cytarabine consolidation dose was an independent determinant of survival in multivariate analysis; age, karyotype, induction protocol, French-American-British classification, and de novo leukemia were not. Comparisons showed that the risk of death was higher in the intermediate-high-dose group 2 (hazard ratio [HR]=4.51; 95% confidence interval [CI]: 1.81-11.21) and the low-dose group 1 (HR=4.43; 95% CI: 1.97-9.96) than in the very-high-dose group 3, with no significant difference between those two groups. Our findings indicated that very-high-dose cytarabine during consolidation in adults with non-promyelocytic AML may improve survival.

Possible benefit of consolidation therapy with high-dose cytarabine on overall survival of adults with non-promyelocytic acute myeloid leukemia.

In adults with non-promyelocytic acute myeloid leukemia (AML), high-dose cytarabine consolidation therapy has been shown to influence survival in selected patients, although the appropriate doses and schemes have not been defined. We evaluated survival after calculating the actual dose of cytarabine that patients received for consolidation therapy and divided them into 3 groups according to dose. We conducted a single-center, retrospective study involving 311 non-promyelocytic AML patients with a median age of 36 years (16-79 years) who received curative treatment between 1978 and 2007. The 131 patients who received cytarabine consolidation were assigned to study groups by their cytarabine dose protocol. Group 1 (n=69) received <1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles. The remaining patients received high-dose cytarabine (≥1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles). The actual dose received during the entire consolidation period in these patients was calculated, allowing us to divide these patients into 2 additional groups. Group 2 (n=27) received an intermediate-high-dose (<27 g/m2), and group 3 (n=35) received a very-high-dose (≥27 g/m2). Among the 311 patients receiving curative treatment, the 5-year survival rate was 20.2% (63 patients). The cytarabine consolidation dose was an independent determinant of survival in multivariate analysis; age, karyotype, induction protocol, French-American-British classification, and de novo leukemia were not. Comparisons showed that the risk of death was higher in the intermediate-high-dose group 2 (hazard ratio [HR]=4.51; 95% confidence interval [CI]: 1.81-11.21) and the low-dose group 1 (HR=4.43; 95% CI: 1.97-9.96) than in the very-high-dose group 3, with no significant difference between those two groups. Our findings indicated that very-high-dose cytarabine during consolidation in adults with non-promyelocytic AML may improve survival.

Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

ABO genotyping in leukemia patients reveals new ABO variant alleles.

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO* variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

Use of gel microcolumn assay for the detection of drug-induced positive direct antiglobulin tests.

Drugs can result in broad variety of hematologic abnormalities including positive direct antiglobulin test. In this study, we evaluated gel microcolumn assay for the detection of drug-induced antibodies. Direct antiglobulin test was performed by conventional tube and by gel microcolumn assay in 139 hospitalized patients. Drug in vitro studies were done in 34 patients with positive direct antiglobulin test by tube test and gel microcolumn assay using serum and eluate. None of them had signs of hemolytic anemia. A total of 1,000 blood samples from donors were used as control group. Gel microcolumn assay was more sensitive than in tube test for direct antiglobulin test (P<0.01). Positive direct antiglobulin test was more frequent in patients than in donors (P<0.01). Drug in vitro studies were positive with at least one drug in 76.5% of patients with positive direct antiglobulin test by immune complex and/or adsorption mechanisms. We found a high incidence of positive drug in vitro tests in positive direct antiglobulin test patients. Gel microcolumn assay showed appropriate results for drug in vitro studies. The combination of tube and gel microcolumn assay can improve detection of drug-induced positive direct antiglobulin tests.

Comparison of conventional tube test technique and gel microcolumn assay for direct antiglobulin test: a large study.

