Chromosome 6p amplification in aqueous humor cell-free DNA is a prognostic biomarker for retinoblastoma ocular survival

Abstract

Aqueous humor (AH) contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma (RB). 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 RB from 12/2014–7/2019 from whom AH was sampled were included. CfDNA was extracted and shallow whole genome sequencing performed to identify highly-recurrent RB SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). One-hundred sixteen AH samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly-recurrent RB 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%; P=0.004). 6p gain in AH cfDNA portended nearly 10-fold increased odds of enucleation (OR=9.87, 95% CI=1.75–55.65, P=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 AH samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for RB.

INTRODUCTION

Retinoblastoma (RB) is a primary intraocular malignancy that forms in the developing retina of young children. Although overall survival rates approach 98% in developed countries (1), ocular survival for advanced eyes (Group D/E or cT2b/3) is far lower. In the field of ocular oncology, there is no known molecular marker to inform the diagnosis, prognosis for eye survival, or treatment of patients with retinoblastoma. This is due, in part, to the inability to directly biopsy tumors for fear that globe entry could provoke tumor seeding outside the eye and lead to orbital relapse (2–8). Because direct tumor biopsy is prohibited in the setting of retinoblastoma, retrospective studies evaluating RB treatment efficacy and corresponding tumor genetics have been limited to analysis of enucleated eyes (9–12). The lack of in vivo molecular data from retinoblastoma patients limits our understanding of tumor biology as well as our ability to develop precision treatment plans and prognosticate treatment outcomes accurately.

In 2017, our group demonstrated that aqueous humor (AH) could be used to evaluate retinoblastoma in vivo at the molecular level (13). From a set of enucleated eyes, we also showed that the molecular findings in AH strongly correlate with genomics of enucleated tumors. With multiple studies now showing that AH can be safely extracted and used to analyze tumor-derived cell-free DNA (cfDNA) throughout the course of treatment, AH has been established as a novel source of surrogate tumor biopsy, or liquid biopsy, for retinoblastoma (13–15). In the context of this liquid biopsy research, we identified a specific highly-recurrent somatic copy number alteration (SCNA), 6p chromosomal gain, as being associated with a 10-fold increased risk of enucleation and significantly decreased rate of ocular salvage (14). This revealed that SCNAs in the AH correlate with clinical outcomes, and highlighted chromosome 6p gain in the AH as a potential prognostic biomarker for retinoblastoma. However, the study was limited by a relatively small cohort of patients, and it was unknown whether this relationship would persist over extended follow-up and with the addition of new patients.

Ultimately, research to establish an AH-based liquid biopsy for retinoblastoma is aimed at addressing the true clinical problems of 1) our inability to biopsy tumor tissue and 2) the lack of prognostic biomarkers for retinoblastoma. Current management decisions for retinoblastoma patients are made based on clinical features that indicate the extent of tumor growth in the eye, the judgment and experience of the treating clinician, and collaboration with the parents. However, identifying biomarkers that inform the prognosis for ocular survival could revolutionize how we plan and care for these children. In this study, we provide a 2-year update—including our initial cohort with extended follow-up (14) and all newly diagnosed patients throughout this 2-year period—in order to more fully explore the relationship between highly-recurrent RB SCNAs (specifically gains on 1q, 2p, 6p; losses on 13q and 16q; and focal MYCN gains (16–19)) and ocular outcomes. More specifically, we explore the possibility of 6p gain as a clinically relevant prognostic biomarker for retinoblastoma.

MATERIALS AND METHODS

This research was conducted under Institutional Review Board approval and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the parents of all participants prior to inclusion in the study. The REMARK (Reporting Recommendations for Tumor Marker Prognostic Studies) guidelines for reporting tumor biomarkers were followed (20).

Patient and specimen characteristics

This study included all patients diagnosed with retinoblastoma between December 2014 and July 2019 at the Children’s Hospital Los Angeles (CHLA) from whom written parental consent and AH sample(s) were obtained; previously published cases now with extended follow up—in addition to cases that were diagnosed since the last study—are included (14). In all cases, samples consisted of approximately 0.1 mL of AH that was extracted via clear cornea paracentesis either 1) during routine clinical therapy with intravitreal melphalan (IVM), 2) at primary or secondary enucleation, or 3) at diagnosis. Consent from both parents was required for patients from whom AH was extracted at the time of diagnosis (i.e., prior to initiation of routine clinical therapy) as it was done as a research-only procedure. RB patients who were treated but did not have AH extracted were excluded from the study. For all participants, retinoblastoma treatment was carried out in a non-randomized manner per CHLA protocol (21,22). As previously (14), genomic testing results remained separate from clinical data until the final retrospective analysis, and thus did not influence patient treatment or the study endpoints.

