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Potential of aqueous humor as a liquid biopsy for uveal melanoma

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Potential of aqueous humor as a liquid biopsy for uveal melanoma

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Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma

by

Jesse L. Berry, M.D.

A Thesis Presented to the
FACULTY OF THE KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CLINICAL, BIOMEDICAL AND TRANSLATIONAL INVESTIGATIONS)

May 2022

Copyright 2022 Jesse L. Berry, M.D.
ii
ACKNOWLEDGEMENTS
I would like to acknowledge Deborah Im, B.S., Chen-Ching Peng, Ph.D., Liya Xu, Ph.D., Mary
E. Kim, B.A., Dejerianne Ostrow, Ph.D., Venkata Yellapantula, Ph.D., Moiz Bootwalla M.S., Jaclyn A
Biegel, Ph.D., Xiaowu Gai, Ph.D., Rishvanth K. Prabakar, Ph.D., Peter Kuhn, Ph.D., and James Hicks,
Ph.D. for their assistance and research in making this thesis possible. We’d like to acknowledge Brianne
Brown, Dilshad Contractor, and Armine Begijianmasihi for the coordination of this study, David Ruble
for technical assistance, as well as Mark Reid, PhD for biostatistical expertise.

iii
TABLE OF CONTENTS

Acknowledgements......................................................................................................................................ii
List of Tables...............................................................................................................................................v
List of Figures.............................................................................................................................................vi
Abbreviations.............................................................................................................................................vii
Abstract ....................................................................................................................................................viii
Chapter 1: Cover Letter…………...............................................................................................................1
Chapter 2: Introduction ...............................................................................................................................2
Chapter 3: Methods and Materials...............................................................................................................4
3.1 Case Series ................................................................................................................................4
3.1.1 Patient and specimen characteristics...........................................................................4
3.1.2 Specimen collection and storage.................................................................................4
3.1.3 Analysis of nucleic acid content in the AH................................................................5
3.1.4 Genomic analysis of samples......................................................................................5
3.1.5 Single Nucleotide Variants (SNV) analysis of samples.............................................5
3.1.6 Statistical analysis.......................................................................................................6
Chapter 4: Results........................................................................................................................................7
4.1 Patient clinical characteristics and demographics......................................................................7
4.2 Evaluation of AH nucleic acid content before and after brachytherapy radiation.....................8
4.3 Circulating tumor DNA in AH..................................................................................................9
Chapter 5: Discussion................................................................................................................................11
Chapter 6: Statements................................................................................................................................16
iv
6.1 Statement of Ethics..................................................................................................................16
6.2 Disclosure Statement...............................................................................................................16
6.3 Funding Sources.......................................................................................................................16
6.4 Author Contributions...............................................................................................................17
References..................................................................................................................................................18

v
LIST OF TABLES
1. Univariate comparison of clinical characteristics between choroidal and ciliary body
tumor UM patients………………………………………………………………………………7
2. Single nucleotide variant analysis of BAP1 and GNAQ in five CB patients………………….10

vi
LIST OF FIGURES
1. Quantification of cell-free nucleic acids in uveal melanoma aqueous humor samples
before (pre-) and after (post-) radiation…………………………………………………………..9
2. Somatic copy number alterations in uveal melanoma post-radiation aqueous humor samples…12

vii
ABBREVIATIONS
AH Aqueous Humor
AJCC American Joint Committee on Cancer
CB Ciliary Body
ctDNA Circulating Tumor DNA
DNA Deoxyribonucleic Acid
dsDNA Double Stranded DNA
FNAB Fine-needle Aspiration Biopsy
GEP Gene Expression Profiles
miRNA MicroRNA
PRAME Preferentially Expressed Antigen in Melanoma
RNA Ribonucleic Acid
SCNAs Somatic Copy Number Alterations
ssDNA Single Stranded DNA
UM Uveal Melanoma

