SETD2 histone modifier loss in aggressive GI stromal tumours.

BACKGROUND: GI stromal tumours (GISTs) are clinically heterogenous exhibiting varying degrees of disease aggressiveness in individual patients. OBJECTIVES: We sought to identify genetic alterations associated with high-risk GIST, explore their molecular consequences, and test their utility as prognostic markers. DESIGNS: Exome sequencing of 18 GISTs was performed (9 patients with high-risk/metastatic and 5 patients with low/intermediate-risk), corresponding to 11 primary and 7 metastatic tumours. Candidate alterations were validated by prevalence screening in an independent patient cohort (n=120). Functional consequences of SETD2 mutations were investigated in primary tissues and cell lines. Transcriptomic profiles for 8 GISTs (4 SETD2 mutated, 4 SETD2 wild type) and DNA methylation profiles for 22 GISTs (10 SETD2 mutated, 12 SETD2 wild type) were analysed. Statistical associations between molecular, clinicopathological factors, and relapse-free survival were determined. RESULTS: High-risk GISTs harboured increased numbers of somatic mutations compared with low-risk GISTs (25.2 mutations/high-risk cases vs 6.8 mutations/low-risk cases; two sample t test p=3.1×10-5). Somatic alterations in the SETD2 histone modifier gene occurred in 3 out of 9 high-risk/metastatic cases but no low/intermediate-risk cases. Prevalence screening identified additional SETD2 mutations in 7 out of 80 high-risk/metastatic cases but no low/intermediate-risk cases (n=29). Combined, the frequency of SETD2 mutations was 11.2% (10/89) and 0% (0/34) in high-risk and low-risk GISTs respectively. SETD2 mutant GISTs exhibited decreased H3K36me3 expression while SETD2 silencing promoted DNA damage in GIST-T1 cells. In gastric GISTs, SETD2 mutations were associated with overexpression of HOXC cluster genes and a DNA methylation signature of hypomethylated heterochromatin. Gastric GISTs with SETD2 mutations, or GISTs with hypomethylated heterochromatin, showed significantly shorter relapse-free survival on univariate analysis (log rank p=4.1×10-5). CONCLUSIONS: Our data suggest that SETD2 is a novel GIST tumour suppressor gene associated with disease progression. Assessing SETD2 genetic status and SETD2-associated epigenomic phenotypes may guide risk stratification and provide insights into mechanisms of GIST clinical aggressiveness.

Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development.

OBJECTIVE: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications. DESIGN: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice. RESULTS: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs. CONCLUSIONS: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.

Costunolide ameliorates MNNG-induced chronic atrophic gastritis through inhibiting oxidative stress and DNA damage via activation of Nrf2.

BACKGROUND: Chronic atrophic gastritis (CAG) is a chronic digestive disease. Modern research has revealed substantial evidence indicating that the progression of CAG is closely linked to the occurrence of oxidative stress-induced DNA damage and apoptosis in the gastric mucosa. Additionally, research has indicated that Costunolide (COS), the primary active compound found in Aucklandiae Radix, a traditional herb, exhibits antioxidant properties. Nevertheless, the therapeutic potential of COS in treating CAG and its molecular targets have not yet been determined. PURPOSE: The objective of this research was to explore the potential gastric mucosal protective effects and mechanisms of COS against N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)-induced CAG. METHODS: Firstly, the MNNG-induced rat CAG model was established in vivo. Occurrence of CAG was detected through macroscopic examination of the stomachs and H&E staining. Additionally, we assessed oxidative stress, DNA damage, and apoptosis using biochemical detection, Western blot, immunohistochemistry and immunofluorescence. Then, an in vitro model was developed to induce MNNG-induced damage in GES-1 cells, and the occurrence of cell damage was determined by Hoechst 33,342 staining and flow cytometry. Finally, the key targets of COS for the treatment of CAG were identified through molecular docking, cellular thermal shift assay (CETSA), and inhibitor ML385. RESULTS: In vivo studies demonstrated that COS promotes the expression of Nrf2 in gastric tissues. This led to an increased expression of SOD, GSH, HO-1, while reducing the production of MDA. Furthermore, COS inhibited DNA damage and apoptosis by suppressing the expression of γH2AX and PARP1 in gastric tissues. In vitro studies showed that COS effectively reversed apoptosis induced by MNNG in GES-1 cells. Additionally, COS interacted with Nrf2 to promote its expression. Furthermore, the expression levels of SOD, GSH, and HO-1 were augmented, while the generation of ROS and MDA was diminished. CONCLUSIONS: Our results indicate that COS exhibits therapeutic effects on CAG through the promotion of Nrf2 expression and inhibition of oxidative stress and DNA damage. Therefore, COS has the potential to provide new drugs for the treatment of CAG.

