Search

BACKGROUND: Intraocular, ciliary body, medulloepithelioma (CBME) is a rare tumor of the nonpigmented ciliary body epithelium, typically presenting in childhood. We describe a case of CBME. MATERIALS AND METHODS: Ocular examination and imaging guided diagnostic and treatment decisions. Aqueous humor (AH) liquid biopsy was collected from the affected eye at eventual enucleation. Whole genome sequencing (WGS) was employed to determine somatic copy number alterations (SCNA) in AH cell-free DNA (cfDNA). Tumor sample was analyzed using various assays to evaluate for oncogenic mutations and SCNAs. Histopathology determined diagnosis. RESULTS: A 5-year-old male with glaucoma and cataract in the left eye (OS) experienced worsening left eye pain and redness. There was no light perception OS and the eye was hypotonus. Anterior segment exam showed complete cataract and rubeosis iridis. Ocular B-scan ultrasound OS revealed an intraocular lesion with calcifications and retinal detachment. Orbital MRI suggested left globe hypercellularity. An infiltrative lesion involving the ciliary body was seen in the left eye on examination under anesthesia. Left eye enucleation was performed in the setting of pain, blindness, and tumor, with anterior chamber paracentesis for AH liquid biopsy collection. SCNA profile of AH cfDNA demonstrated loss of copy of chromosomes 4, 6, and 9. Tumor was negative for clinically significant mutations or SCNAs. Histopathology diagnosed malignant teratoid CBME. CONCLUSIONS: We present a case of CBME and include the unique SCNA profile of AH cfDNA from the enucleated eye. This case suggests utility of AH liquid biopsy in distinguishing between differential diagnoses for intraocular mass lesions.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.

S1. Platelet confirmation with CD61 staining in the SKBR3-spiked NBD sample S2. Enumeration of CTC subtypes S3. Survival analysis using AVPC-MS-CTC status S4. Survival analysis in patients without pc.CTC S5. Treatment decision tree Table S1. Correlation between CTC and clinical characteristics

Metastatic castration-resistant prostate cancer (mCRPC) includes a subset of patients with particularly unfavorable prognosis characterized by combined defects in at least two of three tumor suppressor genes: PTEN, RB1, and TP53 as aggressive variant prostate cancer molecular signature (AVPC-MS). We aimed to identify circulating tumor cells (CTC) signatures that could inform treatment decisions of patients with mCRPC with cabazitaxel–carboplatin combination therapy versus cabazitaxel alone. Liquid biopsy samples were collected prospectively from 79 patients for retrospective analysis. CTCs were detected, classified, enumerated through a computational pipeline followed by manual curation, and subjected to single-cell genome-wide copy-number profiling for AVPC-MS detection. On the basis of immunofluorescence intensities, detected rare cells were classified into 8 rare-cell groups. Further morphologic characterization categorized CTC subtypes from 4 cytokeratin-positive rare-cell groups, utilizing presence of mesenchymal features and platelet attachment. Of 79 cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS–positive with the worst prognosis and minimal benefit from combination therapy. In AVPC-MS–negative patients, its presence showed significant survival improvement from combination therapy. Our findings suggest the presence of pc.CTCs as a predictive biomarker to further stratify AVPC subsets with the worst prognosis and the most significant benefit of additional platinum therapy.Implications:HDSCA3.0 can be performed with rare cell detection, categorization, and genomic characterization for pc.CTC identification and AVPC-MS detection as a potential predictive biomarker of mCRPC.

Metastatic castration-resistant prostate cancer (mCRPC) includes a subset of patients with particularly unfavorable prognosis characterized by combined defects in at least two of three tumor suppressor genes: PTEN, RB1, and TP53 as aggressive variant prostate cancer molecular signature (AVPC-MS). We aimed to identify circulating tumor cells (CTC) signatures that could inform treatment decisions of patients with mCRPC with cabazitaxel–carboplatin combination therapy versus cabazitaxel alone. Liquid biopsy samples were collected prospectively from 79 patients for retrospective analysis. CTCs were detected, classified, enumerated through a computational pipeline followed by manual curation, and subjected to single-cell genome-wide copy-number profiling for AVPC-MS detection. On the basis of immunofluorescence intensities, detected rare cells were classified into 8 rare-cell groups. Further morphologic characterization categorized CTC subtypes from 4 cytokeratin-positive rare-cell groups, utilizing presence of mesenchymal features and platelet attachment. Of 79 cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS–positive with the worst prognosis and minimal benefit from combination therapy. In AVPC-MS–negative patients, its presence showed significant survival improvement from combination therapy. Our findings suggest the presence of pc.CTCs as a predictive biomarker to further stratify AVPC subsets with the worst prognosis and the most significant benefit of additional platinum therapy. Implications: HDSCA3.0 can be performed with rare cell detection, categorization, and genomic characterization for pc.CTC identification and AVPC-MS detection as a potential predictive biomarker of mCRPC.

