Background Current melphalan-based intravitreal regimens for retinoblastoma (RB) vitreous seeds cause retinal toxicity. We assessed the efficacy and toxicity of topotecan monotherapy compared with melphalan in our rabbit model and patient cohort.
Methods Rabbit experiments: empiric pharmacokinetics were determined following topotecan injection. For topotecan (15 μg or 30 µg), melphalan (12.5 µg) or saline, toxicity was evaluated by serial electroretinography (ERG) and histopathology, and efficacy against vitreous seed xenografts was measured by tumour cell reduction and apoptosis induction. Patients: retrospective cohort study of 235 patients receiving 990 intravitreal injections of topotecan or melphalan.
Results Intravitreal topotecan 30 µg (equals 60 µg in humans) achieved the IC90 across the rabbit vitreous. Three weekly topotecan injections (either 15 µg or 30 µg) caused no retinal toxicity in rabbits, whereas melphalan 12.5 µg (equals 25 µg in humans) reduced ERG amplitudes 42%–79%. Intravitreal topotecan 15 µg was equally effective to melphalan to treat WERI-Rb1 cell xenografts in rabbits (96% reduction for topotecan vs saline (p=0.004), 88% reduction for melphalan vs saline (p=0.004), topotecan vs melphalan, p=0.15). In our clinical study, patients received 881 monotherapy injections (48 topotecan, 833 melphalan). Patients receiving 20 µg or 30 µg topotecan demonstrated no significant ERG reductions; melphalan caused ERG reductions of 7.6 μV for every injection of 25 µg (p=0.03) or 30 µg (p<0.001). Most patients treated with intravitreal topotecan also received intravitreal melphalan at some point during their treatment course. Among those eyes treated exclusively with topotecan monotherapy, all eyes were salvaged.
Conclusions Taken together, these experiments suggest that intravitreal topotecan monotherapy for the treatment of RB vitreous seeds is non-toxic and effective.
- animal models
- intravitreal chemotherapy
- vitreous seeds
Data availability statement
Data are available upon reasonable request. For laboratory data, contact email@example.com. For deidentified patient data, contact firstname.lastname@example.org and email@example.com.
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- animal models
- intravitreal chemotherapy
- vitreous seeds
Vitreous seeds have historically been the most difficult-to-treat aspect of intraocular retinoblastoma (RB).1 2 RB tumours with vitreous seeds are those least likely to be salvaged with radiation3 or intravenous chemotherapy.1 2 4 ,5 Newer approaches to delivering chemotherapy, including intra-arterial chemotherapy (IAC)6 and direct intravitreal injection of chemotherapy,7 8 have partially overcome the treatment-resistant nature of vitreous seeds and have improved globe salvage rates for RB.9 10 However, the primary chemotherapeutic agent used in both IAC and intravitreal chemotherapy is melphalan, which has been associated with retinal toxicity.8 11–13 Thus, while intravitreal melphalan may be effective, retinal functional loss is common.12 14 Furthermore, the toxicity is dose-dependent15 and worsens with each subsequent melphalan injection delivered.12 13 16
Recently, topotecan has been explored as an alternative chemotherapy agent, both by intravenous,17 intra-arterial18 and intravitreal19 20 routes. Preliminary laboratory and clinical evidence suggests that topotecan may be less toxic than current standard-of-care melphalan.20 21 However, it is unclear if a non-toxic dose of topotecan is clinically effective.21 Further, it is unclear just how effective topotecan is as monotherapy, as many centres have generally used it in combination with melphalan,22 or have been quick to re-add melphalan back into the regimen if topotecan monotherapy appeared to not achieve adequate tumour control.20 Likewise, the optimal dose of topotecan that best balances efficacy with toxicity as intravitreal monotherapy has not been established.
We recently developed a rabbit xenograft model of RB with vitreous seeds and retinal tumours, which we have used to study the toxicity of IAC, exploring various different IAC drugs.23 24 We have also previously described a complete platform to assess functional and structural retinal toxicity associated with local delivery of various chemotherapeutic agents.11 Here, we use this rabbit model23 and this toxicity evaluation platform,11 to determine the dose of intravitreal topotecan which is effective and non-toxic when delivered as monotherapy. We then corroborate this evidence of non-toxicity with our clinical experience treating RB patients with vitreous seeds with intravitreal topotecan.
