Ivan F. Loncarevic

Trisomy 21 is a recurrent secondary aberration in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion.

TEL/AML1 gene fusion is the most frequent genetic lesion in pediatric acute lymphoblastic leukemia (ALL). It occurs as a consequence of the cryptic chromosomal translocation t(12;21)(p13;q22). In a cohort of 50 RT‐PCR–positive TEL/AML1 patients, karyotype examination by GTG banding and fluorescence in situ hybridization (FISH) allowed us to identify chromosome anomalies in addition to the already existing t(12;21). Secondary aberrations were found in 29 out of 41 patients (71%) at initial diagnosis and in all 9 patients with relapse. Structural rearrangements affected chromosome arms 2p, 2q, 5q, 9p, 12p (n = 2), 6q, 11p (n = 3), and 21q (n = 4). An extra chromosome 21 was found to be the most frequent anomaly. It was detected in 6 out of 41 patients at initial diagnosis (15%) and in 7 out of the 9 patients at relapse. No karyotype with trisomy 21 exceeded 47 chromosomes. Gain of chromosome 21 was the sole anomaly in GTG‐banding analysis in 2/41 patients at initial diagnosis and in 4/9 at relapse. Notably, chromosome painting analysis performed in 11 out of the 13 patients with an extra chromosome 21 revealed duplication of the normal chromosome 21 in 8, and duplication of der(21)t(12;21) in 3 patients. Furthermore, gain of der(21)t(12;21) chromosome was confined exclusively to the relapse patients. Genes Chromosomes Cancer 24:272–277, 1999.

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence

BackgroundDefensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics.ResultsWe find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes.ConclusionsComplexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association.

Heterogenic molecular basis for loss of ABL1‐BCR transcription: Deletions in der(9)t(9;22) and variants of standard t(9;22) in BCR‐ABL1‐positive chronic myeloid leukemia

The objective of this study was to characterize the ABL1‐BCR fusion gene in 76 BCR‐ABL1‐positive chronic myeloid leukemia (CML) patients regarding expression as well as genomic status, to assess the frequency of ABL1‐BCR gene deletion in these patients, which has been reported to be an adverse prognostic factor in Philadelphia chromosome‐positive CML. Patients were analyzed for ABL1‐BCR 1b‐b3 and/or 1b‐b4 transcription by RT‐PCR analysis. ABL1‐BCR gene status was analyzed by FISH in 16 CML patients with no ABL1‐BCR transcript. FISH revealed a partial or total deletion of the ABL1‐BCR gene in 9/16 and localized the 5′ portion of ABL1 and the 3′ portion of BCR at separated loci in 5/16 patients. The latter FISH pattern resulted from a nonreciprocal translocation in two and a complex translocation in three individuals. In 2/16 patients, FISH could not exclude an intact ABL1‐BCR fusion gene. Thus, most CML patients without ABL1‐BCR transcript could be characterized cytogenetically to belong to two major subgroups: a silent ABL1‐BCR gene was attributed to a deletion in der(9)t(9;22) in 56% of the investigated patients or to variants of a standard t(9;22) (∼ 31%). Conversely, none of the 50 patients with an ABL1‐BCR transcript exhibited a variant t(9;22) in GTG‐banding analysis. Thus, genomic aberrations such as deletions or complex genomic rearrangements are the basic and most frequent cause for ABL1‐BCR RNA negativity in CML. The heterogeneity of the underlying molecular mechanisms may explain divergent clinical implications described for patients with an ABL1‐BCR deletion and those with no ABL1‐BCR transcript.

Molecular Dissection of t(11;17) in Acute Myeloid Leukemia Reveals a Variety of Gene Fusions with Heterogeneous Fusion Transcripts and Multiple Splice Variants

The majority of translocations that involve the long arms of chromosomes 11 and 17 in acute myeloid leukemia appear identical on the cytogenetic level. Nevertheless, they are diverse on the molecular level. At present, two genes are known in 11q23 and four in 17q12‐25 that generate five distinct fusion genes: MLL‐MLLT6/AF17, MLL‐LASP1, MLL‐ACACA or MLL‐SEPT9/MSF, and ZBTB16/PLZF‐RARA. We analyzed 14 cases with a t(11;17) by fluorescence in situ hybridization and molecular genetic techniques and determined the molecular characteristics of their fusion genes. We identified six different gene fusions that comprised seven cases with a MLL‐MLLT6/AF17, three with a MLL‐SEPT9/MSF, and one each with MLL‐LASP1, MLL‐ACACA, and ZBTB16/PLZF‐RARA fusions. In the remaining case, a MLL‐SEPT6/Xq24 fusion suggested a complex rearrangement. The MLL‐MLLT6/AF17 transcripts were extremely heterogeneous and the detection of seven different in‐frame transcript and splice variants enabled us to predict the protein domains relevant for leukemogenesis. The putative MLL‐MLLT6 consensus chimeric protein consists of the AT‐hook DNA‐binding, the methyltransferase, and the CXXC zinc‐finger domains of MLL and the highly conserved octapeptide and the leucine‐zipper dimerization motifs of MLLT6. The MLL‐SEPT9 transcripts showed a similar high degree of variability. These analyses prove that the diverse types of t(11;17)‐associated fusion genes can be reliably identified and delineated with a proper combination of cytogenetic and molecular genetic techniques. The heterogeneity of transcripts encountered in cases with MLL‐MLLT6/AF17 and MLL‐SEPT9/MSF fusions clearly demonstrates that thorough attention has to be paid to the appropriate selection of primers to cover all these hitherto unrecognized fusion variants.

