MLL Partial Tandem Duplication Research Articles

Long-term disease-free survivors with cytogenetically normal acute myeloid leukemia and MLL partial tandem duplication: a Cancer and Leukemia Group B study

AbstractThe clinical impact of MLL partial tandem duplication (MLL-PTD) was evaluated in 238 adults aged 18 to 59 years with cytogenetically normal (CN) de novo acute myeloid leukemia (AML) who were treated intensively on similar Cancer and Leukemia Group B protocols 9621 and 19808. Twenty-four (10.1%) patients harbored an MLL-PTD. Of those, 92% achieved complete remission (CR) compared with 83% of patients without MLL-PTD (P = .39). Neither overall survival nor disease-free survival significantly differed between the 2 groups (P = .67 and P = .55, respectively). Thirteen MLL-PTD+ patients relapsed within 1.4 years of achieving CR. MLL-PTD+ patients who relapsed more often had other adverse CN-AML–associated molecular markers. In contrast with previously reported studies, 9 (41%) MLL-PTD+ patients continue in long-term first remission (CR1; range, 2.5-7.7 years). Intensive consolidation therapy that included autologous peripheral stem-cell transplantation during CR1 may have contributed to the better outcome of this historically poor-prognosis group of CN-AML patients with MLL-PTD.

A comparative study of molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 patients with acute myeloid leukemia

The precise relationship between myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is unclear and the role of molecular mutations in leukemic transformation in MDS is controversial. The aim of this study was to clarify the relationship between AML and MDS by comparing the frequency of molecular mutations in the two conditions. We compared the frequency of FLT3-length mutations (FLT3-LM), FLT3-TKD, MLL-partial tandem duplications (MLL-PTD), NRAS, and KITD816 in 381 patients with MDS refractory anemia with excess blasts [RAEB] n=49; with ringed sideroblasts [RARS] n=310; chronic monomyelocytic leukemia [CMML] n=22) and in 4130 patients with AML (de novo: n=3139; secondary AML [s-AML] following MDS: n=397; therapy-related [t-AML]: n=233; relapsed: n=361). All mutations were more frequent in s-AML than in MDS and all but the FLT3-TKD were more frequent in RAEB than in RA/RARS. The higher incidences in s-AML were significant for FLT3-TKD (p=0.032), MLL-PTD (p=0.034), and FLT3-LM (RA/RARS: 0/45; RAEB: 8/293; 2.7%; s-AML: 45/389; 11.6%; p<0.0001). The incidence of NRAS-mutations increased from 17/272 (6.3%) in MDS to 41/343 in s-AML (12.0%) and that of KITD816-mutations from 2/290 (0.7%) to 5/341 (1.5%) (p=n.s.). FLT3-LM-acquisition occurred in 3/22 cases (13.6%) during MDS transformation; NRAS-acquisition occurred in 1/24 (4.2%). FLT3-LM and MLL-PTD were more frequent in AML relapse than in de novo AML or s-AML (p<0.0001). The increase of molecular mutations from low- to high-risk MDS, to s-AML, and to relapsed AML emphasizes the value of these mutations as markers of progressing disease. Finally, we found a low rate of 5q- in the molecularly mutated cases in MDS which might explain the stability of this subtype.

Mutations of the TP53 Gene Occur in 13.4% of Acute Myeloid Leukemia and Are Strongly Associated with a Complex Aberrant Karyotype.

The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others.Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p<0.001). They occur frequently in particular in the subgroup showing a typical pattern of chromosomal deletions (5q, 7q, 17p). TP53 mutations might explain in part the chemoresistance of AML with complex aberrant karyotype. In addition to cytogenetics a rapid diagnostic screening for TP53 mutations could be a valuable tool to identify a subgroup of AML with poor prognosis. This would allow the early assignment of patients to alternative treatment strategies using also options targeting the TP53 pathway.

Different Patterns of Cooperating Mutations between De Novo AML Patients with MLL-Partial Tandem Duplication and MLL Translocations.

