M. Barbieri
HIGH-THROUGHPUT SEQUENCING FOR THE IDENTIFICATION OF DIS3 MUTATIONS IN MULTIPLE MYELOMA [Tesi di dottorato]
Università degli Studi di Milano, 2015-02-04

Multiple myeloma (MM) is an incurable malignancy of mature plasma cells (PCs), and its pathogenesis is only partially understood. Recently, whole exome sequencing studies have identified mutations of DIS3, a catalytic subunit of the human exosome complex, in about 10% of MM patients. This study was aimed at investigating the spectrum of DIS3 mutations indifferent stages of plasma cell dyscrasia. To analyze DIS3 mutations, we investigated by next generation sequencing (NGS) a retrospective cohort of 130 cases with MM at onset and 17 at relapse (of whom 15 were also tested at onset). Moreover, we examined 24 patients with primary PC leukemia (pPCL), 12 with secondary PCL and 20 multiple myeloma cell lines. Deep sequencing of the PIN (exons 1-4) and RNB (exons 10-18) DIS3 functional domains was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. Mutations were validated by conventional Sanger sequencing or independent ultra-deep 454 pyrosequencing. All samples were characterized by fluorescence in situ hybridization (FISH) for the main genomic aberrations, such as IGH translocations, 13q and 17p deletions, hyperdiploidy, 1p33 (CDKN2C) loss and 1q21.3 (CKS1B) gain. In order to verify if the mutations detected on genomic DNA were expressed at transcriptomic level, DIS3 cDNA of mutated cases was subjected to deep sequencing. Global gene expression of MM patients was also profiled, looking for transcriptional patterns possibly related to DIS3 mutations. Additionally, to investigate DIS3 mutation status longitudinally, we analyzed 20 patients whose bone marrow specimens were collected at two different time points. Finally, the association of DIS3 mutations with clinical outcome was tested, comparing wild-type patients (n=12) with those characterized by mutations in PIN or RNB functional domains of DIS3 (n=4). NGS analysis revealed the presence of 41 coding non-synonymous variants, with a mutant allele frequency ranging from 0.38% to 100% of total reads (median depth of coverage 245x, range: 64-1160). Among the 41 tumor-specific mutations, 30 (73%) were single nucleotide variations, and the remaining 11 (27%) were indels. Nine of these variants have been already reported by others, eight of which also specifically in MM patients, while 32 were novel. The great majority of mutations identified in our cohort of patients affected the RNB domain (30/41, 73.2%). The mutations affected 26 MM patients at diagnosis (26/130, 20%), four at relapse (4/17, 23.5%), six pPCL (6/24, 25%), four sPCL (4/12, 33.3%) cases, and three multiple myeloma cell lines (3/20, 15%). We observed a positive association of DIS3 mutations with the occurrence of translocations involving IGH@ locus, particularly with the t(11;14), and a negative association with hyperdiploid cases, but no association with 13q and 17p deletion, gain of chromosome 1q and loss of chromosome 1p. Additionally, we showed that mutant allele frequencies detected on genomic DNA and on retrotranscribed total RNA are linearly correlated. Moreover, in serially analyzed patients, we detected mutations at constant allele frequency at both time points, or acquired/increased variants in the late sample, consistent with the expected positive selection of mutated subclones. Furthermore, global gene expression profiling analysis revealed 119 differentially expressed genes (all up-regulated in mutated cases) between DIS3 mutated and wild-type cases. Finally, we tested the association of DIS3 mutations with clinical outcome in 16 pPCL patients for whom the follow-up was available, showing that mutational events did not show any impact neither on PFS nor OS. Our data confirm DIS3 as frequently mutated in MM, and importantly, seem to indicate an even greater involvement of DIS3 alteration in more advanced stages of PC dyscrasias. Furthermore, these data, although requiring confirmation in independent patients’ series, support the hypothesis that DIS3 may play a role in development and progression of MM. Further studies are required to elucidate the role of this gene in the pathogenesis of MM, as well as its potential use as a drug target.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/259303
NERI, ANTONINO
relatore: A. Neri ; coordinatore: P. Corradini
CORRADINI, PAOLO
Settore MED/15 - - Malattie del Sangue


Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18001930