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Supplementary Table 3 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 9 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Figure 1 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 8 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 5 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 4 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 11 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 2 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Figure 3 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 7 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Methods and Materials, Results, Legends for Tables 1-11 and Figures 1-3 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Table 6 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Supplementary Figure 2 from Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Bin Tean Teh and colleagues report exome sequencing of Opisthorchis viverrini–related cholangiocarcinoma, a fatal bile duct cancer associated with liver fluke infection. Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

Genetic alterations in kinases have been linked to multiple human pathologies. To explore the landscape of kinase genetic variation in gastric cancer (GC), we used targeted, paired-end deep sequencing to analyze 532 protein and phosphoinositide kinases in 14 GC cell lines. We identified 10,604 single-nucleotide variants (SNV) in kinase exons including greater than 300 novel nonsynonymous SNVs. Family-wise analysis of the nonsynonymous SNVs revealed a significant enrichment in mitogen-activated protein kinase (MAPK)-related genes (P < 0.01), suggesting a preferential involvement of this kinase family in GC. A potential antioncogenic role for MAP2K4, a gene exhibiting recurrent alterations in 2 lines, was functionally supported by siRNA knockdown and overexpression studies in wild-type and MAP2K4 variant lines. The deep sequencing data also revealed novel, large-scale structural rearrangement events involving kinases including gene fusions involving CDK12 and the ERBB2 receptor tyrosine kinase in MKN7 cells. Integrating SNVs and copy number alterations, we identified Hs746T as a cell line exhibiting both splice-site mutations and genomic amplification of MET, resulting in MET protein overexpression. When applied to primary GCs, we identified somatic mutations in 8 kinases, 4 of which were recurrently altered in both primary tumors and cell lines (MAP3K6, STK31, FER, and CDKL5). These results demonstrate that how targeted deep sequencing approaches can deliver unprecedented multilevel characterization of a medically and pharmacologically relevant gene family. The catalog of kinome genetic variants assembled here may broaden our knowledge on kinases and provide useful information on genetic alterations in GC. Cancer Res; 71(1); 29–39. ©2011 AACR.

Background & Aims: VEGF is a key stimulator of angiogenesis and promotes the growth colon tumors via increased nutrient and oxygen supply. A recent study demonstrated that colorectal cancer (CRC) cells have increased expression of VEGF and its receptors VEGFR1 & VEGF-R2, which indicates a possibility of direct stimulation of CRC cell growth by VEGF. However, the role of VEGF in CRC cell proliferation by an autocrine mechanism has not been fully explored in depth. We hypothesized that VEGF secreted by CRC cells binds to VEGF-R1 & VEGF-R2 on CRC cells and: 1) promotes proliferation of these cells through an autocrine pathway, 2) inhibits CRC apoptosis. Methods: We used: (1) human CRC cell lines HCT116 & HT29 and (2) normal colonic epithelial cells NCM356 & NCM460 cultured in nutrient media. Studies: 1) VEGF, VEGF-R1 & VEGF-R2 mRNA and protein expression of by Real-Time RT-PCR and immunoblotting; 2) VEGF165 secretion into the culture media by ELISA; 3) cell proliferation by BrdU assay; 4) apoptosis by TUNEL staining and assessment of activated caspases 3 and 9; 5) Inhibition of VEGF receptor function using AAL993 inhibitor, which blocks function of VEGF-R1 and VEGF-R2; 6) assessment of phosphorylation of EGF receptor (EGF-R) to test its transactivation by VEGF. Results: 1) Normal colonic cells do not secrete VEGF (< 35 pg/ml culture media) while CRC cells express and secrete high levels of VEGF (840 1500 pg/ml culture media); 2) Normal colonic epithelial cell lines expressed no or minimal VEGF-R1 & VEGF-R2 mRNA and protein while CRC cell lines exhibit strong expression of VEGF-R1 & VEGF-R2 vs. normal colonic epithelial cells by 14and 24-fold, respectively (all p < 0.001); 3) CRC cell lines have significantly increased cell proliferation by 31% (p < 0.01) and decreased apoptosis vs. normal colonic epithelial cells; 3) Inhibition of both VEGF-R1 & VEGF-R2 receptors using AAL-993 significantly decreased CRC cell proliferation by (2.2-fold) ; 4) Treatment of CRC cells with AAL-993 decreased phosphorylation of EGF-R by 1.9-fold (p < 0.01). Conclusions: 1) VEGF and its VEGF-R1 & VEGF-R2 receptors are increased in CRC cell lines; 2) CRC cells secrete VEGF, which stimulates CRC cell proliferation via an autocrine mechanism by its VEGF-R1 & VEGF-R2 receptors and also inhibits apoptosis; 3) VEGF receptor signaling pathway is critical for activating EGF-R in CRC cells; 4) These studies suggest a cross-talk between VEGF receptor and EGF-R signaling in CRC cells, which promotes these cells' proliferation.

BACKGROUND: Gastric cancer is the second highest cause of global cancer mortality. To explore the complete repertoire of somatic alterations in gastric cancer, we combined massively parallel short read and DNA paired-end tag sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal instability and the other with microsatellite instability. RESULTS: Integrative analysis and de novo assemblies revealed the architecture of a wild-type KRAS amplification, a common driver event in gastric cancer. We discovered three distinct mutational signatures in gastric cancer--against a genome-wide backdrop of oxidative and microsatellite instability-related mutational signatures, we identified the first exome-specific mutational signature. Further characterization of the impact of these signatures by combining sequencing data from 40 complete gastric cancer exomes and targeted screening of an additional 94 independent gastric tumors uncovered ACVR2A, RPL22 and LMAN1 as recurrently mutated genes in microsatellite instability-positive gastric cancer and PAPPA as a recurrently mutated gene in TP53 wild-type gastric cancer. CONCLUSIONS: These results highlight how whole-genome cancer sequencing can uncover information relevant to tissue-specific carcinogenesis that would otherwise be missed from exome-sequencing data.