The primary clinical endpoints were eye salvage (the ability to treat and save the eye using standard chemotherapeutic modalities) versus enucleation (surgical removal of the diseased eye).

Specimen collection and storage

A clear corneal paracentesis with a 32-guage needle was performed to extract 0.1 mL of AH from retinoblastoma eyes (14). This procedure occurred either as part of the standard IVM injection protocol (23), immediately following enucleation of the eye, or at the time of diagnosis following thorough examination under anesthesia. During sampling, needles entered only the anterior chamber via the clear cornea and remained bevel up over the pharmacologically-dilated iris; they did not make contact with the iris, lens, vitreous cavity or tumor. While the anterior chamber shallowed slightly it remained formed during paracentesis, and the needle site was examined after AH extraction for any leakage.

Immediately following specimen extraction, AH samples were stored at −80°C. Beginning on January 1, 2019, RNase inhibitor (Applied Bio N8080119) was added to newly extracted samples to a concentration of 1U/ul. All samples underwent cfDNA isolation and sequencing within 1 month of extraction, as described previously (13,14).

Genomic analysis of AH samples

Analysis of the cfDNA from AH samples was previously outlined in depth and based on established methods of SCNA analysis (13,14,24,25). Briefly, isolated cfDNA was constructed into a whole genome library followed by shallow whole genome sequencing for copy number profiling. As before, SCNAs were considered to be present at 20% deflection from a baseline human genome, consistent with previously established liquid biopsy analyses (14,24,25).

Clinical demographics

To assess the relationship between the SCNAs present in AH samples and clinical features of retinoblastoma, we compiled a data set with clinical characteristics of participants, germline RB1 status (determined from peripheral blood cells as part of the routine retinoblastoma work-up; the results of this clinical test were recorded, it is not part of our research platform), SCNA profiles of AH samples, and matched tumor characteristics for enucleated eyes. Retrospective chart review was performed to determine the following clinical features: age at diagnosis, sex, laterality, IIRC group and TNM staging (26,27), seeding morphology, therapy, clinical outcomes, histopathology when available and follow-up times for all participants.

Statistical analyses

Fisher’s exact test was used to evaluate associations between RB SCNAs and clinical factors or outcomes. Kaplan-Meier survival analyses with log-rank tests were performed for comparisons of eye salvage in treated eyes based on the presence of RB SCNAs. Single variable analyses were performed using logistic regression, followed by the creation of a multivariable model wherein age and the presence of other RB SCNAs were considered covariates; Hosmer-Lemeshow test was used to evaluate the fit of this multivariable model. To compare median amplitudes of 6p gain in enucleated versus salvaged eyes, Mann Whitney U test was used (14). A Cox proportional hazard model was used to estimate hazard ratios of RB SCNAs. JMP Pro 13 (SAS Institute, Inc.) and Stata/SE 14.2 (StataCorp) were used for statistical analyses. We did not assess internal validation by split-sample or cross-validation due to the inherently small sample size of this rare disease.

RESULTS

Clinical characteristics of participants

Forty-six patients were included in the study; 4 patients with bilateral disease had both eyes sampled, so a total of 50 eyes were included in the analysis. For the 26 patients that were included in the previous publication and followed for 2 additional years, case numbers (1–26) remained consistent with the prior study for comparison purposes (14). No patients who entered the study dropped out or withdrew consent over the study period. Demographics and clinical features of all participants are summarized in Supplementary Table S1. The median age at diagnosis for all patients was 13 months. Enucleated eyes tended to be diagnosed at an older age than salvaged eyes, although this difference was not statistically significant (enucleated median 19 months, range 0 to 59 months; salvaged median 9 months, range 2 to 45 months; P = 0.09). Of all eyes, the most commonly diagnosed IIRC group was D (35/50, 70%) and the most common TNM stage was cT2b (36/50, 72%), although less advanced (Groups B and C) eyes were also present (see Supplementary Table S1). Based on the International Retinoblastoma Staging System (28), all salvaged (i.e., non-enucleated) eyes were stage 0 (intraocular disease only), and all enucleated eyes were stage I (intraocular disease only).

