Aurora Kinase A Is Overexpressed in Human Retinoblastoma and Correlates with Histopathologic High-Risk Factors: Implications for Targeted Therapy.

Retinoblastoma (RB) is an intraocular malignancy initiated by loss of RB1 function and/or dysregulation of MYCN oncogene. RB is primarily treated with chemotherapy; however, systemic toxicity and long-term adverse effects remain a significant challenge necessitating the identification of specific molecular targets. Aurora kinase A (AURKA), a critical cell cycle regulator, contributes to cancer pathogenesis, especially in RB1-deficient and MYCN-dysregulated tumors. Our immunohistochemistry study in patient specimens (n = 67) discovered that AURKA is overexpressed in RB, and elevated expression correlates with one or more histopathologic high-risk factors, such as tumor involvement of the optic nerve, choroid, sclera, and/or anterior segment. More specifically, AURKA is ubiquitously expressed in most advanced-stage RB tumors that show a suboptimal response to chemotherapy. shRNA-mediated depletion/pharmacologic inhibition studies in cell lines, patient-derived cells, in vivo xenografts, and enucleated patient specimens confirm that RB cells are highly sensitive to a lack of functional AURKA. In addition, we deciphered that AURKA and MYCN associate with each other to regulate their levels in RB cells. Overall, our results demonstrate a previously unknown up-regulation of AURKA in RB, facilitated by its crosstalk with MYCN, and elevated levels of this kinase may indicate unfavorable prognosis in tumors refractory to chemotherapy. This study provides a rationale and confirms that therapeutic targeting of elevated AURKA in RB could be a potential treatment approach.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

Purpose: To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Methods: Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Conclusions: Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

Lipid Metabolic Reprogramming in Embryonal Neoplasms with MYCN Amplification.

Tumor cells reprogram their metabolism, including glucose, glutamine, nucleotide, lipid, and amino acids to meet their enhanced energy demands, redox balance, and requirement of biosynthetic substrates for uncontrolled cell proliferation. Altered lipid metabolism in cancer provides lipids for rapid membrane biogenesis, generates the energy required for unrestricted cell proliferation, and some of the lipids act as signaling pathway mediators. In this review, we focus on the role of lipid metabolism in embryonal neoplasms with MYCN dysregulation. We specifically review lipid metabolic reactions in neuroblastoma, retinoblastoma, medulloblastoma, Wilms tumor, and rhabdomyosarcoma and the possibility of targeting lipid metabolism. Additionally, the regulation of lipid metabolism by the MYCN oncogene is discussed.

Quantitative background parenchymal enhancement and fibro-glandular density at breast MRI: Association with BRCA status.

OBJECTIVES: To investigate whether MRI-based measurements of fibro-glandular tissue volume, breast density (MRBD), and background parenchymal enhancement (BPE) could be used to stratify two cohorts of healthy women: BRCA carriers and women at population risk of breast cancer. METHODS: Pre-menopausal women aged 40-50 years old were scanned at 3 T, employing a standard breast protocol including a DCE-MRI (35 and 30 participants in high- and low-risk groups, respectively). The dynamic range of the DCE protocol was characterised and both breasts were masked and segmented with minimal user input to produce measurements of fibro-glandular tissue volume, MRBD, and voxelwise BPE. Statistical tests were performed to determine inter- and intra-user repeatability, evaluate the symmetry between metrics derived from left and right breasts, and investigate MRBD and BPE differences between the high- and low-risk cohorts. RESULTS: Intra- and inter-user reproducibility in estimates of fibro-glandular tissue volume, MRBD, and median BPE estimations were good, with coefficients of variation < 15%. Coefficients of variation between left and right breasts were also low (< 25%). There were no significant correlations between fibro-glandular tissue volume, MRBD, and BPE for either risk group. However, the high-risk group had higher BPE kurtosis, although linear regression analysis did not reveal significant associations between BPE kurtosis and breast cancer risk. CONCLUSIONS: This study found no significant differences or correlations in fibro-glandular tissue volume, MRBD, or BPE metrics between the two groups of women with different levels of breast cancer risk. However, the results support further investigation into the heterogeneity of parenchymal enhancement. KEY POINTS: • A semi-automated method enabled quantitative measurements of fibro-glandular tissue volume, breast density, and background parenchymal enhancement with minimal user intervention. • Background parenchymal enhancement was quantified over the entire parenchyma, segmented in pre-contrast images, thus avoiding region selection. • No significant differences and correlations in fibro-glandular tissue volume, breast density, and breast background parenchymal enhancement were found between two cohorts of women at high and low levels of breast cancer risk.

Beyond the learning curve: improving outcomes in Robotic myomectomy compared to laparoscopic myomectomy.