Two-hit model of leukemogenesis has been proposed for AML. In AML with MLL rearrangements (MLL-R), MLL gene is fused to a variety of partner genes through reciprocal chromosomal translocations (MLL/t11q23), or is rearranged to generate a partial tandem duplication (MLL-PTD). The cooperating mutations of AML with MLL-R have not been systematically analyzed. We aimed to determine the cooperating mutations, including receptor tyrosine kinase (RTK) /Ras signaling pathway, NPM1 and myeloid transcription factors in de novo AML with MLL-R. MLL-R was screened by Southern blot analysis. RT-PCR was used to detect common MLL fusion transcripts. cDNA panhandle PCR was used to identify the infrequent or unknown MLL partner genes. Mutational analysis was peformed by DNA/cDNA PCR-GeneScan analysis for FLT3/ITD, by PCR-RFLP followed by direct sequencing for FLT3/TKD, by DNA/cDNA PCR and direct sequencing for N-Ras, K-Ras, c-KIT, c-FMS, PTPN11, NPM1, AML1 and CEBPα. Of the 131 patients with MLL-R, 77 had MLL-PTD and 54 had MLL/t11q23. None of the 131 patients with MLL-R had c-FMS mutations and c-KIT mutation was present in only one patient with MLL/t11q23. NPM1 mutations occurred in one with MLL-PTD and 2 with MLL/t11q23. The frequencies of other cooperating mutations are shown in Table 1. Taken together, cooperating mutations involving RTK/Ras pathway, NPM1, and/or myeloid transcription factors occurred in 71.4% (55/77) of patients with MLL-PTD and 59.3% (32/54) of patients with MLL/t11q23. In MLL-PTD group, coexistence of two mutations occurred in 23 patients. In MLL/t11q23 group, 6 patients had two mutations. Of the 18 patients with MLL-PTD and AML1 mutations, 8 mutations were located in the Runt homology domain (RHD) and 10 in the non-RHD, 15 were frameshift or nonsense mutations and 3 were missense mutations. Fourteen patients with MLL-PTD and AML1 mutations also had mutations of RTK/Ras singling pathway. Three patients with MLL/t11q23 and AML1 mutations, one in the RHD and 2 in the non-RHD, all were missense mutations. Of the 5 patients with MLL-PTD and CEBPα mutations, 3 harbored FLT3/ITD. Patients with MLL-PTD had a significantly higher frequency of cooperating mutations with myeloid transcription factors than patients with MLL/t11q23 (20/77 vs. 3/54, P=0.002), whereas there was no difference in the frequency of mutations involving RTK/Ras pathway between MLL-PTD and MLL/t11q23 groups (51/77 vs. 29/54, P=0.202). Our results showed that patients with de novo AML and MLL-R had a high frequency of cooperating mutations with RTK/Ras signaling pathway, NPM1 or myeloid transcription factors, and the mutation patterns were different between MLL-PTD and MLL/t11q23 groups.Table 1Comparison of cooperating mutations between MLL-PTD and MLL/t11q23 groupsFLT3/ITDFLT3/TKDN-RasK-RasPTPN11AML1CEBPαMLL-PTD35/7711/775/770/773/7718/775/77MLL/t11q232/547/549/5413/541/533/540/54P value<0.00011.0000.085<0.00010.6480.0070.077

The Prognosis of Karyotypic Subgroups in Pediatric Acute Myeloid Leukemia Depends on the Alterations of KIT, MLL and FLT3 Genes: A Report from Japanese Childhood AML Cooperative Study Group.

Karyotypic abnormalities are associated with a prognosis of acute myeloid leukemia (AML). However, there are some patients who had poor prognosis among patients with the same karyotypic abnormalities. Recently, it is revealed that alterations of the genes including tyrosine kinases, lead to poor prognosis in AML. Genetic alterations associated with a poor prognosis remain to be clarified in karytotypic subgroups of AML. We performed the mutation analysis of FLT3, MLL, KIT, RAS and NPM in 158 pediatric AML patients who were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, including 33 with normal karyotype, 46 with t(8;21), 7 with inv(16), 20 with 11q23 translocations, 13 with t(15;17), 10 with Down syndrome (DS) FAB-M7 and 29 other karyotypic abnormalities. In the 33 patients with normal karyotype, 9 FLT3-internal tandem duplication (ITD), 2 FLT3-D835 mutation (D835Mt), 8 MLL-partial tandem duplication (PTD), 2 KIT, 2 NRAS and 3 KRAS mutations were identified. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Notably, NPM gene mutation was not identified in these patients. In the 46 patients with t(8;21), 2 FLT3-ITD, 1 D835Mt, 4 MLL-PTD, 8 KIT, 4 NRAS and 5 KRAS mutations were found. KIT mutations were associated with a poor prognosis. In the 7 patients with inv(16), 2 FLT3-D835Mt and 1 KIT mutations were identified. Only one patient with KIT mutation relapsed. In the 20 patients with 11q23 translocations, 1 D835Mt and 5 MLL-PTD were identified. MLL-PTD was associated with a poor prognosis in patients with 11q23 translocations. In the 13 patients with t(15;17), 3 FLT3-ITD and 3 D835Mt were found. FLT3-ITD was not associated with a poor prognosis. In the 10 patients with DS-M7, KIT and KRAS mutations were found in each one patient, showing no prognostic significance. In the 29 patients with other karyotypic abnormalities, 6 FLT3-ITD, 2 D835Mt, 4 MLL-PTD, 0 KIT, 2 NRAS and 2 KRAS mutations were found. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Fifteen patients (9.5%) who had alterations of 2 of these 4 genes showed a poor prognosis. RAS gene mutations were found in 26 (16.5%) of 158 patients, but were not associated with the prognosis. These results suggest that a new different therapeutic strategy for the patients with these gene alterations in each subgroup, for example KIT mutations in t(8;21)-patients and MLL-PTD in patients with 11q23 translocation, is needed to improve the prognosis of pediatric AML.

