Integrated Proteogenomic Analysis Reveals Distinct Potentially Actionable Therapeutic Vulnerabilities in Triple-Negative Breast Cancer Subtypes.

Triple-negative breast cancer (TNBC) is characterized by an aggressive clinical presentation and a paucity of clinically actionable genomic alterations. Here, we utilized the Cancer Genome Atlas (TCGA) to explore the proteogenomic landscape of TNBC subtypes to see whether genomic alterations can be inferred from proteomic data. We found only 4% of the protein level changes are explained by mutations, while 21% of the protein and 35% of the transcriptomics changes were determined by copy number alterations (CNAs). We found tighter coupling between proteome and genome in some genes that are predicted to be the targets of drug inhibitors, including CDKs, PI3K, tyrosine kinase (TKI), and mTOR. The validation of our proteogenomic workflow using mass spectrometry Clinical Proteomic Tumor Analysis Consortium (MS-CPTAC) data also demonstrated the highest correlation between protein-RNA-CNA. The integrated proteogenomic approach helps to prioritize potentially actionable targets and may enable the acceleration of personalized cancer treatment.

A Multi-Center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System.

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker-defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

Circulating Tumor Cell Transcriptomics as Biopsy Surrogates in Metastatic Breast Cancer.

BACKGROUND: Metastatic breast cancer (MBC) and the circulating tumor cells (CTCs) leading to macrometastases are inherently different than primary breast cancer. We evaluated whether whole transcriptome RNA-Seq of CTCs isolated via an epitope-independent approach may serve as a surrogate for biopsies of macrometastases. METHODS: We performed RNA-Seq on fresh metastatic tumor biopsies, CTCs, and peripheral blood (PB) from 19 newly diagnosed MBC patients. CTCs were harvested using the ANGLE Parsortix microfluidics system to isolate cells based on size and deformability, independent of a priori knowledge of cell surface marker expression. RESULTS: Gene expression separated CTCs, metastatic biopsies, and PB into distinct groups despite heterogeneity between patients and sample types. CTCs showed higher expression of immune oncology targets compared with corresponding metastases and PB. Predictive biomarker (n = 64) expression was highly concordant for CTCs and metastases. Repeat observation data post-treatment demonstrated changes in the activation of different biological pathways. Somatic single nucleotide variant analysis showed increasing mutational complexity over time. CONCLUSION: We demonstrate that RNA-Seq of CTCs could serve as a surrogate biomarker for breast cancer macrometastasis and yield clinically relevant insights into disease biology and clinically actionable targets.

DECUSSATE network with flowering genes explains the variable effects of qDTY12.1 to rice yield under drought across genetic backgrounds.

The impact of qDTY12.1 in maintaining yield under drought has not been consistent across genetic backgrounds. We hypothesized that synergism or antagonism with additive-effect peripheral genes across the background genome either enhances or undermines its full potential. By modeling the transcriptional networks across sibling qDTY12.1-introgression lines with contrasting yield under drought (LPB = low-yield penalty; HPB = high-yield penalty), the qDTY12.1-encoded DECUSSATE gene (OsDEC) was revealed as the core of a synergy with other genes in the genetic background. OsDEC is expressed in flag leaves and induced by progressive drought at booting stage in LPB but not in HPB. The unique OsDEC signature in LPB is coordinated with 35 upstream and downstream peripheral genes involved in floral development through the cytokinin signaling pathway. Results support the differential network rewiring effects through genetic coupling-uncoupling between qDTY12.1 and other upstream and downstream peripheral genes across the distinct genetic backgrounds of LPB and HPB. The functional DEC-network in LPB defines a mechanism for early flowering as a means for avoiding the drought-induced depletion of photosynthate needed for reproductive growth. Its impact is likely through the timely establishment of stronger source-sink dynamics that sustains a robust reproductive transition under drought.

Identification of putative actionable alterations in clinically relevant genes in breast cancer.

BACKGROUND: Individualising treatment in breast cancer requires effective predictive biomarkers. While relatively few genomic aberrations are clinically relevant, there is a need for characterising patients across different subtypes to identify actionable alterations. METHODS: We identified genomic alterations in 49 potentially actionable genes for which drugs are available either clinically or via clinical trials. We explored the landscape of mutations and copy number alterations (CNAs) in actionable genes in seven breast cancer subtypes utilising The Cancer Genome Atlas. To dissect the genomic complexity, we analysed the patterns of co-occurrence and mutual exclusivity in actionable genes. RESULTS: We found that >30% of tumours harboured putative actionable events that are targetable by currently available drugs. We identified genes that had multiple targetable alterations, representing candidate targets for combination therapy. Genes predicted to be drivers in primary breast tumours fell into five categories: mTOR pathway, immune checkpoints, oestrogen signalling, tumour suppression and DNA damage repair. Our analysis also revealed that CNAs in 34/49 (69%) and mutations in 13/49 (26%) genes were significantly associated with gene expression, validating copy number events as a dominant oncogenic mechanism in breast cancer. CONCLUSION: These results may enable the acceleration of personalised therapy and improve clinical outcomes in breast cancer.

EP300 knockdown reduces cancer stem cell phenotype, tumor growth and metastasis in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300 (EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology. METHODS: We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performed in silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC. RESULTS: EP300 KD abolished the CSC phenotype by reducing ABCG2 expression, side population cells and tumorsphere formation capacity in vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells. TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC. CONCLUSION: We report a novel oncogenic role for EP300 in driving CSC phenotype representing a potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Idiopathic Bilateral Ovarian Vein Thrombosis in a Non-Pregnant Healthy Patient: A Case Report and Review of the Literature.

Ovarian vein thrombosis (OVT) is a potentially life-threatening condition, and it is typically related to the peripartum period; however, it is also associated with pelvic inflammatory disease, recent pelvic or abdominal surgery, inflammatory bowel disease, thrombophilia, malignancy, and sepsis. Idiopathic isolated OVT is rare and is usually presented as case reports in the medical literature. In this report, we present a case of bilateral OVT in a postmenopausal female with no identifiable risk factors and normal coagulation profile workup to highlight the importance of considering it as a differential diagnosis in female patients presenting with abdominal pain. Early identification can prevent potentially life-threatening complications. Management is often conservative, and the choice of anticoagulation is based on the patient's medical conditions. In this particular scenario, the patient was managed with low molecular weight heparin (LMWH) and transitioned to direct oral anticoagulant (DOAC) before discharge.

Increased risk of disseminated cryptococcal infection in a patient with multiple sclerosis on fingolimod.

Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS), with an estimated 2.3 million people being affected globally, and is a major cause of permanent disability. About 90 % of the affected patients with MS have relapsing-remitting type. Fingolimod became the first FDA approved oral drug in 2010 with an immunomodulating mechanism to control the relapse rates. However, since its introduction, increased cases of cryptococcal infections have been reported including meningoencephalitis and disseminated infections. Herein, we present the case of a 34-year-old-male with disseminated Cryptococcal and localized varicella zoster virus (VZV) coinfection to highlight the risk of opportunistic infections associated with the long-term use of fingolimod. The objective of this literature review is for clinicians to have a high index of suspicion for cryptococcal infections when dealing with MS patients on Fingolimod, especially those who present with neurological symptoms, as this mimics MS relapse.

A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer.

The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases.

Comparison of TCGA and GENIE genomic datasets for the detection of clinically actionable alterations in breast cancer.

Whole exome sequencing (WES), targeted gene panel sequencing and single nucleotide polymorphism (SNP) arrays are increasingly used for the identification of actionable alterations that are critical to cancer care. Here, we compared The Cancer Genome Atlas (TCGA) and the Genomics Evidence Neoplasia Information Exchange (GENIE) breast cancer genomic datasets (array and next generation sequencing (NGS) data) in detecting genomic alterations in clinically relevant genes. We performed an in silico analysis to determine the concordance in the frequencies of actionable mutations and copy number alterations/aberrations (CNAs) in the two most common breast cancer histologies, invasive lobular and invasive ductal carcinoma. We found that targeted sequencing identified a larger number of mutational hotspots and clinically significant amplifications that would have been missed by WES and SNP arrays in many actionable genes such as PIK3CA, EGFR, AKT3, FGFR1, ERBB2, ERBB3 and ESR1. The striking differences between the number of mutational hotspots and CNAs generated from these platforms highlight a number of factors that should be considered in the interpretation of array and NGS-based genomic data for precision medicine. Targeted panel sequencing was preferable to WES to define the full spectrum of somatic mutations present in a tumor.

RNA-Seq of Circulating Tumor Cells in Stage II-III Breast Cancer.