Gel microcolumn assay (GMA) is a modified serological technique that has been used for ABO and Rh typing, direct antiglobulin test (DAT), detecting alloantibodies, red cell phenotyping, and other applications. However, for DAT, the role of GMA is controversial. The purpose of this large study was to compare the performance of the conventional tube test (CTT) to GMA for detecting potentially significant antibodies coating red blood cells in vivo. From January 1996 to May 2002, we performed DATs by GMA and CTT on 9,862 blood samples submitted to our reference laboratory, using LISS/Coombs cards (DiaMed-Latino America, Lagoa Santa-MG, Brazil) for GMA and polyspecific and monospecific anti-IgG reagents for CTT. Acid eluates were prepared from all positive DAT samples. The specificity of eluates was determined by GMA. We detected nonconcordant results in 2,079 out of 3,163 positive DATs (65.7%). All of these tests were only positive in GMA. Sensitivity and specificity for DATs was 100% and 83.0% for gel, and 50.7% and 97.8% for tube, respectively. Based on this study GMA showed to be more sensitive than CTT for detecting potentially significant antibodies coating red blood cells in vivo.

Comparison of conventional tube test with diamed gel microcolumn assay for anti-D titration.

Anti-D titration is the first step in the evaluation of the RhD-sensitized patient. Traditionally, anti-D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti-D titer needs to be established. Seventy-nine known blood samples with anti-D (titers 1-32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R0r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2-2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti-D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4-fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti-D titration by the gel microcolumn assay.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells.

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

In vitro cytotoxicity of the LDE-daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7 percent of blast cells and 20.2 percent of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues

Immunohematological findings in myelodysplastic syndrome.

BACKGROUND: Few immunohematological studies have been done in myelodysplastic syndrome (MDS). METHODS: Twenty-nine MDS patients were retrospectively evaluated with a direct antiglobulin test (DAT), antibody screening, serum electrophoresis and immunoelectrophoresis. Clinical and laboratory studies (hemoglobin level, reticulocyte count, DHL, total and indirect bilirubin) were done simultaneously, as well as the French-American-British subtype and bone marrow biopsy findings. RESULTS: Alloantibodies were demonstrated in 17 patients (58.6%), autoantibodies in 10 (34.4%) patients and cold agglutinin in 18 (62%) patients. DAT was mediated by only IgG in 8 patients (80%), by IgG and C3 in 1 patient (10%) and by IgG, IgA and C3 in 1 (10%) patient. No hemolytic disease occurred in patients with autoantibodies. Increased serum gammaglobulin was observed in 16 (54.4%) patients. There was no correlation between the incidence of allo-/autoantibodies and the gammaglobulin level (p = 0.937) and the presence of lymphocyte infiltrates in bone marrow biopsies (p = 0.156). No significant difference was observed when the incidence of autoantibodies and number of red blood cell transfusions were compared (p = 0.334). Patients with refractory anemia and refractory anemia with ringed sideroblasts subtypes had a higher incidence of allo-/autoantibodies than other MDS subtypes (p = 0.03). CONCLUSION: Patients with MDS, in particular refractory anemia and refractory anemia with ringed sideroblasts have a high incidence of allo- and autoantibodies, probably related to intrinsic immune disorder, without clinical or laboratory hemolysis.

Easy method for determining the frequency of O(1) and O(2) alleles in Brazilian blood donors by PCR-RFLP analysis.

Serologic ABO blood typing is routinely performed using anti-A and anti-B sera to distinguish four phenotypes (A, B, AB, and O). Restriction fragment length polymorphisms (RFLPs) and DNA sequence studies offer the possibility of direct ABO genotyping. We used polymerase chain reaction-RFLP analysis to determine the frequency of O(1) and O(2) alleles in 82 unrelated blood donors in São Paulo, Brazil, known to be group O. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. Different genotypes (O(1)O(1), O(1)O(2), O(2)O(2)) were identified after digestion with restriction enzymes KpnI, HpaII, and AluI, followed by agarose gel electrophoresis. Of 82 samples analyzed, 74 were O(1)O(1), 7 were O(1)O(2), and 1 was O(2)O(2). These results showed the frequency of O(1)O(1), O(1)O(2), and O(2)O(2) genotypes to be 90.24 percent, 8.53 percent, and 1.22 percent, respectively, in blood donors in São Paulo, Brazil.

Comparison of tube and gel techniques for antibody identification.

