Kooperativer Bibliotheksverbund

In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 109-109

Abstract: Multiple Myeloma (MM) initiation and progression is driven by recurrent cytogenetic events, i.e. multiple trisomies or translocations within the immunoglobulin locus. Gene mutations have been extensively studied, and they are generally involved in late phases of disease development. On the contrary, very little is known about non-recurrent structural variations (SV), which are increasingly emerging as critical driver in several cancers. To determine the extent to which the MM genome is shaped by such events, we performed whole genome sequencing (WGS) on 67 CD138+ purified bone marrow MM samples from 30 patients (median of 2 samples per patient; range 1-4), to which we added 22 previously published cases (Chapman et al, Nature 2011) for a total of 52 patients and 89 tumor samples. We defined SVs as inversions, translocations, internal tandem duplications and deletions, which we analysed using publicly available tools developed at the Wellcome Sanger Institute. We found a stunning 1887 unique SVs in the whole cohort, with a variable distribution across the entire series (median 29 per patient, range 0-156). To derive a homogeneous catalogue of SV across the different MM patients and biological subgroups, we annotated events according to the recently proposed classification on 〉 2500 cancer genomes (Li Y. et al BioRxiv 2018). IGH and MYC translocations were the most frequent recurrent events and accounted for just 5.3% of the entire SV catalogue. We defined as complex events the following SV classes: chromothripsis, chromoplexy, multiple inversions (distinct between Local_n_Jumps and Local_and_distant_n_jumps in case of translocation involvement), templated insertion between more than 2 chromosomes. According to this classification, in 93% of patients a single, private complex SV was responsible for multiple and simultaneous CNAs across different chromosomes, thus providing a novel pathogenetic explanation for many recurrent CNAs in MM. Overall, 136 complex events were observed in 43/52 patients (83%). We found 34 instances of chromotripsis (Korbel J.O. et al., Cell 2013) in 18/52 (34%) patients. The vast majority (30/34) were clonal and conserved during evolution, suggesting an early role in MM pathogenesis. In addition, we observed 5 chromoplexy events (Korbel J.O. et al., Cell 2013) acquired in 5 patients. More interestingly, evidence of templated insertion on more than 2 chromosomes was observed in 13 patients (25%). This event is composed by multiple concatenated translocations causing small CNAs (mostly gains) and in 77% it resulted in a translocation involving an important MM oncogene (8 MYC and 2 CCND1), suggesting that this is a novel relevant driver mechanism in MM. Given the variety of the landscape of SV between patients, we investigated the presence of SV patterns (SV signatures) by the hierarchical dirichlet process (hdp) (https://github.com/nicolaroberts/hdp). Six main SV signatures were extracted: SV signature #1 was characterized by multiple isolated deletions; SV signature #2 was associated with chromotripsis; SV signature #3 was characterized by reciprocal translocation, local_and_distant_n_jumps and templete insertion between 2 chromosomes. Signature #4 was mostly characterized by local_n_jumps; Signature #5 and #6 were associated with temple insertions on multiple chromosomes with or without large tandem duplication, respectively. Different patients showed differential contribution from different signatures, and based on this we observed 5 distinct clusters. Interestingly some of these clusters were associated with distinct and known MM drivers. For example t(4;14)(MMSET;IGH) cases were enriched for SV signature #1. A significant fraction of patients without any recurrent IGH translocation were characterized by high prevalence of chromothripsis. SV signatures #5 and #6 were mostly associated with hyperdiploid patients with MYC translocation and low genomic impairment. In this study, we describe the landscape of SVs and complex events in MM, suggesting that this notation may represent an important step forward in disentangling the genomic complexity and heterogeneity of MM. Disclosures Moreau: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Corradini:Roche: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer; Sandoz: Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board & Lecturer; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Celgene: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Amgen: Honoraria, Other: Advisory Board & Lecturer. Anderson:Celgene: Consultancy; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy. Avet-Loiseau:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director. Bolli:Celgene: Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-112420