Retinoblastoma (RB) is a childhood cancer that forms in the developing retina of young children; this tumor cannot be biopsied due to the risk of provoking extraocular tumor spread, which dramatically alters the treatment and survival of the patient. Recently, aqueous humor (AH), the clear fluid in the anterior chamber of the eye, has been developed as an organ-specific liquid biopsy for investigation of in vivo tumor-derived information found in the cell-free DNA (cfDNA) of the biofluid. However, identifying somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) of the RB1 gene, typically requires either: (1) two distinct experimental protocols—low-pass whole genome sequencing for SCNAs and targeted sequencing for SNVs—or (2) expensive deep whole genome or exome sequencing. To save time and cost, we applied a one-step targeted sequencing method to identify both SCNAs and RB1 SNVs in children with RB. High concordance (median = 96.2%) was observed in comparing SCNA calls derived from targeted sequencing to the traditional low-pass whole genome sequencing method. We further applied this method to investigate the degree of concordance of genomic alterations between paired tumor and AH samples from 11 RB eyes. We found 11/11 AH samples (100%) had SCNAs, and 10 of them (90.1%) with recurrent RB-SCNAs, while only nine out of 11 tumor samples (81.8%) had positive RB-SCNA signatures in both low-pass and targeted methods. Eight out of the nine (88.9%) detected SNVs were shared between AH and tumor samples. Ultimately, 11/11 cases have somatic alterations identified, including nine RB1 SNVs and 10 recurrent RB-SCNAs with four focal RB1 deletions and one MYCN gain. The results presented show the feasibility of utilizing one sequencing approach to obtain SCNA and targeted SNV data to capture a broad genomic scope of RB disease, which may ultimately expedite clinical intervention and be less expensive than other methods.

Introduction: Retinoblastoma (RB), a pediatric retinal malignancy, is unique in that it cannot be biopsied, impeding biomarker discovery. Aqueous humor (AH) is an ocular fluid that is an established source of tumor information, serving as a surrogate liquid biopsy. Extracellular vesicles (EVs) are promising biomarker candidates, and were recently established in RB AH. EVs are membrane-enclosed secretory vesicles carrying bioactive molecular information, which can be phenotyped using validated tetraspanin protein markers. Currently, relationships between EVs and RB clinical features are unknown, but establishing such relationships is necessary to determine the utility of EVs as RB biomarkers. Methods: We quantified and phenotyped EVs in 37 AH samples from 18 RB eyes with varying International Intraocular Retinoblastoma Classification (IIRC) stages and explored clinical correlations. Samples were collected from 10 eyes at diagnosis (DX) and 27 eyes during treatment (Tx). By outcome, 6 DX eyes were enucleated and 4 were salvaged. DX samples were collected from one A, one B, six D and two E eyes by IIRC group. 10μL of unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) analysis for fluorescent-based particle count and immunophenotyping of tetraspanin expression (CD63/81/9). Counts were converted to percentages. Statistics were done with Mann-Whitney U, ANOVA, and Tukey’s tests. Results: Samples from DX eyes (n=10) contained a higher number of EVs than samples from Tx eyes (n=27) (1.68 x 107 ± 4.28 x 107 per mL vs. 2.10x 106 ± 1.33 x 106 per mL) by SP-IRIS analysis. By percentage, DX samples were enriched with CD63/81+ EVs (P Citation Format: Sarah Pike, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6717.

