Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells.

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer.

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.

t(15;21) translocations leading to the concurrent downregulation of RUNX1 and its transcription factor partner genes SIN3A and TCF12 in myeloid disorders.

Through a combined approach integrating RNA-Seq, SNP-array, FISH and PCR techniques, we identified two novel t(15;21) translocations leading to the inactivation of RUNX1 and its partners SIN3A and TCF12. One is a complex t(15;21)(q24;q22), with both breakpoints mapped at the nucleotide level, joining RUNX1 to SIN3A and UBL7-AS1 in a patient with myelodysplasia. The other is a recurrent t(15;21)(q21;q22), juxtaposing RUNX1 and TCF12, with an opposite transcriptional orientation, in three myeloid leukemia cases. Since our transcriptome analysis indicated a significant number of differentially expressed genes associated with both translocations, we speculate an important pathogenetic role for these alterations involving RUNX1.

A novel method for the isolation of single cells mimicking circulating tumour cells adhered on Smart Bio Surface slides by Laser Capture Microdissection.

In recent years, the importance of isolating single cells from blood circulation for several applications, such as non-invasive tumour diagnosis, the monitoring of minimal residual disease, and the analysis of circulating fetal cells for prenatal diagnosis, urged the need to set up innovative methods. For such applications, different methods were developed. All show some weaknesses, especially a limited sensitivity, and specificity. Here we present a new method for isolating a single or a limited number of cells adhered to SBS slides (Tethis S.p.a.) (a glass slide coated with Nanostructured Titanium Dioxide) by Laser Capture Microdissection (LCM) and subsequent Whole Genome Amplification. SBS slides have been shown to have an optimal performance in immobilizing circulating tumour cells (CTCs) from early breast cancer patients. In this work, we spiked cancer cells in blood samples to mimic CTCs. By defining laser parameters to cut intact samples, we were able to isolate genetically intact single cells. We demonstrate that SBS slides are optimally suited for isolating cells using LCM and that this method provides high-quality DNA, ideal for gene-specific assays such as PCR and Sanger sequencing for mutation analysis.

Gene amplification as double minutes or homogeneously staining regions in solid tumors: origin and structure.

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.

Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP).

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Characterization of a hotspot region on chromosome 12 for amplification in ring chromosomes in atypical lipomatous tumors.

Ring chromosomes are cytogenetic hallmarks of genomic amplification in several bone and soft tissue tumors, in particular atypical lipomatous tumors (ALT). In ALT, the ring chromosomes invariably contain amplified material from the central part of the long arm of chromosome 12, mainly 12q12-->15, but often also segments from other chromosomes are involved. Previous studies have shown that one of the recurrent amplicons in ALT, located in 12q13.3-14.1 and harboring the candidate target genes TSPAN31 and CDK4, often has a sharp centromeric border. To characterize this breakpoint region in more detail, 12 cases of ALT with ring chromosomes were analyzed by array comparative genomic hybridization and fluorescence in situ hybridization. In the seven cases showing a sharply delineated amplicon in 12q13.3-14.1, the breakpoint region was further investigated by real time quantitative polymerase chain reaction and Vectorette PCR. The breakpoints clustered to a 146-kb region containing 11 genes. Whereas there was no indication that the breakpoints gave rise to fusion genes, in silico analysis revealed that the breakpoint region was enriched for repeated elements that could be important for ring chromosome formation in ALT.

1q23.1 homozygous deletion and downregulation of Fc receptor-like family genes confer poor prognosis in chronic lymphocytic leukemia.

The identification of chromosome 1 translocations and deletions is a rare and poorly investigated event in chronic lymphocytic leukemia (CLL). Nevertheless, the identification of novel additional molecular alterations is of great interest, opening to new prognostic and therapeutic strategies for such heterogeneous hematological disease. We here describe a patient affected by CLL with a mutated IGHV status, showing a balanced t(1;3)(q23.1;q21.3) translocation and a der(18)t(1;18)(q24.2;p11.32), accompanying the recurrent 13q14 heterozygous deletion in all analyzed cells at onset. By combining whole-genome sequencing, SNP array, RNA sequencing, and FISH analyses, we defined a 1q23.1 biallelic minimally deleted region flanking translocations breakpoints at both derivative chromosome 1 homologues. The deletion resulted in the downregulation of the Fc receptor-like family genes FCRL1, FCRL2, and FCRL3 and in the lack of expression of FCRL5, observed by RT-qPCR. The mutational status of TP53, NOTCH1, SF3B1, MYD88, FBXW7, and XPO1 was investigated by targeted next-generation sequencing, detecting a frameshift deletion within NOTCH1 (c.7544_7545delCT). We hypothesize a loss of tumor suppressor function for FCRL genes, cooperating with NOTCH1 mutation and 13q14 genomic loss in our patient, both conferring a negative prognosis, independently from the known biological prognostic factors of CLL.

Bone marrow ectopic expression of a non-coding RNA in childhood T-cell acute lymphoblastic leukemia with a novel t(2;11)(q11.2;p15.1) translocation.

