Introduction
One of the first applications of genetics in precision medicine is pharmacogenomics informed pharmacotherapy. It promises to personalize medicine by using an individual’s germline genetic makeup, to guide optimal drug and dose selection [1 , 2 3 , 4 5 , 6 7 8 , 9 10 11–13
A prominent barrier preventing widespread adoption of pharmacogenomics panel-based testing is the lack of evidence supporting this approach. Although a number of small, randomized and observational studies have indicated the potential benefits of pharmacogenomics panel-based testing [14–17 18 ClinicalTrials.gov 19 , 20 21
This paper outlines the multiple methodological and logistical challenges that were encountered while designing and operationalizing the PREPARE study, and the solutions developed to overcome these challenges. Similar difficulties are likely to confront other investigators aiming to generate evidence to show the utility of gene panel tests affecting multiple drugs in different therapeutic areas.
Methods
An evaluation of the challenges was undertaken across the following domains: study aims and design, primary endpoint definition and collection of adverse drug events, inclusion and exclusion criteria, target population, pharmacogenomics intervention strategy, and statistical analyses. The final design has previously been published elsewhere [19
Box. 1: PREPARE study design in brief. PREPARE, PREemptive Pharmacogenomic testing for prevention of Adverse drug Reactions.
Results
The following sections discuss challenges and respective solutions considered by the Ubiquitous Pharmacogenomics Consortium when designing the PREPARE study. Figure 1 provides an overview of these challenges and the respective solutions.
Fig. 1: An overview of challenges encountered by the Ubiquitous Pharmacogenomics Consortium and respective solutions utilized.
Study aim and design
In the context of precision medicine, several fundamental options for generating evidence have been suggested such as observational research designed to identify modifiers of the effectiveness of interventions; subgroup analyses and interaction testing in standard randomized controlled trials (RCTs) of intervention effectiveness; or dedicated precision medicine RCTs that directly compare targeted vs. untargeted intervention approaches [22 DPYD )–fluoropyrimidine interaction [23
An RCT can also, of course, be used to generate evidence. Indeed, several RCTs have provided gold-standard evidence showing the clinical utility of individual gene–drug interactions to guide dosing [3 , 24–26 27 , 28 18 29–32 CYP2D6 genotype may only be observed after an estimated 10-year follow-up [33 34
Regardless of the inconvenience, there is still a need for evidence showing patient benefit and cost-effectiveness to enable implementation of pharmacogenomics-guided drug dose and drug choice. Therefore, the Ubiquitous Pharmacogenomics Consortium decided on a novel way to evaluate the (cost-)effectiveness of pharmacogenomics-guided pharmacotherapy. Instead of conducting 51 separate RCTs (one for each DPWG guideline), the consortium set out to quantify the collective clinical utility of a panel of pharmacogenomics-markers (50 variants in 13 pharmacogenes) within one trial (the PREPARE study) for proof-of-concept across multiple potentially clinically relevant gene–drug interactions.
Primary endpoint definition
The final defined primary endpoint is described in Fig. 2 . Since the DPWG guidelines encompass a broad range of gene–drug interactions, the resulting effects on patient outcomes are heterogeneous and therefore difficult to capture within one endpoint. Possible universal endpoints measuring these heterogeneous effects could have been quality of life (QoL) or overall survival (OS). On the other hand, anticipated asymptomatic effects are not captured with QoL, and the follow-up time to capture effects on OS require long follow-up times. Therefore, we decided to define the primary endpoint as the prevention of clinically relevant ADRs as 90% of the DPWG guidelines aim to decrease the risk of ADRs (Table 1 ). Clearly, gene–drug interactions that lead to improved efficacy, such as that associated with CYP2C19 polymorphism and omeprazole efficacy to increase Helicobacter pylori eradication, would not contribute to this defined endpoint. Therefore, these gene–drug interactions were excluded from the study.