Hemolysis has been known to affect the measurement of circulating biomarkers. In this study, clinically applicable procedures for microRNA (miRNA) detection in serum samples of acute myocardial infarction patients were established. The 89 samples from patients admitted to the coronary care unit were collected. These samples obtained from heparin-treated and untreated patients were subjected to heparinase digestion prior to miRNA measurements by multiplex RT-qPCR. The good reproducibility of miRNA detection after heparinase digestion (average R2 = 0.97) indicated that this method can be used routinely for samples regardless of heparin medication. Additionally, the degree of hemolysis in these samples was highly related to the hemoglobin absorbance at 414 nm. Based on the hemoglobin absorbance, five hemolysis-associated miRNAs were identified in our data normalized with respect to both the spike-in control and the RNA amount in a given sample. Using these calibration procedures, miRNAs can be accurately quantified and identified for clinical samples.

The weight losses after bariatric surgery are modulated by multiple factors in people with obesity. MicroRNAs (miRNAs) have been reported to show significant regulatory roles in adipose tissue. However, a serum miRNA signature to serve as a biomarker of sustained weight losses following bariatric surgery has not yet been established.MiRNA microarray was used to identify differentially expressed miRNAs in the serum of patients with an effective response after bariatric surgery compared with those without. Excess weight loss55% at 6 months after surgery was defined as an effective response.Three miRNAs were shown to have a significantly differential expression between patients with or without an effective response following bariatric surgery. The miR-31-5p was downregulated, whereas miR-328-3p and miR-181a-5p were upregulated in the patients with effective responses compared with those without effective responses. Panels of the serum ratios of miR-328-3p/miR-31-5p or miR-181a-5p/miR-31-5p and individual BMI value exhibited good performance in preoperative prediction of treatment effectiveness. Bioinformatic analysis depicted that predicted targets of these miRNAs were involved in the regulation of the AMP-activated protein kinase signaling pathway.A circulating miRNA signature with clinical variables (BMI) can be a clinical biomarker to predict effectiveness following bariatric surgery.

There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is prohibited in these patients. The aqueous humor (AH) forms in a separate compartment from the tumor but is enclosed within the same ocular space. Thus, it is an enriched source of eye-specific tumoral genomic information that can be used as a liquid biopsy or surrogate to tumor biopsy for this disease. This manuscript details a methodology for safely extracting the AH from retinoblastoma eyes via clear corneal paracentesis. Additionally, the steps for genomic analysis, including cell-free DNA isolation and purification, next-generation sequencing, somatic copy number alteration (SCNA) analysis, RB1 single nucleotide variant (SNV) mutation identification, and tumor fraction estimation are presented. The pre-analytical, analytical, and early clinical validity of the AH liquid biopsy platform have been evaluated; however, it is not without limitations. These are largely a consequence of the quantity of cell-free DNA that is required for certain steps of the assay. Compared to other blood-based liquid biopsy platforms currently under investigation for retinoblastoma, an AH-based platform is limited by the volume of biofluid (and thus the quantity of DNA) that can be extracted from the eye; the benefit is that AH is eye-specific. The platform discussed here is unique in that it detects circulating tumor DNA in the AH via two mechanisms (SCNAs and RB1 SNVs), yielding a higher sensitivity for identifying tumoral genomic information. The AH liquid biopsy has the potential for direct clinical application to precision oncology for retinoblastoma patients, with particular importance for patients with bilateral disease as the AH is specific to the tumors in each eye. There is ongoing research with applications of this platform to patients with other ocular tumors as well.

Aqueous humor (AH) liquid biopsy has been established as a surrogate tumor biopsy for retinoblastoma (RB). Previous AH studies have focused on highly recurrent RB somatic copy number alterations (SCNAs) including gain of 1q, 2p, 6p, and loss of 13q and 16q. In this retrospective study, we provide a comprehensive, whole-genome analysis of RB SCNAs and evaluate associated clinical features for 68 eyes of 64 RB patients from whom AH was obtained between December 2014 and October 2020. Shallow whole-genome sequencing of AH cell-free DNA was performed to assess for SCNAs. The prevalence of specific non-highly recurrent SCNAs, such as 20q gain and 8p loss, differed between primarily and secondarily enucleated eyes. Increases in chromosomal instability predict more advanced seeding morphology (p = 0.015), later age of diagnosis (p &lt, 0.0001), greater odds of an endophytic tumor growth pattern (without retinal detachment, p = 0.047), tumor heights &gt, 10 mm (p = 0.09), and containing 6p gain, a biomarker of poor ocular prognosis (p = 0.004). The AH liquid biopsy platform is a high-yield method of whole-genome RB SCNA analysis, and SCNAs are associated with numerous clinical findings in RB eyes. Prospective analyses are encouraged to further elucidate the clinical relevance of specific SCNAs in RB.

