Mutations in the p53 gene in acute myeloid leukemia patients correlate with poor prognosis

Inactivation of tumor suppressor genes, whose products exert an inhibitory influence on cell cycle progression, can lead to neoplastic transformation. In acute myeloid leukemia (AML), the frequency of p53 gene mutations ranges from 4 to 15% in populations from USA and Europe. In an attempt to investigate the frequency of point mutations in the p53 gene in AML Brazilian patients, DNA samples of 35 patients were studied using PCR-SSCP techniques, screening exons 4-10. Mutations were identified in bone marrow DNA in 5 of the 35 AML patients (14.3%), a frequency similar to those reported for Northern American and European populations. The overall survival of patients with mutations in the p53 gene was significantly shorter than for patients without mutations.     

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N- ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American- British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.     

Hypodiploidy is associated with a poor prognosis in childhood acute lymphoblastic leukemia

Leukemic cells from 31 (7.6%) of 409 children with newly diagnosed acute lymphoblastic leukemia (ALL) had a hypodiploid karyotype. The patients' ages ranged from 0.8 to 17 years (median, 5 years) and their initial leukocyte counts from 1.0 to 132 X 10(9)/L (median, 12.7 X 10(9)/L). Modal chromosome numbers for the leukemic stem lines were 45 in 26 cases, 28 in two cases, and 26, 36 and 43 in one case each. Seven cases had one to three additional abnormal lines due to clonal evolution. Chromosome 20 was lost most frequently (nine cases). Structural abnormalities--including chromosomal translocations (21 cases), deletions (ten cases), duplications (two cases), or inversions (one case)--were common findings; the nonrandom translocations consisted of the t(1;19)(q23;p13.3) in two pre-B cases and tdic(9;12)(p1?1;p1?2) in three cases of common ALL. When compared with hyperdiploid cases (greater than 50 chromosomes), ALL with hypodiploidy was found to have a poorer outcome and was more likely to be associated with chromosomal translocations, higher serum lactic dehydrogenase levels, and age less than 2 or greater than or equal to 10 years. Moreover, patients with hypodiploid ALL fared as poorly as those with pseudodiploid karyotypes, even though their leukocyte counts and serum lactic dehydrogenase levels were lower and they had a comparable frequency of leukemic cell translocations. Hypodiploidy is therefore an unfavorable karyotypic feature in childhood ALL.     

Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), chromosomes usually evade detailed cytogenetic analyses because cells poorly respond to the traditionally used set of mitogens. We applied novel technologies, such as stimulation of CLL cells either with CD40 ligand or with a combination of CpG-oligodeoxynucleotides and IL-2, to increase the frequency of metaphase spreads for detailed chromosome analysis in 96 patients with CLL. This approach revealed that translocations occurred in 33 of 96 (34%) of our patients with CLL. The presence of translocations defined a new prognostic subgroup because these patients have significantly shorter median treatment-free survival (24 months vs 106 months; P < .001) and significantly inferior overall survival (OS; median, 94 months) than patients without translocations (346 months; P < .001). In multivariate analysis-including Binet stage, complex karyotype, CD38 expression, and 17p deletions-translocation proved to be the prognostic marker with the highest impact for an unfavorable clinical outcome (P < .001). In summary, we identified a new subgroup of patients with CLL defined by chromosomal trans-locations and poor prognosis. Our data may facilitate the identification of molecular events crucial for transforming activity in this disease and should have implications for risk-adapted clinical management of patients with CLL.     

Analysis of N-ras gene mutations in acute myeloid leukemia by allele specific restriction analysis

N-ras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of N-ras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant N-ras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. N-ras mutations were more common in patients over 65 years of age (P less than 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of N-ras mutations in leukemia by ASRA and include the first identification of three concurrent N-ras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of N-ras mutations in leukemogenesis.     

Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age

MLL gene rearrangements are associated with an extremely poor prognosis in infants with acute lymphoblastic leukemia (ALL), but little is known about their clinical significance in older children. Therefore, we studied 45 cases of childhood ALL with abnormalities of chromosome 11q23 for rearrangement of the MLL gene to determine if this feature confers a uniformly poor prognosis. MLL gene rearrangements were detected in all 18 cases with the common t(4;11), t(9;11) or t(11;19) translocations, whereas only 5 of 12 patients with either unbalanced or uncommon balanced translocations demonstrated a rearrangement. Abnormalities of the MLL gene were not detected in any of the 15 cases with a deletion or inversion of the chromosomes 11q23 region. The presence of an MLL rearrangement was significantly associated with age less than 1 year (P < .001), leukocyte count >50 x 10(9)/L (P = .003), and the absence of leukemic cell CD10 expression (P < .001). In a stratified statistical analysis adjusted for age and treatment protocol, MLL gene rearrangement was correlated with an inferior treatment outcome (P = .028). The 4-year event-free survival estimate (+/- SE) was 10% +/- 6.5% for cases with a rearranged MLL gene and 64% +/- 19.2% for other cases. When infants were excluded from the analysis, MLL rearrangement was still significantly associated with a poor outcome (P = .02), and remained so with the exclusion of t(4;11)- positive cases (P = .05). Thus, regardless of presenting age, MLL gene rearrangement identifies a high-risk subgroup of patients who are not likely to be cured with conventional treatment.     

Actinomycin D in childhood acute lymphocytic leukemia.

Eighteen evaluable children who relapsed with acute lymphocytic leukemia (ALL) were treated with intermittent, high-dose actinomycin D. Objective responses occurred in four of 11 children who had relapsed with chemotherapy which did not contain an anthracycline. The major toxic effects included thrombocytopenia and granulocytopenia. Minor toxic effects included nausea, vomiting, skin rash, and stomatitis. The onset of the maculopapular skin rash coincided with the platelet count nadir. These data suggested that actinomycin D is active in ALL.     

Rare occurrence of N-ras point mutations in Philadelphia chromosome positive chronic myeloid leukemia

Point mutations of the N-ras oncogene are relatively common in acute myelogenous leukemia (AML) cells, occurring in some 25% to 50% of patient samples. We used a technique involving the direct nucleotide sequencing of in vitro amplified N-ras genomic fragments to determine the frequency of N-ras point mutations in chronic myeloid leukemia (CML) cells at various stages of the disease. This approach will detect N-ras point mutations in a mixed population of cells if the mutation is present in 25% or more of the cells. We could not demonstrate any point mutation at N-ras codons 12,13 or 59-63 in any of the 44 CML cases analyzed, which included 21 blast crisis samples. In contrast with AML N-ras point mutations are exceedingly rare in CML.     

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.     

Autoimmune cytopenia does not predict poor prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is characterized by an acquired immune defect that can cause autoimmune complications, including anemia and thrombocytopenia. We conducted an observational study of the epidemiology, clinical presentation and significance of autoimmune complications of CLL/SLL in 132 patients from a large population (>45,000 veterans), in which at least 90% of patients with CLL/SLL have been previously identified. Over a period of 12.5 years, 12 patients (9.1%) had autoimmune complications; of these, 6 (4.5%) had autoimmune hemolytic anemia (AIHA), 5 (3.8%) had immune thrombocytopenia (ITP), and 1 (0.8%) had pure red blood cell aplasia (PRBA). All 6 cases of AIHA had a positive direct immunoglobulin test for IgG and C3d. In 6 patients, CLL/SLL was an incidental finding at the time of presentation with autoimmune cytopenia. Nine out of 10 patients responded to immunosuppressive therapy, which was complicated by serious infection in 7 cases, one of which was fatal. The major cause of mortality in patients with autoimmune complications of CLL/SLL was secondary malignancy. Survival of patients with immune cytopenia was not significantly different from CLL/SLL patients without immune cytopenia. Among patients with anemia or thrombocytopenia, mortality was significantly higher in those with bone marrow failure compared to an autoimmune etiology. We show that in a non-referred population with a high incidence of CLL/SLL, autoimmune cytopenia can occur early in the natural history of the disease. These data suggest that the Rai and Binet classifications for CLL need to be modified for patients with autoimmune cytopenia. .     

Near-haploid acute lymphoblastic leukemia: a unique subgroup with a poor prognosis?

We describe two adolescent girls with acute lymphoblastic leukemia (ALL) whose leukemia cells were near-haploid. Their lymphoblasts stained in a block pattern with periodic acid Schiff and had "common ALL" surface markers confirmed by indirect immunofluorescence. Each patient had two populations of blasts, one near-haploid and one hyperdiploid, which was an exact doubling of the near-haploid karyotype. The first patient had a predominant population of cells with 26 chromosomes and a few with 52, while the second had a predominance of cells with 56 and a minority with 28. Flow cytometric analysis of DNA content initially detected the minor near-haploid population in the second patient, which was confirmed later by cytogenetic review of the marrow sample. In addition to our two patients, only four patients have been reported with near-haploid ALL. Of these six, five were girls, five were adolescents, and five had short survivals (median, 10 mo). All six had disomy of chromosome 21 with or without disomy for chromosomes 10, 14, 18, or X (four patients each). Thus, near-haploid ALL may represent a unique subgroup of ALL with a poor prognosis. To detect these and other possible subgroups, we have included cytogenetic analysis and flow cytometric analysis of DNA content in our initial evaluation of patients with ALL.     