Chromosomal translocations play a crucial role in tumorigenesis, often resulting in the formation of chimeric genes or in gene deregulation through position effects. T-cell acute lymphoblastic leukemia (T-ALL) is associated with a large number of such rearrangements. We report the ectopic expression of the 3' portion of EST DA926692 in the bone marrow of a childhood T-ALL case showing a t(2;11)(q11.2;p15.1) translocation as the sole chromosome abnormality. The breakpoints, defined at the sequence level, mapped within HPS5 (Hermansky Pudlak syndrome 5) intron 1 at 11p15.1, and DA926692 exon 2 at 2q11.2. The translocation was accompanied by a submicroscopic inversion that brought the two genes into the same transcriptional orientation. No chimeric trancript was detected. Interestingly, Real-Time Quantitative (RQ)-PCR detected, in the patient's bone marrow, expression of a 173 bp product corresponding to the 3' portion of DA926692. Samples from four T-ALL cases with a normal karyotype and normal bone marrow used as controls were negative. It might be speculated that the juxtaposition of this genomic segment to the CpG island located upstream HPS5 activated DA92669 expression. RQ-PCR analysis showed expression positivity in 6 of 23 human tissues examined. Bioinformatic analysis excluded that this small non-coding RNA is a precursor of micro-RNA, although it is conceivable that it has a different, yet unknown, functional role. To the best of our knowledge, this is the first report, in cancer, of the activation of a small non-coding RNA as a result of a chromosomal translocation.

Extramedullary molecular evidence of the 5'KIAA1509/3'PDGFRB fusion gene in chronic eosinophilic leukemia.

We report a case of chronic eosinophilic leukemia (CEL), demonstrating for the first time: (i) the association of CEL with the 5'KIAA1509/3'PDGFRB fusion gene as a consequence of a t(5;14)(q33;q32); (ii) the molecular detection of this rearrangement in an extramedullary site; (iii) the cloning and sequencing of the KIAA1509 and PDGFRB genomic breakpoints. The 5'KIAA1509/3'PDGFRB fusion gene is predicted to encode a protein of 2059 amino acids. The genomic breakpoints were localized inside KIAA1509 intron 11 and PDGFRB intron 10. Sequence analysis in correspondence with these breakpoints revealed the presence of repetitive DNA, such Alu elements, which could promote chromosomal rearrangements.

MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

Correction: MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

In the original version of this Article, the affiliation details for Giovanni Martinelli were incorrectly given as 'Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy' and it should have been given as 'Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy and not Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy.'Furthermore, the original version of this Article contained an error in the spelling of the authors Alberto L'Abbate and Pietro D'Addabbo, an acute accent was used instead of an apostrophe for these authors names.These errors have now been corrected in both the PDF and HTML versions of the Article.

Molecular cytogenetic characterization of a complex rearrangement involving chromosomes 9 and 22 in a case of Ph-negative chronic myeloid leukemia.

The "golden path", produced by the Human Genome Project effort, is composed of a collection of overlapping and fully sequenced BAC/PAC clones covering almost completely the human genome. These clones can be advantageously exploited as fluorescence in situ hybridization (FISH) probes for the characterization of rearrangements frequently found in tumors. Breakpoint characterization can be further refined by generating additional smaller FISH probes through LONG-PCR amplification of specific DNA segments, 5-10 kb in size, using appropriate BAC/PAC probes as template. We report here an example of this approach that has been used to characterize a complex Ph-negative chronic myeloid leukemia (CML Ph-) case in which the BCR/ABL fusion gene was found located on chromosome 9.

Two alternatively spliced 5'BCR/3'JAK2 fusion transcripts in a myeloproliferative neoplasm with a three-way t(9;18;22)(p23;p11.3;q11.2) translocation.

Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) are known to harbor alterations of the tyrosine kinase JAK2 (9p24), resulting in the constitutive autoactivation of the encoded protein. Here, we report an unclassifiable MPN case, BCR/ABL1-negative, showing a three-way t(9;18;22)(p23;p11.3;q11.2) translocation, which generates a 5'BCR/3'JAK2 gene by fusing BCR at intron 1 to JAK2 at intron 14 on the derivative chromosome 22. The fusion gene produced two alternatively spliced 5'BCR/3'JAK2 transcripts, fusing in-frame BCR exon 1 to JAK2 exon 15 and exon 17. This is the first report of the simultaneous occurrence of two BCR/JAK2 fusion transcripts in the same sample and of the longer transcript isoform (BCR exon 1 fused to JAK2 exon 15). Notably, both BCR/JAK2 encoded fusion proteins are predicted to juxtapose the coiled-coil dimerization domain of BCR to the catalytically inactive pseudokinase domain (JH2), entirely or partially deprived of the inhibitory region 1 (IR1). Interestingly, IR1 is involved in the auto-inhibitory interaction with the JAK2 kinase domain (JH1), which may result in deregulation of JAK2 activity.

MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene.

Double minutes (dmin)-circular, extra-chromosomal amplifications of specific acentric DNA fragments-are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in approximately 1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of approximately 500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the 'episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplifications.