Investigation of Different Library Preparation and Tissue of Origin Deconvolution Methods for Urine and Plasma cfDNA Methylome Analysis.

Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, p ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; p ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRß-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

PipeIT2: A tumor-only somatic variant calling workflow for molecular diagnostic Ion Torrent sequencing data.

Precision oncology relies on the accurate identification of somatic mutations in cancer patients. While the sequencing of the tumoral tissue is frequently part of routine clinical care, the healthy counterparts are rarely sequenced. We previously published PipeIT, a somatic variant calling workflow specific for Ion Torrent sequencing data enclosed in a Singularity container. PipeIT combines user-friendly execution, reproducibility and reliable mutation identification, but relies on matched germline sequencing data to exclude germline variants. Expanding on the original PipeIT, here we describe PipeIT2 to address the clinical need to define somatic mutations in the absence of germline control. We show that PipeIT2 achieves a > 95% recall for variants with variant allele fraction >10%, reliably detects driver and actionable mutations and filters out most of the germline mutations and sequencing artifacts. With its performance, reproducibility, and ease of execution, PipeIT2 is a valuable addition to molecular diagnostics laboratories.

DNA Methylation Landscapes of Prostate Cancer Brain Metastasis Are Shaped by Early Driver Genetic Alterations.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand-receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation. SIGNIFICANCE: DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

Integrated longitudinal analysis of adult grade 4 diffuse gliomas with long-term relapse interval revealed upregulation of TGF-ß signaling in recurrent tumors.

BACKGROUND: Adult-type diffuse gliomas, CNS WHO grade 4 are the most aggressive primary brain tumors and represent a particular challenge for therapeutic intervention. METHODS: In a single-center retrospective study of matched pairs of initial and post-therapeutic glioma cases with a recurrence period greater than 1 year, we performed whole exome sequencing combined with mRNA and microRNA expression profiling to identify processes that are altered in recurrent gliomas. RESULTS: Mutational analysis of recurrent gliomas revealed early branching evolution in 75% of the patients. High plasticity was confirmed at the mRNA and miRNA levels. SBS1 signature was reduced and SBS11 was elevated, demonstrating the effect of alkylating agent therapy on the mutational landscape. There was no evidence for secondary genomic alterations driving therapy resistance. ALK7/ACVR1C and LTBP1 were upregulated, whereas LEFTY2 was downregulated, pointing towards enhanced Tumor Growth Factor ß (TGF-ß) signaling in recurrent gliomas. Consistently, altered microRNA expression profiles pointed towards enhanced Nuclear Factor Kappa B and Wnt signaling that, cooperatively with TGF-ß, induces epithelial to mesenchymal transition (EMT), migration, and stemness. TGF-ß-induced expression of pro-apoptotic proteins and repression of antiapoptotic proteins were uncoupled in the recurrent tumor. CONCLUSIONS: Our results suggest an important role of TGF-ß signaling in recurrent gliomas. This may have clinical implications since TGF-ß inhibitors have entered clinical phase studies and may potentially be used in combination therapy to interfere with chemoradiation resistance. Recurrent gliomas show high incidence of early branching evolution. High tumor plasticity is confirmed at the level of microRNA and mRNA expression profiles.

Discovery of synthetic lethal interactions from large-scale pan-cancer perturbation screens.

The development of cancer therapies is limited by the availability of suitable drug targets. Potential candidate drug targets can be identified based on the concept of synthetic lethality (SL), which refers to pairs of genes for which an aberration in either gene alone is non-lethal, but co-occurrence of the aberrations is lethal to the cell. Here, we present SLIdR (Synthetic Lethal Identification in R), a statistical framework for identifying SL pairs from large-scale perturbation screens. SLIdR successfully predicts SL pairs even with small sample sizes while minimizing the number of false positive targets. We apply SLIdR to Project DRIVE data and find both established and potential pan-cancer and cancer type-specific SL pairs consistent with findings from literature and drug response screening data. We experimentally validate two predicted SL interactions (ARID1A-TEAD1 and AXIN1-URI1) in hepatocellular carcinoma, thus corroborating the ability of SLIdR to identify potential drug targets.

Transcription factors TEAD2 and E2A globally repress acetyl-CoA synthesis to promote tumorigenesis.

Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.

Siglec-7 represents a glyco-immune checkpoint for non-exhausted effector memory CD8+ T cells with high functional and metabolic capacities.

While inhibitory Siglec receptors are known to regulate myeloid cells, less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state, and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells, which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer.

Multi-omics subtyping of hepatocellular carcinoma patients using a Bayesian network mixture model.