Statement of research ethics
All animal experiments adhered to the Association for Research in Vision and Ophthalmology Statement on Animal Use and were performed under the auspices of the Vanderbilt Institutional Animal Care and Use Committee.
Intravitreal topotecan pharmacokinetics
New Zealand white rabbits (2.8–3.0 kg) were used for all studies. For pharmacokinetic experiments, a 20-gauge valved vitrectomy cannula was inserted 2–3 mm behind the limbus. One microgram topotecan hydrochloride was injected on the opposite side 2–3 mm behind the limbus into the vitreous cavity. Serial vitreous taps were performed through the valved cannula at 30 min, 1 hour, 2 hours, 4 hours and 6 hours. Use of a valved cannula-maintained eye stability and prevented efflux of vitreous contents during manipulations.25 Vitreous samples were immediately placed on dry ice and then stored at −80°C until drug levels were measured.
Vitreous samples were thawed, an internal carbamazepine standard was added and samples were diluted with blank plasma, then deproteinised with acetonitrile. Samples were analysed on a Thermo Scientific TSQ Quantum Ultra mass spectrometer interfaced to a Waters Acquity UPLC system, using methodology we have reported previously.23
Topotecan concentrations were averaged across rabbits at each time point. The resulting mean time-concentration data from each matrix were analysed via non-compartmental analysis (Phoenix WinNonlin V.6.4, Pharsight/Certara USA, Princeton, New Jersey, USA) to determine pharmacokinetic parameters, including half-life.
In vitro determination of dosing
Human WERI-Rb1 RB cells (5×103) were plated in 96-well plates in the presence of various concentrations of topotecan for 16 hours (five half-lives as determined through the above pharmacokinetic experiments). Topotecan-containing media was then removed, and fresh media added. After 7 days, the CellTiter Blue assay (Promega, Madison, Wisconsin, USA) was used to count live cells. Survival curves were graphed with GraphPad, and the IC90 was calculated.
Using the pharmacokinetic parameters determined above, we calculated25 the dose of topotecan that would need to be injected into the eye to achieve the IC90 in the vitreous on the opposite side of the eye for a duration of five half-lives.
Assessment of efficacy of intravitreal topotecan for vitreous seeds in rabbits
Figure 1A depicts our experimental design. RB vitreous seeds were created by intravitreal injection of 1 000 000 WERI-Rb1 cells in 100 µL saline in both eyes of cyclosporine-immunosuppressed rabbits, as we have described previously.23 ,25 After 2 weeks of growth, the right eyes received three weekly injections of either 15 µg/100 µL topotecan or 12.5 µg/100 µL melphalan, while all left eyes received 100 µL saline.
Two weeks after the final injection, all rabbits were euthanised, and the eyes were removed. For five rabbits, the vitreous of each eye was harvested and digested in 0.5 mg/mL hyaluronidase and 1 mg/mL collagenase overnight at 37°C. Live cells were counted by direct microscopy using trypan blue stain. In four additional rabbits from each treatment group, the entire eyes were submitted for histopathology (two rabbits after receiving three injections and two rabbits after receiving a single injection).
Assessment of ocular toxicity of intravitreal topotecan in rabbits
Four cohorts (n=4–6 rabbits/cohort) received either topotecan 30 µg (the calculated IC90), topotecan 15 µg (half the calculated IC90), saline (control) or melphalan 12.5 µg (current standard-of-care).26 In all rabbits within a given cohort, the right eyes received three injections, one injection per week, of the same drug/dose. Figure 1B depicts our experimental design. Electroretinography (ERG; OcuScience, Henderson, Nevada, USA) was performed according to the modified International Standard for Clinical Electrophysiology of Vision protocol for rabbits.27 Intravitreal injections were performed weekly, and always within 1 day following testing. After euthanasia, eyes were harvested and fixed in Davidson’s solution.
Toxicity was defined for every ERG parameter, using our previously published definition.11 ,25 Briefly, toxicity was deemed significant for a given dose in a rabbit group if there was a 25% reduction in average ERG amplitude, or a 25% prolongation of average implicit time comparing the post-treatment parameter values after three injections with the pretreatment values, if the difference was statistically significant.
