The utility of aqueous humor liquid biopsy in retinoblastoma genetic analysis: a systematic review of concordance and influencing factors

Recent studies have shown the presence of human papillomavirus (HPV) genome in retinoblastoma (RB) tumor samples. There is no information on the HPV status in the RB tumors of Indian patients. We studied the presence of HPV genome in RB tumor samples from patients with unilateral tumor. Forty-four fresh RB tumor samples and 30 non-neoplastic donor retinas were analyzed for the presence of HPV 16 and 18 genome by nested and seminested polymerase chain reaction. Tumor tissue sections were also used to assess the expression of the retinoblastoma (Rb) protein. All 30 control tissues were negative for HPV genome. Among the 44 tumor samples, there were 23 tumors with invasion of optic nerve/choroid and 21 tumors with no invasion. HPV DNA was present in 21/44 (47%) RB tumors. Among 21 unilateral RB tumors that were positive for HPV DNA, HPV 16 was detected in 12/21 (57%) tumors. However, HPV 18 was negative in all the tumors. Rb protein was absent in 16 (71%) of 21 tumors that had HPV DNA. However, Rb was also absent in 20 (86%) of 23 tumors that were HPV negative. Children younger than 18 months old were significantly associated with the presence of HPV DNA compared with children above 24 months old (P<0.014). Our study shows the presence of HPV and HPV 16 in a subset of RB tumor samples. However, further studies are in progress to know the role played by HPV in RB.

Purpose: Germline alterations in the RB1 tumor suppressor gene predispose patients to developing retinoblastoma (RB) in both eyes. However, tumors in bilateral RB may not respond identically to treatment. The additional genomic events that occur independently in each eye during tumorigenesis are not well characterized. The aqueous humor (AH) provides a novel source of cell-free tumor-derived DNA (ctDNA) for liquid biopsy, enabling the in vivo study of RB tumors. In this case report, we use our AH liquid biopsy to compare genomic profiles between the right and left eyes of a single patient with heritable RB while also showing that ctDNA longitudinal dynamics correspond to therapeutic response. Methods: One patient with bilateral RB was included. Multiple samples of AH were obtained from each eye during routine intravitreal melphalan therapy and following enucleation of the left eye. Routine clinical blood testing was performed to determine germline RB1 status. CtDNA was isolated from the AH and sequenced on an Illumina platform to assess genome-wide somatic copy number alterations (SCNAs). The same sequencing libraries were used to identify somatic RB1 pathogenic variants using a custom hybridization and next generation sequencing panel targeting RB1. Tumor fraction (TFx) was estimated using ichorCNA software. Results: Five AH samples from both eyes (3 from the right eye and 2 from the left eye) were included. Peripheral blood RB1 testing detected germline 13q and 16p deletions. Targeted RB1 mutational analysis of AH ctDNA identified a different somatic RB1 mutation in each eye. At initial AH sampling, three SCNAs were present in the right eye and these same SCNAs persisted in further samples. Two SCNAs were initially detected in the left eye and were consistently identified in later sampling. Despite the same germline RB1 mutation, the second somatic mutation was different in each eye and there were distinct, non-overlapping patterns of SCNAs in each eye. In addition, the right eye demonstrated a progressive decrease in TFx corresponding with therapeutic responsiveness and ocular salvage. The left eye had persistently larger TFx values and required enucleation due to tumor recurrence. Conclusions: Our AH liquid biopsy detected distinct genomic events between eyes in a patient with bilateral RB and TFx changes corresponding with disease activity. Identifying inter-eye genomic heterogeneity without the need for enucleated tumor tissue may help direct active management of RB, with particular usefulness in bilateral cases. Citation Format: Elyssa Y. Wong, Liya Xu, Lishuang Shen, Mary E. Kim, Ashley Polski, Rishvanth K. Prabakar, Rachana Shah, Rima Jubran, Jonathan W. Kim, Jaclyn A. Biegel, Xiaowu Gai, Peter Kuhn, James Hicks, Jesse L. Berry. Genomic heterogeneity in the aqueous humor cell-free DNA in a patient with bilateral retinoblastoma [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 2247.

