Identificazione di trascritti aberranti nei linfomi anaplastici a grandi cellule regolati da lunghe ripetizioni terminali endogene

Anaplastic Large Cell lymphoma (ALCL) is a highly heterogeneous CD30 positive Non Hodgkin T-cell neoplasm category comprising 2 8% of NHL in adults and 10 15% in children. On the basis of the presence or absence of deregulated expression of Anaplastic Lymphoma Kinase they are divided in ALK positive and ALK negative respectively. While ALK positive ALCL is a defined subclass, ALK negatives are extremely difficult to determine and to classify, because they are a set of different neoplasm not yet characterized. In order to discover genes potentially involved in ALK negative ALCL pathogenesis we apllied a bioinformatic analysis named COPA (Cancer Outlier Profile Analysis) on a Micro-Array Gene Expression Profile data set. COPA is a statistical procedure which identifies outlier in profile of genes expression and it is useful to evidence over-expressed oncogenes in tumor samples subsets. In this case, more than 300 cases of T-NHLs and normal T-cells were analyzed. ERBB4 and COL29A1 were identified as outliers in ALK negative ALCL patients. ERBB4 is a transmembrane Tyrosine Kinase Receptor (RTK) member of Epidermal Growth Factor Receptor (EGFR) family. Alteration in ERBB4 are reported in several neoplasms. COL29A1 (or COL6A5) is alpha 5 chain of VI-type collagen, and it is involved in different tumor case. These two genes resulted aberrantly over-expressed in 33% of cases (8 out 24). Moreover the expression of ERBB4 and COL29A1 was strictly correlated. RT-qPCR analysis showed that ERBB4 transcript lacked of its 5' region. Through Exon-Walking RT-qPCRs we identified two drops of ERBB4 levels, between Exon 20-21 and between 12-13. Performing the 5'RLM-RACE technique (5'RNA ligase mediated rapid amplification of cDNA ends) it was possible to highlight the presence of two different transcripts. Specifically, the first transcript includes the exons 21-28 and the second the exons 13-28. Both of them have an intronic portion (of Intron 20 and 12 respectively) as 5'UTR. We called these two transcripts I20ΔERBB4 e I12ΔERBB4. Final validation came from Whole Transcriptome Sequencing (RNA-seq) of ALK negative ALCL samples data analysis which evidenced lack of transcript's 5'portion. Analysis of a large set of T-NHL identified the presence of the I20ΔERBB4 in the 24% of ALK negative ALCLs, with the exclusion from PTCL-NOS and ALK positive ALCL. High resolution Genome Sequencing did not reveal any evident genetic lesion in ERBB4 which could explain the aberrant expression. Analysis of 5'UTR region revealed that both the TSS are in the middle of endogenous Long Terminal Repeats, which, if de-repressed, could act as ERBB4 aberrant transcripts promoters. Luciferase assays demonstrated the capacity of these LTR promoters to induce transcription, even if the reason why these two intronic UTRs become fused with respective exons remains for the moment unclear. In conclusion, two ERBB4 truncated transcripts, selectively expressed in an ALK negative ALCL subset, have been identified and characterized; experiments were performed to assess whether these two transcripts were oncogenic and possess catalytic activity. Their LTR-driven transcription raise the issue how these LTR are activated. Ongoing studies will allow to analyze epigenetic status of the LTR sequences and to discover if activation of these repetitive elements, which should be silenced, is due to epigenetic alterations as DNA hypomethylation.