viii
ABSTRACT
Tumor biopsy can be used to identify prognostic biomarkers for metastatic uveal melanoma
(UM). However, aqueous humor (AH) biopsy is less invasive and may serve as an adjunct to tumor
biopsy. The objective of this paper is to evaluate whether the AH of UM eyes has sufficient circulating
tumor DNA (ctDNA) to perform genetic analysis. We evaluated a case series of 37 AH samples from 12
choroidal and 8 ciliary body (CB) melanoma eyes. Of 12 choroidal patients, there were 9 paired pre- and
post-radiation, 2 post-radiation only and 1 enucleation without radiation AH samples. There were 8
paired pre- and post-radiation CB AH samples and one CB tumor biopsy wash sample. Samples were
taken between August 2020 - May 2021 at a tertiary care center. Participants included a convenience
sample of 20 UM patients.
AH was analyzed for nucleic acid concentration (Qubit Assay Kits). Shallow whole-genome
sequencing was done on isolated AH DNA and one tumor wash sample followed by 2x150bp paired-end
Illumina sequencing to detect somatic copy number alterations (SCNAs). Hybridization-based capturing
and targeted sequencing were subsequently done using a customized panel that covers all exon regions
of BAP1 and GNAQ genes.
Mean dsDNA, ssDNA, RNA and miRNA concentrations were higher in CB compared to
choroidal AH samples. Post-radiation AH samples had significantly higher concentrations in DNA and
miRNA than paired pre-radiation AH samples for both choroidal and CB tumors (choroidal: ssDNA,
P=0.035 and miRNA P=0.016) (CB: dsDNA, P=0.023 and miRNA, P=0.008). Highly recurrent
UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 (75.0%) post-radiation CB AH
samples. High concordance of copy number alterations was identified in a CB post-radiation AH sample
with its matched tumor sample (r=0.978). Five post-radiation SCNA-positive AH samples underwent
ix
targeted resequencing to identify single nucleotide variants in BAP1 or GNAQ and 3/5 (60%) pathogenic
variants were detected.
We concluded that AH is a valid source of ctDNA in UM eyes, with a higher yield of nucleic
acids post-radiation, particularly in CB eyes. This is the first-time that UM SCNAs and mutations were
identified in ctDNA isolated from the AH. This suggests that AH can serve as a liquid biopsy for UM.
1
CHAPTER 1: COVER LETTER

University of Southern California
1450 San Pablo St. 4
th
Floor, Suite 4700, Los Angeles, CA 90033 • Tel: 323-442-6335 Fax: 323-442-6338
Jesse.Berry@med.usc.edu

Neil M. Bressler, MD
Editor-in-Chief, JAMA Ophthalmology

February 23, 2022

RE: Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma

Deborah Im, Chen-Ching Peng, Liya Xu, Mary E. Kim, Dererianne Ostrow, Venkata Yellapantula, Moiz Bootwalla,
Jaclyn A. Biegel, Xiaowu Gai, Rishvanth K. Prabakar, Peter Kuhn, James Hicks, Jesse L. Berry

Dear Dr. Bressler,

Enclosed please find our manuscript entitled “Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma”,
which we respectfully submit to JAMA Ophthalmology for consideration as an original investigation article.

Uveal melanoma (UM) is the most common primary intraocular cancer in adults. Although radiation and enucleation
(surgical removal of the eye) are available for the treatment of the primary tumor, ~50% of patients experience fatal
metastatic disease. Identifying tumoral biomarkers can stratify the risk of metastatic disease. Tumoral prognostic
molecular biomarkers are available via intraocular tumor biopsy, and can be stratified into gene expression profiles,
chromosomal copy number alterations, and the key mutation in tumor oncogenesis. While once taboo, intraocular
tumor biopsy by fine-needle aspiration is now a part of routine clinical management for UM. However, aqueous humor
(AH) liquid biopsy, is a significantly less invasive alternative and is repeatable for longitudinal monitoring of disease
without returning to the operating room.

However, studies of the AH in UM patients have only previously reported on cytokines and sHLA. To date, the
presence and prognostic significance of genomic biomarkers have not been reported in the AH of UM eyes. Herein, we
present the first prospective report of the detection of genomic biomarkers in the AH of UM eyes detailing 20
eyes of 20 UM patients, who had AH sampled at diagnosis and longitudinally after treatment.

This work in AH as a liquid biopsy for melanoma is derived from our work using the AH for retinoblastoma. The initial
publication on this novel platform for retinoblastoma was published in JAMA Ophthalmology
(https://jamanetwork.com/journals/jamaophthalmology/fullarticle/2656334) and has been cited 42 times in the last 5
years and has an Altimetric score of 83; it is thus fitting that we submit the pilot work on this platform for uveal
melanoma to JAMA Ophthalmology.

Drs. Berry, Xu, and Hicks have filed a provisional patent application entitled, Aqueous Humor Cell Free DNA for
Diagnostic and Prognostic Evaluation of Ophthalmic Disease. Financial support was provided by: National Cancer
Institute of the National Institute of Health Award K08CA232344, Hyundai Hope on Wheels, Childhood Eye Cancer
Trust, Wright Foundation. In kind support from Larry and Celia Moh Foundation, The Institute for Families, Inc.,
Children’s Hospital Los Angeles and Research to Prevent Blindness (an unrestricted departmental grant) and The Dr.
Miriam and Sheldon G. Adelson Medical Research Foundation (J.H., P.K.). This manuscript is original, and no related
papers have been submitted or published elsewhere.