Purpose Retinoblastoma (RB) is the most common intraocular malignancy in children. The diagnosis of RB is mainly based on clinical features and imaging characteristics. Prognosis is based on stage of disease and response to treatment. In salvaged globes, direct tumor biopsy for genetic analysis and prognostication is an absolute contraindication at this point of time for the fear of extraocular tumor spread. Currently, there is a search for surrogate markers to allow accurate diagnosis and for prognostication, to predict the chances of globe salvage in RB. Therefore, biofluids such as plasma or aqueous humor have been studied to detect circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA), respectively, to allow for treatment decision making, monitoring treatment response, and prognostic counselling. Methods A search of electronic databases (PubMed, Google Scholar and MEDLINE) of all articles on liquid biopsy in retinoblastoma published in English was performed. The keywords used for the search included “retinoblastoma”, “liquid biopsy”, “aqueous humor” “circulating tumor cells”, “cell-free DNA”, “cfDNA”, “circulating tumor DNA”, “ctDNA”, “tumor fraction”, “RB1 mutation” and “SNCA”. Additionally, historic articles on the advent of liquid biopsy in medicine were also reviewed. Pertinent cross-references from the studies were reviewed. Retrospective interventional and observational case series, observational case series, prospective cohort studies, reviews, case reports, surgical techniques, invited commentary and letters were included. Results A total of 40 relevant articles were selected. Biomarkers in aqueous humor, serum and cerebrospinal fluid and their clinical applications are discussed. Conclusion Harvesting aqueous humor from eyes with retinoblastoma has been found safe and superior to blood for the detection of chromosomal changes. cfDNA from aqueous can be a surrogate marker to detect somatic copy number alterations and other genetic alterations in RB. ctDNA in plasma also has potential to help in diagnosis and prognosis of RB. Liquid biopsy in RB is an emerging topic, which could pave way for a better understanding of mechanisms for treatment response, resistance and recurrence in RB as well as possibly provide specific therapeutic targets to improve globe salvage.

Simple SummaryDue to prohibition of direct tumor biopsy for patients with retinoblastoma, the prospect of a liquid biopsy for the identification of tumor derived biomarkers for this cancer is enticing. The aqueous humor (AH) is a rich source of eye-specific tumoral genomic information. This is the first prospective study wherein we demonstrate that molecular profiling of the AH at diagnosis and longitudinally throughout therapy has clinical utility for diagnosis, prognosis, and monitoring of treatment response. Tumoral genomic information was detected in 100% of diagnostic aqueous humor samples, including single nucleotide variants in the RB1 tumor suppressor gene and large-scale somatic chromosomal alterations. All eyes that failed therapy and required enucleation had poor prognostic biomarkers for ocular salvage present in the aqueous humor at time of diagnosis. This highlights the potential of the AH liquid biopsy for direct clinical applications to precision oncology to direct genome-specific, personalized treatment for retinoblastoma patients.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.

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.