RUNX1 mutations in cytogenetically normal acute myeloid leukemia are associated with a poor prognosis and up-regulation of lymphoid genes

Background

The RUNX1 (AML1) gene is a frequent mutational target in myelodysplastic syndromes and acute myeloid leukemia. Previous studies suggested that RUNX1 mutations may have pathological and prognostic implications.

Design and Methods

We screened 93 patients with cytogenetically normal acute myeloid leukemia for RUNX1 mutations by capillary sequencing of genomic DNA. Mutation status was then correlated with clinical data and gene expression profiles.

Results

We found that 15 out of 93 (16.1%) patients with cytogenetically normal acute myeloid leukemia had RUNX1 mutations. Seventy-three patients were enrolled in the AMLCG-99 trial and carried ten RUNX1 mutations (13.7%). Among these 73 patients RUNX1 mutations were significantly associated with older age, male sex, absence of NPM1 mutations and presence of MLL-partial tandem duplications. Moreover, RUNX1-mutated patients had a lower complete remission rate (30% versus 73% P=0.01), lower relapse-free survival rate (3-year relapse-free survival 0% versus 30.4%; P=0.002) and lower overall survival rate (3-year overall survival 0% versus 34.4%; P<0.001) than patients with wild-type RUNX1. RUNX1 mutations remained associated with shorter overall survival in a multivariate model including age and the European LeukemiaNet acute myeloid leukemia genetic classification as covariates. Patients with RUNX1 mutations showed a unique gene expression pattern with differential expression of 85 genes. The most prominently up-regulated genes in patients with RUNX1-mutated cytogenetically normal acute myeloid leukemia include lymphoid regulators such as HOP homeobox (HOPX), deoxynucleotidyltransferase (DNTT, terminal) and B-cell linker (BLNK), indicating lineage infidelity.

Conclusions

Our findings firmly establish that RUNX1 mutations are a marker of poor prognosis and provide insights into the pathogenesis of RUNX1 mutation-positive acute myeloid leukemia.Key words: RUNX1, mutations, prognosis, acute myeloid leukemia     

Nucleophosmin mutations in childhood acute myelogenous leukemia with normal karyotype

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein involved in leukemia-associated chromosomal translocations, and it regulates the alternate reading frame (ARF)-p53 tumor-suppressor pathway. Recently, it has been demonstrated that mutations of the NPM1 gene alter the protein at its C-terminal, causing its cytoplasmic localization. Cytoplasmic NPM was detected in 35% of adult patients with primary non-French-American-British (FAB) classification M3 acute myeloid leukemia (AML), associated mainly with normal karyotype. We evaluated the prevalence of the NPM1 gene mutation in non-M3 childhood AML patients enrolled in the ongoing Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP-AML02) protocol in Italy. NPM1 mutations were found in 7 (6.5%) of 107 successfully analyzed patients. NPM1-mutated patients carried a normal karyotype (7/26, 27.1%) and were older in age. Thus, the NPM1 mutation is a frequent abnormality in AML patients without known genetic marker; the mutation may represent a new target to monitor minimal residual disease in AML and a potential candidate for alternative and targeted treatments.     

FLT3 mutations in childhood acute lymphoblastic leukemia

Activating mutations of the FLT3 receptor tyrosine kinase are common in acute myelogenous leukemia (AML) but are rare in adult acute lymphoblastic leukemia (ALL). We have recently shown that FLT3 is highly expressed and often mutated in ALLs with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Because hyperdiploid ALL samples also show high-level expression of FLT3, we searched for the presence of FLT3 mutations in leukemic blasts from 71 patients with ALL. The data show that approximately 25% (6 of 25) of hyperdiploid ALL samples possess FLT3 mutations, whereas only 1 of 29 TEL/AML1-rearranged samples harbored mutations (P =.04, Fisher exact test). Three mutations are novel in-frame deletions within a 7-amino acid region of the receptor juxtamembrane domain. Finally, 3 samples from patients whose disease would relapse harbored FLT3 mutations. These data suggest that patients with hyperdiploid or relapsed ALL might be considered candidates for therapy with newly described small-molecule FLT3 inhibitors.