I will serve as the corresponding author.

Sincerely yours,

Jesse L. Berry, MD
Associate Professor of Ophthalmology
Clinical Scholar
Jesse L. Berry, MD
Associate Professor of Clinical Ophthalmology
Director, Ocular Oncology at Children's Hospital
Los Angeles and USC Roski Eye Institute

2
CHAPTER 2: INTRODUCTION
Uveal melanoma (UM) is the most common primary intraocular cancer in adults.
1
Tumors arise
from the uveal tract and can affect the choroid, iris, and ciliary body, with the latter two lesions being
anatomically closer to the aqueous humor (AH). Conservative treatment consists of radiation, with
enucleation reserved for the most advanced cases. Even when the intraocular tumor is successfully
treated, approximately half of all patients with UM will develop metastases.
2
Unfortunately, metastatic
UM is usually fatal within one year of symptom onset, as it is poorly responsive to chemotherapy and/or
targeted therapy.
It is widely known that identifying tumor biomarkers stratifies the risk of metastatic disease and
may help improve earlier detection of metastases.
3
While once taboo, intraocular tumor biopsy via fine-
needle aspiration biopsy (FNAB) is now part of routine clinical workup for UM. Tumor-derived
prognostic molecular markers can be categorized into 1) gene expression profiles (GEP) 2) somatic copy
number alterations (SCNA), or 3) mutations in key genes for UM oncogenesis. Recent multi-omic work
has used these three categories to divide UM patients into 4 subsets of low (Type A), intermediate (Type
B), and high (Types C and D) metastatic potential.
4
Clinically, Onken et al. found that this translated
into a 4-year risk of metastasis of 3% for Types A and B and upwards of 80% for Types C and D.
5

Cytogenetic characteristics include highly recurrent UM SCNAs such as monosomy 3, losses of
chromosome arms 1p, 6q, 8p and 16q, and gains of 1q, 6p and 8q,
6
with monosomy 3, 8q gain, and 1p
loss being the most prognostically unfavorable.
7
Although American Joint Committee on Cancer
(AJCC) staging of UM does not currently include cytogenetic information and is based only on clinical
observations, it has been shown that addition of these molecular subsets yields a significant
improvement in prognostication compared to AJCC stage alone.
8-12
While FNAB is considered a safe
procedure, there remain small risks of retinal detachment, subretinal hemorrhage, and vitreous
3
hemorrhage, sometimes requiring further surgical intervention to repair.
13,14
Furthermore, direct tumor
biopsy is often not repeatable without returning to the operating room. Given these risks, liquid biopsy
has emerged as a less invasive alternative that also offers the ability to track genomic changes
longitudinally over time without the need for return to the operating room.
Most liquid biopsy research in UM has focused on the blood as a biofluid source of circulating
tumor cells and/or circulating tumor DNA (ctDNA). However, the low tumor fraction found in the blood
due to the blood-ocular barrier limits the detection of prognostic biomarkers; thus, blood liquid biopsies
for UM may be better utilized to detect systemic disease.
15-18
AH liquid biopsies have been investigated
as an eye-specific alternative. However, previous studies of the AH in UM patients have only reported
on cytokines and soluble HLA.
19-23
To our knowledge, no genomic prognostic biomarkers have been
found in UM AH. Given that we have not only demonstrated the presence but also established the
clinical utility of diagnostic and prognostic biomarkers in the AH of retinoblastoma eyes,
24,25
we
hypothesized that the AH of UM eyes may similarly harbor tumor-derived nucleic acids to serve as a
surrogate tumor biopsy in UM. The presence and prognostic significance of ctDNA isolated from the
AH of UM patients has yet to be evaluated. Furthermore, eye-specific biopsy allows for repeatable
testing near the primary tumor and may allow for detection of local recurrence, new avenues for
prognostication, and objective markers of tumoral regression post-therapy.

4
CHAPTER 3: MATERIALS AND METHODS
This investigation was a case series study at a tertiary care hospital (University of Southern
California Roski Eye Institute). Samples were taken between August 2020 and May 2021. The study
was approved by the Institutional Review Board at the University of Southern California Keck School of
Medicine (HS-19-00293) and at Children’s Hospital Los Angeles (CHLA-20-00167). The research
conformed to the requirements of the United States Health Insurance Portability and Accountability Act.