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 3107-3107

Abstract: Cereblon (CRBN) was recently identified as a protein binding immunomodulatory drugs (IMiDs) (Ito et al., 2010, Lopez-Girona et al., 2011) essential for the activity of thalidomide, lenalidomide and pomalidomide in patients suffering from multiple myeloma (MM) (Zhu et al., 2011 & 2012). It is known that some patients develop resistance to these drugs, raising the question of a possible role of CRBN alterations (mutations, deletions, loss of transcription...) in the development of such treatment escapes (Broyl et al., 2012, Heintel et al, 2013). Ito et al (2010) showed that loss of the C-ter fragment of CRBN or missense mutations (Y384A and W386A) in the thalidomide binding domain (TBD) indeed impair the efficacy of this drug. We investigated whether the loss of CRBN expression, particularly of full length isoforms including exons 10 and 11 encoding TBD, or missense mutations could be detected by polymerase chain reaction (PCR) followed by fragment-length analysis or Sanger bidirectional sequencing. This method was applied to a cohort of 19 patients issued from a consecutive cohort of 45 elderly patients treated with lenalidomide and dexamethasone for relapsed or refractory MM (Touzeau et al., 2012). RNA was extracted from plasma cells purified by CD138 immunomagnetic sorting (STEM CELLS® beads) at the time of diagnosis. Sanger sequencing performed on RT-PCR products revealed no missense mutations but disclosed a high frequency of alternative splicing of CRBN in samples from MM patients (Lodé et al 2013). We thus developed a new PCR strategy to detect and semi-quantify alternative spliced isoforms of CRBN involving or not a loss of CRBN specific domains, focusing on exons 10 and 11. Two PCR were performed with custom-made primers: PCR-1 was designed to encompass the whole coding sequence of CRBN and PCR-2 was specifically designed to cover TBD. Fragment-length analysis of fluorescent PCR-2 products was obtained by capillary electrophoresis on an Applied Biosystems 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA) allowing for precise sizing and semi-quantification by assessing the height of peaks. This PCR strategy was highly efficient to detect both unspliced CRBN transcripts (PCR-1 and PCR-2 fragments detected at 1562 nucleotides (nt) and 568nt, respectively) and CRBN alternatively spliced variants. The sizing strategy chosen allowed to identify exon skipping. For instance, PCR-2 fragments without exon 10 were visualized as peaks of 234nt, 319nt and 436nt depending on the presence or not of exons 7, 8 and 9. Alternative CRBN splicing in TBD was seen in nearly all patients of this cohort. Examples of representative splicing patterns are shown in figure 1. Semi-quantification of CRBN spliced transcripts showed that 58% of patients had lost more than 50% of CRBN full-length isoform and that 37% had lost more than 50% of exon10. Of note, two patients with a very short duration of response to lenalidomide and dexamethasone (16 days and 58 days respectively) had lost more than 95% of full-length CRBN and more than 90% of exon 10 (see MM6179 in figure1) in initial samples in spite of proper detection of an actin fragment of 858nt.Figure1Figure1. The fragment-length PCR analysis presented here demonstrates that CRBN splicing profiles in MM patients are very heterogeneous. A correlation with response to treatment was not established in this rather small cohort of patients, but this could be due to the fact that diagnosis samples were collected too early in relation to the time of initiating lenalidomide treatment. Indeed, CRBN splicing could be therapy-induced. A prospective study is needed to determine whether the semi-quantification of CRBN alternatively spliced variants would indeed correlate with, or even predict, clinical response to IMiDs. Moreover, further mutation studies are needed to elucidate the heterogeneity of splicing profiles which could reflect the presence of subclonal mutations in genes encoding the splicing machinery. Disclosures: Moreau: CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.3107.3107