Eye-sparing treatment modalities were non-randomized and included the following: 3-drug intravenous chemotherapy (CEV) (21,22), intra-arterial chemotherapy, intravitreal chemotherapy for seeding (IVM) (22,29), laser consolidation, and cryotherapy. No patients were treated with external beam radiation therapy or plaque brachytherapy. Twenty-seven eyes were salvaged with one or more of the above treatments during the study period. Twenty-three eyes required enucleation either 1) primarily as initial treatment (n = 8 eyes), 2) secondarily due to tumor recurrence (n = 10 eyes), or 3) secondarily due to tumor persistence (i.e., poor response to initial chemotherapy; n = 5 eyes) (Figure 1). It should be noted that 2 eyes (cases 15 and 25) that were initially considered salvaged in the previous study (14) were both subsequently enucleated after 22 months of conservative treatment due to intraocular retinal recurrence; the recurrence was not associated with the paracentesis site and there was no orbital or metastatic disease for these or any patients in the study. No enucleated patient developed any subsequent tumor spread or recurrence throughout the study period.

Figure 1.

Flow diagram summarizing the treatments given and the presence or absence of 6p gain in each treatment group.

Altogether, clinical follow-up from diagnosis to final evaluation ranged from 6 to 64 months (median, 28.5 months). There was no significant difference in length of follow-up between enucleated (median 24 months, range 6 to 64 months) and salvaged (median 34 months, range 7 to 63 months) eyes (T-test, P = 0.93).

Genomic changes in tumor-derived cfDNA of the AH

Genomic analysis included a total of 116 AH samples, of which 5 (4.3%) were removed for quality control due to poor reads alignment ratio (<2%). Of the remaining 111 AH samples, 91 were extracted at the time of IVM injection, 15 immediately after enucleation (primary or secondary), and 5 at the time of diagnosis in eyes that were treated conservatively for salvage. Ten eyes had a sample taken at some point during treatment and also at the time of enucleation. No participant had adverse effects or complications secondary to AH extraction—including no infection, bleeding, iris trauma, or cataract.

After genomic evaluation, 33 out of 50 sampled eyes (66%) were positive for one or more of the highly-recurrent RB SCNAs (Supplementary Table S1), and 39/50 (78%) were positive for any measurable copy number alteration (CNA). Gain of 6p was the most frequently observed RB SCNA among all 50 eyes (50%), followed by 1q gain (38%), 16q loss (30%), 2p gain (including 3 eyes with focal MYCN gains; 18%), and 13q loss (10%). Of note, all 3 eyes with focal MYCN gains (cases 10, 28, and 31) had no other SCNAs and ultimately required enucleation, and all demonstrated biallelic RB1 mutations on post-enucleation tissue analysis. The frequency of 6p gain in the profiles was associated with 1q gain (Fisher’s exact; P = 0.02), and the presence of 1q gain was also associated with 16q loss (Fisher’s exact; P = <0.0001). 6p gain and 16q loss were not significantly associated; however, there was a trend toward significance (Fisher’s exact; P = 0.062). Age was positively associated with the frequency of SCNAs, as older patients had more SCNAs (P < 0.0001).

RB SCNAs and 6p gain in the AH predict enucleation

When the SCNA profiles for enucleated versus salvaged eyes were compared, the presence of any highly-recurrent RB SCNAs were significantly more common in enucleated eyes (21/23, 91.3%) than in salvaged eyes (12/27, 44.4%; Fisher’s exact, P = 0.0007). Altogether, the presence of any RB SCNAs within the AH portended at least 13-fold greater odds of an eye requiring enucleation (OR = 13.13, 95% CI = 2.56–67.46, P = 0.002).