3.1 Case Series
3.1.1 Patient and specimen characteristics
This study included a convenience sample of 20 UM patients at the University of Southern
California Roski Eye Institute from whom written informed consent for an AH sample was obtained. All
samples consisted of ~0.1 mL of AH extracted via clear cornea paracentesis at the end of surgery for
brachytherapy plaque placement (pre-radiation), brachytherapy plaque removal (post-radiation) or after
enucleation without radiation. We include 37 AH samples from 20 UM eyes: a total of 17 matched AH
samples pre-radiation and post-radiation, two AH samples post-radiation only, and one AH sample after
enucleation without radiation. Radiation methods in the treatment of UM have been detailed and
published previously.
26
Genomic testing results were coded and maintained separately from clinical data
and thus did not alter patient treatment for all participants.

3.1.2 Specimen collection and storage
A clear corneal paracentesis with a 30-gauge needle was performed to extract ~0.1 mL of AH
from UM eyes during clinically indicated surgery to treat UM. The extraction method has been
described in detail and published previously by our group for specimen collection from retinoblastoma
5
eyes.
27
Briefly, needles only entered the anterior chamber via the clear cornea at the limbus and did not
make contact with the iris, lens, vitreous, or UM tumor. Samples were stored on dry ice immediately and
transferred to -80ºC within hours of extraction. Routine FNAB with either a 25- or 27-gauge needle was
done on 14 patients for mutational analysis and 15 patients for GEP and preferentially expressed antigen
in melanoma (PRAME) status which was performed at Castle Biosciences (Phoenix, AZ). In one
patient, the same tumor biopsy needle was washed with basic saline solution (BSS) separately to obtain
a tumor wash sample.

3.1.3 Analysis of nucleic acid content in the AH
Nucleic acids (dsDNA, ssDNA, RNA, and miRNA) were assayed using Qubit Assay Kits
(Thermo Fischer), which measures concentration of the assayed nucleic acid with the Qubit Fluorometer
following the manufacturer’s manual.

3.1.4 Genomic analysis of samples
All samples underwent DNA isolation, sequencing, and analysis within 1 month of collection, as
consistent with established methods of SCNA analysis.
27-29
Briefly, cell-free DNA of AH was isolated
with the QIAamp circulating nucleic acid kit (QIAGEN), and DNA from FNAB was isolated with the
QIAamp DNA blood mini kit (QIAGEN). Isolated DNA was used to prepare whole-genome libraries
with the QIAseq Ultralow Input Library Kit (QIAGEN) followed by 2X150 bp paired end shallow 0.1-
0.3x whole genome sequencing (WGS) for copy-number alteration profiling. SCNAs were considered to
be present at 20% deflection from a baseline human genome, which is based on liquid biopsy analyses
that have been previously established.
27-29

6
3.1.5 Single Nucleotide Variants (SNV) analysis of samples
For five SCNA-positive AH samples (UM_005, 007, 012, 013 and 019), the same sequencing
libraries then underwent targeted resequencing for mutation detection using a customized hybridization
panel laboratory developed test (Twist Bioscience, San Francisco, CA) at the Children’s Hospital Los
Angeles Center for Personalized Medicine that covers BAP1 and GNAQ gene exon regions.
Bioinformatics analysis was done in parallel and blind to the clinically available tumor testing (Castle
Biosciences). Results were compared to tumor SNV results in all but one patient for whom AH analysis
was done but there was no matched clinical analysis as insurance refused to cover the Castle testing.

3.1.6 Statistical analysis
To describe the patient population, categorical variables were compared using the Fisher's exact
test or linear-by-linear association test as indicated. To evaluate changes in concentration levels, the
continuous variables were summarized as the mean ± standard error and percentages. Non-normally
distributed variables were compared by the Mann–Whitney U test. To evaluate concordance between
tumor and liquid biopsy genomic alterations, the Pearson’s coefficient was calculated by comparing the
segmented ratio to medians at 5k bins. All statistical tests were 2-tailed, and P < 0.05 was considered
statistically significant. P-values are represented as: *, P < 0.05; **, P < 0.01; ***, P < 0.001. All
statistical analyses and plots were conducted using the Prism 8 (GraphPad).

7

CHAPTER 4: RESULTS
4.1 Patient clinical characteristics and demographics
Overall, 37 AH and one tumor wash sample from 20 UM patients (20 eyes) were evaluated.
Patient demographics and clinical characteristics are summarized in Table 1. A total of twelve (60%)
choroidal and eight (40%) ciliary body (CB) tumor patients were included. All choroidal tumors (100%)
were AJCC stage I or IIA and did not have concomitant CB involvement. Of CB tumors, 5/8 (62.5%)
were AJCC stage I or IIA, while the other three were more advanced. A significant number of CB
tumors were diagnosed at a more advanced AJCC stage than choroidal tumors (P=0.003; Table 1). Of
note, one choroidal UM patient underwent primary enucleation due to the tumor surrounding the optic
nerve head. Results from clinically indicated tumor biopsy, including UM mutation, PRAME status, and
GEP class are included (Table 1).