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 4212-4212

Abstract: Progress in the treatment of multiple myeloma (MM) has increased extent and frequency of response, as well as prolonged progression-free (PFS) and overall survival (OS). Today complete remission (CR) rates up to 70% are achieved with new drug combinations. This has lead to development of sensitive next generation sequencing (NGS) -based methods to predict deeper responses that may more accurately predict survival outcomes in MM. Our large recent study has confirmed the clinical impact of achieving MRD- status in MM. Here we are evaluating the genomic alterations that may predict attainment of MRD negative status in MM. MRD status was evaluted in 279 patients from IFM/DFCI 2009 trial. We obtained gene expression by RNA-seq, and copy number profile by cytoScan HD array to evaluate genomic differences between MRD negative and MRD positive groups. We generated copy number data for 175 / 279 patients (72 MRD- and 103 MRD+) with Affymetrix Cytoscan HD array and compared genome wide copy number alterations. We observed statistically significant copy number alterations in chromosome 1p, 2, 4q, 11q, 13, 14 and 20 between MRD- and MRD+ patients. However, the extent of alterations in these regions is limited. The largest difference was on chromosome 11q arm where MRD- patients had 2.2 copies on average and MRD+ had 2.4 (p value 〈 0.001). Similarly, we generated gene expression profiles with RNAseq for 69 MRD- patients and 92 MRD+ patients to study gene expression alterations that may predict attainment of MRD negative status and to examine possible biological pathways. Although first two component of principle component analysis (PCA) showed that two groups have similar expression profile, we were able to identify 586 differentially expressed genes; 333 of those were up and 253 were down regulated in MRD+ compared to MRD- groups. We found that seven oncogenes (CCND1, CD79B, IDH1, PATZ1, PAX5, POU2AF1, RUNX1) were significantly high in MRD+ and two (CCND2 and MYCN) were high in MRD-. Additional genes that were high in MRD+ samples were enriched in genes regulated by NF-kB in response to TNF, P53 pathway, KRAS signaling and genes down-regulated in response to ultraviolet (UV) radiation. Genes that were high in MRD- compared to MRD+ were also enriched in genes up-regulated by STAT5 in response to IL2 stimulation, p53 pathways and networks, and genes up-regulated in response to ultraviolet (UV) radiation pathways. Finally, we have created a signature to predict MRD+ and MRD- in MM samples from differentially expressed genes. We used 40 genes that has at least 2 fold change difference between MRD+ and MRD- groups as a predictor and we randomly separated 161 RNAseq samples into train (n=99) and test group (n=62). We developed our classifiers with diagonal discriminant analysis and we achieved 0.79 classifier performance on test dataset. Then we tested our signature against 1000 random signature and it was significantly different than random signatures (Figure). In conclusion, we here report a first genomic landscape predictive of minimal residual disease (MRD) in Multiple Myeloma (MM). This analysis will help understand genomic and molecular correlates of achieving minimal residula disease and confirms feasibility of using RNAseq data from diagnosis sample to predict MRD status. The ongoing integration of other genomic correlates such as copy number status as well as alternate splicing may allow further improvement in the performance of prediction. Figure 1. Figure 1. Disclosures Anderson: Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; BMS: Consultancy; Millennium: Consultancy. Attal:jansen: Honoraria; celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.4212.4212

