Numerous large-scale genomic studies of matched tumor-normal samples have established the somatic landscapes of most cancer types. However, the downstream analysis of data from somatic mutations entails a number of computational and statistical approaches, requiring usage of independent software and numerous tools. Here, we describe an R Bioconductor package, Maftools, which offers a multitude of analysis and visualization modules that are commonly used in cancer genomic studies, including driver gene identification, pathway, signature, enrichment, and association analyses. Maftools only requires somatic variants in Mutation Annotation Format (MAF) and is independent of larger alignment files. With the implementation of well-established statistical and computational methods, Maftools facilitates data-driven research and comparative analysis to discover novel results from publicly available data sets. In the present study, using three of the well-annotated cohorts from The Cancer Genome Atlas (TCGA), we describe the application of Maftools to reproduce known results. More importantly, we show that Maftools can also be used to uncover novel findings through integrative analysis.

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3'-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified novel significantly mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to previously discovered ones (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Moreover, our approaches uncovered many novel druggable candidates, and XPO1 was further explored as a therapeutic target because of its mutation and protein overexpression. Together, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

The Per1 gene is a core clock factor that plays an essential role in generating circadian rhythms. Recent data reveal that major biological pathways, including those critical to cell division, are under circadian control. We report here that Per1 provides an important link between the circadian system and the cell cycle system. Overexpression of Per1 sensitized human cancer cells to DNA damage-induced apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human cancer cell lines led to significant growth reduction. Finally, Per1 levels were reduced in human cancer patient samples. Our results highlight the importance of circadian regulation to fundamental cellular functions and support the hypothesis that disruption of core clock genes may lead to cancer development.

Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.

Loss of heterozygosity (LOH), either with or without accompanying copy-number loss, is a cardinal feature of cancer genomes that is tightly linked to cancer development. However, detection of LOH is frequently hampered by the presence of normal cell components within tumor specimens and the limitation in availability of constitutive DNA. Here, we describe a simple but highly sensitive method for genomewide detection of allelic composition, based on the Affymetrix single-nucleotide-polymorphism genotyping microarray platform, without dependence on the availability of constitutive DNA. By sensing subtle distortions in allele-specific signals caused by allelic imbalance with the use of anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers, even in the presence of up to 70%-80% normal cell contamination. The performance of the new algorithm, called "AsCNAR" (allele-specific copy-number analysis using anonymous references), was demonstrated by detecting the copy-number neutral LOH, or uniparental disomy (UPD), in a large number of acute leukemia samples. We next applied this technique to detection of UPD involving the 9p arm in myeloproliferative disorders (MPDs), which is tightly associated with a homozygous JAK2 mutation. It revealed an unexpectedly high frequency of 9p UPD that otherwise would have been undetected and also disclosed the existence of multiple subpopulations having distinct 9p UPD within the same MPD specimen. In conclusion, AsCNAR should substantially improve our ability to dissect the complexity of cancer genomes and should contribute to our understanding of the genetic basis of human cancers.

Somatic mutations in the spliceosome gene ZRSR2 — located on the X chromosome — are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3΄ splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.