Alpinia oxyphylla Miq. volatile oil ameliorates depressive behaviors and inhibits neuroinflammation in CUMS-exposed mice by inhibiting the TLR4-medicated MyD88/NF-κB signaling pathway.

This study aimed to explore the antidepressant effect and underlying mechanism of the Alpinia oxyphylla Miq. volatile oil (AOVO) in mice exposed to chronic unpredictable mild stress (CUMS). C57BL/6 mice were grouped and administered with different dosages of AOVO (0.25, 0.50, 1.00, or 2.00 mL/kg body weight, i.g.), TAK242 (a TLR4 inhibitor, 0.75 mg/kg body weight, i.p.), or TAK242 (0.75 mg/kg body weight, i.p.) + AOVO (0.50 mL/kg body weight, i.g.) for 21 days. Depression-like symptoms in the mice were then evaluated through their body weight gain (BW), the open field test (OFT), the sucrose preference test (SPT), the novelty-suppressed feeding test (NSFT), and forced swimming test (FST). The concentrations of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and 5-hydroxytyrptamine (5-HT) in the mice were determined using ELISA kits. Hematoxylin and eosin (HE) dying were performed for histopathological examination. The expression of inflammatory proteins was assessed through western blotting (WB) and immunofluorescence staining. AOVO was found to improve the behavioral indexes of CUMS-exposed mice behavioral and synergize TAK242 to mitigate both their depressive symptoms and neuroinflammation. Moreover, AOVO was found to inhibit the hippocampal damage, decrease inflammatory cytokines (Reduced IL-1ß, IL-6, and TNF-α by 19.97 %, 22.87 %, and 24.13 %, respectively), and downregulate the expression of TLR4/MyD88/NF-κB signaling pathway-related proteins in the hippocampus of CUMS-exposed mice (Reduced TLR4, MyD88, and NF-κB by 46.14 %, 42.48 %, and 38.08 %, respectively). These findings demonstrate that AOVO can ameliorate depressive behaviors and mitigate neuroinflammation in the CUMS-exposed mice via suppressing the TLR4-medicated MyD88/NF-κB signaling pathway.

A network pharmacology approach to investigate dehydrocostus lactone inhibits the proliferation and epithelial-mesenchymal transition of human gastric cancer cells via regulating the PI3K/Akt and extracellular signal-regulated kinases/mitogen-activated protein kinase signalling pathways.

OBJECTIVES: Dehydrocostus lactone (DHE), a sesquiterpene lactone, has been proven the significant inhibition of multiple cancer cells. However, there are limited reports on the activity of DHE in gastric cancer (GC). In this research, Network pharmacology predicted the anti-GC mechanism of DHE, and the prediction was verified by in-vitro experiments. METHODS: Network pharmacology confirmed the major effect signalling pathway of DHE in treating GC. Cell viability assay, colony formation assay, wound healing assay, cell migration and invasion assay, apoptosis assay, western blot and real-time quantitative polymerase chain reaction verified the mechanism of DHE in GC cell lines. KEY FINDINGS: The results showed that DHE inhibited the growth and metastasis of MGC803 and AGS GC cells. Mechanistically, the analysis results indicated that DHE significantly induced the apoptosis process by suppressing the PI3K/protein kinase B (Akt) signalling pathway, and inhibited epithelial-mesenchymal transition by suppressing the extracellular signal-regulated kinases (ERK)/MAPK signalling pathway. The Akt activator (SC79) inhibited DHE induced apoptosis, and DHE had similar effects with the ERK inhibitor (FR180204). CONCLUSIONS: All results suggested that DHE was a potential natural chemotherapeutic drug in GC treatment.