The MLL partial tandem duplication in acute myeloid leukaemia

Mixed lineage leukaemia gene-partial tandem duplications (MLL-PTD) characterise acute myeloid leukaemia (AML) with trisomy 11 and AML with a normal karyotype. MLL-PTD confer a worse prognosis with shortened overall and event free survival in childhood and adult AML. In spite of these clinical observations, the leukaemogenic mechanism has, so far, not been determined. This review summarises clinical studies on MLL-PTD positive AML and recent experimental findings on the putative leukaemogenic role of MLL-PTD.

Mll partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll(PTD/WT) mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll(PTD/WT) mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.

Lifelong persistence of AML associated MLL partial tandem duplications (MLL-PTD) in healthy adults

AML-associated MLL-PTD contribute to leukemogenesis by a gain of function and confer an unfavorable prognosis. Like other leukemia associated aberrations they are also present in healthy adults. To delineate the leukemogenic mechanism we tracked down MLL-PTD in normal hematopoiesis and investigated cord blood samples. MLL-PTD were observed in 56/60 (93%) of all cord bloods. In contrast to AML, the transcript frequency in cord blood was four log scales lower as determined by real-time PCR. The CD34+ progenitor cell, CD33+ myeloid, CD19+ B-lymphoid and CD3+ T-lymphoid subfractions were positive. The ubiquitous presence of MLL-PTD in cord blood implicates a lifelong exposure, not an accumulation during lifetime. Since also present in the stem cell subfraction, these factors seem not to be major determinants in MLL-PTD leukemogenesis.

Study on Clinical and Biological Characteristics of Childhood Acute Leukemia with MLL Gene Rearrangements.

The rearrangement of MLL gene is reported in 70%~80% of infant and in 5%~10% of older children (under the age of 15) with acute leukemia (AL). The biological features associated with alterations in MLL gene are hyperleukocytosis, CD10−/CD19+ phenotype and very poor prognosis. To explore the MLL rearrangement in details in our AL children patients and obtain more information on the relationship between the MLL gene abnormality and clinical outcomes. The following study has been conducted. A total of 298 patients with AL attended The Affiliated Children's Hospital of Soochow University, including 16 cases with MLL rearrangements, were recruited in this study. Of the cohort, 11 were diagnosed as ALL, 5 were AML. 9 of 16 patients were in infant age (up to 2 year) and the rest were between the age of 2 to 13 years. Fluorescence in situ hybridization (FISH) analysis using LSI MLL dual color probe. Multiplex reverse transcriptase- polymerase chain reaction (multiplex RT-PCR) were used to discriminate 13 different fusion transcripts. These results were analyzed together with R banding karyotyping and immunolphenotyping determined by flow cytometry. We have found MLL rearrangements in 16 cases of childhood AL which were accounted for 5.4% of 298 AL patients, and 56.3% of infant AL. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangement was found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6, MLL/ELL combined with MLL/AFX or HOX11 in one of each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1. In addition an activated HOX11 gene was found in 1 case.. In 27 cases assayed by FISH, MLL gene rearrangements have been detected in 9 cases (36.0%). In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases, presenting as t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), and t(9;11) in one of each, respectively, trisomy 11 in 2 and 11q- in 3 cases. Among these 16 patients, 11 were B-ALL, including Pro-B and Pre-B ALL; 5 of AML-M5, 3 of these 5 M5 patients were CD7+ and CD2+. Of these 16 patients 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients eventually gave up treatment. Our results demonstrated that multiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements, including chromosome translocation, deletion and duplication. Our findings lead to the detection of novel rearrangements at molecular genetic level. These findings regarding the MLL rearrangement provide most important information in guiding therapy and predicting prognosis in childhood AL. Besides our results also provide evidence in support of the value of 11q23/MLL in WHO classification categories.

Tandem Duplications of the FLT3 and MLL Are Independent Poor Prognostic Factors in Pediatric Acute Myeloid Leukemia. AML99 Study in Japan.