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 2989-2989

Abstract: Long intergenic non-coding RNA (lincRNA) are transcripts longer than 200 nucleotides which have a diverse sets of regulatory functions but do not get translated into protein. lincRNAs are located between the protein coding genes and do not overlap exons of either protein-coding or other non-lincRNA. However precise role of individual lincRNA in disease biology remains unclear. Here, we have evaluated the lincRNA expression and their potential biological functions in MM. We performed RNA-seq on CD138+ MM cells from 296 newly diagnosed patients and 16 normal bone marrow plasma cells (NBM) and analyzed for lincRNA expression. Data from paired-end RNAseq reads were mapped to the latest human genome, differentially expressed lincRNAs were identified and for each expressed lincRNA event free survival was examined with univariate cox regression model and support vector machine. Finally, we identified protein coding genes that are strongly correlated (cor 〉 0.5) with lincRNAs with significant altered expression in MM and impact on EFS to identify their biological role. lincRNA and protein coding genes that have more than 10 reads/million reads for at least 15 normal samples or 62 MM samples (20% all MM samples) were included in the analysis. We identified 60 differentially expressed lincRNA (adj p value 〈 0.05), 51 of those had at least 1.5 fold change difference. The differentially expressed lncRNAs were in close proximity of Ig-related genes, genome stability related genes, hosting miRNAs such as mir222 and mir22 and previously reported for other cancers (PVT and TTY15). We evaluated relation of these lincRNAs with event free survival (EFS) and observed 6 lincRNAs associated with shorter EFS. We have developed multivariate signature model to predict EFS by using these 6 lincRNAs. We divided our dataset into training (n=99) and test (n=156) dataset and we utilized support vector machine classification to divide samples into 2 groups using six lincRNAs. This model was able to predict good and poor survival groups in training dataset (p val 〈 0.001) as well as test dataset (p val = 0.002) (Figure). We examined genome wide correlation between these six differentially expressed and prognostically significant lincRNAs to expressed protein coding genes to identify their biological functions in MM. Four of these lincRNAs strongly correlated with 47 to 504 genes (abs(cor) 〉 0.5), affecting immune system pathways and pathways in cancer including Jak-STAT signaling pathway. We also found that these lincRNAs are also highly correlated with tumor development genes such as TNFRSF1B,FGR,TP53BP2,TNF and T or B cells related genes PIK3CD, BCL6. In addition, two of these lincRNAs (LINC00936 and CTB-61M7.2) were found highly correlated with their protein coding neighbor genes ATP2B1(cor = 0.45) and FCAR (cor = 0.95) respectively and MIR22HG was host gene for mir22 which may indicate lincRNAs are using different machinery in MM to regulate protein coding genes. In summary, we report that lincRNA is differentially expressed and prognostically significant in myeloma and may function through their impact on immune system and tumor progression. Our ongoing integrative approach will provide further evidence of their regulatory role in MM with potential therapeutic application. Figure 1. Figure 1. Disclosures Anderson: acetylon pharmaceuticals: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Oncocorp: Equity Ownership; Millennium: Consultancy; BMS: Consultancy. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.2989.2989

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 1816-1816

Abstract: Abstract 1816 Multiple Myeloma (MM) is characterized by several recurrent chromosomal abnormalities, some of them driving the outcome of the patients, especially t(4;14), del(17p), and hyperdiploidy. On the other hand, recent data based on whole genome sequencing showed that MM is characterized by many gene mutations, some of them being recurrent. In order to try to reconcile these two types of genetic abnormalities, we performed whole exome sequencing (WES) in 53 newly-diagnosed patients characterized by high or low risk at the chromosomal level. We selected 16 patients with del(17p) in at least 60% of the clonal plasma cells, 5 patients with del(12p), including 3 with associated t(4;14), 2 patients with t(14;16), 1 patient with both del(17p), del 12p), and t(4;14), considered as the poor risk group (24 patients), 24 patients with hyperdiploidy (including chromosome 5 gain), and 2 patients with a normal SNParray profile, considered as the good risk group (26 patients, and 3 patients with hyperdiploidy and either del(17p) and/or del(12p), considered as the uncertain risk group. A total of 3621 non-silent mutations were observed through the 53 tumor genomes. The median number of mutations per patient was 79 (31–462). We did not observe differences in the number of mutations according to cytogenetic risk. Regarding specific gene mutations, 376 genes presented 2 mutations, and 128 genes presented at least 3 mutations (3–16). Comparison with recently published sequencing data (Chapman, Nature, 2011) revealed only a few common mutated genes (NRAS 26%, KRAS 23%, TP53 13%, BRAF 11%, and FAM46C 10%). In contrast, many recurrently mutated genes were identified in this series, but not in the published one. This could be related to the relatively low number of sequenced cases in both series. Very interestingly, quite a high number of strictly identical mutations involving many genes were observed in different patients (139 cases), suggesting that those mutations are more probably driver rather than passenger events. To conclude, in this large comprehensive study, we did not find any significant correlation between recurrent chromosomal changes and gene mutations, suggesting that these two events occur independently. We cannot address the issue of the prognostic value of the gene mutations because of the low number of patients sequenced so far. This question will definitely have to be addressed in future larger series on newly diagnosed patients with MM. Disclosures: Facon: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V118.21.1816.1816

