Chromothripsis and progression-free survival in metastatic colorectal cancer

Metastatic dissemination of the primary tumor is the major cause of death in colorectal cancer (CRC) patients. Multiple chromosomal breaks and chromothripsis, a phenomenon involving multiple chromosomal fragmentations occurring in a single catastrophic event, are associated with cancer genesis, progression and developing of metastases. The aim of this study was to evaluate the effect of chromothripsis and total breakpoint count (breakpoint instability index) on progression-free survival (PFS). A total of 19 patients with metastatic CRC (mCRC) receiving FOLFOX first‑line palliative chemotherapy between August, 2011 and October, 2012 were selected for this study. The results indicated that the highest breakpoint count was observed in chromosomes 1, 2 and 6. Chromothripsis was detected in 52.6% of the study patients. Furthermore, chromothripsis was associated with an increased median PFS (mPFS; 14 vs. 8 months, respectively; P=0.03), but an association with overall survival was not identified. The present study demonstrated that chromothripsis affected CRC patient survival, suggesting a role for this event as a prognostic and predictive marker in mCRC treatment.publishersversionPeer reviewe

The shaping and functional consequences of the dosage effect landscape in multiple myeloma

Background: Multiple myeloma (MM) is a malignant proliferation of plasma B cells. Based on recurrent aneuploidy such as copy number alterations (CNAs), myeloma is divided into two subtypes with different CNA patterns and patient survival outcomes. How aneuploidy events arise, and whether they contribute to cancer cell evolution are actively studied. The large amount of transcriptomic changes resultant of CNAs (dosage effect) pose big challenges for identifying functional consequences of CNAs in myeloma in terms of specific driver genes and pathways. In this study, we hypothesize that gene-wise dosage effect varies as a result from complex regulatory networks that translate the impact of CNAs to gene expression, and studying this variation can provide insights into functional effects of CNAs. Results: We propose gene-wise dosage effect score and genome-wide karyotype plot as tools to measure and visualize concordant copy number and expression changes across cancer samples. We find that dosage effect in myeloma is widespread yet variable, and it is correlated with gene expression level and CNA frequencies in different chromosomes. Our analysis suggests that despite the enrichment of differentially expressed genes between hyperdiploid MM and non-hyperdiploid MM in the trisomy chromosomes, the chromosomal proportion of dosage sensitive genes is higher in the non-trisomy chromosomes. Dosage-sensitive genes are enriched by genes with protein translation and localization functions, and dosage resistant genes are enriched by apoptosis genes. These results point to future studies on differential dosage sensitivity and resistance of pro- and anti-proliferation pathways and their variation across patients as therapeutic targets and prognosis markers. Conclusions: Our findings support the hypothesis that recurrent CNAs in myeloma are selected by their functional consequences. The novel dosage effect score defined in this work will facilitate integration of copy number and expression data for identifying driver genes in cancer genomics studies. The accompanying R code is available at http://www.canevolve.org/dosageEffect/

Classify Hyperdiploidy Status of Multiple Myeloma Patients Using Gene Expression Profiles

Multiple myeloma (MM) is a cancer of antibody-making plasma cells. It frequently harbors alterations in DNA and chromosome copy numbers, and can be divided into two major subtypes, hyperdiploid (HMM) and non-hyperdiploid multiple myeloma (NHMM). The two subtypes have different survival prognosis, possibly due to different but converging paths to oncogenesis. Existing methods for identifying the two subtypes are fluorescence in situ hybridization (FISH) and copy number microarrays, with increased cost and sample requirements. We hypothesize that chromosome alterations have their imprint in gene expression through dosage effect. Using five MM expression datasets that have HMM status measured by FISH and copy number microarrays, we have developed and validated a K-nearest-neighbor method to classify MM into HMM and NHMM based on gene expression profiles. Classification accuracy for test datasets ranges from 0.83 to 0.88. This classification will enable researchers to study differences and commonalities of the two MM subtypes in disease biology and prognosis using expression datasets without need for additional subtype measurements. Our study also supports the advantages of using cancer specific characteristics in feature design and pooling multiple rounds of classification results to improve accuracy. We provide R source code and processed datasets at www.ChengLiLab.org/software

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants

Chromothripsis in acute myeloid leukemia: Biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix\uae) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology

Heterogeneity of genomic evolution and mutational profiles in multiple myeloma.

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment

Complex Reorganization and Predominant Non-Homologous Repair Following Chromosomal Breakage in Karyotypically Balanced Germline Rearrangements and Transgenic Integration

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations

Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples

<p>Abstract</p> <p>Background</p> <p>Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome.</p> <p>Methods</p> <p>We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays.</p> <p>Results</p> <p>Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%.</p> <p>We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the <it>SLC45A3-ELK4 </it>e4-e2 TIC to <it>ERG</it>-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines.</p> <p>Conclusions</p> <p>Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as <it>MSMB-NCOA4</it>, may play functional roles in cancer.</p