Uterine myomas are benign tumours frequently seen in women of reproductive age. Myomectomy remains a viable option for treating this condition in women who wish to preserve their uterus. We undertook this study to compare the peri-operative surgical outcomes of Robotic myomectomy (RM) with laparoscopic myomectomy (LM) in Indian patients of uterine myomas after the initial learning curve of RM was achieved. A retrospective chart review was performed for the patients who underwent RM or LM for the treatment of uterine myomas. A total of 177 patients, 116 in the RM group and 61 in the LM group, were included in the study. The mean age in the RM and LM group was 34.31 ± 5.40 years and 33.54 ± 4.96 years, respectively (p = 0.355). The mean total operative time was marginally more in RM group (127.37 ± 110.67 vs. 120.66 ± 44.27, p = 0.650) but the difference was not statistically significant. Patients in the RM group had significantly less blood loss (115.43 ± 79.43 vs. 340.98 ± 453.9 ml, p = < 0.0001), hospital stay (1.28 ± 0.49 vs. 1.92 ± 1.05 days, p = < 0.0001), requirement of blood transfusion (93.97 vs. 81.97%, p = 0.031) and requirement of intravenous (IV) analgesia (41.38 vs. 34.43%, p = 0.019) as compared to the patients in the LM group. The Robotic myomectomy significantly reduces blood loss, the duration of hospital stay, and requirement of blood transfusions and IV analgesia as compared to the laparoscopic myomectomy.

Comprehensive Analysis of Serum Small Extracellular Vesicles-Derived Coding and Non-Coding RNAs from Retinoblastoma Patients for Identifying Regulatory Interactions.

The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 1011 ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.

Capacity of Candida species to produce acetaldehyde at various concentrations of alcohol.

Background: Along with tobacco use, alcohol consumption is one of the crucial factors for oral cancer. Acetaldehyde (ACH), a byproduct of alcohol, is reported as carcinogenic. One of the producers of ACH from alcohol is Candida species. The aim of the study was to quantify the ACH produced by Candida species at various concentrations of alcohol. Materials and Methods: Clinical isolates of Candida, namely Candida albicans, Candida krusei and Candida tropicalis and C. albicans ATCC 18,804, were subjected to various concentrations of alcohol. Alcohol dehydrogenase and ACH were estimated using spectrophotometry and headspace gas chromatography, respectively. Results: Out of all three clinical isolates, C. tropicalis produced more ACH (412.1 µM) at 10 mM alcohol concentration by 105colony-forming unit/ml followed by C. albicans (233 µM) and C. krusei (53.7 µM). C. albicans of clinical isolate and ATCC species (222 µM) did not show much difference. Conclusion: The study results conclude that Candida species are capable of producing carcinogenic levels of ACH on exposure to various concentrations of alcohol.

The Oncogene MYCN Modulates Glycolytic and Invasive Genes to Enhance Cell Viability and Migration in Human Retinoblastoma.

Retinoblastoma is usually initiated by biallelic RB1 gene inactivation. In addition, MYCN copy number alterations also contribute to RB pathogenesis. However, MYCN expression, its role in disease progression and correlation with RB histological risk factors are not well understood. We studied the expression of MYCN in enucleated RB patient specimens by immunohistochemistry. MYCN is overexpressed in RB compared to control retina. Our microarray gene expression analysis followed by qRT-PCR validation revealed that genes involved in glucose metabolism and migration are significantly downregulated in MYCN knockdown cells. Further, targeting MYCN in RB cells using small molecule compounds or shRNAs led to decreased cell survival and migration, increased apoptosis and cell cycle arrest, suggesting that MYCN inhibition can be a potential therapeutic strategy. We also noted that MYCN inhibition results in reduction in glucose uptake, lactate production, ROS levels and gelatinolytic activity of active-MMP9, explaining a possible mechanism of MYCN in RB. Taking clues from our findings, we tested a combination treatment of RB cells with carboplatin and MYCN inhibitors to find enhanced therapeutic efficacy compared to single drug treatment. Thus, MYCN inhibition can be a potential therapeutic strategy in combination with existing chemotherapy drugs to restrict tumor cell growth in RB.

Aurora Kinase B Inhibition: A Potential Therapeutic Strategy for Cancer.

Aurora kinase B (AURKB) is a mitotic serine/threonine protein kinase that belongs to the aurora kinase family along with aurora kinase A (AURKA) and aurora kinase C (AURKC). AURKB is a member of the chromosomal passenger protein complex and plays a role in cell cycle progression. Deregulation of AURKB is observed in several tumors and its overexpression is frequently linked to tumor cell invasion, metastasis and drug resistance. AURKB has emerged as an attractive drug target leading to the development of small molecule inhibitors. This review summarizes recent findings pertaining to the role of AURKB in tumor development, therapy related drug resistance, and its inhibition as a potential therapeutic strategy for cancer. We discuss AURKB inhibitors that are in preclinical and clinical development and combination studies of AURKB inhibition with other therapeutic strategies.