This association appears to be primarily driven by the presence of 6p gain. Of the RB SCNAs, 6p gain in particular was more prevalent in enucleated eyes (17/23, 73.9%) than in salvaged eyes (8/27, 29.6%; Fisher’s exact, P = 0.004). There were no significant differences in the prevalence of other individual SCNAs (at 1q, 2p, 13q, or 16q) between salvaged and enucleated eyes (Table 1). Given that enucleation after a year of conservative therapy is less likely (21,22), we also performed a more strict evaluation in which only eyes with ≥12 months of follow-up were considered salvaged. Overall, a total of 39 eyes (21 salvaged) had ≥12 months of follow-up in our study. Using this stricter criterion for the salvaged group, 6p gain was still significantly more prevalent in the enucleated group (17/23, 73.9%) than in the modified salvaged group (7/21, 33.3%; Fisher’s exact, P = 0.014).

Table 1.

Frequencies of highly-recurrent retinoblastoma (RB) somatic copy number alterations (SCNAs), including gain of 1q, 2p, and 6p, and loss of 13q and 16q.

	1q	2p	6p	13q	16q	Any RB SCNA	Totals
Salvaged eyes	29.6%	11.1%	29.6%	7.4%	25.9%	44.4%	27
Enucleated eyes	47.8%	21.7%	73.9%	13.0%	34.8%	91.3%	23
P-value	0.2465	0.4442	0.0041	0.6507	0.548	0.0007
IVM during CTx for persistent seeds	57.1%	28.6%	57.1%	14.3%	50.0%	92.9%	14
IVM after CTx for recurrent seeds	23.8%	9.5%	33.3%	14.3%	23.8%	38.1%	21
P-value	0.075	0.1907	0.1871	1	0.1534	0.0015

Abbreviations: CTx, chemotherapy; IVM, intravitreal melphalan; RB, retinoblastoma; SCNA, somatic copy number alteration.

Considered as a single variable, the presence of 6p gain in the AH was associated with a nearly 7-fold increased odds of enucleation (OR = 6.73, 95% CI = 1.94–23.36, P = 0.003); no other RB SCNA demonstrated a statistically significant association. Based on univariable Kaplan-Meier analysis (Figure 2), the risk of enucleation in eyes with 6p gain was 3.8 times greater after one year than in eyes without this chromosomal alteration (82.8% of eyes without 6p gain remain, versus 34.1% of eyes with 6p gain; χ² (1) = 11.90; P = 0.0006). After 3 years, 68.3% of eyes without 6p were salvaged, versus only 27.3% of eyes with 6p gain in the AH. Overall, the hazard of enucleation over time is 4.27 times greater in those patients with 6p gain (95% CI = 1.65–11.07; P = 0.003).

Figure 2.

Kaplan-Meier analysis demonstrating a 3.8-times greater risk of enucleation in eyes with 6p gain compared to eyes without a 6p gain after 1 year.

Overall, enucleated eyes also demonstrated a significantly greater median amplitude of 6p gain compared to salvaged eyes (1.46 in enucleated eyes versus 1.02 in salvaged eyes, P = 0.005; Figure 3).To determine whether greater median amplitudes of 6p gain are more predictive of enucleation, we compared all eyes with median amplitudes at 6p of ≥1.5 to eyes with median amplitudes at 6p of <1.5. Gains of 6p with median amplitudes of ≥1.5 were significantly more prevalent in enucleated eyes (14/23, 60.9%) than in salvaged eyes (4/27, 14.8%; Fisher’s exact, P = 0.001). Considered as a single variable, the presence of 6p gain with a median amplitude of ≥1.5 in the AH was associated with a nearly 9-fold increased odds of enucleation (OR = 8.94, 95% CI = 2.31–34.58, P = 0.001).

Figure 3.

A, Composite somatic copy number alteration (SCNA) profile of cell-free DNA (cfDNA) sampled from the aqueous humor (AH) of enucleated (Enuc., red) and salvaged (Salv., blue) eyes. B, Box plots demonstrating the range of ratio to median amplitudes for SCNAs in enucleated (Enuc.) and salvaged (Salv.) eyes (median, black bar; mean, green bar). The median of the ratio to the median amplitude of chromosome 6p gain is significantly greater in enucleated eyes compared to salvaged eyes (P = 0.005).