Table 1.
Table 1 Univariate comparison of clinical characteristics between choroidal and ciliary body tumor UM patients
Choroidal, n=12 Ciliary body tumor, n=8 p value
Sex (Fisher), n (%) 0.197
Females 5 (41.7) 6 (75.0)
Males 7 (58.3) 2 (25.0)
Eye (Fisher), n (%) 0.650
OD 5 (41.7) 5 (62.5)
OS 7 (58.3) 3 (37.5)
Age at diagnosis, mean (± SD) (MWU) 60.8 (12.5) 54.0 (15.5) 0.438
Eye Color (Fisher), n (%) 0.999
Light (blue, gray, green, hazel) 8 (66.7) 6 (75.0)
Dark (brown) 4 (33.3) 2 (25.0)
Ciliary Body Involvement (Fisher), n (%) <0.001
Yes 0 (0) 8 (100)
No 12 (100) 0 (0)
AJCC Stage (Linear-by-Linear association), n (%) 0.003
I 9 (75.0) 1 (12.5)
IIA 3 (25.0) 4 (50.0)
IIB 0 (0) 2 (25.0)
IIIA, IIIB, IIIC 0 (0) 1 (12.5)
IV 0 (0) 0 (0)
PRAME Status, known in 15 cases (Fisher), n (%) 0.999
Negative 7 (100) 7 (87.5)
Positive 0 (0) 1 (12.5)
GEP Class, known in 15 cases (Linear-by-Linear association), n (%) 0.876
1A 5 (71.4) 6 (75.0)
1B 0 (0) 0 (0)
2 2 (28.6) 2 (25.0)
Tumor Stage (Linear-by-Linear association), n (%) 0.159
T1 9 (75.0) 4 (50.0)
T2 3 (25.0) 3 (37.5)
T3 0 (0) 1 (12.5)
T4 0 (0) 0 (0)
Characteristic
AJCC, American Joint Committee in Cancer; Fisher, Fisher’s exact test; GEP, gene expression profile; MWU, Mann-Whitney U
test; PRAME, preferentially expressed antigen in melanoma; SD, standard deviation
8

4.2 Evaluation of AH nucleic acid content before and after brachytherapy radiation
Analysis of AH samples revealed measurable levels of nucleic acids in the majority of eyes with
choroidal and CB melanomas, with the exception of RNA which was only detectable in post-radiation
AH from four CB tumors (Figure 1). Mean nucleic acid concentrations of all analytes were higher in CB
compared to choroidal UM samples (Figure 1, A-D). Notably, four CB post-radiation AH samples
(UM_005, 007, 012, and 013) contained the highest nucleic acid concentration among all 37 AH
samples.
Paired pre- and post-radiation AH samples from nine choroidal and eight CB melanoma eyes
were analyzed. In both choroidal and CB AH, post-radiation AH samples had significantly higher
concentrations of DNA and miRNA (choroidal: ssDNA, P=0.035 and miRNA, P=0.016) (CB: dsDNA,
P=0.023 and miRNA, P=0.008) (Figure 1, E, F and H).

9

Figure 1.

4.3 Circulating tumor DNA in AH
To determine the presence of ctDNA in the AH, shallow WGS was completed to profile SCNAs
in all 37 AH samples. All pre-radiation AH SCNA profiles were neutral. SCNAs were found only in AH
collected after brachytherapy radiation, with significantly more found in CB post-radiation AH samples
(6/8, 75%) than choroidal post-radiation AH samples (0/11, P=0.001; Figure 2B). Highly recurrent UM
SCNAs of monosomy 3, 6p gain, 6q loss, and 8q gain were identified in SCNA-positive post-radiation
AH samples (Figure 2C). A tumor wash (UM_019) was done in a single case to determine whether this
would be a feasible mechanism to obtain tumor samples for research (instead of a repeat tumor biopsy).
There was high concordance of SCNA alterations between the post-radiation AH sample and the tumor
wash collected before radiation (Pearson’s r=0.978; Figure 2D).
Figure 1. Quantification of cell-free nucleic acids in uveal melanoma aqueous humor samples
before (pre-) and after (post-) radiation