Aurora Kinase B Expression, Its Regulation and Therapeutic Targeting in Human Retinoblastoma.

Purpose: Aurora kinase B (AURKB) plays a pivotal role in the regulation of mitosis and is gaining prominence as a therapeutic target in cancers; however, the role of AURKB in retinoblastoma (RB) has not been studied. The purpose of this study was to determine if AURKB plays a role in RB, how its expression is regulated, and whether it could be specifically targeted. Methods: The protein expression of AURKB was determined using immunohistochemistry in human RB patient specimens and immunoblotting in cell lines. Pharmacological inhibition and shRNA-mediated knockdown were used to understand the role of AURKB in cell viability, apoptosis, and cell cycle distribution. Cell viability in response to AURKB inhibition was also assessed in enucleated RB specimens. Immunoblotting was employed to determine the protein levels of phospho-histone H3, p53, p21, and MYCN. Chromatin immunoprecipitation-qPCR was performed to verify the binding of MYCN on the promoter region of AURKB. Results: The expression of AURKB was found to be markedly elevated in human RB tissues, and the overexpression significantly correlated with optic nerve and anterior chamber invasion. Targeting AURKB with small-molecule inhibitors and shRNAs resulted in reduced cell survival and increased apoptosis and cell cycle arrest at the G2/M phase. More importantly, primary RB specimens showed decreased cell viability in response to pharmacological AURKB inhibition. Additional studies have demonstrated that the MYCN oncogene regulates the expression of AURKB in RB. Conclusions: AURKB is overexpressed in RB, and targeting it could serve as a novel therapeutic strategy to restrict tumor cell growth.

Antimicrobial peptides in human corneal tissue of patients with fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is the leading cause of unilateral blindness in the developing world. Antimicrobial peptides (AMPs) have been shown to play an important role on human ocular surface (OS) during bacterial, viral and protozoan infections. In this study, our aim was to profile a spectrum of AMPs in corneal tissue from patients with FK during the active pase of infection and after healing. METHODS: OS samples were collected from patients at presentation by impression cytology and scraping. Corneal button specimens were collected from patients undergoing therapeutic penetrating keratoplasty for management of severe FK or healed keratitis. Gene expression of human beta-defensin (HBD)-1, -2, -3 and -9, S100A7, and LL-37 was determined by quantitative real-time PCR. RESULTS: Messenger RNA expression (mRNA) for all AMPs was shown to be significantly upregulated in FK samples. The levels of HBD-1 and -2 mRNA were found to be elevated in 18/20 FK samples. Whereas mRNA for HBD-3 and S100A7 was upregulated in 11/20 and HBD9 was increased in 15/20 FK samples. LL-37 mRNA showed moderate upregulation in 7/20 FK samples compared with controls. In healed scar samples, mRNA of all AMPs was found to be low and matching the levels in controls. CONCLUSION: AMP expression is a consistent feature of FK, but not all AMPs are equally expressed. HBD-1 and -2 are most consistently expressed and LL-37 the least, suggesting some specificity of AMP expression related to FK. These results will help to identify HBD sequence templates for designing FK-specific peptides to test for therapeutic potential.

An automated approach for the optimised estimation of breast density with Dixon methods.

OBJECTIVE: To present and evaluate an automated method to correct scaling between Dixon water/fat images used in breast density (BD) assessments. METHODS: Dixon images were acquired in 14 subjects with different T1 weightings (flip angles, FA, 4°/16°). Our method corrects intensity differences between water (W) and fat (F) images via the application of a uniform scaling factor (SF), determined subject-by-subject. Based on the postulation that optimal SFs yield relatively featureless summed fat/scaled-water (F+WSF) images, each SF was chosen as that which generated the lowest 95th-percentile in the absolute spatial-gradient image-volume of F+WSF . Water-fraction maps were calculated for data acquired with low/high FAs, and BD (%) was the total percentage water within each breast volume. RESULTS: Corrected/uncorrected BD ranged from, respectively, 10.9-71.8%/8.9-66.7% for low-FA data to 8.1-74.3%/5.6-54.3% for high-FA data. Corrected metrics had an average absolute increase in BD of 6.4% for low-FA data and 18.4% for high-FA data. BD values estimated from low- and high-FA data were closer following SF-correction. CONCLUSION: Our results demonstrate need for scaling in such BD assessments, where our method brought high-FA and low-FA data into closer agreement. ADVANCES IN KNOWLEDGE: We demonstrated a feasible method to address a main source of inaccuracy in Dixon-based BD measurements.

Normalised quantitative polymerase chain reaction for diagnosis of tuberculosis-associated uveitis.