Ocular toxicity and efficacy of intravitreal topotecan versus melphalan in patients
Medical records of all patients treated with intravitreal injections at Memorial Sloan-Kettering Cancer Center and Vanderbilt University Medical Center were reviewed. Patients receiving intravitreal topotecan were identified. A second cohort of all patients receiving melphalan as monotherapy were included as a comparator group. Injection number, drug and dose were recorded. ERGs were performed using a previously published, and validated, abbreviated ERG protocol.28–30 For efficacy, we included all treated patients for which complete records were available. Our toxicity analyses included only those monotherapy injections for which this ERG protocol was performed prior to the intravitreal injection, as well as subsequent to the injection, and patients who also received concomitant IAC between the two ERGs were excluded. Ocular and systemic adverse events, as well as clinical outcomes, were recorded.
Statistical analyses of rabbit and human efficacy and ERG data
For univariate analysis to compare toxicity in patients, the Wilcoxon signed-rank test was used. For multivariable analysis, to evaluate the toxicity of each drug at different dosages, a linear mixed-effects model was fitted with treatment groups and the repeated measurements (pre or post) for each parameter and each test. Using model-based (least-square) means, the average adjusted change from pretreatment versus post-treatment and the difference in change between different treatment groups (difference-of-differences) were estimated and compared with the Wald test. Our predefined definition of toxicity (see earlier) was used in the rabbit analyses. Data were transformed to better meet normality assumptions and adjusted for heteroscedasticity when necessary. To account for multiple comparisons, Bonferroni-adjusted p values were reported (two-tailed), with adjusted p values less than 0.05 considered statistically significant. The analyses were performed using R V.3.6.3 including packages ‘nlme’ and ‘emmeans’. For experiments in rabbits, ‘pre’ and ‘post’ were defined before and after the three injections. For human experiments, because of the variability between dosing and interval of injections, ‘pre’ and ‘post’ were defined on a ‘per injection’ basis without minimum reduction limits, and inter-eye/intra-patient and intra-eye correlations were taken into account in our modelling.
For efficacy experiments in rabbits comparing paired right eyes receiving topotecan (or melphalan) and contralateral left eyes receiving saline, the paired t-test was used. Relative reduction of cell counts was analysed to compare the difference between two independents groups (topotecan and melphalan rabbit cohorts) using Welch two sample t-test.
In vivo topotecan pharmacokinetics and in vitro determination of expected effective in vivo dose
Peak concentration at the opposite side of the eye was achieved at 2 hours postinjection (figure 2A). The average Cmax at the opposite side of the eye was 0.31 μmol/L. However, the theoretical Cmax (calculated as 1 µg/1.4 mL rabbit vitreous volume) is 1.56 μmol/L. Therefore, compared with the theoretical Cmax, the actual empiric Cmax at the opposite side of the eye achieved 2 hours after injection was ~20% of expected, likely due to rapid efflux during this period of slow diffusion of topotecan across the vitreous. The half-life of topotecan in the rabbit eye was 3.27 hours. We therefore exposed WERI-Rb1 human RB cells to various doses of topotecan for five vitreous half-lives (~16 hours total) and measured live cells 7 days later. The IC90 was 300 nM (figure 2B). We calculated that we would need to inject 30 µg topotecan to sustain this IC90 concentration for 16 hours (five half-lives) at the opposite side of the rabbit eye.
Relative efficacy of intravitreal topotecan versus melphalan to treat RB vitreous seeds in vivo in rabbits
Three weekly injections of 15 µg topotecan killed 96% of vitreous seed tumour cells, compared with saline-treated contralateral eyes (p=0.004, figure 3). Three weekly injections of 12.5 µg melphalan (corresponding to the clinically used dose of 25 µg in patients) killed 88% of cells, compared with saline-treated contralateral eyes (p=0.004; topotecan vs melphalan: p=0.15, figure 3). For additional rabbits in each cohort, the entire eyes were harvested and submitted for histopathology. Residual RB cells in the topotecan-treated eyes were TUNEL positive, suggesting that the ~4% of ‘remaining’ cells counted in the vitreous seed quantitation assay were likely in the process of dying as well.
