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

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 ∼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 ∼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 amplification

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

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 12q 12 -> 15, but often also segments from other chromosomes are involved. Previous studies have shown that one of the recurrent amplicons in ALT, located in 12q 13.3-14.1 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 12q 13.3-14.1, 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. (C) 2009 Wiley-Liss, Inc

The transcriptome plasticity of genomic amplification in cancer

Genomic amplification, in the form of homogeneously staining regions, double minutes, and ring/giant rod-shaped markers, is a pivotal event in many tumors. It was recently shown that amplifications as extra-chromosomal DNA are present in nearly half of all tumors, representing a driving force towards their accelerated evolution. To achieve a better understanding of the implications of genomic amplifications we focused on their structure and impact upon transcription. Amplified cancer-associated genes are often overexpressed as a direct consequence of the copy number gain. We analyzed the whole genome (WGS) and transcriptome (RNA-seq) sequencing data of nine small lung carcinoma (SCLC), seven neuroblastoma (NB) and three well- differentiated liposarcoma (WDLPS) cell lines, all carrying genomic amplifications. A widespread heterogeneity was detected in the amplicon arrangement of many cell lines, disclosing the progressive evolution of their structure through cell division. By integrating the WGS (structural variation calling) and RNA-seq (chimeras detection) data we detected a burst of chimeric transcripts partially derived from post- transcriptional events (i.e cis- or trans-splicing) in most of the analyzed cell lines. Notably, we found PVT1 and RLF as hotspots for cis- or trans-splicing events in SCLC and NB cell lines with MYC and MYCL1 amplifications, respectively. In WDLPS cell lines we found fusion genes originated by extremely complex genomic rearrangements, such as those involving three partner genes or assembled by multiple interposed non-contiguous, non-collinear genomic fragments (spliced out in the mature transcript). Our results strongly indicate that the “amplification/overexpression” paradigm does not cover all aspects of the genomic amplification impact upon transcription. The extraordinary transcriptome plasticity herein described, enriching the genetic repertoire of cancer cells with genomic amplifications, likely provides a selective advantage and might have a crucial role in cancer establishment and progression

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

A rare but recurrent t(8;13)(q24;q14) translocation in B-cell chronic lymphocytic leukaemia causing MYC up-regulation and concomitant loss of PVT1, miR-15/16 and DLEU7

B-cell chronic lymphocytic leukaemia (B-CLL) is the most common haematological malignancy in Western countries. Trisomy 12 (12%), and deletions of 13q14 (50%), 11q23 (18%) and 17p13 (7%) are the most frequent chromosomal aberrations (Palamarchuk et al, 2010). 13q14 losses, associated with better prognosis, are primary changes resulting in loss of MIR15A/MIR16-1 and DLEU7 (Palamarchuk et al, 2010). Notably, the 10% of these deletions occur together with unbalanced translocations with multiple partners (Puiggros et al, 2014). Two cases with t(8;13)(q14;q24) translocations have been described, although not characterized at molecular level (Gardiner et al, 1997; Put et al, 2012). 8q24 rearrangements are generally rare in CLL and their prognostic significance is unclear.This work was supported by the AIRC (Associazione Italiana per la Ricerca sul Cancro).Peer Reviewe