Improved definition of chromosomal breakpoints using high-resolution multicolour banding

Abstract. Characterisation of chromosome rearrangements using conventional banding techniques often fails to determine the localisation of breakpoints precisely. In order to improve the definition of chromosomal breakpoints, the high-resolution multicolour banding (MCB) technique was applied to identify human chromosome 5 breakpoints from 40 clinical cases previously assessed by conventional banding techniques. In 30 cases (75%), at least one breakpoint was redefined, indicating that MCB markedly improves chromosomal breakpoint localisation. The MCB pattern is highly reproducible and, in contrast to conventional banding pattern, is consistent in both short and elongated chromosomes. This might be of fundamental interest for the detection of chromosomal abnormalities, especially in tumour cells. Moreover, MCB even allows the detection of abnormalities that remain cryptic in GTG-banding analysis.

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drugs action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

Two‐Photon Multicolor FISH: A Versatile Technique to Detect Specific Sequences with in Single DNA Molecules in Cells and Tissues

Two-photon multicolor fluorescence in situ hybridisation (FISH) is presented as an advanced single molecule detection technique. Based on a two-photon excitation process it enables pinhole-free 3D laser scanning microscopy. Nonresonant two-photon absorption in a sub-femtoliter excitation volume was realized with a femtosecond laser scanning microscope equipped with a high numerical aperture objective. Nonlinear near infrared laser excitation and multicolor FISH has been used to image a variety of specific DNA regions. This innovative single molecule technique was applied to visualize genomic regions in fiber-DNA, human metaphase chromosomes, interphase nuclei and histological sections. Intense 170 fs laser pulses at 800 nm and GW/cm2 intensities have been employed to induce visible fluorescence of a variety of FISH fluorophores coupled to DNA probes. In particular, two-photon excited FITC-labeled 40 kb probes and multicolor labeled centromeric probes that bind to repetitive sequences of 0.340-2.000 kb have been used to visualize subtelomeric and different centromeric regions in metaphase chromosome spreads. Spectrum Orange and Spectrum Green labeled bcr and abl gene probes that target a stretch of 300 kb and 650 kb, respectively, have been imaged in a single fiber-DNA molecule with an estimated number of fluorescent molecules of 75 - 225 μm-1. Using the advantages of pinhole-free optical sectioning with submicron spatial resolution and multi-fluorophore single-wavelength-excitation, 3D images of multiple labeled centromeric regions of amniotic fluid cells in interphase have been obtained and used in diagnosis of trisomy 18. In addition, first 3D two-photon studies on centromer distribution in human kidney biopsies by labeling chromosomes X, Y and 4 with the FISH-fluorophores Spectrum Blue, Spectrum Green and Spectrum Orange have been performed. As demonstrated, two-photon Multicolor FISH has the potential to perform Multi-Gene-Imaging with high spatial resolution also in turbid tissue layers.

Characteristic genomic imbalances in pediatric pheochromocytoma

Pheochromocytoma (PCC) in children is rare, genetically not well described, and often related to a poor prognosis. We detected genomic imbalances in all 14 tumors from children analyzed by comparative genomic hybridization. A combinatorial loss of chromatin from 3p and 11p was a common feature in 10 of 14 (72%) patients, which was a result of either a loss of a total chromosome 3 and a total chromosome 11 in 6 of 10 patients, or confined deletions of their p arms in 4 of 10 patients. All patients exhibiting a loss of 3p and 11p carried VHL mutations. The VHL mutations were constitutive in 9 cases and somatic and restricted to tumor DNA in the remaining tumor. On the other hand, VHL mutations were absent in 4 patients, 2 who had other familial syndromes (NF1, SDHD) and 2 with unknown etiology. Our data show that the pattern of imbalances in the tumor DNA of PCC patients strongly correlated with an underlying familial VHL mutation. Furthermore, we show that true sporadic PCC is rare in childhood. Thus, children with PCC should be checked for a related predisposing gene. This would also identify familial syndrome patients requiring long‐term monitoring for other syndrome‐related malignancies.

Number of genomic imbalances correlates with the overall survival for adrenocortical cancer in childhood.

Adrenocortical tumours (ACT) in children are rare and, if malignant, often associated with poor prognosis. Relevant cytogenetic factors for prognosis are hardly available.

Analyses of minimal residual disease based on Flt3 mutations in allogeneic peripheral blood stem cell transplantation

PurposeActivating Flt3 mutations are observed in about 30% of patients with acute myeloid leukaemia (AML) and individual Flt3 mutations are applicable for minimal residual disease (MRD) analyses.MethodsWe investigated the MRD status in four AML patients carrying different Flt3 mutations (three patients with Flt3 length mutations of the juxtamembrane domain, one patient carrying a mutation of the Flt3 tyrosine kinase domain, i.e. Flt3-TKD mutation) who underwent allogeneic peripheral blood stem cell transplantation (PBSCT). Residual leukaemia cells were retrospectively determined by real-time PCR at different time points.ResultsWe can demonstrate a good correlation between the course of MRD status and clinical events in all four investigated patients.ConclusionThese examples demonstrate the potential impact of Flt3 based MRD status not only after but also prior to allogeneic PBSCT.