Background Acute kidney injury (AKI) is a common complication of acute myocardial infarction (AMI), and is associated with adverse outcomes. The study aimed to identify a miRNA signature for the early diagnosis of post-AMI AKI. Methods A total of 108 patients admitted to a coronary care unit (CCU) were divided into four subgroups: AMI−AKI−, AMI+AKI−, AMI+AKI+, and AMI−AKI+. Thirty-six miRNA candidates were selected based on an extensive literature review. Real-time quantitative RT-PCR analysis was used to determine the expression levels of these miRNAs in the serum collected on the day of CCU admittance. TargetScan 7.1 and miRDB databases were used for target prediction and Metacore 6.13 was used for pathway analysis. Results Through a stepwise selection based on abundance, hemolytic effect and differential expression between four groups, 9 miRNAs were found to have significantly differential expression levels as potential biomarkers for post-AMI AKI specifically. Noticeably, the expression levels of miR-24, miR-23a and miR-145 were significantly down-regulated in AMI+AKI+ patients compared to those in AMI+AKI− patients. Combination of the three miRNAs as a panel showed the best performance in the early detection of AKI following AMI (AUC = 0.853, sensitivity 95.65%), compared to the analysis of serum neutrophil gelatinase-associated lipocalin (AUC = 0.735, sensitivity 63.16%). Furthermore, bioinformatic analysis indicated that these three miRNAs regulate the transforming growth factor beta signaling pathway and involve in apoptosis and fibrosis in AKI. Conclusions For the first time, this study identify a unique circulating miRNA signature (miR-24-3p, miR-23a-3p, miR-145-5p) that can potentially early detect AKI following AMI and may be involved in renal injury and fibrosis in post-AMI AKI pathogenesis. Electronic supplementary material The online version of this article (10.1186/s12967-019-1890-7) contains supplementary material, which is available to authorized users.

Because direct tumor biopsy is prohibited for retinoblastoma (RB), eye-specific molecular biomarkers are not used in clinical practice for RB. Recently, we demonstrated that the aqueous humor (AH) is a rich liquid biopsy source of cell-free tumor DNA. Herein, we detail clinically-relevant molecular biomarkers from the first year of prospective validation data. Seven eyes from 6 RB patients who had AH sampled at diagnosis and throughout therapy with ≥12 months of follow-up were included. Cell-free DNA (cfDNA) from each sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing for pathogenic variants using a RB1 and MYCN custom hybridization panel. Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the seven diagnostic AH samples, 5/7 were positive for RB SCNAs. Mutational analysis identified RB1 variants in 5/7 AH samples, including the 2 samples in which no SCNAs were detected. Two eyes failed therapy and required enucleation, both had poor prognostic biomarkers (chromosome 6p gain or MYCN amplification) present in the AH at the time of diagnosis. In the context of previously established pre-analytical, analytical, and clinical validity, this provides evidence for larger, prospective studies to further establish the clinical utility of the AH liquid biopsy and its applications to precision oncology for RB.