To find poor prognostic factors other than karyotypes is desired to improve the treatment outcome in pediatric acute myeloid leukemia (AML). Total three hundread and eighteen patients were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, from January 2000 to December 2002. We performed the mutation analysis of FLT3 and MLL in 158 patients (13 of 29 with FAB-M3, 10 of 49 with Down syndrome, 135 of 240 with other type of AML) Patients with FAB-M3 and patients with Down syndrome were treated on different protocol. Seventeen (12.6%) of 135 patients had FLT3-internal tandem duplication (ITD). The differences between AML patients with or without FLT3-ITD were significant in 4-year overall survival (OS) (35.3% vs. 86.1%, p<0.00001), disease free survival (DFS) (38.5% vs. 68.1%, p=0.0013), and relapse rate (RR) (53.8% vs. 28.5%, p=0.0029). Furthermore, MLL-partial tandem duplication (PTD) was found in 21 (15.6%) of 135 patients. The differences between patients with or without MLL-PTD were significant in 4y-OS (56.3% vs. 81.4%, p=0.013), DFS (41.7% vs. 68.3%, p=0.017), and RR (53.9% vs. 27.5%, p=0.0078). Out of 18 patients with MLL-PTD who received allo-SCT, 9 patients survived (6 of 8 in 1st CR, 3 of 4 in 2nd CR, 0 of 6 in non CR). Out of 12 patients with FLT3-ITD who received allo-SCT, 4 patients survived (4 of 6 in 1st CR, 0 of 6 in non CR). Allo-SCT is considered to be effective for the patients with TD of both genes. Coduplication of both genes was observed in 3 patients. FLT3 kinase domain mutation was found in 7.0% and MLL translocations were found in 11.4% of the patients, but both aberrations did not show poor outcome in this study. FLT3-ITD and MLL-PTD were found in 9 (25.7%) and 8 (22.9%) of 35 patients with normal karyotype. FLT3-ITD and MLL-PTD are considered to be independent poor prognostic factors of pediatric AML patients, especially in patients with normal karyotype. The different therapeutic approach including allo-SCT will be needed for these patients.

Differential Antigenic Profile of High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Expressed by 11q23-Positive Acute Leukemia: An Immunotherapeutic Target.

The poor clinical response of 11q23-positive [also known as mixed lineage leukemia (MLL)] acute leukemia (AL) to chemotherapy-containing regimens has stimulated interest in developing alternative therapeutic strategies. Among them is immunotherapy. Since no leukemia-specific antigen has been identified in 11q23-positive AL, we are developing an antibody-based immunotherapeutic strategy which targets the HMW-MAA. This antigen, which is a membrane bound proteoglycan, represents an attractive target because of its high expression on the surface of 11q23-positive AL blasts and its restricted distribution in normal tissues. Taking advantage of a unique panel of monoclonal antibodies (mAb) recognizing 7 distinct and spatially distant antigenic determinants, we have analyzed the antigenic profile of HMW-MAA by flow cytometry in samples from 15 adult and 14 pediatric patients with 11q23-positive AL. Our results demonstrate a differential expression of the HMW-MAA antigenic determinants and that their expression pattern correlates with cytogenetic subgroups. Specifically, all the determinants were expressed on 6 adult samples [3 t(11;19)(q23;p13), 2 t(4;11)(q21;q23), and 1 t(10;11)(p12;q23)]. In contrast only 3 determinants were detected on 8 adult samples [3 t(9;11)(p22;q23), and 1 each with t(6;11)(q27;q23), t(11;12)(q23;q13), t(11;14)(q23;p11.2), inv(11)(q21q23.2) and add(11)(q23)]. No antigenic determinant was detected on leukemic cells from the adult patient with t(2;11)(p21;q23). Interestingly, the antigenic profile of HMW-MAA expressed on leukemic cells from pediatric patients was different, since all the determinants were expressed on leukemic cells from 6 t(4;11), 1 t(9;11), 1 t(11;1)(1;13;9)(q23;q25p34;q14.3;p13), 2 t(11;19) and 1 del(11)(q14q23). On the other hand no determinant was detectable on the leukemic cells from 3 children [1 with both t(1;11)(p32;q23) and t(4;11), 1 inv(11)(p15q23) and 1 add(11)(q23)]. Whether the difference in the antigenic profile of HMW-MAA expressed by adult and pediatric 11q23-positive AL cells reflects the different pathogenesis of AL in adults and children remains to be determined. Our data show that the differential expression of antigenic determinants of HMW-MAA on 11q23-positive AL cells does not reflect structural differences in the HMW-MAA expressed by various types of 11q23-positive AL as indicated by the results of Western blotting analysis. Further, the differential expression does not correlate with MLL gene rearrangement, since fluorescent in-situ hybridization (FISH) performed on 15 adult samples detected MLL gene rearrangement in 4 of the 6 samples that express all the determinants and in 6 of the 8 samples that express only 3 determinants. In addition, the pediatric sample with inv(11) that does not express any determinant, has MLL gene rearrangement by FISH. Finally, the differential expression does not correlate with the presence of MLL partial tandem duplication, since it was detected in 1 sample that expresses all the antigenic determinants and in 2 samples that express only 3 determinants. These findings emphasize the need to use more than one HMW-MAA-specific mAb to phenotype 11q23-positive AL and to select patients to be treated with HMW-MAA-specific antibody-based immunotherapy.