Toxicity of various doses of intravitreal topotecan compared with melphalan in rabbits
While there was no worsening of ERG parameters in the saline control group, melphalan caused significant worsening of almost all ERG parameters, with reductions in ERG amplitudes between 42% and 79% (figure 4A). These ERG changes occurred in every rabbit within the cohort, with a median of 9 (IQR: 7–9, out of 18) parameters affected per rabbit. Similarly, implicit times were prolonged. Histopathology demonstrated severe atrophy affecting all retinal layers, worst near the injection sites (figure 4B–D).
In contrast, rabbits in the 15 µg and 30 µg topotecan cohorts did not experience any statistically or clinically meaningful worsening of ERG parameters (figure 4A). Even at twice the clinically effective dose, multiple repeated weekly intravitreal topotecan injections did not cause retinal toxicity. No other signs of toxicity were observed on clinical examination, and histopathology showed none of the retinal damage that was seen in the melphalan-treated groups, with retinas of eyes treated with topotecan being histologically indistinguishable from saline-treated control eyes (figure 4B–F).
Comparative toxicity in patients receiving intravitreal topotecan at various doses compared with melphalan
In 41 patients, 108 intravitreal injections of topotecan were given to 42 eyes. Of these 108 injections, 48 injections consisted of topotecan as intravitreal monotherapy, at dosages of either 30 µg (18 injections), 20 µg (29 injections) or 10 µg (one injection). In general, the lower dose of 20 µg was used until ~2017, and 30 µg was used beginning in mid-2017, when it was felt that the efficacy-versus-toxicity balance warranted an increase in dose (the single treatment with 10 µg was given in 2014). Preinjection and postinjection ERG data were available for 40 topotecan injections. Nine ‘undetectable’ pretreatment ERGs (<5 μV) were excluded. Six additional injections were excluded because they were still receiving concomitant IAC. Ultimately, this left 25 topotecan monotherapy injections (from 14 eyes) with evaluable ERGs for analysis (11 at 20 µg and 14 at 30 µg).
In the comparator group, 882 intravitreal melphalan injections were given to 210 eyes of 194 patients. Of these, 833 injections (205 eyes) consisted of melphalan as intravitreal monotherapy (99 injections at 25 µg, 732 injections at 30 µg, 2 injections at 40 µg). Preinjection and postinjection ERG data using the previously described and validated28–30 abbreviated clinical protocol were available for 384 injections. Injections were excluded if they were still receiving concomitant IAC, if pretreatment ERGs were ‘undetectable’ (<5 μV), or if multiple injections occurred between the preinjection and postinjection ERGs. Thus, the final group included for analysis of toxicity and ERGs included 225 intravitreal melphalan monotherapy injections (66 at 25 µg, 159 at 30 µg).
In a univariate analysis, patients receiving melphalan experienced a 7.29 μV reduction in ERG amplitude, per injection (p<0.001), with no significant difference in the amount of reduction between those receiving 25 µg or 30 µg of melphalan. In contrast, patients receiving topotecan experienced no reduction in ERG amplitude, in either the 20 µg subcohort or the 30 µg subcohort (figure 5).
In a mixed effect model, patients receiving melphalan experienced a 7.55 μV reduction in ERG amplitude, per injection (p<0.001), consisting of 7.58 μV reduction per 25 µg injection (p=0.03), and 7.57μV reduction per 30 µg injection (p<0.001). In contrast, in the mixed effect models, patients receiving topotecan at either 20 µg or 30 µg experienced no reduction in ERG amplitude (figure 5).