Introduction: Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the cornea which enables use of AH as a liquid biopsy for intraocular disease. Retinoblastoma (Rb), an intraocular cancer in children, is unique in that direct tumor biopsy is prohibited, thus liquid biopsy has great clinical potential. cfDNA in the AH as a biomarker for Rb patients has been investigated and extracellular vesicles (EVs) are detectable in the AH from adults. However, AH EVs in Rb have previously never been explored. We know very little about the heterogeneity of AH EV populations in ocular cancers. Materials and Methods: 27 AH samples from 19 patients from 13 tumor-free eyes and 11 Rb eyes were collected. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9). Results: NTA demonstrated the concentration of AH EVs is 3.11 x 109-1.38 x 1010 vesicles/mL; majority sized 76.8-103 nm. Study group comparisons showed that non-tumor containing eyes had a smaller nanoparticle mean size compared to Rb containing eyes (P = 0.002). More EVs were significantly detected in Rb_Tn containing eyes compared to Rb_Tx containing eyes (P = 0.022), suggesting the possible presence of tumor-derived EVs in Rb_Tn which were subsequently eliminated by treatment. SP-IRIS revealed distinct patterns of tetraspanin expression of AH small EVs (sEVs). Mono-CD63+ EVs were identified to be the most dominant sEV subpopulation from AH across non-tumor and Rb_Tx eyes. However, more diverse sEV subpopulation profile was detected in Rb_Tn AH samples. Significantly lower percentage of mono-CD63+ EVs could be determined in Rb_Tn eyes compared to Rb_Tx eyes (70.3% vs. 96.1%, P = 0.001). A significant accumulation of CD9/CD63 (4.3% vs. 1.1%, P = 0.035), CD63/CD81 (20.7% vs. 2.0%, P = 0.012) and CD9/CD63/CD81 (4.8% vs. 0.8%, P = 0.022) subpopulations in Rb_Tn was also observed. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of Rb there was a more heterogeneous population of sEVs which normalized with treatment. Conclusions: Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ subpopulation in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of retinoblastoma-derived EV heterogeneity by chemotherapy. These novel finding suggests a potential clinical application of measurement of sEV subpopulations in AH samples to monitor response to therapy. Citation Format: Chen-Ching Peng, Deborah Im, Shreya Sirivolu, Bibiana Reiser, Aaron Nagiel, Paolo Neviani, Liya Xu, Jesse L. Berry. Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3416.

Introduction: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and arises from the uvea (iris, ciliary body and choroid). Although there are prognostic biomarkers available, identifying them requires intraocular tumor biopsy. Blood biopsy has emerged as a significantly less invasive alternative that also offers the ability to longitudinally track disease. However, blood biopsy has been unsuccessful for clinical use in identifying prognostic biomarkers in UM due to low detection rates. Thus, eye-specific aqueous humor (AH) biopsy may be a better alternative. Methods: This case series includes 20 primary UM patients with 13 (65%) choroid and 7 (35%) ciliary body (CB) tumors. AH samples were collected prior to brachytherapy plaque placement (n = 17), after brachytherapy (n = 19) and at enucleation (n = 1). A tumor wash sample (n = 1) was collected after routine fine needle aspiration biopsy. Nucleic acids ([NA], dsDNA, RNA, ssDNA and miRNA) were quantitated by Qubit Assay Kits. All AH samples and cells from tumor washing underwent DNA isolation and was followed by shallow whole-genome sequencing for copy-number profiling. Results: Comparison of pre- and post-radiation samples revealed significantly higher concentration of ssDNA (n = 10 pairs, P = 0.039) and miRNA (n = 10 pairs, P = 0.008) in choroid post- samples and significantly higher concentration of dsDNA (n = 7 pairs, P = 0.031) and miRNA (n = 7 pairs, P = 0.016) in CB post- samples. Between choroid (n = 12) and CB (n = 7) post- samples, there was a significantly higher concentration of dsDNA (P = 0.022) and RNA (P = 0.009) in post- CB samples, suggesting NA content is released from the disturbance of tumor cells by radiation and due to the proximity of the tumor to the AH in CB. Highly recurrent UM somatic copy number alterations (SCNAs) of monosomy 3, 6p gain, 6q loss and 8q gain were identified in post- AH samples. There was a significant difference between altered SCNA profiles in CB and choroid (P= 0.038). Altered SCNA profiles were found in 1/12 (8.3%) and 4/7 (57.1%) post-radiation AH samples of choroid and CB, respectively. No SCNAs were found in the same eyes’ pre-radiation sample. A high concordance of DNA profiles between a tumor wash and post- AH sample was identified (r = 0.978) and UM mutations GNAQ and BAP1 were detected in 3/6 (50%) post- AH with 2/5 (40%) matching tumor samples, demonstrating AH dsDNA’s tumor-origin. Conclusions: The AH is a source of cfDNA, with a higher yield of NAs after radiation in UM. Given the significant increase in NA concentration after radiation and in CB compared to choroid tumors, we hypothesize that all detected NAs post-radiation are tumor-derived due to apoptosis of tumor cells and subsequent release of cfDNA and due to the proximity of the CB tumor to AH. These results represent the first time that UM SCNAs and mutations were identified in cfDNA isolated from the AH, and this suggests that the AH can serve as a liquid biopsy for UM, especially in CB tumors. Citation Format: Deborah H. Im, Chen-Ching Peng, Liya Xu, Mary E. Kim, Dejerianne Ostrow, Venkata Yellapantula, Moiz Bootwalla, Jaclyn A. Biegel, Xiaowu Gai, Peter Kuhn, James Hicks, Jesse L. Berry. Potential of aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1967.

