Carin Lassen

Microarray-based classification of a consecutive series of 121 childhood acute leukemias: prediction of leukemic and genetic subtype as well as of minimal residual disease status.

Gene expression analyses were performed on 121 consecutive childhood leukemias (87 B-lineage acute lymphoblastic leukemias (ALLs), 11 T-cell ALLs and 23 acute myeloid leukemias (AMLs)), investigated during an 8-year period at a single center. The supervised learning algorithm k-nearest neighbor was utilized to build gene expression predictors that could classify the ALLs/AMLs according to clinically important subtypes with high accuracy. Validation experiments in an independent data set verified the high prediction accuracies of our classifiers. B-lineage ALLs with uncharacteristic cytogenetic aberrations or with a normal karyotype displayed heterogeneous gene expression profiles, resulting in low prediction accuracies. Minimal residual disease status (MRD) in T-cell ALLs with a high (>0.1%) MRD at day 29 could be classified with 100% accuracy already at the time of diagnosis. In pediatric leukemias with uncharacteristic cytogenetic aberrations or with a normal karyotype, unsupervised analysis identified two novel subgroups: one consisting mainly of cases remaining in complete remission (CR) and one containing a few patients in CR and all but one of the patients who relapsed. This study of a consecutive series of childhood leukemias confirms and extends further previous reports demonstrating that global gene expression profiling provides a valuable tool for genetic and clinical classification of childhood leukemias.

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph+) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34+ cells and also in cord blood CD34+ cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph−) and leukemic (Ph+) cells within the CML CD34+CD38− cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34+CD38−IL1RAP+ cells were Ph+, whereas CML CD34+CD38−IL1RAP− cells were almost exclusively Ph−. By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph+ and Ph− candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34+CD38− cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph+ from Ph− candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.

Fusion of the BCR and the fibroblast growth factor receptor-1 (FGFR1) genes as a result of t(8;22)(p11;q11) in a myeloproliferative disorder: The first fusion gene involving BCR but not ABL

Constitutive activation of tyrosine kinases as a consequence of chromosomal translocations, forming fusion genes, plays an important role in the development of hematologic malignancies, in particular, myeloproliferative syndromes (MPSs). In this respect, the t(9;22)(q34;q11) that results in the BCR/ABL fusion gene in chronic myeloid leukemia is one of the best‐studied examples. The fibroblast growth factor receptor 1 (FGFR1) gene at 8p11 encodes a transmembrane receptor tyrosine kinase and is similarly activated by chromosomal translocations, in which three alternative genes—ZNF198 at 13q12, CEP110 at 9q34, and FOP at 6q27—become fused to the tyrosine kinase domain of FGFR1. These 8p11‐translocations are associated with characteristic morphologic and clinical features, referred to as “8p11 MPS.” In this study, we report the isolation and characterization of a novel fusion gene in a hematologic malignancy with a t(8;22)(p11;q11) and features suggestive of 8p11 MPS. We show that the breakpoints in the t(8;22) occur within introns 4 and 8 of the BCR and FGFR1 genes, respectively. On the mRNA level, the t(8;22) results in the fusion of BCR exons 1–4 in‐frame with the tyrosine kinase domain of FGFR1 as well as in the expression of a reciprocal FGFR1/BCR chimeric transcript. By analogy with data obtained from previously characterized fusion genes involving FGFR1 and BCR/ABL, it is likely that the oligomerization domain contributed by BCR is critical and that its dimerizing properties lead to aberrant FGFR1 signaling and neoplastic transformation.

Gene expression profiling of leukemic cell lines reveals conserved molecular signatures among subtypes with specific genetic aberrations

Hematologic malignancies are characterized by fusion genes of biological/clinical importance. Immortalized cell lines with such aberrations are today widely used to model different aspects of leukemogenesis. Using cDNA microarrays, we determined the gene expression profiles of 40 cell lines as well as of primary leukemias harboring 11q23/MLL rearrangements, t(1;19)[TCF3/PBX1], t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC], t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10], t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed that hematopoietic cell lines of diverse origin, but with the same primary genetic changes, segregated together, suggesting that pathogenetically important regulatory networks remain conserved despite numerous passages. Moreover, primary leukemias cosegregated with cell lines carrying identical genetic rearrangements, further supporting that critical regulatory pathways remain intact in hematopoietic cell lines. Transcriptional signatures correlating with clinical subtypes/primary genetic changes were identified and annotated based on their biological/molecular properties and chromosomal localization. Furthermore, the expression profile of tyrosine kinase-encoding genes was investigated, identifying several differentially expressed members, segregating with primary genetic changes, which may be targeted with tyrosine kinase inhibitors. The identified conserved signatures are likely to reflect regulatory networks of importance for the transforming abilities of the primary genetic changes and offer important pathogenetic insights as well as a number of targets for future rational drug design.

IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN TLS-CHOP CARRYING MYXOID LIPOSARCOMAS

Myxoid liposarcomas (MLS) carry a t(12;16) or, more rarely, a t(12;22) resulting in fusion of the transcription factor gene CHOP on chromosome 12 with TLS/FUS on chromosome 16 or EWS on chromosome 22. The chimeric TLS‐CHOP or EWS‐CHOP proteins most probably function as abnormal transcription factors, causing transcriptional de‐regulation of several target genes and relaxation of functions critical for growth and differentiation control. A PCR‐based subtractive hybridization technique was used to identify genes that are differentially expressed in TLS‐CHOP‐carrying MLS but not in normal fat tissue. Six myxoid‐liposarcoma‐associated transcripts, MLAT, were isolated. The genes identified as MLAT can be divided into 2 groups. MLAT1, 2 and 6 show high similarity to glia‐derived nexin, neuronatin and the RET oncogene, respectively, all normally involved in development of tissues of neural origin. MLAT3 to MLAT5 represent new genes. Int. J. Cancer 83:30–33, 1999.

Clinical impact of internal tandem duplications and activating point mutations in FLT3 in acute myeloid leukemia in elderly patients

Background:  The FLT3 gene is frequently mutated in acute myeloid leukemia (AML), either by an internal tandem duplication (ITD) of the juxtamembrane domain or by activating point mutations in the second tyrosine kinase domain (ATKD). Only a few investigations have focused on the prognostic significance of FLT3 alterations in AML among the elderly, yielding conflicting results. In the present study, the frequency and clinical relevance of FLT3 abnormalities were ascertained in a cohort of elderly AML patients.

Characteristic sequence motifs at the breakpoints of the hybrid genes FUS/CHOP , EWS/CHOP and FUS/ERG in myxoid liposarcoma and acute myeloid leukemia

We have sequenced the breakpoint regions in one acute myeloid leukemia (AML) with t(16;21)(p11;q22) resulting in the formation of a FUS/ERG hybrid gene and in four myxoid liposarcomas (MLS), three of which had the translocation t(12;16) (q13;p11) and a FUS/CHOP fusion gene and one with t(12;22;20)(q13;q12;q11) and an EWS/CHOP hybrid gene. The breakpoints were localized to intron 7 of FUS, intron 1 of CHOP, an intronic sequence of ERG and intron 7 of EWS. In two MLS cases with t(12;16) and in the AML, the breaks in intron 7 of FUS had occurred close to each other, a few nucleotides downstream from a TG dinucleotide repeat region. The break in the two MLS had occurred in the same ATGGTG hexamer and in the AML 40 nucleotides upstream from the hexamer. The third case of t(12;16) MLS had a break upstream and near a TC-dinucleotide repeat region and a sequence similar to the chi bacterial recombination element was found to flank the breakpoint. In the MLS with the EWS/CHOP hybrid gene, the break in intron 7 of EWS had occurred close to an Alu sequence. Similarly, in all 4 MLS, the breaks in intron 1 of CHOP were near an Alu sequence. No Alu or other repetitive sequences were found 250 bp upstream or downstream from the break in the ERG intron involved in the AML case. In the AML, the MLS with ESW/CHOP and in one MLS with FUS/CHOP there were one, two and six, respectively, nucleotide identity between the contributing germline sequences in the breakpoint. In the other two MLS cases, two and three extra nucleotides of unknown origin were inserted between the FUS and CHOP sequences. At the junction and/or in its close vicinity, identical oligomers, frequently containing a trinucleotide TGG, were found in both partner genes. Our data thus show that all four genes-FUS, EWS, CHOP and ERG-contain characteristic motifs in the breakpoint regions which may serve as specific recognition sites for DNA-binding proteins and have functional importance in the recombination events taking place between the chromosomes. Different sequence motifs may, however, play a role in each individual case.