Comprehensive molecular characterization of cancer subtypes is essential for predicting clinical outcomes and searching for personalized treatments. We present bnClustOmics, a statistical model and computational tool for multi-omics unsupervised clustering, which serves a dual purpose: Clustering patient samples based on a Bayesian network mixture model and learning the networks of omics variables representing these clusters. The discovered networks encode interactions among all omics variables and provide a molecular characterization of each patient subgroup. We conducted simulation studies that demonstrated the advantages of our approach compared to other clustering methods in the case where the generative model is a mixture of Bayesian networks. We applied bnClustOmics to a hepatocellular carcinoma (HCC) dataset comprising genome (mutation and copy number), transcriptome, proteome, and phosphoproteome data. We identified three main HCC subtypes together with molecular characteristics, some of which are associated with survival even when adjusting for the clinical stage. Cluster-specific networks shed light on the links between genotypes and molecular phenotypes of samples within their respective clusters and suggest targets for personalized treatments.

The Role of Chronic Liver Diseases in the Emergence and Recurrence of Hepatocellular Carcinoma: An Omics Perspective.

Hepatocellular carcinoma (HCC) typically develops from a background of cirrhosis resulting from chronic inflammation. This inflammation is frequently associated with chronic liver diseases (CLD). The advent of next generation sequencing has enabled extensive analyses of molecular aberrations in HCC. However, less attention has been directed to the chronically inflamed background of the liver, prior to HCC emergence and during recurrence following surgery. Hepatocytes within chronically inflamed liver tissues present highly activated inflammatory signaling pathways and accumulation of a complex mutational landscape. In this altered environment, cells may transform in a stepwise manner toward tumorigenesis. Similarly, the chronically inflamed environment which persists after resection may impact the timing of HCC recurrence. Advances in research are allowing an extensive epigenomic, transcriptomic and proteomic characterization of CLD which define the emergence of HCC or its recurrence. The amount of data generated will enable the understanding of oncogenic mechanisms in HCC from the CLD perspective and provide the possibility to identify robust biomarkers or novel therapeutic targets for the treatment of primary and recurrent HCC. Importantly, biomarkers defined by the analysis of CLD tissue may permit the early detection or prevention of HCC emergence and recurrence. In this review, we compile the current omics based evidence of the contribution of CLD tissues to the emergence and recurrence of HCC.

PipeIT2: Somatic Variant Calling Workflow for Ion Torrent Sequencing Data.

The detection of somatic genetic variants forms the cornerstone of precision oncology. Molecular diagnostics laboratories require economical and rapid protocols to generate accurate variant calls for clinical purposes. The Ion Torrent sequencing platform is frequently used but lacks robust analysis workflows tailored to the needs of custom sequencing panels. We present PipeIT2, a somatic variant calling workflow for both matched tumor-germline and tumor-only analyses. PipeIT2 is enclosed in a Singularity container to ensure reproducibility and ease of deployment.

Genomic analysis of focal nodular hyperplasia with associated hepatocellular carcinoma unveils its malignant potential: a case report.

Background: Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence. Methods: A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone. Results: Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events. Conclusion: To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases.

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-ß-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome.

Alterations in homologous recombination repair genes in prostate cancer brain metastases.

Improved survival rates for prostate cancer through more effective therapies have also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. Here we investigate the repertoire of somatic genetic alterations present in brain metastases from 51 patients with prostate cancer brain metastases (PCBM). We highlight the clonal evolution occurring in PCBM and demonstrate an increased mutational burden, concomitant with an enrichment of the homologous recombination deficiency mutational signature in PCBM compared to non-brain metastases. Focusing on known pathogenic alterations within homologous recombination repair genes, we find 10 patients (19.6%) fulfilling the inclusion criteria used in the PROfound clinical trial, which assessed the efficacy of PARP inhibitors (PARPi) in homologous recombination deficient prostate cancer. Eight (15.7%) patients show biallelic loss of one of the 15 genes included in the trial, while 5 patients (9.8%) harbor pathogenic alterations in BRCA1/2 specifically. Uncovering these molecular features of PCBM may have therapeutic implications, suggesting the need of clinical trial enrollment of PCBM patients when evaluating potential benefit from PARPi.

GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers.

Synthetic lethal interactions, where the simultaneous but not individual inactivation of two genes is lethal to the cell, have been successfully exploited to treat cancer. GATA3 is frequently mutated in estrogen receptor (ER)-positive breast cancers and its deficiency defines a subset of patients with poor response to hormonal therapy and poor prognosis. However, GATA3 is not yet targetable. Here we show that GATA3 and MDM2 are synthetically lethal in ER-positive breast cancer. Depletion and pharmacological inhibition of MDM2 significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoids/xenograft (PDOs/PDX) harboring GATA3 somatic mutations. The synthetic lethality requires p53 and acts via the PI3K/Akt/mTOR pathway. Our results present MDM2 as a therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy.