Efficacy of intravitreal topotecan compared with melphalan in patients
There were 23 patients (23 eyes) who were treated with intravitreal topotecan for whom a complete clinical course was available for review (follow-up: 23.5±18.8 months). Of these six, five were treated with 30 µg (all after October 2017), and one was treated with 20 µg. The comparator group consisted of 66 patients (70 eyes) whose entire intravitreal treatment course consisted solely of melphalan monotherapy, receiving a mean of 4.0±2.4 injections (follow-up: 30.9±26.0 months). In this melphalan monotherapy group, seed eradication and globe salvage was achieved in 65/70 (92.9%) of eyes. It is difficult to evaluate the true efficacy of topotecan in this cohort as the majority of patients who received intravitreal topotecan also received intravitreal melphalan at some point during the course of treatment, and so we cannot definitively attribute the seed eradication to the topotecan in those cases. Only six patients received topotecan monotherapy exclusively throughout their intravitreal treatment course, and while the vitreous seeds were successfully eradicated in all of these patients (having received a mean of 1.8±0.75 injections), it is possible that there might have been selection bias whereby the patients with the least significant vitreous tumour burden were most likely to receive only topotecan. Future randomised studies are needed to evaluate the relative efficacy of topotecan versus melphalan.
To assess the efficacy, toxicity and optimal therapeutic dose of intravitreal topotecan monotherapy for vitreous seeds, we performed several in vitro experiments, in vivo experiments in our rabbit model, and we report our clinical experience using intravitreal topotecan in RB patients. Our pharmacokinetic experiments and in vitro experiments calculated an optimal dose range of 15–30 µg in rabbits (equivalent to 30–60 µg in the larger human eye). Our in vivo efficacy experiments in our rabbit xenograft model demonstrate that 15 µg topotecan is highly effective at eradicating vitreous seeds, with efficacy equivalent to standard dose melphalan. Our in vivo toxicity experiments in rabbits demonstrate that multiple injections of topotecan, even up to 30 µg, do not cause retinal functional or structural toxicity, in contrast to melphalan. Finally, in the clinical study, our experience with intravitreal topotecan monotherapy confirms that 30 µg in humans (equivalent to 15 µg in the smaller rabbit eye) does not cause retinal toxicity in patients.
Evidence from animal models11 12 31 32 and clinical experience,8 12 33 34 suggests that currently used melphalan may be associated with retinal toxicity. Topotecan has been proposed as an alternative agent with efficacy against RB, and it has been incorporated into chemotherapy regimens by intravenous,17 intra-arterial18 and recently intravitreal routes.19 20 However, topotecan has always been used in combination with other drugs for intravenous17 35 and intra-arterial regimens.9 18 36 When topotecan was initially explored for intravitreal use, it was likewise combined with melphalan in an effort to increase efficacy in recalcitrant eyes,22 but Nadelmann et al have shown that combining topotecan with melphalan still causes the expected toxicity from melphalan.20 Recently, single-agent topotecan has been proposed for vitreous seeds.19 While some have reported good results, there is much variability in the doses used and many reports still ultimately include melphalan in combination, presumably because of a perceived lack of adequate efficacy at the doses selected for topotecan. A previous evaluation21 of the toxicity of intravitreal topotecan in an animal model selected a 5 µg dose (equivalent to 10 µg in humans), far less than the doses currently used in clinical practice, six-times less than the dose that we calculate to achieve the IC90, and three-times less than the dose we demonstrate to be clinically effective. Importantly, we demonstrate no toxicity with 15 µg or even 30 µg of intravitreal topotecan.
We took an evidence-based approach, rather than an exploratory trial-and-error approach, to determine the ideal dose of topotecan to study. Since each individual injection can only be given at a single location within the globe, while seeds are often diffuse throughout the vitreous cavity, the goal was to identify the concentration required at the farthest-most side of the vitreous to eradicate vitreous seeds at this farthest location. There are different factors to consider, including the rate of diffusion and the rate of efflux. The amount of drug present at the opposite side of the eye at various time-points following injection was therefore determined empirically. In vitro cytotoxicity experiments were then performed to determine the minimum concentration necessary at that location based on the pharmacokinetic parameters found in vivo, and we then calculated the initial dose that would have been required to be injected to achieve the desired concentration for a sustained length of time (five half-lives) at that farthest point. This systematic approach to dose-finding is superior to selecting several doses in a more random fashion. Since this approach identifies the required concentration at the end of five-half-lives, and at the farthest location in the eye with the lowest exposure levels, this likely represents a high-end estimate—most of the vitreous cavity is exposed to higher concentrations, and indeed at earlier time points the concentration is higher at all locations than it is at the end of the fifth half-life. In addition, this calculated effective dose assumes a single injection, whereas in practice, one would always give multiple injections. Therefore, we also explored half the calculated dose (15 µg instead of the full 30 µg). Since 15 µg was shown in our rabbit model to be as effective as current melphalan doses, we then explored toxicity at the full 30 µg in our rabbit model as well, and we demonstrate that there is a wide therapeutic window with topotecan.