A B
C D
0
200
400
600
6000
8000
dsDNA conc. (ng/mL)
dsDNA
Choroid-pre (10)
Choroid-post (11)
CB-pre (8)
CB-post (8)
Choroid (21) CB (16)
54.9 (± 8.2) 591.1 (± 463.4)
P = 0.094
005
013
007
012
Mean (± S.E.M)
0
1000
2000
3000
RNA conc. (ng/mL)
RNA
Choroid-pre (10)
Choroid-post (11)
CB-pre (8)
CB-post (8)
Choroid (21) CB (16)
0 (± 0) 417.8 (± 204.3)
P = 0.028
005
013
007
012
Mean (± S.E.M)
0
1000
2000
10000
20000
ssDNA conc. (ng/mL)
ssDNA
Choroid-pre (10)
Choroid-post (11)
CB-pre (8)
CB-post (8)
Choroid (21) CB (16)
153.4 (± 22.6) 1631.0 (± 1315.0)
P = 0.069
005
013
007
012
Mean (± S.E.M)
0
1000
2000
10000
20000
miRNA conc. (ng/mL)
miRNA
Choroid-pre (10)
Choroid-post (11)
CB-pre (8)
CB-post (8)
Choroid (21) CB (16)
147.8 (± 32.6) 1274.0 (± 999.9)
P = 0.399
005
013
007
012
Mean (± S.E.M)
Mean 44.6 55.3 49.8 1132.0
S.E.M. 8.9 14.0 9.1 914.6
Mean 108.2 170.6 148.9 3113.0
S.E.M. 24.2 36.6 23.9 2604.0
Choroid-pre (9)
Choroid-post (9)
CB-pre (8)
CB-post (8)
0
200
400
600
800
10000
20000
ssDNA conc. (ng/mL)
ssDNA
P = 0.035
F
P = 0.055
Choroid-pre (9)
Choroid-post (9)
CB-pre (8)
CB-post (8)
0
200
400
600
800
4000
6000
8000
dsDNA conc. (ng/mL)
dsDNA
P = 0.023 P = 0.461
E
10
Due to limited DNA concentration, five SCNA-positive AH samples (UM_005, 007, 012, 013,
and 019) were further evaluated for the presence of tumor variants in the BAP1 and GNAQ genes. We
analyzed the presence of SNVs using a pan-cancer predisposition panel and identified UM mutations in
BAP1 and GNAQ in 3/5 (60%) post-radiation AH samples (UM_005, 007 and 013, Table 2). These were
concordant with clinical tumor SNV testing from Castle Biosciences in two patients (UM_007 and 013).
A mutation was identified in the AH from patient UM_005; however, this patient did not have FNAB
results available from Castle Biosciences to determine concordance (Table 2).

Table 2. Mutation detection assay in UM AH samples after radiation compared to clinical tumor single
nucleotide variant (SNV) testing. UM mutations BAP1 and GNAQ were detected in 3/5 (60%) post-
radiation AH. These were concordant with clinical tumor tissue SNV testing from Castle Biosciences in
2 patients with available clinical testing of tumor tissue samples.

Table 2 Single nucleotide variant analysis of BAP1 and GNAQ in five CB patients
BAP1 (VAF%) GNAQ (VAF%)
ciliary body tumor
UM_005_Tumor NA NA
UM_005_AH ND c.626A>T (42.9)
UM_007_Tumor ND c.626A>T (23.2)
UM_007_AH ND c.626A>T (40.9)
UM_012_Tumor ND c.626A>T (53.0)
UM_012_AH ND ND
UM_013_Tumor c.830_831del (68.0) ND
UM_013_AH c.830_831del (81.8) ND
UM_019_Tumor c.375+1G>A (93.8) c.626A>C (47.3)
UM_019_AH ND ND
VAF, variant allele frequency; NA, not-available, tumor biopsy not done; ND, non-detectable
Sample
11
CHAPTER 5: DISCUSSION
We examined 37 AH samples from 20 UM patients to investigate whether the AH liquid biopsy
can serve as a surrogate to tumor biopsy. We hypothesized that the AH may contain ctDNA and may
serve as a liquid biopsy for UM; these hypotheses arose from our work on another ocular cancer,
retinoblastoma, wherein we and others have demonstrated that the AH is an enriched source of
ctDNA.
27,30-33
In this pilot study, we first demonstrated that there were measurable concentrations of
nucleic acids (dsDNA, ssDNA, RNA and miRNA) in the small volumes of AH that can be extracted
from UM patients during plaque brachytherapy or enucleation. This is the first-time nucleic acids were
quantified and characterized in the AH of UM patients. Mean pre- and post-radiation combined nucleic
acid concentrations were compared between choroidal and CB tumors. Mean nucleic acid concentrations
of all analytes were higher in CB compared to choroidal UM, significantly for RNA (Figure 1, A-D).
Interestingly, the AH samples with the highest nucleic acid content among all 37 samples were found in
the same 4 CB tumors’ (005, 007, 012 and 013) post-radiation samples (Figure 1, A-D). We hypothesize
that the higher mean concentration of nucleic acids in CB AH compared to choroidal AH is likely due to
the proximity of the tumor to the AH in these more anteriorly located neoplasms. When comparing pre-
and post-radiation AH samples, there was a significantly higher concentration of evaluated nucleic acids
in post-radiation AH samples, most notably in patients with CB tumors (Figure 1, E-H). We hypothesize
that the increased nucleic acids in post-radiation AH are tumor-derived due to necrosis and lysis of
tumor cells after radiation. Importantly, 17/20 (85%) UM tumors included herein were AJCC stage I or
IIA tumors, suggesting utility in AH liquid biopsy even for smaller, early-stage UM.
Next, we determined whether the AH DNA was tumor derived. Copy number variation profiling
confirmed the presence of highly recurrent UM SCNAs
34
in post-radiation CB AH samples including
monosomy 3, 6p gain, 6q loss, and 8q gain (Figure 2A). No SCNAs were found in post-radiation
12
choroidal AH samples or any pre-radiation AH samples. Altered SCNA profiles were found in 0/11 and
6/8 (75.0%) post-radiation AH samples of choroidal and CB melanomas respectively, a significant
difference (P=0.001) (Figure 2B). This suggests that AH liquid biopsy may be more useful for tumors
that form anteriorly in the eye in proximity to the anterior chamber, thus allowing an increased amount
of ctDNA to diffuse into the AH after radiation induced necrosis of tumor cells.