Overexpression of the ETS-Related Gene, ERG, Predicts a Worse Outcome in Acute Myeloid Leukemia With Normal Karyotype: A Cancer and Leukemia Group B Study

To test the prognostic significance of ETS-related gene (ERG) expression in cytogenetically normal primary acute myeloid leukemia (AML). Pretreatment blood samples from 84 cytogenetically normal AML patients aged less than 60 years, who were characterized for BAALC expression, FLT3 internal tandem duplication (ITD), and MLL partial tandem duplication (PTD) and uniformly treated on Cancer and Leukemia Group B 9621 protocol, were analyzed for ERG expression by real-time reverse transcriptase polymerase chain reaction. Patients were divided into quartiles according to ERG levels and were compared for clinical outcome. High-density oligonucleotide arrays were used to identify genes differentially expressed between high and low ERG expressers. With a median follow-up of 5.7 years, patients with the upper 25% of ERG expression values had a worse cumulative incidence of relapse (CIR; P < .001) and overall survival (OS; P = .011) than the remaining patients. In a multivariable analysis, high ERG expression (P < .001) and the presence of MLL PTD (P = .027) predicted worse CIR. With regard to OS, an interaction was observed between expression of ERG and BAALC (P = .013), with ERG overexpression predicting shorter survival only in low BAALC expressers (P = .002). ERG overexpression was an independent prognostic factor even when the unfavorable group of FLT3 ITD patients lacking an FLT3 wild-type allele was included. High ERG expression was associated with upregulation of 112 expressed-sequenced tags and named genes, many of which are involved in cell proliferation, differentiation, and apoptosis. ERG overexpression in AML patients with normal cytogenetics predicts an adverse clinical outcome and seems to be associated with a specific molecular signature.

The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy

MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLL(PTD/WT) genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLL(PTD/WT) blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.

Clues for Novel Pathways in Leukemogenesis: Global Gene Expression Profiling in Acute Myeloid Leukemia Patients Negative for a Comprehensive Series of Molecular Alterations.

Acute leukemias are remarkably heterogeneous as evidenced by the increasing number of recurring genetic and epigenetic abnormalities, especially the presence or absence of balanced translocations, which have been shown to be of independent prognostic significance. Thus, In a recent single center study we analyzed 250 AML patients for a series a genetic alteration and found that balanced translocations (identified by a multiplex PCR reaction; Pallisgaard et al, BLOOD, 1998 and Olesen et al, Brit. J. Hem. in press) were present in 17% of the patients, FLT3 internal tandem duplication in 24%, and MLL partial tandem duplication in 4%. In addition, we delineated the presence of promoter hypermethylation for the p15 (71%), MDR1 (4%), E-cadherin (CDH1) (64%), and Estrogen receptor (ER) (40%) genes by bisulfite DGGE (Aggerholm, Cancer Res. 1999) as well as the abnormal expression by RQ-PCR of genes related to increased chemotherapy resistance (MDR1 and MRP1) as well as resistance to undergo apoptosis (FAS, Bcl2, BAX and CASPASE3). Here we have performed gene expression profiling focusing on patients negative for all these molecular lesions. Out of the 250 patients, 8 were determined to be molecularly negative. Global gene expression analysis (Affymetrix human genome chip (U133A)) was performed on the 6 samples, from which high-quality RNA could be harvested. [Display omitted] As will be seen from the Figure, all samples could be easily distinguished from TEL/AML pre-B ALL samples, and also in 4/6 cases clustered differently from AML/ETO+ and CBF/MYH11+ AML groups. Interestingly, as seen from the dendrogram, the 6 samples separated in three clusters, one also containing a CBF/MYH11+ patient (4 cases), one consisting of one single patient, while the last patient clustered together with the AML/ETO cases. While these data suggest that the gene expression in the molecularly negative patients can be different from that in CBF leukemias, it gives no clues for the leukemogenetic events in these patients. To that end, we analyzed the gene expression data and found 24 genes to be more than 5-fold overexpressed and 26 genes to be underexpressed compared to CBF AMLs. Of special notice was the increased usage of the prostaglandin/leukotriene metabolism pathway in two patients with strikingly similar global gene expression (arrows 3 and 4 from right on Fig.) including the prostaglandin I2 (prostacyclin) synthase, and the prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) genes, which were more than 10-fold increased. Also noteworthy was the upregulation of LR8 protein IQ motif containing GTPase activating protein 1 in the remaining 4 cases. Applied together with novel global cytogenetic techniques, these data will form a platform for the identification of leukemogenesis in AML patients with no demonstrable genetic aberrations.ßßß

Chemotherapy-Induced T-MDS/AML-Associated Genetic Aberrations.