Fusion gene-mediated truncation of RUNX1 as a potential mechanism underlying disease progression in the 8p11 myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome (EMS) is a chronic myeloproliferative disorder molecularly characterized by fusion of various 5′ partner genes to the 3′ part of the fibroblast growth factor receptor 1 (FGFR1) gene at 8p, resulting in constitutive activation of the tyrosine kinase activity contained within FGFR1. EMS is associated with a high risk of transformation to acute myeloid leukemia (AML), but the mechanisms underlying the disease progression are unknown. In the present study, we have investigated a case of EMS harboring a t(8;22)(p11;q11)/BCR‐FGFR1 rearrangement as well as a t(9;21)(q34;q22) at the time of AML transformation. FISH and RT‐PCR analyses revealed that the t(9;21) leads to a fusion gene consisting of the 5′ part of RUNX1 (exons 1–4) fused to repetitive sequences of a gene with unknown function on chromosome 9, adding 70 amino acids to RUNX1 exon 4. The t(9;21) hence results in a truncation of RUNX1. No point mutations were found in the other RUNX1 allele. The most likely functional outcome of the rearrangement was haploinsufficiency of RUNX1, which thus may be one mechanism by which EMS transforms to AML.

Combined high-resolution array-based comparative genomic hybridization and expression profiling of ETV6/RUNX1 -positive acute lymphoblastic leukemias reveal a high incidence of cryptic Xq duplications and identify several putative target genes within the commonly gained region

Seventeen ETV6/RUNX1-positive pediatric acute lymphoblastic leukemias were investigated by high-resolution array-based comparative genomic hybridization (array CGH), gene expression profiling and fluorescence in situ hybridization. Comparing the array CGH and gene expression patterns revealed that genomic imbalances conferred a great impact on the expression of genes in the affected regions. The array CGH analyses identified a high frequency of cytogenetically cryptic genetic changes, for example, del(9p) and del(12p). Interestingly, a duplication of Xq material, varying between 30 and 60 Mb in size, was found in 6 of 11 males (55%), but not in females. Genes on Xq were found to have a high expression level in cases with dup(Xq); a similar overexpression was confirmed in t(12;21)-positive cases in an external gene expression data set. By studying the expression profile and the proposed function of genes in the minimally gained region, several candidate target genes (SPANXB, HMGB3, FAM50A, HTATSF1 and RAP2C) were identified. Among them, the testis-specific SPANXB gene was the only one showing a high and uniform overexpression, irrespective of gender and presence of Xq duplication, suggesting that this gene plays an important pathogenetic role in t(12;21)-positive leukemia.

Clinical and genetic studies of ETV6/ABL1-positive chronic myeloid leukaemia in blast crisis treated with imatinib mesylate

Summary. Most chronic myeloid leukaemia (CML) patients are genetically characterized by the t(9;22)(q34;q11), generating the BCR/ABL1 fusion gene. However, a few CML patients with rearrangements of 9q34 and 12p13, leading to ETV6/ABL1 chimaeras, have also been reported. Here we describe the clinical and genetic response to imatinib mesylate treatment of an ETV6/ABL1‐positive CML patient diagnosed in blast crisis (BC). A chronic phase was achieved after acute myeloid leukaemia induction therapy. Then, treatment with imatinib mesylate (600 mg/d) was initiated and the effect was assessed clinically as well as genetically, including by repeated interphase fluorescence in situ hybridization studies. Until d 71 of imatinib mesylate therapy, stable improvements in the clinical and laboratory features were noted, and the frequency of ABL1‐rearranged peripheral blood cells decreased from 56% to 11%. At d 92, an additional t(12;13)(p12;q13), with the 12p breakpoint proximal to ETV6, was found. The patient relapsed into BC 126 d after the start of the imatinib mesylate treatment and succumbed to the disease shortly afterwards. No mutations in the tyrosine kinase domain of ABL1 of the ETV6/ABL1 fusion were identified in the second BC. However, whereas the ETV6/ABL1 expression was seemingly the same at diagnosis and at second BC, the expression of ETV6 was markedly lower at the second BC. This decreased expression of wild‐type ETV6 may have been a contributory factor for the relapse.