LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma.

BACKGROUND & AIMS: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity. METHODS: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. RESULTS: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth. CONCLUSIONS: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models. IMPACT AND IMPLICATIONS: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Overexpression of the energy metabolism transcriptome within clonal plasma cells is associated with the pathogenesis and outcomes of patients with multiple myeloma.

Altered energy metabolism and changes in glycolytic and oxidative phosphorylation pathways are hallmarks of all cancer cells. The expression of select genes associated with the production of various enzymes and proteins involved in glycolysis and oxidative phosphorylation were assessed in the clonal plasma cells derived from patients with newly diagnosed multiple myeloma (NDMM) enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass data set. A scoring system consisting of assigning a point for every gene where their fragments per kilobase of transcript per million (FPKM) was above the median yielded a minimum of 0 and a maximum of 12 for the set of genes in the glycolytic and oxidative phosphorylation pathways to create a total energy metabolism molecular signature (EMMS) score. This EMMS score was independently associated with worse progression free survival (PFS) and overall survival (OS) outcomes of patients with NDMM. A higher EMMS score was more likely to be present in clonal plasma cells derived from Multiple myeloma (MM) patients than those from patients with monoclonal gammopathy of undetermined significance (MGUS). This was functionally confirmed by the clonal plasma cells from MM patients having a higher rate of mitochondrial and glycolysis-derived ATP formation than clonal plasma cells from MGUS patients. Thus, this study provides evidence for the effect of energy metabolism within clonal plasma cells on pathogenesis and outcomes of patients with MM. Exploiting the energy-producing metabolic pathways within clonal plasma cells for diagnostic and therapeutic purposes in MM should be explored in the future.

Mass cytometry identifies expansion of double positive and exhausted T cell subsets in the tumour microenvironment of patients with POEMS syndrome.

We sought to dissect the tumour microenvironment in a small cohort (N = 10) of patients with POEMS at diagnosis and after therapy using mass cytometry. We included 10 MGUS patients as controls. We identified 29 immune cell subsets in the CD45+ and CD3+ compartments. Double positive T cells and PD-1 positive CD4 T cells were expanded and naïve CD4 T cells were decreased in the bone marrow of patients with newly diagnosed/progressing POEMS. These findings provide evidence for possible antigenic-driven selection as a driver of disease pathogenesis in POEMS.

Rho and NusG suppress pervasive antisense transcription in Escherichia coli.

Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation.

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2-/- mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2-/- (double knockout) mice resembled Phlpp1-/- mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1-/- and Phlpp1/2-/- chondrocytes had similarities and were significantly different from WT and Phlpp2-/- chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

Comprehensive profiling of cancer neoantigens from aberrant RNA splicing.

BACKGROUND: Cancer neoantigens arise from protein-altering somatic mutations in tumor and rank among the most promising next-generation immuno-oncology agents when used in combination with immune checkpoint inhibitors. We previously developed a computational framework, REAL-neo, for identification, quality control, and prioritization of both class-I and class-II human leucocyte antigen (HLA)-presented neoantigens resulting from somatic single-nucleotide mutations, small insertions and deletions, and gene fusions. In this study, we developed a new module, SPLICE-neo, to identify neoantigens from aberrant RNA transcripts from two distinct sources: (1) DNA mutations within splice sites and (2) de novo RNA aberrant splicings. METHODS: First, SPLICE-neo was used to profile all DNA splice-site mutations in 11,892 tumors from The Cancer Genome Atlas (TCGA) and identified 11 profiles of splicing donor or acceptor site gains or losses. Transcript isoforms resulting from the top seven most frequent profiles were computed using novel logic models. Second, SPLICE-neo identified de novo RNA splicing events using RNA sequencing reads mapped to novel exon junctions from either single, double, or multiple exon-skipping events. The aberrant transcripts from both sources were then ranked based on isoform expression levels and z-scores assuming that individual aberrant splicing events are rare. Finally, top-ranked novel isoforms were translated into protein, and the resulting neoepitopes were evaluated for neoantigen potential using REAL-neo. The top splicing neoantigen candidates binding to HLA-A*02:01 were validated using in vitro T2 binding assays. RESULTS: We identified abundant splicing neoantigens in four representative TCGA cancers: BRCA, LUAD, LUSC, and LIHC. In addition to their substantial contribution to neoantigen load, several splicing neoantigens were potent tumor antigens with stronger bindings to HLA compared with the positive control of antigens from influenza virus. CONCLUSIONS: SPLICE-neo is the first tool to comprehensively identify and prioritize splicing neoantigens from both DNA splice-site mutations and de novo RNA aberrant splicings. There are two major advances of SPLICE-neo. First, we developed novel logic models that assemble and prioritize full-length aberrant transcripts from DNA splice-site mutations. Second, SPLICE-neo can identify exon-skipping events involving more than two exons, which account for a quarter to one-third of all skipping events.

Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis.

Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45+ cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al.1.

IL-17 signaling in primary sclerosing cholangitis patient-derived organoids.

BACKGROUND: The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study, we aimed to investigate and characterize the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. METHODS: Cholangiocytes obtained from patients with PSC and without PSC by endoscopic retrograde cholangiography were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing. RESULTS: Unsupervised clustering of all integrated single-cell RNA sequencing data identified 8 cholangiocyte clusters that did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, as noted by an increased number of differentially expressed genes by transcriptomics and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, genome sequencing identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. CONCLUSIONS: PSC and non-PSC patient-derived ECO respond differently to IL-17 stimulation, implicating this pathway in the pathogenesis of PSC.

YAP-TEAD inhibition is associated with upregulation of an androgen receptor mediated transcription program providing therapeutic escape.

Cholangiocarcinoma (CCA) is a highly aggressive form of liver cancer and is an increasing cause of cancer-related death worldwide. Despite its increasing incidence globally and alarming mortality, treatment options for CCA have largely remained unchanged, stressing the importance of developing new effective therapies. YAP activation is common in CCA, and its major transcriptional signaling partners are the TEAD proteins. CA3 is a small-molecule YAP-TEAD disrupter discovered utilizing a TEAD reporter assay. Utilizing CCA, gastric cancer cell lines, and patient-derived xenograft models (PDX), we demonstrate that CA3 is effective in inducing cell death and delaying tumor growth in both FGFR2 fusion and wild-type models. CA3 was associated with on-target decreases in YAP-TEAD target gene expression, TEAD reporter activity, and overall TEAD levels. Hippo pathway signaling was not altered as there was no change in YAP phosphorylation status in the cells exposed to CA3. RNA sequencing of gastric cancer and CCA models demonstrated upregulation of an androgen receptor-mediated transcriptional program following exposure to CA3 in five unique models tested. Consistent with this upstream regulator analysis, CA3 exposure in CCA cells was associated with increased AR protein levels, and combinatorial therapy with CA3 and androgen receptor blockade was associated with increased cancer cell death. CA3 behaves functionally as a YAP-TEAD disrupter in the models tested and demonstrated therapeutic efficacy. Exposure to CA3 was associated with compensatory androgen receptor signaling and dual inhibition improved the therapeutic effect.

Utility of Targeted Sequencing Compared to FISH for Detection of Chronic Lymphocytic Leukemia Copy Number Alterations.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

Cell-specific cross-talk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor-initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

Single cell atlas of human gastric muscle immune cells and macrophage-driven changes in idiopathic gastroparesis.

Gastrointestinal immune cells, particularly muscularis macrophages (MM) interact with the enteric nervous system and influence gastrointestinal motility. Here we determine the human gastric muscle immunome and its changes in patients with idiopathic gastroparesis (IG). Single cell sequencing was performed on 26,000 CD45+ cells obtained from the gastric tissue of 20 subjects. We demonstrate 11 immune cell clusters with T cells being most abundant followed by myeloid cells. The proportions of cells belonging to the 11 clusters were similar between IG and controls. However, 9/11 clusters showed 578-11,429 differentially expressed genes. In IG, MM had decreased expression of tissue-protective and microglial genes and increased the expression of monocyte trafficking and stromal activating genes. Furthermore, in IG, IL12 mediated JAK-STAT signaling involved in the activation of tissue-resident macrophages and Eph-ephrin signaling involved in monocyte chemotaxis were upregulated. Patients with IG had a greater abundance of monocyte-like cells. These data further link immune dysregulation to the pathophysiology of gastroparesis.

IL-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids.

The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. Highlights: Periventricular GBM is more malignant and disrupts neurogenesis in a rodent model.Cell-specific proteomics elucidates tumor-promoting crosstalk between GBM and NPCs.NPCs induce upregulated CTSB expression in GBM, promoting tumor progression.GBM stalls neurogenesis and promotes NPC senescence via CTSB.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

Transcriptomic Analysis of Cirrhosis-Like Hepatocellular Carcinoma Reveals Distinct Molecular Characteristics and Pathologic Staging Implications.