Language: English

Publisher: American Society of Hematology

Publication Date: 2011

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 117, No. 6 ( 2011-02-10), p. 2009-2011

Abstract: Many trials in myeloma are stratified on cytogenetic abnormalities. Among them, the most commonly chosen are the t(4;14), the del(17p), and the t(14;16). If data are well established for t(4;14) and del(17p), very few data support the use of t(14;16). To address this issue, we retrospectively analyzed 1003 patients with newly diagnosed myeloma for this abnormality. We identified 32 patients with the t(14;16). Compared with patients lacking the t(14;16), we did not observe any difference in overall survival (P = .28). Moreover, in multivariate analyses, the t(14;16) was not prognostic (P = .39). In conclusion, our data do not support the use of t(14;16)-specific probes in the diagnostic panels of multiple myeloma.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2010-07-295105

Language: English

Publisher: American Society of Hematology

Publication Date: 2011

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 118, No. 3 ( 2011-07-21), p. 675-678

Abstract: Multiple myeloma (MM) develops from a premalignant plasma cell proliferative disorder, and with time can progress to a more aggressive disease in extramedullary locations. The gradually clinical evolution is supported by clonal expansion of cells that acquire genetic lesions over years. This model of cancer evolution based on ongoing genomic instability mechanism may apply to development of most MM cases. However, in a small fraction of newly diagnosed MM who relapse quickly and finally die within 2 years, the gradual model appears to be untenable. Analysis of high resolution copy number profiles obtained using single nucleotide polymorphism array data from 764 newly diagnosed MM identified large numbers of genomic rearrangements with the hallmarks of chromothripsis in 1.3% of samples. Moreover, this catastrophic event confers a poor outcome. Because chromothripsis appears to occur in a single crisis, our results suggest that high-risk MM patients use this novel way of cancer evolution.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2011-03-344069