BACKGROUND: We characterized the whole transcriptome of circulating tumor cells (CTCs) in stage II-III breast cancer to evaluate correlations with primary tumor biology. METHODS: CTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). CTCs, PB, and fresh tumors were profiled using RNA-seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA-seq and NanoString PAM50 assays with risk of recurrence (ROR) scores. RESULTS: CTCs were detected in 29/33 (88%) patients. We selected 21 cases to attempt RNA-seq (median number of CTCs = 9). Sixteen CTC samples yielded results that passed quality-control metrics, and these samples had a median of 4,311,255 uniquely mapped reads (less than PB or tumors). Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA-seq subtype assessed by the PAM50 classification genes was highly discordant, both with the subtype predicted by ER/PR/HER2 and by PAM50 tumors. Two patients died of metastatic disease, both of whom had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators, and molecular interaction networks comparing CTCs by various clinical factors. We also identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium datasets. CONCLUSION: It is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.

Challenges in using liquid biopsies for gene expression profiling.

Circulating tumor cells (CTCs) have potential utility as a surrogate biomarker of tumor biology via a liquid biopsy. The aim of this study was to evaluate if the nCounter NanoString assay could be used for accurate gene expression profiling of CTCs using the PAM50 research-use-only CodeSet. Analysis was performed on CTCs isolated by the ANGLE Parsortix system from healthy blood spiked with the breast cancer cell lines Hs578T, SkBr3, MDA-MB-231 or MCF7. Using cell lines as gold standard positive controls and Parsortix processed blood without spiking (unspiked) as negative controls, we found an average of 12 significantly differentially expressed genes among spiked samples versus unspiked controls. We validated our findings with the NanoStringDiff differential expression statistical method. The NanoString recommended targeted pre-amplification introduced false positive results due to pre-amplification bias, and the amplification of non-cancer genes from normal leukocytes confounded gene expression profiling of CTCs. Pre-amplification bias is a concern for other similar assays that may be used as discovery tools or target validation of transcripts of interest in gene expression profiling of CTCs. We recommend the use of an unspiked negative control when evaluating CTC technologies regarding gene expression profiling. Given that the molecular profiling of CTCs as a liquid biopsy may have clinical ramifications for potential treatment selection in future clinical trials, our study emphasizes cautious consideration of pre-analytical variables such as amplification bias in the context of liquid biopsy studies.

On genome annotation of Brucellaphage Gadvasu (BpG): discovery of ORFans for integrated systems biology approaches.

Brucellaphage Gadvasu (BpG) is a lytic phage infecting Brucella spp. Brucellaphages contain dsDNA as genetic material and are short-tailed particles with host-specificity. Here, we report the challenges on annotation in the complete genome sequence of BpG when compared with that of a recent broad host-range brucellaphage Pr, an original reference genome. The extracted DNA was subjected to genome sequencing with Illumina technology and assembled using SSAKE/Velvet. A significant number of genes were found to be similar between the phages with sequence analysis revealing conserved open reading frames that correspond to 33 gene ontology classifiers, transcriptional terminators and a few putative transcriptional promoters. The analyses revealed that the genome constitutes 1269 contigs and 275 genes encoding 260 proteins. The sequence comparison from the reference data indicated that the genome shares an approximately 70 % nucleotide similarity and differs mainly in the region encoding proteins. We bring this commentary providing an overview of how this exemplar genome can allow us to understand these known unknown regions in brucellaphages.

Synthesis of novel antimicrobial aryl himachalene derivatives from naturally occurring himachalenes.

Five new 2,9,9-trimethyl-6,7,8,9-tetrahydro-benzocyclohepten-5-ylidene-amine derivatives (16a-16e) were synthesized from α-dehydro-ar-himachalene (11) that was originally prepared from an isomeric mixture of α, ß and γ himachalenes (10), the abundant sesquiterpenes of Cedrus deodara essential oil. In addition, different aryl himachalenes derivatives (9, 12, 14 and 15) were also formed from 11. The structures of the synthesized compounds were confirmed on the basis of their NMR, IR and mass spectral data. The prepared compounds were tested against a group of sixteen organisms including gram positive and gram negative bacterial and fungal strains. The introduction of a series of substituted imine groups into aryl himachalenes at 5(th) position (16a-16e) enhanced antimicrobial activity as compared to the aromatized derivatives (9, 12, 14 and 15) against gram-positive bacteria Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus, and mycotoxigenic fungi Aspergillus parasiticus, A. ochraceous and A. sydowii. graphical Abstract, Figure 1(Fig. 1).

The epigenome as a potential mediator of cancer and disease prevention in prenatal development.