Background: The mutation, selection, and adaptation of tumor cells along disease progression exhibits a spectrum of phenotypic and genotypic heterogeneity. The importance of distinguishing phenotypic states of CTC in addition to genomic alterations has been addressed for identifying predictive biomarkers and understanding CTC biology. Current liquid biopsy usually relies on only one phenotypic state of CTCs without further genomic validation of cancer cell identity. Here, we developed HDSCA3.0, a multi-omic platform, which could distinguish various phenotypic states of CTCs followed by genomic characterization. Methods: Paired peripheral blood (PB) and bone marrow aspirate (BMA) samples were collected prospectively from 80 metastatic castrate resistant prostate cancer (mCRPC) patients for retrospective analysis. Seventy-nine of them were part of Cabazitaxel With or Without Carboplatin Trial (NCT01505868) and one independent index patient was included with aggressive disease and unfavorable prognosis. CTCs were detected, classified, enumerated through a four-channel immunostaining assay (DAPI|Cytokeratin|Vimentin|CD45/CD31) and a computational pipeline followed by manual curation, and subjected to single-cell copy-number profiling for clonality analysis and aggressive variant prostate cancer molecular signature (AVPC-MS) detection i.e. 2+ defects in PTEN, RB1, and TP53 genes. Results: CTC subtypes were categorized from Cytokeratin-positive rare cell groups based on the presence of mesenchymal features and platelet attachment. Of 79 trial cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS-positive with the worst prognosis. In AVPC-MS-negative patients, its presence showed significant survival improvement from combination therapy. In index patient, we uniquely identified genetically clonal mesenchymal-like CTCs (mes.CTCs) and their presence was significantly associated with one subclone emerged along clonal lineage. Meanwhile, differences of CTC abundance and phenotypic diversity were observed between paired PB and BMA as well as genomic variations. Conclusion: Our findings suggest pc.CTCs and AVPC-MS in CTCs as a multi-omic predictive biomarker to stratify mCRPC subpopulations with the worst prognosis and the most significant benefit of additional platinum therapy and illustrate a robust approach to analyze intra-patient CTC genotypic and phenotypic heterogeneity and association. Citation Format: Shoujie Chai, Nicholas Matsumoto, Ryan Storgard, Chen-Ching Peng, Ana Aparicio, Benjamin Ormseth, Kate Rappard, Cunningham Kate, Anand Kolatkar, Rafael Nevarez, Kai Han Tu, Ching-Ju Hsu, Amin Naghdloo, Paymaneh Malihi, Liya Xu, Paul Corn, Amado Zurita-Saavedra, James Hicks, Carmen Ruiz-Velasco, Peter Kuhn. Dissecting CTC phenotypic heterogeneity for predictive biomarker identification and its association with clonal lineage through single-cell multi-omic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1957.

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.

Hemolysis has been known to affect the measurement of circulating biomarkers. In this study, clinically applicable procedures for microRNA (miRNA) detection in serum samples of acute myocardial infarction patients were established. The 89 samples from patients admitted to the coronary care unit were collected. These samples obtained from heparin-treated and untreated patients were subjected to heparinase digestion prior to miRNA measurements by multiplex RT-qPCR. The good reproducibility of miRNA detection after heparinase digestion (average R

Background Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC). Methods Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5’azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator’s binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay. Results A tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region − 62 to − 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration. Conclusions CLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression. Electronic supplementary material The online version of this article (10.1186/s13046-018-0754-y) contains supplementary material, which is available to authorized users.

