aqueous-humor-as-liquid-biopsy-for-retinoblastoma-clear-corneal

Aqueous Humor as a Liquid Biopsy for Retinoblastoma: Clear Corneal Paracentesis and Genomic Analysis

There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is ...

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal  amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes ( = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes ( = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes ( = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH ( = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy. The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. .

Genomic cfDNA Analysis of Aqueous Humor in Retinoblastoma Predicts Eye Salvage: The Surrogate Tumor Biopsy for Retinoblastoma.

We present SMURF-seq, a protocol to efficiently sequence short DNA molecules on a long-read sequencer by randomly ligating them to form long molecules. Applying SMURF-seq using the Oxford Nanopore MinION yields up to 30 fragments per read, providing an average of 6.2 and up to 7.5 million mappable fragments per run, increasing information throughput for read-counting applications. We apply SMURF-seq on the MinION to generate copy number profiles. A comparison with profiles from Illumina sequencing reveals that SMURF-seq attains similar accuracy. More broadly, SMURF-seq expands the utility of long-read sequencers for read-counting applications.

SMURF-seq: efficient copy number profiling on long-read sequencers.

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1 ). RB can be hereditary (germline RB1 pathogenic allele is present) or non-hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell-free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non-hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non-hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly-recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non-hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.

Variability in retinoblastoma genome stability is driven by age and not heritability.

Aqueous humor contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma. We previously associated somatic copy-number alteration (SCNA) 6p gain with a 10-fold increased risk of enucleation. Here we provide a 2-year update to further explore 6p gain as a prognostic biomarker for ocular survival. Patients diagnosed with retinoblastoma from December 2014 to July 2019 from whom aqueous humor was sampled were included. cfDNA was extracted and shallow whole-genome sequencing performed to identify highly recurrent retinoblastoma SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). 116 aqueous humor samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly recurrent retinoblastoma SCNAs were found in 66% eyes. 6p gain was the most prevalent SCNA (50% eyes). It was particularly more prevalent in enucleated eyes (73.9%) than in salvaged eyes (29.6%;  = 0.004). 6p gain in aqueous humor cfDNA portended nearly 10-fold increased odds of enucleation (OR = 9.87; 95% confidence interval = 1.75-55.65;  = 0.009). In the enucleated eyes, 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures. With extended follow-up and nearly double the aqueous humor samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for retinoblastoma. IMPLICATIONS: Aqueous humor is a high-yield source of tumor-derived DNA in retinoblastoma eyes. Detection of 6p gain in the aqueous humor allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with aqueous humor sampled from all eyes at diagnosis are warranted to validate these findings.

Chromosome 6p Amplification in Aqueous Humor Cell-Free DNA Is a Prognostic Biomarker for Retinoblastoma Ocular Survival.

Detection of germline  mutations is critical for risk assessment of retinoblastoma (RB) patients. Assessment of somatic copy number alterations (SCNAs) is also critically important because of their prognostic significance. Herein we present a refined approach for the simultaneous identification of  variants and SCNAs in the aqueous humor (AH) of RB eyes.

Simultaneous identification of clinically relevant  mutations and copy number alterations in aqueous humor of retinoblastoma eyes.

An amendment to this paper has been published and can be accessed via the original article.

Author Correction: SMURF-seq: efficient copy number profiling on long-read sequencers.

The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumor-derived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA-including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude-correspond to therapeutic response.

Cell-Free DNA Tumor Fraction in the Aqueous Humor Is Associated With Therapeutic Response in Retinoblastoma Patients.