Introduction: The development of liquid biopsy testing based on sequencing analysis of cell-free DNA (cfDNA) has the potential to transform cancer care in pediatric tumors, which are characterized by a wide array of genomic alterations including copy number alterations (CNAs), sequence variants, epigenetic alterations, and structural rearrangements in tumor-associated genes. In patients with retinoblastoma where direct tumor biopsy is not possible due to the risk of tumor spread, aqueous humor (AH) is a rich source of cfDNA. Comprehensive cfDNA testing, combining a low pass whole genome sequencing (LP-WGS) assay to detect CNAs and a custom targeted sequencing panel (TSP) to detect mutations, can provide a highly effective platform to inform diagnosis, risk stratification, response to therapy, and surveillance in patients with retinoblastoma. LP-WGS Results: We validated a liquid biopsy LP-WGS assay, LBSeq4Kids, to detect CNAs in cfDNA from plasma, cerebrospinal fluid, or AH. Clinical testing has been performed for the past two years for diagnostic evaluation and monitoring response to therapy using AH from retinoblastoma patients. Thirty-six children with unilateral or bilateral retinoblastoma (44 eyes) underwent LBSeq4Kids LP-WGS analysis. CNAs were detected in 30/32 (94%) samples at diagnosis or recurrence. Serial sampling of 22/44 (50%) eyes was performed using a range of 1-10 samples per patient. Seventeen of those 22 (77%) eyes demonstrated clearance of CNAs with treatment. Five eyes showed persistence of abnormal profiles, necessitating enucleation of 2 eyes. Treatment and evaluation are ongoing for the remaining three eyes. Targeted Sequencing Panel Results: We are validating a custom cfDNA TSP to detect single nucleotide variants and small indels in the coding sequence of 136 genes and fusions involving EWSR1, FOXO1, and BRAF for patients with ocular disease, solid tumors and brain tumors. To date, 17 children (21 eyes) diagnosed with retinoblastoma have had TSP testing using AH. All eleven (100%) patients with germline variants demonstrated the same RB1 alterations in the AH using the panel. Six of these patients (55%) had a second somatic alteration in RB1 detected with the panel. Two of 11 (18%) with known germline RB1 mutations also demonstrated mutations in BCOR, ARID1A, or MSH6. Somatic variants in RB1, ARID1A and BCOR were detected in the remaining six patients with sporadic retinoblastoma. Targeted sequencing of the AH surveillance samples from these patients is in progress. Conclusion: Here we demonstrate clinical utility for aqueous humor liquid biopsy molecular testing as a complementary means of diagnosis and monitoring treatment response in patients with retinoblastoma. Continued surveillance of these patients is ongoing to monitor disease status and rule out recurrence, as well as identify novel prognostic biomarkers. Citation Format: Laura A. Kagami, Eirini Christodoulou, Venkata Yellapantula, Dong Xu, Cindy Fong, Dejerianne Ostrow, Liya Xu, Jesse Berry, Jaclyn A. Biegel. Implementation of a comprehensive clinical cfDNA analysis platform for aqueous humor liquid biopsy in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1977.

Purpose: Aqueous humor (AH) is a novel source of tumor-derived cell-free DNA (cfDNA) and can serve as a surrogate tumor biopsy, or liquid biopsy, for retinoblastoma (RB). We previously identified a somatic copy number alteration (SCNA) gain of chromosome 6p as associated with a 10-fold increased risk of enucleation. In this study, we provide a 2-year extended update to further explore 6p gain as a prognostic biomarker for ocular survival. Methods: Patients diagnosed with RB from 12/2014 - 7/2019 from whom AH was sampled were included. AH was extracted via clear corneal paracentesis at diagnosis, during intravitreal injection of chemotherapy or enucleation. CfDNA was extracted; shallow whole genome sequencing was performed to assess for cfDNA tumor fractions and highly recurrent RB SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). Patient demographics, treatment regimen and globe salvage were recorded. Results: 50 eyes from 46 patients were included: 27 eyes were salvaged and 23 were enucleated. Most eyes (36/50,72%) were TNM stage cT2b. 116 samples of AH were analyzed. There were no cases of orbital relapse or metastatic spread. The median follow-up was 30 months (range 6-64 mos). After genomic evaluation, 33 / 50 eyes (66%) demonstrated one or more highly-recurrent RB SCNAs and 39/50 (78%) demonstrated any measurable SCNA. Gain of 6p was the most prevalent RB SCNA among all 50 eyes (50%), followed by 1q gain (38%), 16q loss (30%), 2p gain (including 3 focal MYCN gains; 18%), and 13q loss (16%). RB SCNAs were significantly more prevalent in enucleated eyes (21/23, 91.3%) than in salvaged eyes (12/27, 44.4%; Fisher's exact, P=0.0007). Of these, 6p gain was particularly more prevalent in enucleated eyes (17/23, 73.9%) than in salvaged eyes (8/27, 29.6%; Chi-squared, P=0.002) Of all the RB SCNAs, the presence of 6p gain in the cfDNA in the AH portended nearly 10 times greater odds of being enucleated (OR=9.87,95% CI=1.75-55.65, P=0.009). No single other SCNA had a statistically significant power to predict enucleation although there was a trend towards significance for 2p/focal MYCN gain (P=0.09) limited by the few number of eyes with this SCNA. Conclusions: Although we previously identified chromosomal gain of 6p as a potential indicator of an aggressive phenotype in RB, that study included a smaller cohort of patients and limited follow-up. With extended follow-up and nearly double the number of eyes and AH samples, the data continue to demonstrate that AH is a high-yield surrogate tumor biopsy, and the presence of RB-specific SCNAs–specifically gain of 6p–has the potential to serve as a prognostic biomarker for RB. Citation Format: Jesse L. Berry, Liya Xu, Ashley Polski, Rishvanth Prabakar, Mark Reid, Jonathan Kim, Peeter Kuhn, James Hicks. Gain of chromosome 6p is a molecular biomarker for prognostication of retinoblastoma ocular survival: The aqueous humor surrogate tumor biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 186.