Epigenetic events establish a particular gene expression signature for each cell type during differentiation and fertilization. Disruption of these epigenetic programs in response to environmental stimuli during prenatal exposure dysregulates the fetal epigenome, potentially impacting susceptibility to disease later in life (the fetal basis of adult disease). Maternal dietary modifications during gestation and lactation play a pivotal role in the period of fetal (re)programming. Recently, many studies have demonstrated the impact of maternal nutrition on the fetal epigenome. This review discusses the complex interplay among various environmental factors and epigenetic mechanisms that have been found to affect offspring in human and animal models. Further, it summarizes the impact of various dietary phytochemicals capable of modulating the epigenome with regard to diverse human cancers and childhood cancer, specifically those with potential environmental etiology through maternal consumption during pregnancy and lactation. Other dietary agents that are still untested as to their effectiveness in transplacental studies are also discussed. The recent developments discussed herein enhance current understanding of how chemopreventive agents act and their potential to impact the prenatal epigenome; they may also aid efforts to identify dietary interventions that can be beneficial in treating and preventing disease.

Naturally occurring limonene to cinnamyl-type gamma-butyrolactone substituted aldol condensation derivatives as antioxidant compounds.

A method has been developed for the synthesis of gamma-butyrolactone substituted cinnamyl type Aldol condensation products under milder proline and triethylamine basic conditions. The antioxidant activity of all the synthesized compounds was assessed using three different assays. The cinnamyl type derivatives of gamma-butyrolactone bearing an -OH group in the benzene ring exhibited excellent antioxidant activity.

Naturally occurring himachalenes to benzocycloheptene amino vinyl bromide derivatives: as antidepressant molecules.

A new series of benzocycloheptene amino vinyl bromide derivatives (9a-9m) were synthesized from isomeric mixture of himachalenes through two-step synthesis. The unusual structure of benzocycloheptene amino vinyl bromide derivative (9a) was confirmed by NMR and X-ray crystallography analyses. The newly synthesized amino vinyl bromide derivatives of benzocycloheptene were further evaluated for their antidepressant activities. The compound 9c had shown significant reduction in the immobility period.

An efficient microwave assisted extraction of phenolic compounds and antioxidant potential of Ginkgo biloba.

Flavonoid glycosides are a significant group of compounds found in Ginkgo biloba leaves, but the long extraction procedures in existing methods are a challenging problem. In this work, a microwave-assisted extraction (MAE) method has been developed for extracting bioactive compounds from G. biloba. Several variables were optimized, such as extracting solvent, microwave power, and extraction time that can potentially affect the extraction yield. The total phenolic content, antioxidant activity (using DPPH, ABTS and FRAP assays) and flavonoid glycosides of different extracts using RP-HPLC were assessed. The antioxidant capacity was found to be highest with MAE using 60% aq. ethanol as extracting solvent and microwave power of 120W for 20 min.

Epigenetic silencing of sFRP1 activates the canonical Wnt pathway and contributes to increased cell growth and proliferation in hepatocellular carcinoma.

The Wnt pathway is a key regulator of embryonic development and stem cells, and its aberrant activation is associated with human malignancies, most notably hepatocellular carcinoma (HCC). Epigenetic deregulation of the genes encoding the secreted frizzled-related proteins (sFRPs), the Wnt signalling antagonists, has been linked with aberrant hyperactivation of the Wnt signalling in HCC cells; however, the precise underlying mechanism remains elusive. We investigated the methylation profiles of Wnt antagonists in liver samples of different stages of HCC development and liver cancer cell lines and studied the functional impact of aberrant epigenetic silencing of sFRPs on the canonical Wnt pathway and cell viability. We found that the sFRP1 gene encoding the subunit is a frequent target of aberrant DNA hypermethylation and silencing in HCC tumours, whereas other extracellular Wnt antagonists, WIF1 and Dkk3, exhibited no methylation in tumour cells, consistent with the notion that aberrant methylation events in cancer cells are non-randomly distributed among the genes and that there is a strong preference for hypermethylation of specific genes in HCC. In addition, by comparing sFRP1 methylation status in HCC tumours with normal, cirrhotic and chronic hepatitis liver tissues, we identified sFRP1 gene as a potential early marker of HCC. The restoration of sFRP1 expression in cancer cells by ectopic expression inhibited Wnt activity accompanied with destabilization of ß-catenin and downregulation of c-Myc and cyclin D1, the known downstream targets of Wnt pathway. Importantly, restoring sFRP1 levels in cancer cells inhibited cell growth and induced apoptotic cell death. This study supports the critical role for sFRP1 silencing in hepatocellular carcinoma and reinforces the importance of the Wnt antagonists in preventing oncogenic stabilization of ß-catenin and chronic activation of the canonical Wnt pathway, suggesting that sFRP1 may be an attractive target for early cancer detection and therapeutic intervention.