Utility of SCNAs in the prediction of enucleation

Previously univariate analyses were done to evaluate the association between RB SCNAs and ocular outcomes (14). With the addition of new patients, samples, and longer overall follow-up, a more accurate model can be constructed. As shown in Table 2, 6p gain is most strongly predictive of enucleation, after controlling for age, sex, IIRC group and the presence of other SCNAs (P = 0.01). Notably, 2p gain (including the 3 patients with focal MYCN gain) is marginally predictive of enucleation in this model (P = 0.08). Evaluated together, this model correctly classifies 74% of patients, with 87% sensitivity for predicting enucleation and 70% specificity for predicting salvage. This model cannot be rejected for issues related to goodness of fit, as evaluated using the Hosmer-Lemeshow test (P = 0.21).

Table 2.

Odds of enucleation given SCNA variations.

SCNA	Odds Ratio	95% CI	P-Value
1q Gain	1.06	0.18 – 7.11	0.90
2p Gain	5.12	0.82 – 32.11	0.08
6p Gain (≥1.15)	9.61	1.62 – 57.07	0.01
13q Loss	1.12	0.14 – 9.22	0.92
16q Loss	0.62	0.07 – 5.27	0.66
Age (Covariate)	1.00	0.93 – 1.06	0.93
Sex (Covariate)	1.22	0.28 – 5.27	0.79
Group (Covariate)	2.10	0.60 – 7.33	0.66

When the criterion for 6p gain is made stricter (≥1.5 times normal values), a trade-off of greater specificity for lower sensitivity emerges. More patients are correctly classified (80%). Further, this model is 78% sensitive and 81% specific for the prediction of enucleation or salvage, respectively. This model also cannot be rejected for fit issues (Hosmer-Lemeshow Goodness-of-Fit P = 0.36).

Other clinical associations with RB SCNAs

RB SCNAs in the AH are associated with persistent tumor seeding

Over the course of the study, 35 eyes required intravitreal melphalan (IVM) injections for seeding; 21 of these eyes received IVM for recurrent seeds (i.e., seeding that resolved with treatment and then subsequently returned) and 14 received IVM for persistent seeds (i.e., seeding that never fully responded to systemic or intra-arterial chemotherapy). RB SCNAs were significantly more prevalent amongst eyes with persistent seeds (13/14, 92.9%) than in eyes with recurrent seeds that initially responded to treatment (8/21, 38.1%; Fisher’s exact, P = 0.001). There was also a trend toward an association between 1q gain and eyes with persistent seeding compared to eyes with recurrent seeding, although this difference was not statistically significant (Fisher’s exact, P = 0.075; Table 1). Each increase in seeding class (from none to dust, from dust to sphere, and from sphere to cloud) was associated with 2-fold increased odds of having a 6p gain present in the AH (P = 0.04).

6p gain is associated with aggressive histopathologic features

Seven of the 23 enucleated eyes (30.4%) had classic higher risk features on histopathologic analysis (30); 6p gain was present in 5 of these eyes (4 eyes had >3 mm of choroidal invasion and 1 eye demonstrated post-laminar optic nerve invasion; see Supplementary Table S1). None of the eyes in this study had massive extrascleral or anterior segment invasion. All of the enucleated tumors with 6p gain had mild to severe nuclear anaplasia ranging from 3% to 60% of the tumor area studied, while the tumors without 6p gain ranged from none to moderate anaplasia. Eighteen of the enucleated eyes had vitreous and/or subretinal tumor seeds present on histopathologic analysis, and 16 of these (88.9%) had 6p gain. The tumors with 6p gain had more necrosis (range 1–70% of the tumor) than the tumors without 6p gain (none – 25% of the tumor). Finally, only cases with 6p gain (8 eyes) had focal invasion of the optic nerve head/lamina cribrosa, choroid (<3 mm), or anterior segment. None of the eyes without 6p gain had focal invasion of any ocular structures. MYCN amplification was seen in 3 AH samples; 2 of these associated tumors were poorly differentiated with scant rosettes, and one had massive (7 mm) choroidal invasion. No specific nuclear features (round bland nuclei with nucleoli and no neuroendocrine-type chromatin) typically associated with MYCN amplification in wild-type (18) retinoblastoma were seen in these tumors.

Minimum region of 6p gain in the AH

We evaluated the boundaries of 6p gain in all AH samples in order to determine the minimum region of gain (MRG) for this alteration. 6p gain was seen in 50 AH samples from 25 eyes. Of these, 35 (70%) demonstrated gain of the entire 6p arm of the chromosome, consistent with an isochromosome 6p. In a single sample (case 11), the MRG was localized to a 19 Mbp region of the distal 6p arm that included DEK but not E2F3. This 19 Mbp gain was present in only 1 of the 50 AH samples; in all other samples, DEK and E2F3 were both included within the regions of 6p gain regardless of the specific lengths of the gains (Figure 4).