Purpose: Metastatic Uveal Melanoma (UM) is usually fatal within one year of symptom onset. There are a variety of biomarkers that can stratify patients' metastatic risk, but identifying them requires intraocular tumor biopsy. Liquid biopsy offers a less invasive alterative without the risks of retinal detachment and intraocular hemorrhage. We investigated whether the aqueous humor (AH) has sufficient tumor-derived cell-free DNA (cfDNA) to function as a liquid biopsy for UM. Methods: This case series includes 12 AH samples from 7 eyes of 7 UM patients from whom AH was sampled during routine brachytherapy plaque placement and/or removal. Double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, and microRNA (miRNA) were quantified with respective Qubit High Sensitivity Assay Kits (Thermo Fischer) for each sample. The lower limit of detection was 0.01 ng/µL for dsDNA, 0.05 ng/µL for ssDNA, 0.25 ng/µL for RNA, and 0.05 ng/µL for miRNA. CfDNA was then isolated and sequenced on an Illumina platform to assess genome-wide somatic copy number alterations (SCNAs). Results: Aqueous humor derived cfDNA was detected in all samples, ranging from 0.085-28.8 ng/µL (mean = 2.7 ng/µL). RNA was detected in 2 of 12 samples (mean = 1.6 ng/µL, range = 0.7-2.5 ng/µL), and miRNA was detected in 11 of 12 samples (mean = 1.7 ng/µL, range = 0.094-16.2 ng/µL). There was a significantly higher cfDNA concentration in post-radiation samples than pre-radiation samples, (median = 0.082 ng/µL in pre-radiation, 0.21 ng/µL in post-radiation, W = 1, z = -2.55, p < 0.05, Wilcoxon Signed-rank test). This finding was similar when comparing all nucleic acid concentrations (dsDNA, ssDNA, RNA, and miRNA) between pre- and post-radiation (median = 0.085 ng/µL in pre-radiation, 0.333 ng/µL in post-radiation, W = 1, z = -3.46, p = 0.00027). Confirmed UM genomic alterations of 6p gain, 6q loss, and 8q gain were identified in one post-radiation AH sample from an eye with an iridociliary body tumor. These SCNAs are characteristic of UM. No SCNAs were found in the AH from the pre-radiation sample in the same eye, or any of the other AH samples sequenced to date. Conclusions: The AH is a source of cfDNA in eyes with UM, with a higher yield of all nucleic acids detected after brachytherapy radiation treatment. UM associated SCNAs were identified in the AH of one eye with an iridociliary tumor after radiation only. We hypothesize this may be due to intratumoral necrosis from the radiation as well as the proximity of the primary tumor to the AH in the case where SCNAs were identified. These results represent the first time that UM SCNAs have been identified in cfDNA isolated from the AH, and this provides preliminary evidence that the AH can serve as a liquid biopsy for UM. Citation Format: Mary E. Kim, Liya Xu, Chen-Ching Peng, Peter Kuhn, James Hicks, Jesse L. Berry. Aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 554.

Purpose: All previous studies of retinoblastoma (RB) aqueous humor (AH) analysis for tumor-derived cell-free DNA (cfDNA) have focused on sampling AH from eyes undergoing therapy; results from AH liquid biopsy at the time of diagnosis have not yet been published. Herein, we detail the diagnostic and prognostic genomic biomarkers found in the AH cfDNA from 7 RB eyes at diagnosis, each with longitudinal evaluation of over 12 months in follow-up. Methods: Subjects included 7 eyes of 6 RB patients who underwent AH sampling at diagnosis. CfDNA from each AH sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing and mutation detection using a custom hybridization panel for RB1 and MYCN. Results were compared to peripheral blood RB1 testing and matched tumor samples, when available. Tumor fraction (TFx) was calculated using ichorCNA. Results: Five of 7 diagnostic AH samples contained highly recurrent RB SCNAs, 4 with large scale alterations and 1 with a focal RB1 gene deletion. Mutational analysis of AH cfDNA identified pathogenic somatic variants in 5 diagnostic AH samples with high variant allele frequency, while the remaining 2 diagnostic AH samples had either a high TFx from SCNAs or a focal MYCN amplification. Taken together, somatic tumoral genomic information was detected in all 7 diagnostic AH samples. The 2 eyes that required enucleation had poor prognostic biomarkers (chromosome 6p gain and MYCN amplification) present at the time of diagnosis. TFx from longitudinal AH sampling corresponded to treatment response over time in all cases for which sequential AH samples were available. Conclusions: This study demonstrates that AH sampling at diagnosis is both feasible and safe. Molecular profiling of AH provides a plethora of diagnostic and prognostic information from only a single 0.1 mL AH sample. 1 Citation Format: Liya Xu, Mary E. Kim, Ashley Polski, Rishvanth K. Prabakar, Chen-Ching Peng, Patricia Chévez-Barrios, Jonathan W. Kim, Rachana Shah, Rima Jubran, Peter Kuhn, David Cobrinik, James Hicks, Jesse L. Berry. Enhancing the molecular diagnosis, prognosis, and treatment monitoring of retinoblastoma using cell free DNA in aqueous humor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 638.