Similarly, the ERG data of topotecan-treated patients corroborated our rabbit findings that doses of 20 µg or even 30 µg did not cause retinal toxicity or ERG reductions. In contrast, in our mixed effect model, melphalan caused a per-injection reduction in retinal function equivalent to 7.55 μV for every melphalan injection, consistent with previous publications by our group.12 37 38 There are two commonly used formulations of melphalan: (traditional) melphalan hydrochloride and captisol-stabilised propylene glycol-free melphalan. In the rabbit experiments, all rabbits were treated with traditional melphalan hydrochloride. In the patient cohort, patients treated up until 2015 (at VUMC) and up until 2016 (at MSKCC) were treated with melphalan hydrochloride, while all patients treated after those dates were treated with the newer propylene glycol-free formulation. We have previously shown that the efficacy and the toxicity of both formulations do not differ.38 However, it should be pointed out that not all eyes receiving melphalan will necessarily experience worsened visual function. As seen in figure 5, while there was a reduction in ERG amplitudes on average, some eyes experienced little or no ERG reductions. It should also be pointed out that macular toxicity (including cystoid macular oedema), which has been reported to occur occasionally with intravitreal injections,39 might not result in measurable reduction in retina-wide, full-field ERG. The specific factors influencing retinal toxicity in a given patient are not clear.
Taken together, our preclinical and clinical findings support that topotecan 30 µg (equivalent to 15 µg in our rabbit models) appears to cause no retinal toxicity in the rabbit model or in patients. Our rabbit model data indicate that topotecan might be equally effective to melphalan, supporting the need for future clinical studies that directly compare the efficacy in patients.
Data availability statement
Data are available upon reasonable request. For laboratory data, contact firstname.lastname@example.org. For deidentified patient data, contact email@example.com and firstname.lastname@example.org.
The Institutional Review Board approval was obtained at both Vanderbilt University Medical Center and Memorial Sloan-Kettering Cancer Center. Informed consent was obtained from patients for all procedures performed. This study adhered to the tenets of the Declaration of Helsinki and was performed in accordance with the Health Insurance Portability and Accountability Act. Data are available from the investigators upon request.
Twitter @KaczmarekMD, @AnthonyBDaniels
Contributors Designed the study: JMP, DLF, AR, ABD. Performed the experiments: CMB, JVK, JMP, KLB, MWC, DHA, JHF, ABD. Analysed the data: CMB, JVK, JMP, S-cC, KLB, MWC, TMB, CWL, JN, AL, TH, DHA, JHF, DLF, AR, ABD. Prepared the manuscript: DLF, AR, ABD.
Funding This work was supported by a Career Development Award from the Research to Prevent Blindness Foundation (ABD) (no specific grant number), by the National Eye Institute grant NIH/NEI K08EY027464 (ABD), by an unrestricted departmental grant from Research to Prevent Blindness to the Vanderbilt Department of Ophthalmology and Visual Sciences (no specific grant number), by a Department of Veterans Affairs Senior Research Career Scientist Award IK6BX005225 (AR), and the Vanderbilt Ingram Cancer Center Support Grant (P30 CA68485) and the Memorial Sloan-Kettering Cancer Center Support Grant (P30 CA008748) for core facilities. It was also supported by P41 GM103391 and by the National Center for Research Resources, Grant UL1RR024975, now at the National Center for Advancing Translational Sciences (2 UL1 TR000445). The content is the sole responsibility of the authors and does not necessarily represent the official views of the NIH.
Competing interests ABD and DLF have a patent with Vanderbilt University Medical Center. ABD has received research funding from Spectrum Pharmaceuticals (now Acrotech Biopharma) through an investigator-initiated study separate from the data presented in this manuscript. None of the other authors has any conflicts of interest or financial disclosures.
Provenance and peer review Not commissioned; externally peer reviewed.
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