Figure 4.

Chromosome 6 heatmap showing the lengths of 6p gain (red) in 25 eyes with retinoblastoma. Most eyes demonstrated a gain of the entire 6p arm. The minimum region of gain (MRG) was localized to a 19 Mbp region that included a gain of DEK (Case 11).

DISCUSSION

Herein, we present an analysis of retinoblastoma tumor-derived DNA from 111 separate AH samples that were extracted from 50 eyes either at the time of diagnosis, post-diagnosis but prior to IVM therapy, and/or after primary or secondary enucleation. The goal of this study is to refine our understanding of molecular biomarkers in the AH and identify clinically impactful targets for prognostication of tumor response to therapy and ocular survival. Although we previously identified chromosomal gain of 6p as a potential indicator of aggressive retinoblastoma, the study included a smaller cohort of patients and limited follow-up period (14). Now with extended follow-up and nearly double the number of eyes and AH samples, the data continue to demonstrate that 1) AH sampling is safe (i.e., the AH extraction procedure caused no complications in any of our study participants), 2) AH is a reliable source of tumor cfDNA and 3) the presence of RB-specific SCNAs—specifically gain of 6p—has the potential to serve as a prognostic biomarker for retinoblastoma. Historically, clinicians have been very limited in their ability to provide targeted prognostic information to parents of children with retinoblastoma, given the lack of eye-specific molecular biomarkers due to the contraindication to direct tumor biopsy (2–8).

With a larger cohort of patients and increased follow-up we have demonstrated additional findings that are clinically impactful. First, we have demonstrated that the AH harbors detectable levels of tumor-derived cfDNA not only in advanced (Groups D and E) eyes but also in less advanced (Groups B and C) eyes. While the majority of sampled eyes in our study were relatively advanced with seeding (because most AH samples were extracted during IVM treatment for intravitreal seeding), the presence of cfDNA in less advanced eyes suggests that advanced intraocular disease is not required for meaningful AH cfDNA analyses. This is an important factor to consider so that these findings can be applied to all patients regardless of extent of intraocular disease.

In a prior analysis, we demonstrated a high concordance between SCNAs in tumor tissue and in the AH for 13 eyes (please see Figure 2 from Berry et al 2018) (14). Now we continue to show with nearly double the number of total AH samples that AH cfDNA can be used to reliably generate profiles of SCNAs that are consistent with the expected genomic landscape of retinoblastoma tumors. Additionally, a recent study from an independent group demonstrated the feasibility of evaluating tumor DNA in the AH in vivo to identify tumor RB1 mutations (15). In our study, the majority (33/50 eyes; 66%) of our sampled eyes contained at least one of the highly-recurrent RB SCNAs, and these alterations were particularly prevalent in enucleated eyes compared to salvaged eyes (P = 0.0007). RB SCNAs have been hypothesized to confer a growth advantage to cells by activating oncogenes involved in tumorigenesis and/or inactivating tumor suppressor genes that would normally protect tissues from dysregulated proliferation (31,32). These genomic alterations are widely accepted as crucial events in the progression of retinoblastoma (13,14,16,17,31–34). However, it is important to note that the exact molecular roles and whether there is a stepwise progression of SCNAs in RB development are not yet known. Additionally, 17/50 (34%) eyes in our study displayed SCNAs but none of the highly recurrent ones associated with retinoblastoma, and 11/50 (22%) had a complete absence of any SCNAs throughout the genome. Similar to tissue-based studies, these findings suggest that although SCNAs seem to influence the progression or advancement of retinoblastoma, they are not required for tumor formation (31). It should be noted that in the 22% of eyes without any SCNAs, the lack of detectable alterations in AH could be 1) due to a true absence of SCNAs in the tumor or 2) theoretically confounded by a lower tumor burden leading to lower tumor-derived cfDNA levels (i.e., tumor fraction) in the AH. Research is ongoing to develop a reliable method of tumor fraction estimation for the AH, in addition to the SCNA analyses that we currently employ.