Circulating Cell-Free DNA Captures the Intratumor Heterogeneity in Multinodular Hepatocellular Carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, with more than 40% of patients initially diagnosed with multinodular HCCs. Although circulating cell-free DNA (cfDNA) has been shown to effectively detect somatic mutations, little is known about its utility to capture intratumor heterogeneity in patients with multinodular HCC undergoing systemic treatment. MATERIALS AND METHODS: Tumor biopsies and plasma were synchronously collected from seven prospectively recruited patients with HCC before and during systemic therapy. Plasma-derived cfDNA and matched germline were subjected to high-depth targeted sequencing with molecular barcoding. The mutational profile of the cfDNA was compared with whole-exome sequencing from matched tumor biopsies. RESULTS: Genomic data revealed that out of the seven patients, five were considered intrahepatic metastasis and two multicentric HCCs. cfDNA captured the majority of mutations in the tumors and detected significantly more mutations than tumor biopsies. Driver mutations such as CTNNB1 S33C, NRAS Q61R, ARID1A R727fs, and NF1 E2368fs as well as standard-of-care biomarkers of response to targeted therapy were detected only in cfDNA. In the two patients with multicentric HCC, cfDNA detected mutations derived from the genetically independent and spatially distinct nodules. Moreover, cfDNA was not only able to capture clonal mutations but also the subclonal mutations detected in only one of the multiple biopsied nodules. Furthermore, serial cfDNA detected variants of tumor origin emerging during treatment. CONCLUSION: This study revealed that the genetic analysis of cfDNA captures the intratumor heterogeneity in multinodular HCC highlighting the potential for cfDNA as a sensitive and noninvasive tool for precision medicine.

Epigenetic priming in chronic liver disease impacts the transcriptional and genetic landscapes of hepatocellular carcinoma.

Hepatocellular carcinomas (HCCs) usually arise from chronic liver disease (CLD). Precancerous cells in chronically inflamed environments may be 'epigenetically primed', sensitising them to oncogenic transformation. We investigated whether epigenetic priming in CLD may affect HCC outcomes by influencing the genomic and transcriptomic landscapes of HCC. Analysis of DNA methylation arrays from 10 paired CLD-HCC identified 339 shared dysregulated CpG sites and 18 shared differentially methylated regions compared with healthy livers. These regions were associated with dysregulated expression of genes with relevance in HCC, including ubiquitin D (UBD), cytochrome P450 family 2 subfamily C member 19 (CYP2C19) and O-6-methylguanine-DNA methyltransferase (MGMT). Methylation changes were recapitulated in an independent cohort of nine paired CLD-HCC. High CLD methylation score, defined using the 124 dysregulated CpGs in CLD and HCC in both cohorts, was associated with poor survival, increased somatic genetic alterations and TP53 mutations in two independent HCC cohorts. Oncogenic transcriptional and methylation dysregulation is evident in CLD and compounded in HCC. Epigenetic priming in CLD sculpts the transcriptional landscape of HCC and creates an environment favouring the acquisition of genetic alterations, suggesting that the extent of epigenetic priming in CLD could influence disease outcome.

The Genomic Landscape of Serrated Lesion of the Colorectum: Similarities and Differences With Tubular and Tubulovillous Adenomas.

Serrated lesions of the colorectum are the precursors of 15-30% of colorectal cancers (CRCs). These lesions have a peculiar morphological appearance, and they are more difficult to detect than conventional adenomatous polyps. In this study, we sought to define the genomic landscape of these lesions using high-depth targeted sequencing. Eight sessile serrated lesions without dysplasia (SSL), three sessile serrated lesions with dysplasia (SSL/D), two traditional serrated adenomas (TSA), and three tubular adenomas (TA) were retrieved from the files of the Institute of Pathology of the University Hospital Basel and from the GILAB AG, Allschwil, Switzerland. Samples were microdissected together with the matched normal counterpart, and DNA was extracted for library preparation. Library preparation was performed using the Oncomine Comprehensive Assay targeting 161 common cancer driver genes. Somatic genetic alterations were defined using state-of-the-art bioinformatic analysis. Most SSLs, as well as all SSL/Ds and TSAs, showed the classical BRAF p.V600E mutation. The BRAF-mutant TSAs showed additional alterations in CTNNB1, NF1, TP53, NRAS, PIK3CA, while TA showed a consistently different profile, with mutations in ARID1A (two cases), SMAD4, CDK12, ERBB3, and KRAS. In conclusion, our results provide evidence that SSL/D and TSA are similar in somatic mutations with the BRAF hotspot somatic mutation as a major driver of the disease. On the other hand, TAs show a different constellation of somatic mutations such as ARID1A loss of function.

Multi-omics data integration reveals novel drug targets in hepatocellular carcinoma.

BACKGROUND: Genetic aberrations in hepatocellular carcinoma (HCC) are well known, but the functional consequences of such aberrations remain poorly understood. RESULTS: Here, we explored the effect of defined genetic changes on the transcriptome, proteome and phosphoproteome in twelve tumors from an mTOR-driven hepatocellular carcinoma mouse model. Using Network-based Integration of multi-omiCS data (NetICS), we detected 74 'mediators' that relay via molecular interactions the effects of genetic and miRNA expression changes. The detected mediators account for the effects of oncogenic mTOR signaling on the transcriptome, proteome and phosphoproteome. We confirmed the dysregulation of the mediators YAP1, GRB2, SIRT1, HDAC4 and LIS1 in human HCC. CONCLUSIONS: This study suggests that targeting pathways such as YAP1 or GRB2 signaling and pathways regulating global histone acetylation could be beneficial in treating HCC with hyperactive mTOR signaling.