Polymerase chain reaction (PCR)-based diagnosis of tuberculosis-associated uveitis (TBU) in TB-endemic countries is challenging due to likelihood of latent mycobacterial infection in both immune and non-immune cells. In this study, we investigated normalised quantitative PCR (nqPCR) in ocular fluids (aqueous/vitreous) for diagnosis of TBU in a TB-endemic population. Mycobacterial copy numbers (mpb64 gene) were normalised to host genome copy numbers (RNAse P RNA component H1 [RPPH1] gene) in TBU (n = 16) and control (n = 13) samples (discovery cohort). The mpb64:RPPH1 ratios (normalised value) from each TBU and control sample were tested against the current reference standard i.e. clinically-diagnosed TBU, to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of mpb64:RPPH1 ratio (0.011) for diagnosing TBU was identified from the highest Youden index. This cut-off value was then tested in a different cohort of TBU and controls (validation cohort, 20 cases and 18 controls), where it yielded specificity, sensitivity and diagnostic accuracy of 94.4%, 85.0%, and 89.4% respectively. The above values for conventional quantitative PCR (≥1 copy of mpb64 per reaction) were 61.1%, 90.0%, and 74.3% respectively. Normalisation markedly improved the specificity and diagnostic accuracy of quantitative PCR for diagnosis of TBU.

Inhibition of Pyruvate Dehydrogenase Kinase as a Therapeutic Strategy against Cancer.

Cancer cells alter their metabolism to support the uninterrupted supply of biosynthetic molecules required for continuous proliferation. Glucose metabolism is frequently reprogrammed in several tumors in addition to fatty acid, amino acid and glutamine metabolism. Pyruvate Dehydrogenase Kinase (PDK) is a gatekeeper enzyme involved in altered glucose metabolism in tumors. There are four isoforms of PDK (1 to 4) in humans. PDK phosphorylates E1α subunit of pyruvate dehydrogenase complex (PDC) and inactivates it. PDC decarboxylates pyruvate to acetyl CoA, which is further metabolized in mitochondria. Overexpression of PDK was observed in several tumors and is frequently associated with chemotherapy related drug resistance, invasion and metastasis. Elevated expression of PDK leads to a shift in glucose metabolism towards glycolysis instead of oxidative phosphorylation. This review summarizes recent literature related to the role of PDKs in cancer and their inhibition as a strategy. In particular, we discuss the role of PDK in tumor progression, metabolic reprogramming in stem cells, and their regulation by miRNAs and lncRNAs, oncogenes and tumor suppressors. Further, we review strategies aimed at targeting PDK to halt tumor growth and progression.

A Novel Mutation in Helical Domain 2 of NOD2 in Sporadic Blau Syndrome.

We report a 12-year-old girl who presented with bilateral granulomatous anterior uveitis accompanied by boggy arthritis of knee and ankle joints, intermittent fever, and nodular skin rash. She was diagnosed with sporadic Blau syndrome (early-onset sarcoidosis) based on above clinical signs and presence of non-necrotising granuloma on iris biopsy. DNA sequencing revealed a previously unreported heterozygous mutation consisting of a G>A transition in exon 4 of the NOD2 gene. This resulted in a glutamic acid to lysine substitution in helical domain 2 of the nucleotide binding and oligomerization (NACHT) region, possibly reducing efficiency of auto-inhibition in NOD2 signaling. Interestingly, the ocular inflammation resolved completely following therapeutic vitrectomy in both eyes whereas the systemic symptoms of fever and arthritis continued to wax and wane while on treatment with oral methotrexate and corticosteroids.

Clinicopathological characteristics of desmoplastic ameloblastoma: A systematic review.

The aim of the present review was to systematically present the clinicopathological data of desmoplastic ameloblastoma (DA) from articles published in the literature. A comprehensive search of the databases (PubMed, Medline, SCOPUS, Web of Science, and Google Scholar) for published articles on DA was conducted. A total of 238 cases were identified and analyzed from 76 published papers. DA showed a slight male predilection (male: female=1.07:1) with a predominance in the fourth and fifth decades of life. Mandibular involvement (52.55%) was most commonly seen with a marked tendency for the anterior region (mandible: 40.9%, maxilla: 48.07%). The size of the lesion ranged from .5 cm to 20.4 cm, with the majority of cases measuring more than 3 cm in size (53.84%). Radiologically, most of the lesions presented mixed radiolucency and radiopacity (62%), and root resorption was observed in only seven cases. The majority of the lesions showed ill-defined margins upon radiographic examination (65.78%). Most of the cases were treated with resection (78.57%), and five of the 10 recurrent cases were treated by enucleation/curettage. DA is characterized by the unique presentation of clinicopathological parameters. It is not possible to comment on its aggressive/recurrent nature and best treatment modality due to inadequate follow-up data.