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.

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.

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.

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.

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.

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.

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.

Antifungal sesquiterpenes from Cedrus deodara.

Two new sesquiterpenes, (E)-(2S,3S,6R)-atlantone-2,3-diol (1) and (E)-(2S,3S,6S)-atlantone-2,3,6-triol (2), along with two known sesquiterpenes, atlantolone (3) and (E)-α-atlantone (4), were isolated from Cedrus deodara Loud. The structures of the new compounds were elucidated on the basis of UV, IR, NMR, HR­ESI-QTOF­MS, and EI mass spectral studies. The n-hexane and chloroformextracts of sawdust and compounds 3 and 4 from the plant exhibited antifungal activity against Aspergillus flavus, A. niger, A. ochracoeus, A. parasiticus, and A. sydowii. A weak activity was also recorded against A. parasiticus and A. sydowii for compound 1, while Trichophyton rubrum was inhibited by compound 2 and the extracts.

Spatial and temporal variation of secondary metabolite profiles in Ginkgo biloba leaves.

Terpene trilactones (TTLs) are the main bioactive constituents of Ginkgo biloba used for the preparation of drugs for several ailments. Flavonoid glycosides (FGs) are the significant group of compounds found in Ginkgo leaves used in food and healthcare products. The variation in the content of bioactive constituents, as well as antioxidant activity, with respect to change in altitude and the time of sample collection was investigated. The amount of TTLs varied from 0.218-0.709% w/w, whereas FGs were observed in the range of 0.130-0.209% w/w. Ginkgolides J and A showed significant variation (p ≤ 0.05) with the change in altitude, while other components remained more or less unaffected. The amount of TTLs and FGs were not affected significantly by the time of sample collection. The antioxidant capacity (by using DPPH, ABTS, and FRAP assays) of ginkgo extracts was highly dependent on the altitude, and was at maximum in the samples collected from 1000-2000 m, followed by the samples collected from altitudes lower than 1000 m and higher than 2000 m. Thus, Ginkgo harvested from 1000-2000 m of altitudinal range irrespective of the season could provide better nutraceutical formulations, whereas that from below 1000 m may yield a terpene trilactone-enriched fraction.

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.

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).

DNA methylation of hepatitis B virus (HBV) genome associated with the development of hepatocellular carcinoma and occult HBV infection.

Recent studies have identified the presence of methylation in the hepatitis B virus (HBV) genome and have suggested that it may be an important mechanism regulating transcription and replication of the HBV virus. However, it remains unclear whether this phenomenon is associated with occult hepatitis. Here, we performed a detailed analysis of DNA methylation in the HBV genome in liver samples of patients at different stages of hepatocarcinoma development and in in vitro infected hepatocytes and found discrete CpG sites in the HBV genome that are recurrently hypermethylated in cancer but not in chronic hepatitis tissue.

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.

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.

CYP17 gene polymorphism and its association in north Indian prostate cancer patients.

BACKGROUND: The CYP17 gene codes for the cytochrome P450c17a enzyme, which mediates two key steps in sex steroid synthesis In this study, the association between CYP17 polymorphism and the risk of prostate cancer in comparison to benign prostatic hyperplasia (BPH) in a north Indian population was investigated. PATIENTS AND METHODS: This study included 157 prostate cancer patients and 170 BPH patients as controls. A 451-bp fragment encompassing the polymorphic site was amplified by PCR and treated with the restriction enzyme MspA1. The undigested allele was recognized as A1 and the MspA1-digested variant allele was designated as the A2 allele. RESULTS: Men with the A2/A2 CYP17 genotype had an increased risk of prostate cancer (OR=3.56; 95% CI=1.49-8.53; p=0.004) compared with those with the A1/A1 genotype. A significantly increased risk of prostate cancer was also found in smokers as well as non-vegetarians by four-fold as compared to their counterparts. There was a significant association between the CYP17 genotype and the tumour status (stage) of prostate cancer. The A2 allele showed a 1.90- (95% CI=1.09-3.32; p=0.02) and a 1.51- (95% CI=1.08-2.13; p=0.017) fold increased risk of prostate cancer in localized and metastatic prostate cancer cases respectively. CONCLUSION: The A2 allele of the CYP17 polymorphism is associated with an increased risk of prostate cancer and has a role in the development of prostate cancer in smokers and non-vegetarians.

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.

Plasma lipid peroxidation and antioxidant status of Parkinson's disease patients in the Indian population.

Present study was to assess lipid peroxidation and antioxidant enzymes in the blood of the Parkinson's disease (PD) patients in the Indian population. It may be useful to develop peripheral markers, for the diagnosis and prognosis of Parkinson's disease during lifetime. Malondialdehyde content was increased in patients with PD (2 fold), with respect to the activity of superoxide-dismutase (p<0.001). The levels of glutathione (p<0.001) and blood thiols were decreased. No changes were observed in gamma-GTP, glutathione peroxidase and glutathione reductase. Increased lipid peroxidation, decreased glutathione levels and increased superoxide dismutase activity in the blood of PD patients indicate oxidative stress.

Interleukin 1 beta gene polymorphism and risk of cervical cancer.

OBJECTIVE: To determine whether a polymorphism at position +3953 in exon 5 of the lL-1beta gene (IL-1beta +3953), a condition associated with an increased risk for a number of inflammatory diseases, is also involved in the development of cervical cancer. METHOD: We isolated DNA from peripheral blood in 150 women with cervical cancer and 200 healthy controls, and IL-1beta +3953 allele polymorphism was determined by polymerase chain reaction. RESULTS: Genotypes A1/A2 and A2/A2+A1/A2 were associated with increased risk of cervical cancer (odds ratio [OR], 2.88; 95% confidence interval [CI], 1.78-4.67; P<0.001 and OR, 2.85; 95% CI, 1.77-4.6; P<0.001, respectively). The risk in a passive smoker with A2/A2 or A1/A2 genotype was increased more than 5-fold (OR, 5.69; 95% CI, 2.61-12.50; P<0.001) compared with a nonsmoker with the A1/A1 genotype. CONCLUSION: This study provides evidence of an association between lL-1beta +3953 polymorphism and risk of cervical cancer.