There are several methods for antibody detection and each technique has advantages and limitations. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and gel test technique for antibody identification. From January to May 1999, we performed antibody screening tests by gel and tube techniques on 10,123 random blood samples submitted to our reference laboratory. Six hundred and twenty-eight (6.2%) reactive samples were tested for antibody specificity by both methods. One hundred and ninety-six were reactive only by gel: 25 anti-D, 33 anti-C, 76 anti-E, 13 anti-c, 5 anti-e, 18 anti-K, 7 anti-Jka, 2 anti-Dia, 3 anti-S, 8 combination Rh antibodies (1 with anti- K), and 6 other antibody specificities. Two samples were reactive only by PEG-IAT: 1 anti-K and 1 anti-Dia. Four hundred and thirty were positive by the two methods: 156 anti-D, 9 anti-C, 68 anti-E, 15 anti-c, 6 anti-e, 61 anti-K, 12 anti-Jka, 17 anti-Dia, 5 anti-S, 73 combination Rh antibodies (2 with anti-K), and 8 other antibody specificities. Based on this study, the gel test is more sensitive (p <.01) than the tube test for identifying potentially clinically significant antibodies.

Mutational analysis of N-RAS and GAP-related domain of the neurofibromatosis type 1 gene in chronic myelogenous leukemia.

RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.

K phenotyping using a PK-7200 automated analyzer.

K (Kell) is one of the most immunogenic of the red blood cell (RBC) antigens. In order to select K- RBC units, we developed K phenotyping on the Olympus PK-7200 equipment to save labor, time, and costs. The Olympus PK-7200 is fully automated equipment used primarily for blood typing and syphilis screening. We tested 3,587 blood donor samples in EDTA using a commercial anti-K serum diluted in HP Hemagen Power Solution(1:40). The equipment was set to prepare a 1.7% RBC suspension in bromelain and to dispense 25 microL of the mixture (diluted serum and HP Hemagen Power Solution) in terraced microplates. After mixing, the microplates were incubated for 1 hour at 30 degrees C. Reading was performed by a C.C.D. camera and the results were automatically transferred to the mainframe computer. We found 185 K+ blood samples and 3,402 K- samples. Four samples, K+ by the PK-7200, were confirmed as K- by tube test. The use of bromelain with the PK-7200 may have caused the falsely positive tests. The Olympus PK-7200, used for K phenotyping, saves labor time and costs. It also reduces handling and thus promotes less contamination risk for laboratory personnel.

Leptomeningeal involvement in chronic lymphocytic leukemia identified by polymerase chain reaction in stored slides: a case report.

The use of polymerase chain reaction (PCR) for routine detection of clonal immunoglobulin heavy-chain (IgH) gene rearrangements represents an attractive alternative to Southern hybridization analysis not only because PCR protocols are quicker and simpler, but also because of the ability to analyze very small population of cells in search of minimal residual disease. This can be especially important for the detection of clonal malignant cells in locations other than bone marrow or peripheral blood. We describe a case in which central nervous system involvement, a very rare complication of chronic lymphocytic leukemia, was confirmed by PCR analysis for IgH genes rearrangement of the lymphocytes found in cerebrospinal fluid. The cerebrospinal fluid and the peripheral blood lymphocytes (obtained from archival cytospins stored at the time of diagnosis, 5 years before) presented an identical IgH gene rearrangement, as shown by sequence analysis. Thus, the use of PCR for IgH genes rearrangement can be very useful in the detection of monoclonality in samples with a small number of cells and in the confirmation of the common origin of B cells in different specimens of the same patient.

Aspergillary bronchopneumonia: an unusual cause of atelectasis and asphyxia in a leukemic patient.

A 22-year-old man in his first relapse of T-acute lymphoblastic leukemia developed fever and a pulmonary infiltrate after 23 days of granulocytopenia. Although having been under amphotericin B for 10 days, productive purulent cough ensued, with right lobe atelectasis and acute ventilatory failure that resolved after the elimination of a thick gelatinous bronchial plug. Sputum cultures yielded Candida Albicans and Staphylococcus epidermidis, and microscopic examination of the sputum plug disclosed Aspergillus hyphae. The patient died 9 days after, of a disseminated Aspergillus infection, confirmed by necropsy.