Language: English

Publisher: American Society of Hematology

Publication Date: 2011

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2088-2088

Abstract: INTRODUCTION: Symptomatic multiple myeloma (MM) is preceded by an indolent expansion of clonal plasma cells, known as monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Despite initial efforts using karyotype, copy number arrays and whole exome sequencing, the full catalogue of genomic events associated with SMM/MGUS progression to MM has not been investigated before in a comprehensive way. MATERIALS AND METHODS: We utilized paired bone marrow samples collected from 11 patents at initial presentation (SMM n=10 and MGUS n= 1) and subsequently at the time of their progression to MM. DNA from purified CD138+ plasma cells from these samples along with a matched normal sample was subjected to whole genome sequencing (WGS) using HiSeq X Ten machine. Somatic mutations, structural variation and copy number changes were analyzed using standard analysis pipeline at the Wellcome Trust Sanger Institute. RESULTS: The total number of somatic variants (substitutions and indels) observed at the asymptomatic stage [5780 (range 2599-7760)] was not significantly different than what observed in the corresponding MM samples [5954 (ranges 2824-8227), p-value = 0.1] . Clonal mutations in driver genes such as NRAS, BRAF and DIS3 were found in SMM/MGUS samples, suggesting they can represent early lesions in MM pathogenesis. Similarly, IGH translocations (3/11), hyperdiploidy (7/11) and recurrent chromosomal aneuploidies (i.e del1p and gain 1q) were found at asymptomatic stage with similar frequencies to MM, suggesting that SMM may share many genomic features with MM. To have a reliable estimate of the evolution of the subclonal structure of samples we used a Bayesian hierarchical Dirichlet process after correcting the VAF for the percentage of contaminating normal cells (Bolli et al, Nature Comms 2014) to group mutations with similar adjusted VAF into clusters that reflect the sub-clonal composition of the tumor. Using this approach, we found that all patients presented one or more clusters of sub-clonal variants, reflecting spontaneous evolution of the disease before diagnosis, even in SMM/MGUS phases of the disease. In the 11 patients we observed two main patterns of progression to MM. In the first, the sub-clonal architecture shifted significantly because of loss of sub-clone(s) and gain of other(s), in a branching evolution pattern likely driven by spontaneous acquisition of new genetic lesions conferring competitive proliferative advantage to a previously minor sub-clone. In the second pattern, there was no change of the sub-clonal architecture of the sample despite a clinical progression to a symptomatic stage. Interestingly, the pattern of change of genomic rearrangements and copy number events was in broad concordance with that of point mutations, suggesting that these classes of events act in a concerted way during tumor evolution. Overall, we found that 7/11 SMM/MGUS progressed to MM showing spontaneous evolution of the subclonal structure of the tumor, and those where generally characterized by a longer time to progression as compared to samples showing no evolution. Interestingly, we did not observe any correlation between the total number of substitutions, rearrangements, indels, copy numbers changes and time to transformation. CONCLUSIONS: We observed two different models of SMM/MGUS progression to MM. Cases characterized by no significant change in the clonal architecture of the tumor likely represented disease which had already acquired the required mutational change/s for disease proliferation, where only time was needed for the disease to reach the burden required to manifest as clinically symptomatic disease. Early identification of such cases could provide a rationale for early treatment in the SMM phase that could be clinically beneficial. Conversely, cases showing subclonal change representing an example of spontaneous Darwinian evolution where the clinical progression is driven by spontaneous acquisition of further genomic lesions in competing subclones. Here, the effects of early treatment would have to be weighed against the risk of hastening the outgrowth of chemoresistant clones. Disclosures Munshi: OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V128.22.2088.2088

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-01), p. 2375-2375

Abstract: Gene expression profiling has provided new insights into the understanding of mature B cell neoplasms by relating each one to its normal counterpart, so that they can be to some extent classified according to the corresponding normal B-cell stage. Thus, diffuse large B cell (DLBCL) and follicular lymphoma (FL) have been related to a germinal center precursor whereas mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL) are more likely to derive from naïve and memory B cell, respectively. However, little is still known about the physiopathology of B-cell lymphomas and particularly the deregulated pathways involved in their oncogenesis. To further investigate that point, we performed laser capture microdissection (LCM) of the three anatomic lymphoid compartments (i.e germinal center, mantle zone and marginal zone) taken from nine normal spleens and lymph nodes and magnetic cell separation of the four normal B cell subpopulations (i.e naïve B cells, centroblasts, centrocytes and memory B cells) purified from twelve normal tonsils for gene expression profiling by cDNA microarray. These molecular profiles have been compared to those of the four most frequent mature B cell neoplasms in adult (i.e DLBCL, FL, MZL and MCL), each one isolated from five previously untreated patients. Unsupervised analysis by hierarchical clustering of the normal anatomic and cellular populations could discriminate the germinal from the extra-germinal populations by genes involved in cell proliferation (e.g. E2F5, CCNB2, BUB1B and AURKB), DNA repair (e.g. PCNA and EXO1), cytokine secretion (e.g. IL8, IL10RB, IL4R and TGFBI) and apoptosis (e.g. CASP8, CASP10, BCL2 and FAS). Supervised analysis of the comparison between each B-cell lymphoma and its anatomic and cellular physiologic equivalent identified molecular deregulations concerning several genes’families characterizing the different histologic subtypes. Genes associated with cellular adhesion and ubiquitin cycle were significantly up-regulated in MCL (FCGBP, ITGAE, USP7, VCAM1) and MZL (CTGF, CDH1, ITGAE) whereas germinal center derived lymphomas (i.e. DLBCL and FL) mainly showed up-regulation of genes involved in cell proliferation (TNFRSF17, SEPT8) and immune response (FCER1G, XBP1, IL1RN). Few deregulated genes were common to the four subtypes, principally associated with cell proliferation (CYR61, GPNMB), cytosqueleton organization (EPB41L3) and carbohydrates metabolism (GNPDA1), suggesting potential similar oncogenic pathways. Those preliminary results are compatible with both subtype-specific and overall mechanisms of lympomagenesis and should be verified in a wider range of samples to confirm the oncogenic events involved in this heterogeneous disease.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V108.11.2375.2375