Although we have demonstrated a significant correlation between 6p gain and increased risk of enucleation, the specific molecular pathways that influence this relationship are uncertain. The ultimate remaining question is what genes and/or corresponding molecular processes are involved to make 6p gain a viable biomarker for retinoblastoma in the first place. Previous studies have mapped the common region of 6p gain to band 6p22, a central region of the short arm of chromosome 6 that contains numerous known genes (16,31,32,35). Of these, the oncogenes DEK and E2F3 have been identified as possible drivers of retinoblastoma, as they demonstrate both RNA and protein overexpression in the setting of 6p gains and promote abnormal cell proliferation when overexpressed (31,32,34). Using cfDNA from AH samples, our study localized the minimum region of 6p gain to a 19 Mbp region of the distal 6p arm, within the much larger MRG identified in previous studies (31,32,35). We also found that E2F3 and DEK are almost always included within the region of 6p gain, regardless of the width of the alteration. While DEK and E2F3 remain promising retinoblastoma candidate genes, 6p gains are highly non-focal in nature and can cover tens to hundreds of different genes in addition to DEK and E2F3—making it difficult to distinguish between true driver genes in this region versus passenger genes that are not affecting cancer development (16,17,31). We and others have shown that 6p gains often span the entire region of 6p—consistent with the formation of an isochromosome, in which misdivision at the centromere leads to abnormal gain of the entire 6p arm (32). This isochromosome is the most common underlying cause of 6p gain in retinoblastoma and is rarely seen in other ocular malignancies (32). From our research and the body of research on RB SCNAs (31,34,35), it remains unclear whether a particular gene (or genes) on 6p is driving aggressive tumor activity, or whether 6p gain is simply a measurable molecular marker of other underlying processes within the tumor genome. In conjunction with AH sampling, expression analyses as well as functional assays with cultured RB cell lines may be useful for further elucidation of potential 6p candidate genes and their possible roles in retinoblastoma progression (31,36).

While the localization of genomic changes is crucial for identifying potential driver genes involved, elucidating the relative sequence of these changes would be a useful step in better understanding retinoblastoma progression. Prior to the development of AH sampling for retinoblastoma, Bowles et al (33) proposed a molecular model for the progression of SCNA events using frequency studies of chromosomal gains and losses in enucleated eyes. Based on the assumption that more prevalent SCNAs likely occur earlier in tumor development, they inferred that 1q gain (which was the most common SCNA in their study and was closely associated with 6p gain) was one of the earliest genomic events in the progression of retinoblastoma, followed subsequently by gain of 6p. While we, too, demonstrated a significant association between 1q and 6p gains in sampled eyes, 6p gain (rather than 1q gain) was by far the most common SCNA both in our study and in others’ (14,16,17,31,35). Interestingly, loss of 16q was also associated with a 1q gain—consistent with studies of breast carcinoma that have identified simultaneous 1q gains and 16q losses as early tumor changes (37). Unlike the Bowles et al model, our findings suggest that 6p gain may be a relatively earlier event than 1q gain given its predominance in sampled eyes. While the exact chronology of SCNAs in retinoblastoma is still poorly understood, our novel liquid biopsy provides the first approach in which the relative frequencies of chromosomal changes could be studied in both enucleated and salvaged eyes.

Because a specific prognostic marker for retinoblastoma in the AH could change how clinicians and parents make decisions regarding retinoblastoma management, our interest was especially focused on the somatic gain of chromosome 6p and its potential predictive value for tumor outcomes. As demonstrated previously (14), we found that 6p gain is not only the most common chromosomal alteration in the AH (present in 50% of all sampled eyes); but it is also the only individual SCNA that is significantly more prevalent in enucleated eyes compared to salvaged eyes (P = 0.004). With both uni- and multivariate analyses we demonstrated that the presence of 6p gain in the AH portends between a 7- and 10-fold increased odds of enucleation (univariate OR = 6.73, 95% CI = 1.94–23.36, P = 0.003, multivariate analyses controlling for age and the presence of other SCNAs OR = 9.87, 95% CI = 1.75 – 55.65, P = 0.009). These odds ratios are highly clinically impactful; as a point of reference, the OR for developing breast cancer in the setting of a positive BRCA mutation is 5.91 (CI = 5.25 – 6.67) (38). While our findings strongly suggest that 6p gain—when present—is a reliable predictive biomarker of ocular survival, we emphasize that the majority of AH samples with 6p gain were extracted either during active IVM treatment or immediately following enucleation. Therefore, many of the eyes were already at fairly advanced stages of disease at the time a 6p gain was discovered. While we have now started to collect AH at diagnosis (before any treatment is initiated) under a novel IRB-approved research protocol, a larger prospective study of AH evaluation at diagnosis is required in order to elucidate whether 6p gain is present and prognostic at the time of initial diagnosis and not just later in the disease process.