Therapy-related solid and hematological neoplasias occur according to the treatment regimen and intensity. The cumulative incidence of therapy related myelodysplastic syndromes and acute myeloid leukemias(t-MDS/AML) ranges between 5 and 15% in non-myeloablative and myeloablative treatment protocols respectively. t-MDS/AML share characteristic genetic aberrations which include translocations (e.g. involving 11q23, MLL and t(9;22), inv 16, t(8;21), t(3;21), t(15;17) and cytogenetic aberrations (e.g. 5q-, 7q-). We have investigated if these genetic aberrations are present in patients who underwent chemotherapy. Since we and others have detected aberrations in aleukemic hematopoiesis (Baesecke et al. Blood . 2002; 100:2267–2268) we also wanted to determine if t-MDS/AML early recognition can be performed by this screening. We enrolled patients with Non Hodgkin Lymphoma, either treated by conventional or high dose chemotherapy, included in the MegaChoep protocoll of the German high grade lymphoma study group. Samples were taken as fresh peripheral blood stem cells (PBSCT), bone marrow aspirates (BM) or peripheral blood (PB) after informed consent according to the convention of Helsinki. PBSC of healthy adult donors and cord blood of healthy newborn were used as control. Blood samples were submitted to RT- or Real-Time PCR (sensitivity 10–4 to 10–5) of t(9;22), inv 16, t(8;21), t(15;17) and MLL-partial tandem duplications (MLL-PTD). RNA preparation, reverse transcription, and PCR were performed in separate laboratories. Cytogenetic analysis was performed by FISH (probes EGR1-del5, p53, cep7-del 7, MLL and cep8-tris 8). The results of the ongoing study are as follows. In the cord blood control group, positive samples were t(15;17) 1.7% (1/60 samples), inv 16 5.6% (3/54), t(8;21) 2.8% (1/36), t(9;22) 3.4% (2/59), MLL-PTD primary PCR 0% (0/34). No positive results were observed in PBSC samples of healthy donors (22 samples). 53 samples of patients of which 21 were PBSC and 32 were BM or PB have been investigated so far. 34/53 patients (64.2%) underwent a high dose chemotherapy. In 9.4% (5/53) the translocation t(15;17) was detected. All five samples were PBSC. No amplification of inv 16, t(8;21) and t(9;22) was observed. In FISH analysis two of 18 patient PBSC samples (11%) exhibited aberrations in the p53 locus, which were classified as abnormal but still non-clonal. Our results confirm the existence of AML-associated translocations in cord blood at frequencies, which by far exceed the incidence of AML in healthy individuals. Compared to these findings the incidence of t-AML- associated aberrations in patients who underwent chemotherapy-induced genotoxic stress is lower than expected. Positive PCR-results of this group may thus be more informative concerning the detection of a leukemic clone and the risk of attracting a t-MDS/AML. Positive patients are currently under observation and the number of samples and aberrations in the ongoing study is increased.

High BAALC Expression as an Independent Adverse Risk Factor in 309 Patients with Acute Myeloid Leukemia (AML) and Normal Cytogenetics: Results of the SHG'96 Study.

High mRNA expression levels of the human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) have been shown to be an adverse risk factor in newly diagnosed AML patients (pts; Blood 2003; 102:1613). The first study included 86 de novo AML pts under the age of 60 with normal cytogenetics and a more favorable FLT3 mutation status, which were uniformly treated on the Cancer And Leukemia Group B protocol 9621. An independent confirmation of these data is necessary, to confirm the impact of BAALC expression in intermediate risk AML pts. Here we present the results of a Süddeutschen Hämoblastosegruppe (SHG) study including 309 adult pts diagnosed with de novo (n=280) and secondary AML (n=29), normal cytogenetics, age <60 years that were uniformly treated on the SHG'96 protocol. Treatment consisted of 2 cycles of induction therapy followed by intensive consolidation including autologous as well as allogeneic stem cell transplantation. Median follow-up for patients alive was 28.7 months (range: 0–86 months). BAALC expression was measured in pretreatment peripheral blood blasts by comparative real-time RT-PCR. Pts having BAALC expression values within the lower 50% were classified as low BAALC and pts having BAALC expression values within the upper 50% were classified as high BAALC. Whereas no correlation was seen between BAALC expression and the prevalence of FLT3 internal tandem duplications (ITD; P=0.16), high BAALC patients had a significantly higher FLT3 mutant/wild-type (wt) ratio as determined by Genescan analysis (mean mutant/wt ratio: 0.59 vs. 0.17; P=0.012). There was no significant difference in the prevalence of MLL partial tandem duplications between the high and the low BAALC group. High BAALC expression was significantly more frequent in FAB groups M0/M1 as compared to the other subtypes (P=0.002). Pts with high BAALC expression levels had a significantly lower CR rate (62.3% vs. 73.5%; P=0.039) and a higher relapse rate (43.8% vs. 28.9%; P=0.030). The overall survival (OS) was significantly shorter for pts with high BAALC expression (OS at 4 years: 29.8% vs. 53.3%; P=0.0018), event-free survival (EFS, at 4 years: 21.6% vs. 45.8%; P=0.0001), and disease-free survival (DFS, at 4 years: 30.4% vs. 57.6%; P=0.0018). Pts with a high FLT3 mutant/wt ratio (greater than 0.8) had a significantly shorter OS and DFS. For patients with a low FLT3 mutant/wt ratio, high BAALC expression allowed identification of pts with a high risk of treatment failure. A multivariate analysis confirmed high BAALC expression and a high mutant/wt FLT3 ratio as the only independent adverse risk factors. For pts with high BAALC expression the hazard ratio of death was 1.7 (95% CI 1.18–2.48; P=0.005), and the hazard ratios for EFS and DFS were 1.8 (95% CI 1.3–2.6; P=0.0003) and 2.0 (95% CI 1.2–3.3; P=0.004), respectively. In conclusion, this independent and larger study strengthens high BAALC expression as one of the most relevant adverse prognostic risk factors in intermediate risk AML pts with normal cytogenetics. Thus, determination of BAALC expression should be considered for risk adaptive treatment strategies.

CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations.

To assess the prognostic relevance of mutations in the CEBPA gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha) in a large prospective series of younger adults with acute myeloid leukemia (AML) and normal cytogenetics. The entire CEBPA coding region was sequenced in diagnostic samples from 236 AML patients 16 to 60 years of age with normal cytogenetics who were uniformly treated on two consecutive protocols of the AML Study Group Ulm, and CEBPA mutation status was correlated with clinical outcome. CEBPA mutations were detected in 36 (15%) of 236 patients. Twenty-one (9%) of 236 patients had mutations predicted to result in loss of C/EBP alpha function. Remission duration and overall survival (OS) were significantly longer for the 36 patients with CEBPA mutations (P =.01 and P =.05, respectively). On multivariate analysis, wild-type CEBPA was an independent prognostic marker affecting remission duration (hazard ratio, 2.85; P =.01) and OS (hazard ratio, 1.87; P =.04). Analysis of cooperating mutations (both types of activating FLT3 mutations and MLL partial tandem duplications) showed that FLT3 mutations had no significant prognostic influence in patients with CEBPA mutations. Furthermore, there was no significant overlap between the subgroup of patients with CEBPA mutation with predicted loss of C/EBP alpha function and patients with FLT3 or MLL mutations, suggesting that CEBPA loss-of-function mutations define a distinct biologic subclass of AML with normal cytogenetics. Mutant CEBPA predicts favorable prognosis and may improve risk stratification in AML patients with normal cytogenetics.

Malignant hematopoietic cell lines: in vitro models for the study of MLL gene alterations.

Human tumor cell lines are powerful tools for investigating basic and applied aspects of cell biology. Leukemia-lymphoma cell lines have been instrumental in the cytogenetic and molecular analysis of recurring chromosome rearrangements, notably translocations and inversions, thus illuminating the pathogenesis of hematological malignancy. Chromosomal translocations targeting the MLL gene at 11q23 have come to represent a paradigm in acute leukemias. These translocations result in the in-frame joining of the MLL gene with a partner gene to generate unique fusion proteins of putatively novel function. More than 30 partner genes that participate with MLL in the more than 60 known 11q23 translocations have been reported. Cell lines provide territory to both explore the detailed structures of 11q23 translocations and investigate the leukemogenic activities of MLL fusion proteins. We review here the leukemia cell lines that have been described to carry 11q23 translocations and MLL fusion genes. Except for the t(10;11)(p12;q23), each of the following relatively frequent 11q23/MLL translocations is represented by one or more cell lines: 16 cell lines with t(4;11)(q21;q23), two cell lines with t(6;11)(q27;q23), seven cell lines with t(9;11)(p22;q23), and eight cell lines with t(11;19)(q23;p13). For each of three rare translocations, one cell line has been reported: t(5;11)(q15;q23), t(11;16)(q23;p13), and t(X;11)(q13;q23). Of these 36 cell lines with 11q23 translocations, 17 have been made available to us; we confirmed the occurrence of the alterations reported in these cell lines at the chromosomal and/or gene level. A second type of MLL gene alteration is the partial tandem duplication (PTD), which occurs in acute myeloid leukemia (AML). We found four AML cell lines with an MLL PTD; one acute lymphoblastic leukemia-derived cell line was reported to show a partial nontandem duplication. Finally, a third rearrangement involves intrachromosomal amplification of the unrearranged MLL gene leading to multiple copies of the gene and (presumably) increased expression. Three cell lines carrying such MLL amplifications have been described. The availability of these cell lines as model systems provides the opportunity to explore the altered expression or functions of MLL genes and their partners in oncogenesis.