Background: Precision medicine has revolutionized cancer treatment, but its application in retinoblastoma (RB) has been limited due to the risks associated with traditional tumor biopsies, which can lead to the spread of cancer outside the eye. Blood plasma has not been effective in intraocular RB management because the blood-brain barrier (BBB) prevents tumor-derived material from entering the bloodstream, and in cases of bilateral RB—occurring in over 40% of patients—each eye may harbor distinct genomic alterations, complicating the use of blood-based biomarkers. Given these challenges, the discovery of tumor-derived genomic material in the aqueous humor (AH) offers a promising and safer alternative—a liquid biopsy that provides localized and specific insights into the tumor's genetic makeup. Although AH liquid biopsies have been valuable in identifying genomic changes in RB, they have yet to guide treatment decisions. This is primarily due to the rarity of the disease, the heterogeneity of treatment modalities, and the unclear definition of in vivo RB subtype classifications. Consequently, translating these genomic insights into actionable, personalized treatment strategies has been challenging. Now, we present the first application of AH-monitored treatment in a 35-month-old male with unilateral RB, characterized by TP53 alteration and ecDNA-MDM4 amplification, highlighting a significant advancement in the precision management of RB. Methods: AH samples were obtained via clear corneal paracentesis and analyzed for copy number alterations and somatic variants using shallow whole-genome sequencing and targeted sequencing. The patient initially received systematic CEV, followed with intravitreal melphalan (IVM) monotherapy. Due to resistance, treatment was escalated to a combination of intravitreal melphalan and topotecan (IVM+IVT), monitored by the AH liquid biopsy genomic findings. Results:The patient initially showed resistance to monotherapy with intravitreal melphalan (IVM), but dual therapy with melphalan and topotecan (IVM+IVT) significantly reduced tumor fraction (TFx) from 81.61% to 26.63% and decreased tumor size. Continued dual therapy and subsequent topotecan monotherapy resulted in complete regression of vitreous seeds and stabilization of the disease, as indicated by a final AH TFx of 0%. At our institution, Children's Hospital Los Angeles (CHLA), the majority of RB cases are treated using IAC or IVM, with dual chemotherapy regimens rarely employed. Conclusions: This case underscores the importance of tailoring treatment protocols to genetic profiles identified through AH liquid biopsy in managing aggressive RB. The successful combination of genomic profiling and personalized therapy has potential to significantly improve clinical outcomes, marking a critical step toward real precision management of RB. Further clinical collaboration is essential to validate and refine protocols for TP53-altered and ecDNA-MDM4-driven RB. Citation Format: Liya Xu, Elaine Huang, Jesse Lee Berry. Advancing precision management in retinoblastoma via aqueous humor liquid biopsy: A case of TP53 and MDM4 alterations [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B030.

The inevitable emergence of acquired resistance is a major limitation to the clinical benefit of precision medicine strategies. Single-lesion tumor biopsies have long been the mainstay of understanding acquired resistance, but recent data suggest tumor biopsies may under-represent the molecular heterogeneity of acquired resistance. Alternatively, studies have suggested that liquid biopsy approaches analyzing cell-free DNA (cfDNA) may offer significant advantages, but extensive prospective comparisons of matched liquid vs. tumor biopsies obtained at the time of acquired resistance are lacking. Here, we assess systematic liquid biopsy upon acquired resistance to targeted therapy in 44 patients across seven molecularly defined subtypes of gastrointestinal cancers. Liquid biopsy at disease progression identified at least one functionally validated molecular mechanism of resistance in 75% of patients, wherein 52% exhibited &amp;gt;1 resistance alteration (range 2-9, median 3 per patient). In 23 patients in whom a matched post-progression tumor biopsy could be obtained, tumor biopsy was less effective than liquid biopsy in identifying resistance mechanisms, with resistance alterations detected in only 48% of patients, and multiple resistance mechanisms detected in only 9% of cases. In matched cases, liquid biopsy detected at least one resistance alteration not detected in tumor biopsy in 78% of cases. Targeted analysis and whole-exome sequencing of serial cfDNA, multiple post-progression biopsies, and rapid autopsy specimens from select cases revealed key insights into the geographic and complex characteristics of heterogeneity captured by liquid biopsy in the setting of acquired resistance. These data illustrate that acquired resistance is characterized by frequent and profound tumor heterogeneity, and suggests that liquid biopsy may more effectively identify heterogeneous clinically relevant resistance alterations compared to standard tumor biopsy. Citation Format: Aparna R. Parikh, Ignaty Leshchiner, Liudmila Elagina, Lipika Goyal, Chaya Levovitz, Giulia Siravegna, Dimitri Livitz, Kahn Rhrissorrakrai, Liz Martin, Emily E. Van Seventer, Megan Hanna, Kara Slowik, Filippo Utro, Christopher J. Pinto, Alicia Wong, Brian P. Danysh, Ferran Fece de la Cruz, Isobel J. Fetter, Brandon Nadres, Heather A. Shahzade, Jill N. Allen, Lawrence S. Blaszkowsky, Jeffrey W. Clark, Bruce Giantonio, Janet E. Murphy, Ryan D. Nipp, Eric Roeland, David P. Ryan, Colin D. Weekes, Eunice L. Kwak, Jason E. Faris, Francois Aguet, Ipsita Guha, Mehlika Hazar-Rethinam, Dora Dias-Santagata, David T. Ting, Andrew X. Zhu, Theodore S. Hong, Todd R. Golub, A J. Iafrate, Viktor Adalsteinsson, Alberto Bardelli, Laxmi Parida, Dejan Juric, Gad Getz, Ryan B. Corcoran. Liquid biopsy versus tissue biopsy to assess acquired resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-257.

Retinoblastoma (RB) is the most common primary intraocular malignancy in children, whereas Uveal melanoma (UM) is the most common intraocular malignancy in adults [1,2]. Tissue biopsy is the standard gold technique for diagnosing the malignant neoplasm but an incisional tissue biopsy or fine needle aspiration biopsy (FNAB) is contraindicated for the intraocular malignancy [3]. Clinical diagnosis and imaging study are the only way to diagnose the intraocular malignancy due to the risk and fear of extraocular spread [4]. Recently, liquid biopsy has gained in popularity in the ophthalmic field. Liquid biopsy allows retinoblastoma diagnosis and a better understanding of the metastatic spread of uveal melanoma. Recently, the USA Food and Drug Administration (FDA) approved the make use of liquid biopsy (LB) as an appropriate diagnosis, prognosis, and also for monitoring tool in non-small cell lung carcinoma to keep away from invasive tissue biopsy in designated cases [5-7]. Liquid biopsy (LB) utilizes biofluid to evaluate for tumor-derived cells or cell-free DNA. LB is a relatively non-invasive technique rather than a tissue biopsy. In LB, material collected from multiple body fluids such as aqueous humor (AH), blood, cerebrospinal fluid, urine, and saliva for molecular diagnosis [8] and detecting of cancer biomarkers such as circulating tumor cells (CTC), tumor derived cell free DNA (ct-DNA), circulating tumor RNA (ct-RNA), microRNA (miRNA), tumorrelated exosomes (TREs), and extracellular vescicles (EVs) [7]. Aqueous humor samples for RB (Ocular LB) and Venous blood samples for UM (systemic LB) are utilizing for analyzing the molecular characteristics [8]. In others ophthalmic malignancies like conjunctival melanoma or squamous cell carcinoma, the role of LB is still not studied because tissue biopsy is routinely done for confirming the diagnosis and also for mutational status [9-11].