In addition to the correlation between RB SCNAs and enucleated eyes, we also found that 6p gain was associated with other features of retinoblastoma that are clinically associated with aggressive disease. On histopathologic analysis, 6p gain was associated with aggressive and high-risk findings, including higher levels of necrosis and focal invasion of intraocular structures. 6p gain was also significantly more prevalent in eyes with persistent tumor seeding that did not respond to therapy (Fisher’s exact, P = 0.001). Intraocular seeding of tumor cells is a well-known clinical indicator of advanced disease and a common cause of treatment failure and loss of the eye, even with targeted intravitreal chemotherapy (23,39). In separate studies, Berry and others found that seeds with a cloud-like morphology were particularly difficult to eradicate due to suboptimal response to IVM injections, compared to eyes with seeding spheres or dust (39–41). In our study, we found that an eye with cloud type seeding was 2 times more likely to have gain of 6p than eyes with dust or sphere morphology of seeding, or no seeding (OR = 2.09, 95% CI = 1.02–4.31, P = 0.04). The association between 6p gain and more aggressive tumor activity is not unique to retinoblastoma. Gains at 6p have been associated with advanced and even metastatic disease in multiple non-retinal cancers, including colorectal, hepatocellular, bladder, and ovarian carcinomas; although the exact molecular influence of 6p gain on these malignancies is not entirely understood (32,42–45). While a better understanding of the specific mechanism for this biomarker in retinoblastoma would be ideal, it does not diminish the value of 6p as a reliable indicator of aggressive disease.

Future studies are ongoing to explore the possibility of additional biomarkers, besides 6p gain, within the AH of retinoblastoma eyes. A possible indicator of aggressive retinoblastoma that deserves further investigation is the focal MYCN gain. Numerous studies have identified primary focal MYCN amplifications (i.e., in patients without any RB1 mutations) as being independent drivers of high-risk disease (18,46). However, focal MYCN gains can also occur secondarily (i.e., in patients with known RB1 mutations) (47). In our study, we identified a total of three 2p gains that were confined to the MYCN region (case 10 was reported previously (14), cases 28 and 31 were recruited since the previous publication). Cases 10 and 28 were both positive for blood RB1 mutations and all 3 cases demonstrated biallelic RB1 mutations in tumor tissue, suggesting that their MYCN gains were secondary in nature rather than the primary MYCN-amplified subtype of retinoblastoma that was previously described (18,46). In all 3 of these cases, focal MYCN gain was the only SCNA present in tumor-derived AH cfDNA, and all 3 eyes ultimately required enucleation due to either severity, persistence, and/or recurrence of disease. This is consistent with studies in pediatric neuroblastoma that found an association between MYCN overexpression and more aggressive disease (48,49). Given that 2p alterations are relatively rare in retinoblastoma—and focal MYCN gains are rarer still—we encourage larger-scale studies of this chromosomal site within AH cfDNA in the future.

The novel use of AH as a liquid biopsy is a promising yet restricted approach to retinoblastoma evaluation. At this time, there is no accepted clinical protocol for using genomic information obtained from the AH to influence diagnostic or treatment decision-making. However, the ability to sample AH in vivo and detect biomarkers like 6p gain at diagnosis and throughout the course of a patients’ disease—including at the end of conservative therapy or in the setting of a clinical recurrence—allows for the possibility of an improved therapeutic approach in the future. In this study, we established 6p as a viable prognostic biomarker of retinoblastoma tumors. A prospective, multicenter study with AH sampled at diagnosis and longitudinally throughout therapy is warranted to determine whether AH analysis can become a standard component of retinoblastoma prognostication and management.

Implications:

AH is a high-yield source of tumor-derived DNA in RB eyes. Detection of 6p gain in the AH allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with AH sampled from all eyes at diagnosis are warranted to validate these findings.