Language: English

Publisher: American Society of Hematology

Publication Date: 2006

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 2846-2846

Abstract: Abstract 2846 Poster Board II-822 Background: A prominent feature of most cancers is striking genetic instability and ongoing accrual of mutational changes associated with tumor progression, including acquisition of invasiveness, drug resistance, and metastasis. Methods: We first utilized single nucleotide polymorphism (SNP) arrays (Affymetrix) to evaluate genome-wide gains and losses in copy number and heterozygosity in CD138+ multiple myeloma (MM) cells collected from 14 patients at two time points at least 6 months apart. To estimate the extent of genomic instability in each patient, the number of events leading to copy number or heterozygosity changes throughout the genome were calculated. An event was defined as detectable change in copy number or heterozygosity in three or more consecutive SNPs. Two cases were also investigated for genome-wide rearrangements utilizing a paired-end approach on next generation sequencing. Results: In a period of six months, all MM patients analyzed acquired multiple new mutational events including changes in copy number and heterozygosity, ranging from 0.021 - 2.674 %, indicating a wide range of genetic instability. Although the rate of mutation varied, the majority (71%) of MM patients had acquired 〉 100 mutational events within the six months period, thus indicating a striking genetic instability. Chromosomes 1, 13, and X were unique with respect to copy number changes and showed large areas of change, spanning the entire length of a chromosome in several patient samples analyzed. Chromosomes 1 and 13 also showed large areas of loss or gain of heterozygosity in several patients, indicating areas of recurrent changes. We were also able to correlate genomic changes with changes in expression of corresponding genes. In two cases, we investigated genome-wide rearrangements utilizing a massively parallel sequencing approach. Short insert (400bp) libraries from two samples collected 6 months apart were constructed and subjected to paired-end sequencing utilizing 37bp readlengths on the Illumina GAII instrument. Approximately 80 million reads were generated for each of the 4 samples. Read pairs were mapped back to the reference genome, and those mapping aberrantly (incorrect orientation, different chromosomes, incorrect genomic distance) were further analyzed. Bespoke PCR assays defining each breakpoint were designed and used to verify the somatic nature of the mapped rearrangement. Further, PCR fragments spanning somatic genomic rearrangements were sequenced to generate base-pair resolution of breakpoints. To date, 29 somatic rearrangements have been sequenced, including three that were present only in the second sample. One of these was on chromosome 13. Breakpoint sequencing revealed a 64.9Kb homozygous (no wild-type readpairs found) deletion removing the first two exons of the RB1 gene. No reads spanning this breakpoint were found in the matching sample taken six months earlier. Conclusions: This is the first study utilizing massively parallel sequencing to investigate the MM genome and provides important insight into the pathogenesis of disease progression .as well as confirms the potential of whole genome sequencing to inform biology of the disease that may affect the therapeutic approach in future. Disclosures: Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Celgene: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Novartis : Consultancy, Honoraria, Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V114.22.2846.2846

Language: English

Publisher: American Society of Hematology

Publication Date: 2009

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7