Figure 2.
In one patient, we attempted a tumor wash sample wherein the needle that had performed the
tumor biopsy was washed with BSS after it had already been flushed for clinical GEP analysis for Castle
Biosciences. We were able to effectively identify ctDNA for research purposes without impacting the
Castle Biosciences analysis required for the clinical care of this patient. We demonstrated near complete
Figure 2. Somatic copy number alterations in uveal melanoma post-radiation aqueous humor
samples

Diagnosis
Choroidal Ciliary body tumor
SCNA
Gain Loss Neutral
A
Diagnosis
1p
1q
2p
2q
3p
3q
4p
4q
5p
5q
6p
6q
7p
7q
8p
8q
9p
9q
10p
10q
11p
11q
12p
12q
13p
13q
14p
14q
15p
15q
16p
16q
17p
17q
18p
18q
19p
19q
20p
20q
21p
21q
22p
22q
1
2
3
4
6
10
11
15
16
17
18
20
5
7
8
9
12
13
14
19
Case
B
Choroidal, n=11 Ciliary body tumor, n=8 P value
SCNA (Fisher), n (%) 0.001
Altered 0 (0.0) 6 (75.0)
Neutral 11 (100.0) 2 (25.0)
Fisher (Fisher's extract test)
Characteristic
Fisher (Fisher’s exact test)
13
concordance in presence and amplitude of SCNAs between the genomic profiles from the post-radiation
AH and tumor wash samples (Pearson’s r=0.978; Figure 2D).
Finally, five post-radiation AH samples that harbored SCNAs (thus, a higher fraction of ctDNA)
were evaluated for canonical UM mutations in BAP1 and GNAQ. Common UM mutations GNAQ/11 are
thought to be an early event in the development of UM,
35
and other mutations such as BAP1 portend
worse prognosis.
1
We identified tumor-derived de novo UM mutations in 3/5 (60%) post-radiation AH
samples in BAP1 or GNAQ (Table 2). These were concordant with clinical tumor SNV testing results
from Castle Biosciences in two patients with available clinical testing of tumor samples (Table 2).
Both the presence of SCNAs and SNVs in post-radiation AH samples provides strong evidence
that dsDNA isolated from the AH is tumor-derived. SCNAs can be visualized by shallow WGS with
lower nucleic acid input requirements with a lower limit of detection at ~5% tumor fraction.
24
Targeted
next-generation sequencing for mutation detection requires higher nucleic acid input, which is a
potential explanation for why some SCNA-containing samples with a known tumor SNV mutation was
not detected. While further optimization is needed, these results suggest that the AH may serve as a
valid surrogate biopsy to identify not only SCNAs and for specific UM mutations. As AH paracentesis
is repeatable and can be done in clinic, this may also facilitate evaluation of other eye-specific
biomarkers and longitudinal evaluation of local disease.
We identified quantifiable amounts of dsDNA, ssDNA, miRNA and RNA in the AH, however
the levels were much higher after radiation. In contrast to retinoblastoma, it appears that UM, known to
be less necrotic, does not shed into the AH at the same level – and may require radiation or other
interventions to cause cell death and shedding of ctDNA.
25,27
This may be somewhat confounded by
tumor size, as the majority of choroidal tumors in this cohort were relatively small. The larger and more
anteriorly located CB tumors had a higher concentration of nucleic acids in post-radiation AH samples,
14
which facilitated detection of SCNAs and SNVs. Research has shown benefit in identifying tumor-
derived prognostic molecular markers and even that eye color may play a role in the interaction of these
biomarkers.
36
Thus, an AH liquid biopsy may serve as an adjunct to FNAB, for example a biopsy done
at plaque placement and a paracentesis done at the time of plaque removal, so that the complex
prognostic association of tumoral biomarkers including GEP, SCNA, and mutations in key UM-related
genes can be better understood and utilized.
A limitation of this study is that only 20 UM patients were evaluated, with the majority of
choroidal tumors being relatively small. It is possible that with a larger cohort and with larger tumors
there may be higher concentrations of DNA and other nucleotides pre-radiation therapy. Additionally,
because there are some risks associated with tumor biopsy, tumor was not available for research only
analysis. We relied instead on clinically available information from Castle Biosciences testing. This was
available for most, but not all patients. Future studies should maximize the availability of tumor as well
as include larger study populations and larger choroidal tumors. Most liquid biopsy platforms center on
identifying ctDNA, which was the aim of our study as well. However, we are also investigating other
nucleic acids as potential biomarkers in UM. MiRNA has been detected in plasma, enveloped in
extracellular vesicles, and discovered here in the AH; miRNA has been investigated as a significant
target in many cancers, and future studies should explore its role in UM.
37