Table 1: An overview of actionable Dutch Pharmacogenetics Working Group guideline and their primary anticipated effects when used to guide dose and drug selection based on the systematic review of literature underlying the guidelines
Fig. 2: The composite primary outcome is the occurrence of causal (definite, probable or possible), clinically relevant (classified as CTCAE grades 2, 3, 4, or 5), drug–genotype associated ADR, attributable to the index drug, within 12 weeks of index drug initiation. For oncology patients receiving fluorouracil, capecitabine, tegafur, or irinotecan, only hematological toxicities of CTCAE grades 4–5 and nonhematological toxicities of CTCAE grades 3–5 will be considered clinically relevant. CTCAE, Common Terminology Criteria for Adverse Events.
Most of the DPWG guidelines recommend a dose decrease. Thus, theoretically the risk of ADRs will decrease in the intervention arm and as a result, one might be suspicious of unintended decreased efficacy because of this dose lowering. Therefore, more generally, one may argue that by using this endpoint we are unable to conclude a reduction of ADRs in the absence of decreased efficacy unless the lack of efficacy is perceived as an ADR. In this context, an ideal control arm would be one where the doses of randomly selected controls, of which genotypes are unknown, are also decreased. This is; however, considered unethical. Nevertheless, since the DPWG dose adjustments are calculated based on pharmacokinetic studies, the anticipated drug exposure among those with variant genotypes treated with a guideline-recommended lowered dose, are expected to have similar drug exposures to those with wildtype genotypes treated with normal doses. This was confirmed in a pharmacokinetic sub-study among patients carrying DPYD variants who were treated with a lower fluoropyrimidine dose [23
Simply defining a composite of ADR occurrence as an endpoint is insufficiently sensitive to measure the effect of pharmacogenomics-guided prescribing for two reasons. First, since patients enrolled are often on multiple drugs, we need to distinguish between ADRs related and unrelated to the drug of enrollment. Secondly, measuring only the ADR incidence, in the absence of severity, is less meaningful clinically. To improve sensitivity of the composite endpoint to these two factors, we incorporated both causality and severity assessments (Fig. 2 ). To minimize measurement error, we selected systematic and validated methodologies for assessments and decided upon thresholds for clinical relevance.
The composite endpoint includes also the third assessment, further aiming to increase sensitivity to the endpoint to measure the intended effects of the pharmacogenomics intervention. If we were to include all reported ADRs in the composite endpoint, it would dilute the effects of the pharmacogenomics intervention, as other ADRs, which may not be preventable by the intervention, would be included. Therefore, to avoid dilution, the composite endpoint only includes drug-genotype associated ADRs where the increase in ADR incidence is known to be associated with a genotype, in keeping with the DPWG guidelines (Table 2 ). As an example, TMPT poor metabolizers or intermediate metabolizers who receive a lower dose of thiopurines have a 10-fold lower risk of severe leucopenia [26
Table 2: An overview of actionable Dutch Pharmacogenetics Working Group guideline and their primary anticipated effect based on the literature underlying the Dutch Pharmacogenetics Working Group guidelines
The time between drug initiation and the onset of ADRs was also considered when defining the time-window for the primary endpoint. A literature review showed that a 12-week window would cover the majority of drug–genotype associated ADRs. Additionally, when not dose-titrated, all drugs included will have reached steady-state at 12 weeks, making it likely that most intrinsic ADRs would have occurred. Nevertheless, the selection of the time-window was complicated by the fact that we expect both on-target (type A) and off-target (or idiosyncratic) (type B) ADRs to be prevented by the pharmacogenomics intervention. On-target ADRs are often related to the causal drug’s pharmacology and therefore the time-of-onset is, in general, more predictable than idiosyncratic ADRs. As an example, on-target ADRs associated with amitriptyline such as anticholinergic effects and cardiotoxicity would be expected to be preventable within the time-frame of the primary end-point [35 36 37 38
Primary endpoint collection
The operationalization of data collection to support the severity and causality assessments required additional consideration because of to the broad range of anticipated ADRs. This was in context of the fact that extracting ADR data retrospectively from EMRs can be difficult. Prospective collection of data in PREPARE after assessment of the patient was therefore felt to be the most accurate method. To capture as much information as possible on the occurrence of ADRs, we use three sources and concurrently collect information to support severity and causality assessments. Information is obtained from patients during scheduled follow-up (both online surveys and interviews), clinician reports, and extraction from the EMR. Patient interviews consist of open-ended questions, designed to uncover any ADRs, which the patients may have endured. On the other hand, a limitation of this methodology is the inability to uncover ADRs which are asymptomatic, such as citalopram-induced corrected QT-interval (QTc) prolongation, as ECGs are not routinely performed, potentially underestimating the occurrence of ADRs. However, once the ADR manifests symptomatically, such as Torsade de Pointes, it will contribute to the endpoint.