Currently, clinical characterization of metastatic breast cancer is based on tissue samples taken at time of diagnosis. However, tissue biopsies are invasive and tumors are continuously evolving, which indicates the need for minimally invasive longitudinal assessment of the tumor. Blood-based liquid biopsies provide minimal invasive means for serial sampling over the course of treatment and the opportunity to adjust therapies based on molecular markers. Here, we aim to identify cellular changes that occur in breast cancer over the lifespan of an affected patient through single-cell proteomic and genomic analysis of longitudinally sampled solid and liquid biopsies. Three solid and 17 liquid biopsies from peripheral blood of an ER/HER2 metastatic breast cancer patient collected over 4 years and eight treatment regimens were analyzed for morphology, protein expression, copy-number alterations, and single-nucleotide variations. Analysis of 563 single morphometrically similar circulating tumor cells (CTCs) and 13 cell-free DNA (cfDNA) samples along with biopsies of the primary and metastatic tumor revealed progressive genomic evolution away from the primary tumor profiles, along with changes in ER expression and the appearance of resistance mutations. Both the abundance and the genomic alterations of CTCs and cfDNA were highly correlated and consistent with genomic alterations in the tissue samples. We demonstrate that genomic evolution and acquisition of drug resistance can be detected in real time and at single-cell resolution through liquid biopsy analytes and highlight the utility of liquid biopsies to guide treatment decisions.

Treatment response and tumor evolution: lessons from an extended series of multianalyte liquid biopsies in a metastatic breast cancer patient.

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  and  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  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  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.

Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma: The Aqueous Humor Liquid Biopsy.

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 ( = 0.015); later age of diagnosis ( < 0.0001); greater odds of an endophytic tumor growth pattern (without retinal detachment;  = 0.047); tumor heights >10 mm ( = 0.09); and containing 6p gain, a biomarker of poor ocular prognosis ( = 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.

Comprehensive Somatic Copy Number Analysis Using Aqueous Humor Liquid Biopsy for Retinoblastoma.

Germline alterations in the RB1 tumor suppressor gene predispose patients to develop retinoblastoma (RB) in both eyes. While similar treatment is given for each eye, there is often a variable therapeutic response between the eyes. Herein, we use the aqueous humor (AH) liquid biopsy to evaluate the cell-free tumor DNA (ctDNA) from each eye in a patient with bilateral RB. Despite the same predisposing germline RB1 mutation, AH analysis identified a different somatic RB1 mutation as well as separate and distinct chromosomal alterations in each eye. The longitudinal alterations in tumor fraction (TFx) corresponded to therapeutic responses in each eye. This case demonstrates that bilateral RB tumors develop separate genomic alterations, which may play a role in tumorigenesis and prognosis for eye salvage. Identifying these inter-eye differences without the need for enucleated tumor tissue may help direct active management of RB, with particular usefulness in bilateral cases.

Article	Year
Genomic cfDNA Analysis of Aqueous Humor in Retinoblastoma Predicts Eye Salvage: The Surrogate Tumor Biopsy for Retinoblastoma. Molecular cancer research : MCR\| PubMed ID: 30061186
SMURF-seq: efficient copy number profiling on long-read sequencers. Genome biology\| PubMed ID: 31287019	2019
Variability in retinoblastoma genome stability is driven by age and not heritability. Genes, chromosomes & cancer\| PubMed ID: 32390242
Chromosome 6p Amplification in Aqueous Humor Cell-Free DNA Is a Prognostic Biomarker for Retinoblastoma Ocular Survival. Molecular cancer research : MCR\| PubMed ID: 32434859
Simultaneous identification of clinically relevant mutations and copy number alterations in aqueous humor of retinoblastoma eyes. Ophthalmic genetics\| PubMed ID: 32799607
Author Correction: SMURF-seq: efficient copy number profiling on long-read sequencers. Genome biology\| PubMed ID: 32843071	2020
Cell-Free DNA Tumor Fraction in the Aqueous Humor Is Associated With Therapeutic Response in Retinoblastoma Patients. Translational vision science & technology\| PubMed ID: 33062393	2020
Treatment response and tumor evolution: lessons from an extended series of multianalyte liquid biopsies in a metastatic breast cancer patient. Cold Spring Harbor molecular case studies\| PubMed ID: 33203646	2020
Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma: The Aqueous Humor Liquid Biopsy. Cancers\| PubMed ID: 33805776	2021
Comprehensive Somatic Copy Number Analysis Using Aqueous Humor Liquid Biopsy for Retinoblastoma. Cancers\| PubMed ID: 34283049	2021
Inter-eye genomic heterogeneity in bilateral retinoblastoma via aqueous humor liquid biopsy. NPJ precision oncology\| PubMed ID: 34316014	2021

Rishvanth K. Prabakar

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