New cytokine-dependent acute myeloid leukemia cell line MUTZ-11 with disomic chromosome rearrangement t(16;17)

Continuous human leukemia–lymphoma (LL) cell lines represent a rich resource of abundant, accessible and manipulable living cells contributing significantly to a better understanding of the pathophysiology of hematopoietic tumors. In particular, classical and molecular cytogenetics have benefitted enormously from the availability of LL cell lines with specific chromosomal abnormalities. Such aberrations may be the portal to the discovery of novel oncogene rearrangements for which positive cell lines provide a resource for both discovery and functional studies. The new continuous leukemia cell line MUTZ-11 was established in 1994 from the peripheral blood of a 60-year-old woman with acute myeloid leukemia (AML) M4 (following 2 years with myelodysplastic syndromes). DNA fingerprinting confirmed the authenticity and derivation of the cell line. The immunoprofile as determined by flow cytometry was as follows: positive for myelocytic markers (CD13, CD15, CD33, CD65 and CD68), negative for T-cell (except for CD4 and CD7), B-cell and erythroid-megakaryocytic markers. The cell line is constitutively cytokine-dependent and growth depends on externally added cytokines. With regard to cytokine receptor expression, the cell line was found to be positive for GM-CSFRα (granulocyte-macrophage colony-stimulating factor receptor, CD116), Kit (CD117) and IL-3Rα (interleukin-3 receptor, CD123). The cytokine response profiles as determined by [ 3 H ]-thymidine incorporation assay were: 2-to-12 fold growth stimulation of MUTZ-11 by GM-CSF, IFN-α (interferon), IFN-β, IFN-γ, IL-3 and SCF (stem cell factor); growth inhibition by TGF-β1 (transforming growth factor), TNF-α (tumor necrosis factor) and TNF-β. Cytogenetic analysis showed the following consensus karyotype: 46, XX, der(16)t(16;17)(p13.3;q23)x2. Previous molecular biological analysis documented that MUTZ-11 cells carry both an FLT3 internal tandem duplication (ITD) and an MLL partial tandem duplication (PTD). The scientific significance of MUTZ-11 lies (i) in the absolute cytokine-dependency and the proliferative response to various cytokines, (ii) in the unique cytogenetic (disomic t(16;17)) and (iii) molecular biological alterations ( FLT3 ITD + MLL PTD). In summary, the new cytokine-dependent AML-derived cell line MUTZ-11 displays unique novel features and emphasizes the need for comprehensive analysis of new LL cell lines which may lead to the discovery of important pathogenetic alterations.

Acute myeloid leukemia with MLL rearrangements: clinicobiological features, prognostic impact and value of flow cytometry in the detection of residual leukemic cells.

The MLL gene, located at 11q23 band, is frequently disrupted by different chromosomal rearrangements that occur in a variety of hematological malignancies. MLL rearrangements are associated with distinct clinical features and a poor prognosis. The aim of this study was to analyze the incidence and the prognostic significance of MLL rearrangements in a consecutive series of adult AML patients and to determine the immunophenotypic features of these cases. The identification of abnormal immunophenotypes could be used for the detection of minimal residual disease (MRD). Ninety-three adult patients with de novo acute myeloid leukemia (AML) were analyzed by Southern blot in order to detect MLL rearrangements (MLL+). RT-PCR and genomic long-range PCR were performed to further characterize MLL partial tandem duplication (PTD) in those patients in whom conventional karyotype did not show 11q23 chromosomal translocations. All the patients were homogeneously immunophenotyped at diagnosis. MLL rearrangements were detected in 13 (14%) patients. Four patients (5%) showed 11q23 translocations by karyotypic conventional analysis. Nine patients (10%) revealed PTD of MLL and one patient showed a MLL cleavage pattern. The MLL+ patients usually expressed myeloid and monocytic antigens CD33 (12/13 cases), CD13 (9/13), CD117 (9/13), CD64 (11/13) and in some cases CD14 (4/11). HLA-DR was also positive in (12/13). Eight out of 13 cases expressed the stem cell marker CD34. Only one patient revealed lymphoid marker reactivity (CD7) and CD56 was expressed in 5/13 cases. All the MLL+ patients showed at least one aberrant phenotype at diagnosis, which allowed us to set out a simple panel for the MRD studies. Twenty-seven samples from eight patients in morphologic complete remission (CR) were analyzed using the aberrant immunologic combinations detected at diagnosis. Phenotypically abnormal cells were detected in all the patients who subsequently relapsed, whereas only one patient with MRD+ remained in CR. Owing to the high level of residual leukemic cells, the MLL+ patients showed a short CR duration and a poor survival. In conclusion, immunophenotyping may be a suitable approach to investigating MRD status in AML patients with PTD of the MLL gene.