To ensure systematic assessment of the defined composite endpoint, we utilized standardized methods for assessment of severity and causality; the Common Terminology Criteria for Adverse Events (CTCAE) and Liverpool Causality Assessment Tool (LCAT), respectively.
In order to undertake severity assessment, using CTCAE, we require different types of data depending on the ADR reported. For example, to assess severity of a drug-induced dry mouth, we require data on the intervention required (grade 1: symptomatic without dietary alteration; grade 2: oral intake alterations; grade 3: tube feeding), whereas when assessing the severity of neutropenia, we require data on neutrophil counts (grade 3: ANC<1000/mm3 ; grade 4: life-threatening consequences; grade 5: death). This content is collected both directly from patients during follow-up (e.g. How much did the side effect interfere with your usual daily activities? Did it limit your self-care activities?) and from the EMR. To minimize the impact of recall bias, patients are contacted at t = 4 and t = 12 weeks after index drug initiation. We note that a previous study indicated that a recall period of 1 week corresponded best to daily reporting, but it concluded that a 4-week recall was also acceptable [39 t = 4-week follow-up is within the acceptable range, and we expect most ADRs to be reported within this time.
Causality assessment was formalized with the LCAT, which requires information on the start- and end-date of both the index drug and the ADR, the severity of the ADR and the outcome of the re-challenge, when applicable. As with the severity assessment, data to support the causality assessment is collected from direct questioning of patients after the report the adverse event during scheduled follow-up (e.g. Did you lower the dose or temporarily stop taking the index drug after having the side effect? Did this reduce the side effect?).
In contrast to severity and causality assessment, there is no standardized method available for assessment of drug-genotype association. Therefore, to ensure unbiased assessment, an algorithm will be used to perform the analysis at study completion, to ensure consistent pharmacogenomics literature across assessments. This algorithm is being created by an expert panel, blinded to patient allocation. It is based on ADRs associated with certain genotypes in the literature underlying the DPWG (Table 1 ) and the ADRs reported in a drug’s Summary of Product Characteristics.
Study design and blinding
To account for center-heterogeneity and time-dependent differences, PREPARE is a cluster-randomized cross-over trial comparing pharmacogenomics-guided strategy with standard care using a single cross-over moment. Clusters are formed by the countries participating in the study. The choice of a single cross-over time-point, instead of many, is dictated by the substantial logistic effort to switch between strategies. Without randomization, all countries could have started with standard of care followed by pharmacogenomics-guided prescribing synchronously. This could potentially introduce time-dependent differences, for example, healthcare professional (HCP) awareness and knowledge of pharmacogenomics may increase over time. Risk of protocol violation of the crossover approach results from centers randomized to begin with the pharmacogenomics-strategy, being unable to switch back to standard of care, resulting in an absence of a site-specific control arm. In our project, this has not occurred. A statistically more powerful alternative, for cluster randomization, would be individual randomization at the patient level. This would likely be logistically challenging and error-prone for the participating clinicians, who would need to follow different strategies for different patients simultaneously and the design was therefore excluded in the design phase of this study.