Purpose: To evaluate whether blood demonstrates similar potential as aqueous humor (AH) to be used as a liquid biopsy for retinoblastoma (RB) and whether tumor-derived cell-free DNA (cfDNA) can be isolated as effectively from the blood as AH. Methods: AH was extracted via clear corneal limbal paracentesis from RB eyes at diagnosis or during intravitreal injection of chemotherapy. Matched peripheral venous blood samples were drawn. Shallow whole-genome sequencing was performed to assess for cell-free tumor DNA fractions and highly recurrent somatic copy number alterations (SCNAs) in the blood and AH samples. Results: Seven samples of AH taken at diagnosis and 13 samples at the time of intravitreal injection were compared to matched blood samples. The presence of any detectable SCNA in the AH was 11/20 and 0/20 in the blood (p=&amp;lt;0.001). The median size distribution of cfDNA molecules in the AH was 157.5 bp versus 181.5 bp in the blood (p=&amp;lt;0.001). Conclusions: The AH appears to be superior to the blood as a source of cell-free tumor DNA for retinoblastoma, thus a better target for development as a liquid biopsy for this cancer. Citation Format: Liya Xu, Jesse L Berry, Ashley Polski, Rima Jubran, Peter Kuhn, Jonathan W. Kim, James Hicks. Aqueous humor is superior to blood as a liquid biopsy for retinoblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A70.

Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

A Multicenter Analysis of Nucleic Acid Quantification Using Aqueous Humor Liquid Biopsy in Retinoblastoma: Implications for Clinical Testing.

Purpose: Previous studies of retinoblastoma (RB) aqueous humor (AH) analysis for tumor-derived cell-free DNA (cfDNA) have shown that the somatic copy number alteration (SCNA) profiles derived from the AH are highly concordant to profiles derived from the tumor in most but not all cases. As one of the reasons for the discordance in certain cases, it was suggested that these patients had multiple retinal tumors that had developed different subsets of SCNAs. The AH was therefore speculated to be a heterogenous mixture of cfDNA from each tumor clone, a heterogeneity that could not be captured in a single tumor biopsy after enucleation. In such cases that do not show high concordance, analyzing the genomic profile of tumor seeds in the VH can help us determine the degree of heterogeneity between the different subclones in the tumor. As single tumor cells from the vitreous humor (VH) have not been studied in RB before, this study demonstrates a technique that could be used to investigate the SCNA profiles of these cells with the aim to further understand the possible origins of AH cfDNA. Methods: The subject of this study is a 24-month-old male diagnosed with advanced unilateral RB (Group D/Stage CT2B) in the left eye and treated with primary enucleation after seven days. VH was collected from the enucleated eye, cfDNA was isolated from the AH and VH, and tumor DNA was extracted from tumor biopsy. Thirty live VH single cells were isolated using fluorescence-activated cell sorting using DAPI staining. The cells were then amplified using GenomePlex® Single Cell Whole Genome Amplification Kit (WGA4). Tumor DNA and single-cell WGA amplicons were constructed using NEBNext Ultra II FS Kit, and whole genome libraries of cfDNA from AH and VH were constructed using QIAseq Ultralow Input Library Kit. Shallow WGS of these libraries were sequenced using the IIlumina paired-end (2 X 150bp) platform to assess genome-wide SCNAs. Heatmap with hierarchical clustering was generated in R using the heatmap.2 function in the ggplots package. Cells were clustered by Ward’s method with Manhattan distance by their median centered data. Cutoffs for gains and losses were 1.2 and 0.8 over the median respectively. Results: There was high concordance in SCNA profiles across AH cfDNA, VH cfDNA, and tumor DNA, showing highly recurrent RB SCNAs such as gains on 1q, 2p, and 6p, in addition to gains on 2p, 5p, 7q, 9q, 10q, 13q, 15q, 17q and loss on 3q. The SCNA profiles of the 30 VH seeds demonstrated the highly recurrent RB SCNAs (gains on 1q, 2p, and 6p) that were consistent with the tumor SCNA signature. The heatmap of the VH single cell SCNA profiles demonstrated low levels of heterogeneity between the seeds. Conclusions: Our results suggest that VH seeds are tumor cells. Analyzing genomic material of the VH seeds using single cell SCNA profiling provides a valuable platform to study tumor heterogeneity in RB. Citation Format: Shreya Sirivolu, Liya Xu, Peter Kuhn, James Hicks, Jesse Berry. Single cell profiling of vitreous humor seeds in retinoblastoma [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 6052.

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.