In conclusion, this study demonstrates that the AH is a source of ctDNA in UM, with a
statistically significantly higher yield of nucleic acids after radiation. Given the significantly higher
concentration of nucleic acids in CB AH compared to choroidal AH, AH biopsy may be more useful in
anteriorly located tumors. To our knowledge, this is the first study determining that (1) tumor nucleic
acids are present and quantifiable in the AH of UM eyes and that (2) UM SCNAs and mutations can be
identified from the AH. These results suggest that the AH can serve as a liquid biopsy for UM,
15
especially in CB tumors. With further investigations, this novel AH liquid biopsy platform may allow
clinicians to better prognosticate, monitor disease progression, and investigate local intraocular
biomarkers for UM.

16

CHAPTER 6: STATEMENTS
6.1 Statement of Ethics
This study was approved by the Institutional Review board at CHLA / USC (IRB number HS-19-
00293 and CHLA-20-00167), and it conformed to the requirements of the United States Health
Insurance Portability and Accountability Act. Research was conducted ethically in accordance with the
World Medical Association Declaration of Helsinki. Written informed consent was obtained from all
subjects.

6.2 Disclosure Statement
Drs. Berry, Xu and Hicks have filed a patent application entitled: Aqueous humor cell free DNA
for diagnostic and Prognostic evaluation of Ophthalmic Disease.

6.3 Funding Sources
In kind, financial support was provided by:
National Cancer Institute of the National Institute of Health Award Number K08CA232344
Hyundai Hope on Wheels
Childhood Eye Cancer Trust
Wright Foundation
The Larry and Celia Moh Foundation
The Institute for Families, Inc., Children's Hospital Los Angeles
An unrestricted departmental grant from Research to Prevent Blindness
The Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (J.H., P.K.)
17

The sponsors had no role in the study design; the collection, analysis, and interpretation of data;
in the writing of the report; or in the decision to submit the article for publication.

6.4 Author Contributions
J.B. and L.X. conceived of the presented idea. M.K. and L.X. carried out the data collection and
data analysis. M.R. helped perform the computations and verify the analytical methods. D.I. and C.P.
prepared the original draft. D.I., C.P., L.X., M.K., D.O., V.Y., M.B., J.A. B., X.G., R.P., P.K., J.H., J.B.
contributed to original draft editing.

18

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Asset Metadata

Creator Berry, Jesse L. (author)

Core Title Potential of aqueous humor as a liquid biopsy for uveal melanoma

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Degree Conferral Date 2022-05

Publication Date 10/09/2022

Defense Date 04/09/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cell-free DNA,circulating tumor DNA,liquid biopsy,Melanoma,OAI-PMH Harvest,uvea

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kuhn, Peter (committee chair), Patino-Sutton, Cecilia (committee chair), Hicks, James (committee member)

Creator Email jesse.berry@med.usc.edu,jesse.berrymd@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC110920048

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