Additional file 1: Figure S1. Study design. Figure S2. Summary of total reference numbers, disease distribution and sample type analysis of the miRNA candidates. Table S3. Kruskal–Wallis test and post hoc Dunn’s multiple comparisons of the 17 miRNAs among all groups (N = 108).

Table S2. Identified proteins from CLDN11-3flag Co-IP by LC-MS/MS. (PDF 222Â kb)

Figure S3. Sequence of CLDN11 promoter (- 1000~ + 200). The promoter sequence of CLDN11, transcription factor binding sites [GATA1(- 90), GATA1/2(- 60), GATA2(+ 184)] and transcription start sites (+ 1) are indicated. (PDF 44Â kb)

Supplementary Materials and Methods. (DOC 39Â kb)

Figure S4. Co-immunoprecipitation of interacting proteins of Flag-tagged CLDN11. (a) Co-immunoprecipitation assays were performed using anti-Flag M2 beads (Sigma) on cell lysates transfected with either vector (Vec, V) or CLDN11-3F (C) expressing plasmid. The immunoprecipitated protein samples were separated in 12% SDS-PAGE. The gel was stained with Coomassie Blue (left panel). (b) For Western blot analysis, the SDS-PAGE was loaded with 1% input lysate, 25% immunoprecipitated lysate, and 1% flow through (FT), and was detected by anti-Flag antibody. (PDF 165Â kb)

Table S1. Sequence of primers in this study. (PDF 105Â kb)

Figure S5. Tubulin polymerization assays. (a) Tubulin polymerization assays were performed in standard condition (TUB), TUB with purified GST, TUB with purified GST-CLDN11, and TUB with 10Â ÂľM nocodazole (tubulin polymerization inhibitor). Tubulin polymerization was monitored using an ELISA reader (340nm) every 30 s for 60 min (upper panel). (b) The rate of tubulin polymerization (?mOD/min) was plotted (lower panel) and the Vmax for each reaction is indicated by an arrow. (c) The purified GST and GST-CLDN11 protein were detected by western blotting using either anti-GST or anti-CLDN11 antibody. (PDF 203Â kb)

// Hsin-Pai Li 1,2,3,* , Chen-Ching Peng 1,* , I-Che Chung 2 , Mei-Yuan Huang 2 , Shao-Tung Huang 1 , Chia-Chun Chen 2 , Kai-Ping Chang 4 , Cheng-Lung Hsu 5 , Yu-Sun Chang 1,2 1 Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 2 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 3 Department of Microbiology and Immunology Medical School, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 4 Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital, Lin-Kou, Taiwan, ROC 5 Division of Hematology-Oncology, Chang Gung Memorial Hospital, Lin-Kou, Taiwan, ROC * These authors contributed equally to this work. Correspondence: Hsin-Pai Li , email: // Keywords : HOXA2, DNA methylation, NPC, MMP9 Received : September 4, 2013 Accepted : November 2, 2013 Published : November 4, 2013 Abstract Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2) , which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5’Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2 . In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which helps explain such observation. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Our results reveal a novel mechanism wherein HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 may use as biomarkers for monitoring NPC treatment. Citation Format: CHEN-CHING PENG. Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2015-4772

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment. Note: This abstract was not presented at the meeting. Citation Format: Chen-Ching Peng. Silencing of the transcription repressor HOXA2 by aberrant hypermethylation enhances invasion via MMP-9 activation in NPC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2300. doi:10.1158/1538-7445.AM2014-2300

This paper assesses the impact of earnings quality on market responses to annual earnings announcements. Earnings quality is measured by the ratio of earnings to funds from operations. The difference in the association between forecast errors and excess returns across the high/low quality earnings subsamples is found to be statistically significant; there is a greater market response to earnings announcements of high-quality firms than to low-quality firms. Hence, earnings quality as measured by the ratio of earnings to funds from operations, is found to have pricing implications. The results are robust across two regression models: OLS on returns ordered in announcement time and SUR/GLS on returns ordered in calendar time.