OBJECTIVES: Cirrhosis-like hepatocellular carcinoma (CL-HCC) is a rare hepatocellular malignancy characterized by multiple tumor nodules that clinically, radiologically, macroscopically, and microscopically mimic cirrhosis. We aimed to elucidate the molecular biology of CL-HCC and determine tumor nodule clonality. METHODS: We performed RNA sequencing on formalin-fixed, paraffin-embedded tissue from confirmed CL-HCC cases (n = 6), along with corresponding nonneoplastic hepatic tissue (n = 4) when available. Transcriptomes from our previous work on steatohepatitic hepatocellular carcinoma and The Cancer Genome Atlas (TCGA) were used for comparison purposes. RESULTS: Histologically, CL-HCC displayed innumerable nodules and extensive vascular invasion. Intratumoral nodule comparison indicated that the multiple nodules were all clonally related, not independent primaries. The unique histomorphologic appearance corresponded with a distinct transcriptome compared with other HCCs, including fibrolamellar HCC (n = 6), steatohepatitic HCC (n = 8), and conventional HCC in TCGA (n = 424). Tumor-normal gene expression analysis revealed consistent overexpression of several genes involved in degradation of tissue matrix. No recurrent translocations or point mutations were identified. CL-HCC showed a gene expression profile indicative of zone 2 hepatocytes. CONCLUSIONS: CL-HCC's distinctive clinicopathologic features correspond to a unique gene expression profile, increased expression of invasive markers, features of zone 2 hepatocytes, and features suggestive of intratumoral nodule monoclonality.

Determining mutational burden and signature using RNA-seq from tumor-only samples.

BACKGROUND: Traditionally, mutational burden and mutational signatures have been assessed by tumor-normal pair DNA sequencing. The requirement of having both normal and tumor samples is not always feasible from a clinical perspective, and led us to investigate the efficacy of using RNA sequencing of only the tumor sample to determine the mutational burden and signatures, and subsequently molecular cause of the cancer. The potential advantages include reducing the cost of testing, and simultaneously providing information on the gene expression profile and gene fusions present in the tumor. RESULTS: In this study, we devised supervised and unsupervised learning methods to determine mutational signatures from tumor RNA-seq data. As applications, we applied the methods to a training set of 587 TCGA uterine cancer RNA-seq samples, and examined in an independent testing set of 521 TCGA colorectal cancer RNA-seq samples. Both diseases are known associated with microsatellite instable high (MSI-H) and driver defects in DNA polymerase ɛ (POLɛ). From RNA-seq called variants, we found majority (> 95%) are likely germline variants, leading to C > T enriched germline variants (dbSNP) widely applicable in tumor and normal RNA-seq samples. We found significant associations between RNA-derived mutational burdens and MSI/POLɛ status, and insignificant relationship between RNA-seq total coverage and derived mutational burdens. Additionally we found that over 80% of variants could be explained by using the COSMIC mutational signature-5, -6 and -10, which are implicated in natural aging, MSI-H, and POLɛ, respectively. For classifying tumor type, within UCEC we achieved a recall of 0.56 and 0.78, and specificity of 0.66 and 0.99 for MSI and POLɛ respectively. By applying learnt RNA signatures from UCEC to COAD, we were able to improve our classification of both MSI and POLɛ. CONCLUSIONS: Taken together, our work provides a novel method to detect RNA-seq derived mutational signatures with effective procedures to remove likely germline variants. It can leads to accurate classification of underlying driving mechanisms of DNA damage deficiency.

Transcriptomic and Proteomic Analysis of Steatohepatitic Hepatocellular Carcinoma Reveals Novel Distinct Biologic Features.

OBJECTIVES: Steatohepatitic hepatocellular carcinoma is a distinct variant of hepatocellular carcinoma strongly associated with underlying nonalcoholic steatohepatitis. The molecular biology of steatohepatitic hepatocellular carcinoma is not fully elucidated, and thus we aimed to investigate the molecular underpinnings of this entity. METHODS: Transcriptomic analysis using RNAseq was performed on eight tumor-nonneoplastic pairs of steatohepatitic hepatocellular carcinoma with comparison to conventional hepatocellular carcinoma transcriptomes curated in The Cancer Genome Atlas. Immunohistochemistry was used to validate key RNA-level findings. RESULTS: Steatohepatitic hepatocellular carcinoma demonstrated a distinctive differential gene expression profile compared with The Cancer Genome Atlas curated conventional hepatocellular carcinomas (n = 360 cases), indicating the distinctive steatohepatitic hepatocellular carcinoma morphology is associated with a unique gene expression profile. Pathway analysis comparing tumor-nonneoplastic pairs revealed significant upregulation of the hedgehog pathway based on GLI1 overexpression and significant downregulation of carnitine palmitoyltransferase 2 transcript. Glutamine synthetase transcript was significantly upregulated, and fatty acid binding protein 1 transcript was significantly downregulated and immunohistochemically confirmed, indicating steatohepatitic hepatocellular carcinoma tumor cells display a zone 3 phenotype. CONCLUSIONS: Steatohepatitic hepatocellular carcinoma demonstrates a distinctive morphology and gene expression profile, phenotype of zone 3 hepatocytes, and activation of the hedgehog pathway and repression of carnitine palmitoyltransferase 2, which may be important in tumorigenesis.

Molecular Genetic Landscape of Sclerosing Pneumocytomas.

OBJECTIVES: Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. METHODS: To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). RESULTS: Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. CONCLUSIONS: Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.