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis.

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5 + lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33 high niche fibroblasts. Ablation of IL-5 + lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A + type 3/17 lymphocytes, predominantly γδ T cells. In contrast, concurrent ablation of IL-5 + and IL-17A + lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients.

Genome-wide analysis of cell-free DNA methylation profile is a promising approach for sensitive and specific detection of many cancers. However, scaling such assays for clinical translation is impractical because of the high cost of whole-genome bisulfite sequencing. We show that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors most of the cancer-specific methylation signature. Here, we report on the simple and effective heat enrichment of CpG-rich regions for bisulfite sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation and quantitative tracking of colorectal cancer patient's response to treatment at much reduced sequencing cost suitable for frequent monitoring. We also show tumor epigenetic subtyping using Heatrich-BS, which could enable patient stratification. Heatrich-BS holds great potential for highly scalable screening and monitoring of cancer using liquid biopsy.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer.

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden.

Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we develop a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions is strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based on just 6 regulatory regions, could accurately predict ctDNA levels in colorectal cancer patients. Strikingly, a model restricted to blood-specific regulatory regions could predict ctDNA levels across both colorectal and breast cancer patients. Using compact targeted sequencing (<25 kb) of predictive regions, we demonstrate how the approach could enable quantitative low-cost tracking of ctDNA dynamics and disease progression.

Detection of genomic alterations in breast cancer with circulating tumour DNA sequencing.

Analysis of circulating cell-free DNA (cfDNA) has opened new opportunities for characterizing tumour mutational landscapes with many applications in genomic-driven oncology. We developed a customized targeted cfDNA sequencing approach for breast cancer (BC) using unique molecular identifiers (UMIs) for error correction. Our assay, spanning a 284.5 kb target region, is combined with a novel freely-licensed bioinformatics pipeline that provides detection of low-frequency variants, and reliable identification of copy number variations (CNVs) directly from plasma DNA. We first evaluated our pipeline on reference samples. Then in a cohort of 35 BC patients our approach detected actionable driver and clonal variants at low variant frequency levels in cfDNA that were concordant (77%) with sequencing of primary and/or metastatic solid tumour sites. We also detected ERRB2 gene CNVs used for HER2 subtype classification with 80% precision compared to immunohistochemistry. Further, we evaluated fragmentation profiles of cfDNA in BC and observed distinct differences compared to data from healthy individuals. Our results show that the developed assay addresses the majority of tumour associated aberrations directly from plasma DNA, and thus may be used to elucidate genomic alterations in liquid biopsy studies.

Multiregion ultra-deep sequencing reveals early intermixing and variable levels of intratumoral heterogeneity in colorectal cancer.

Intratumor heterogeneity (ITH) contributes to cancer progression and chemoresistance. We sought to comprehensively describe ITH of somatic mutations, copy number, and transcriptomic alterations involving clinically and biologically relevant gene pathways in colorectal cancer (CRC). We performed multiregion, high-depth (384× on average) sequencing of 799 cancer-associated genes in 24 spatially separated primary tumor and nonmalignant tissues from four treatment-naïve CRC patients. We then used ultra-deep sequencing (17 075× on average) to accurately verify the presence or absence of identified somatic mutations in each sector. We also digitally measured gene expression and copy number alterations using NanoString assays. We identified the subclonal point mutations and determined the mutational timing and phylogenetic relationships among spatially separated sectors of each tumor. Truncal mutations, those shared by all sectors in the tumor, affected the well-described driver genes such as APC, TP53, and KRAS. With sequencing at 17 075×, we found that mutations first detected at a sequencing depth of 384× were in fact more widely shared among sectors than originally assessed. Interestingly, ultra-deep sequencing also revealed some mutations that were present in all spatially dispersed sectors, but at subclonal levels. Ultra-high-depth validation sequencing, copy number analysis, and gene expression profiling provided a comprehensive and accurate genomic landscape of spatial heterogeneity in CRC. Ultra-deep sequencing allowed more sensitive detection of somatic mutations and a more accurate assessment of ITH. By detecting the subclonal mutations with ultra-deep sequencing, we traced the genomic histories of each tumor and the relative timing of mutational events. We found evidence of early mixing, in which the subclonal ancestral mutations intermixed across the sectors before the acquisition of subsequent nontruncal mutations. Our findings also indicate that different CRC patients display markedly variable ITH, suggesting that each patient's tumor possesses a unique genomic history and spatial organization.

Individualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgery.

Circulating tumour DNA (ctDNA) has the potential to be a specific biomarker for the monitoring of tumours in patients with colorectal cancer (CRC). Here, our aim was to develop a personalised surveillance strategy to monitor the clinical course of CRC after surgery. We developed patient-specific ctDNA assays based on multiplexed detection of somatic mutations identified from patient primary tumours, and applied them to detect ctDNA in 44 CRC patients, analysing a total of 260 plasma samples. We found that ctDNA detection correlated with clinical events - it is detectable in pre-operative but not post-operative plasma, and also in patients with recurrent CRC. We also detected ctDNA in 11 out of 15 cases at or before clinical or radiological recurrence of CRC, indicating the potential of our assay for early detection of metastasis. We further present data from a patient with multiple primary cancers to demonstrate the specificity of our assays to distinguish between CRC recurrence and a second primary cancer. Our approach can complement current methods for surveillance of CRC by adding an individualised biological component, allowing us not only to point to the presence of residual or recurrent disease, but also attribute it to the original cancer.

Whole-exome sequencing studies of parathyroid carcinomas reveal novel PRUNE2 mutations, distinctive mutational spectra related to APOBEC-catalyzed DNA mutagenesis and mutational enrichment in kinases associated with cell migration and invasion.

CONTEXT: Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC). OBJECTIVE: To identify additional genetic abnormalities in PCs. DESIGN: Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumor and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA). RESULTS: PC had an average of 51 somatic variants/tumor (range 3-176) with approximately 58% of variants occurring as nonsynonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of the mutant CDC73 allele. Furthermore, recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC. CONCLUSION: This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.

Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

BACKGROUND: Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. OBJECTIVE: The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. DESIGN, SETTING, AND PARTICIPANTS: Eight seminomas and matched normal samples were surgically obtained from eight patients. INTERVENTION: DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. RESULTS AND LIMITATIONS: The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. CONCLUSIONS: Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. PATIENT SUMMARY: We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96 new genes in which mutations occurred during seminoma development, some of which might contribute to cancer development or progression. The study also showed that the rates of DNA mutations during seminoma development are higher than previously thought, but still lower than for other common solid-organ cancers. Such low rates are also observed among other cancers that, like seminomas, show excellent rates of disease remission after chemotherapy.

Mutation signatures implicate aristolochic acid in bladder cancer development.

BACKGROUND: Aristolochic acid (AA) is a natural compound found in many plants of the Aristolochia genus, and these plants are widely used in traditional medicines for numerous conditions and for weight loss. Previous work has connected AA-mutagenesis to upper-tract urothelial cell carcinomas and hepatocellular carcinomas. We hypothesize that AA may also contribute to bladder cancer. METHODS: Here, we investigated the involvement of AA-mutagenesis in bladder cancer by sequencing bladder tumor genomes from two patients with known exposure to AA. After detecting strong mutational signatures of AA exposure in these tumors, we exome-sequenced and analyzed an additional 11 bladder tumors and analyzed publicly available somatic mutation data from a further 336 bladder tumors. RESULTS: The somatic mutations in the bladder tumors from the two patients with known AA exposure showed overwhelming AA signatures. We also detected evidence of AA exposure in 1 out of 11 bladder tumors from Singapore and in 3 out of 99 bladder tumors from China. In addition, 1 out of 194 bladder tumors from North America showed a pattern of mutations that might have resulted from exposure to an unknown mutagen with a heretofore undescribed pattern of A > T mutations. Besides the signature of AA exposure, the bladder tumors also showed the CpG > TpG and activated-APOBEC signatures, which have been previously reported in bladder cancer. CONCLUSIONS: This study demonstrates the utility of inferring mutagenic exposures from somatic mutation spectra. Moreover, AA exposure in bladder cancer appears to be more pervasive in the East, where traditional herbal medicine is more widely used. More broadly, our results suggest that AA exposure is more extensive than previously thought both in terms of populations at risk and in terms of types of cancers involved. This appears to be an important public health issue that should be addressed by further investigation and by primary prevention through regulation and education. In addition to opportunities for primary prevention, knowledge of AA exposure would provide opportunities for secondary prevention in the form of intensified screening of patients with known or suspected AA exposure.

Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers.

The impact of different carcinogenic exposures on the specific patterns of somatic mutation in human tumors remains unclear. To address this issue, we profiled 209 cholangiocarcinomas (CCAs) from Asia and Europe, including 108 cases caused by infection with the liver fluke Opisthorchis viverrini and 101 cases caused by non-O. viverrini-related etiologies. Whole-exome sequencing (n = 15) and prevalence screening (n = 194) identified recurrent somatic mutations in BAP1 and ARID1A, neither of which, to our knowledge, has previously been reported to be mutated in CCA. Comparisons between intrahepatic O. viverrini-related and non-O. viverrini-related CCAs demonstrated statistically significant differences in mutation patterns: BAP1, IDH1 and IDH2 were more frequently mutated in non-O. viverrini CCAs, whereas TP53 mutations showed the reciprocal pattern. Functional studies demonstrated tumor suppressive functions for BAP1 and ARID1A, establishing the role of chromatin modulators in CCA pathogenesis. These findings indicate that different causative etiologies may induce distinct somatic alterations, even within the same tumor type.

Genome-wide mutational signatures of aristolochic acid and its application as a screening tool.

Aristolochic acid (AA), a natural product of Aristolochia plants found in herbal remedies and health supplements, is a group 1 carcinogen that can cause nephrotoxicity and upper urinary tract urothelial cell carcinoma (UTUC). Whole-genome and exome analysis of nine AA-associated UTUCs revealed a strikingly high somatic mutation rate (150 mutations/Mb), exceeding smoking-associated lung cancer (8 mutations/Mb) and ultraviolet radiation-associated melanoma (111 mutations/Mb). The AA-UTUC mutational signature was characterized by A:T to T:A transversions at the sequence motif A[C|T]AGG, located primarily on nontranscribed strands. AA-induced mutations were also significantly enriched at splice sites, suggesting a role for splice-site mutations in UTUC pathogenesis. RNA sequencing of AA-UTUC confirmed a general up-regulation of nonsense-mediated decay machinery components and aberrant splicing events associated with splice-site mutations. We observed a high frequency of somatic mutations in chromatin modifiers, particularly KDM6A, in AA-UTUC, demonstrated the sufficiency of AA to induce renal dysplasia in mice, and reproduced the AA mutational signature in experimentally treated human renal tubular cells. Finally, exploring other malignancies that were not known to be associated with AA, we screened 93 hepatocellular carcinoma genomes/exomes and identified AA-like mutational signatures in 11. Our study highlights an unusual genome-wide AA mutational signature and the potential use of mutation signatures as "molecular fingerprints" for interrogating high-throughput cancer genome data to infer previous carcinogen exposures.

Exome sequencing of liver fluke-associated cholangiocarcinoma.

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.

Janus kinase 3-activating mutations identified in natural killer/T-cell lymphoma.

UNLABELLED: The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identified Janus kinase 3 (JAK3) somatic-activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs. SIGNIFICANCE: Gene mutations causing NKTCL have not been fully identified. Through exome sequencing, we identified activating mutations of JAK3 that may play a significant role in the pathogenesis of NKTCLs. Our findings have important implications for the management of patients with NKTCLs.