Certainly, blinding both patients and clinicians from the pharmacogenomics-test results would optimize scientific rigor and prevent potential information bias [40 41 42
Inclusion and exclusion criteria
Some drugs are more frequently prescribed than others in routine care. To avoid overrepresentation of those drugs, which are frequently prescribed within the patient sample, a number of potential solutions were considered. First, imposing a cap to limit the maximum portion (10%) of patients enrolled in a particular drug. This option is feasible and would have a positive effect on clinical relevance since it would provide the opportunity for a minimum of 10 eligible drugs to be enrolled in the study. Second, reflecting the enrolment of drugs in the study to prescription frequencies in the European population. This option would be feasible, but drugs, which are uncommonly prescribed, will not be equally represented as commonly prescribed drugs thereby decreasing the generalizability of the results. Third, to only select gene–drug interactions that we anticipate having the greatest effect size and to maximize the proportion of patients enrolled on the corresponding drug. Selecting this option; however, would synthetically increase the effect size of the intervention, thereby leading to a biased outcome with low generalizability. After consideration, the first option was chosen and implemented since it was most feasible and is likely to have a positive effect on the generalizability of results across all 41-index drugs (Fig. 3 ). In order to avoid ethical problems and maintain equipoise, countries already implementing specific gene–drug interactions as part of routine care are exempt from enrolling patients on the corresponding drugs. For example, pre-emptive routine DPYD testing is standard of care in the Netherlands and therefore patients initiating fluoropyrimidines are not being recruited in the Netherlands, but are being recruited in other countries where DPYD testing is not standard of care.
Fig. 3: Index drugs are capped at 10% of the total sample, 5% for each arm. This 5% is in turn equally divided over two groups of sites (2.5% in each), equally providing all sites with the opportunity to enroll patients on certain index drugs; the first group being those starting with the intervention arm and the second group being those staring with the control arm. Index drug enrollment is monitored centrally in real-time to ensure cessation of enrolment of index drugs once their cap is reached.
Target setting and population
Whether pharmacogenomics-guided pharmacotherapy is effective in reducing ADRs is partially dependent on the health-care system in which it is applied, as the following vary across healthcare systems beliefs and knowledge about pharmacogenomics amongst HCPs [43 44
Whether pharmacogenomics-guided pharmacotherapy is effective in reducing ADRs is also dependent on the intersection of variants included in the panel and the ethnicity of the target population. Allele frequencies in pharmacogenes vary significantly between different ethnic groups [45 46 47 HLA-B *15:02 and HLA-A *31:01 status. However, the tagging SNPs only appeared suitable in subjects of Asian origin and not in a cohort of subjects of predominantly Caucasian origin. As a result, these tagging SNPs were removed from the panel and carbamazepine was removed as a drug of enrolment.
Pharmacogenomics intervention strategy
As literature underlying pharmacogenomics accumulates over time, the DPWG guidelines are regularly updated. In the PREPARE study, these updates are also implemented regularly, as we felt it unethical not to treat patients with the most up-to-date knowledge. However, one may argue that this imposes limitations on the scientific rigor of determining the effect of the intervention since it is not constant over time. However, it is inevitable that the guidelines will be updated regularly even after completion of the PREPARE study. Therefore, updating the guidelines within the study reflects the real-world aspects of the intervention more accurately than using out-of-date guidelines for the sake of scientific rigor. Examples of this during the study are the removals of oxycodone and clozapine as drugs of enrolment. At study initiation, the CYP2D6– oxycodone and CYP1A2– clozapine interactions were classified as actionable interactions requiring pharmacotherapy adjustment for at least one phenotype category. However, after preplanned updates of these guidelines by the DPWG, both were no longer considered actionable. Therefore, these drugs cannot result in a pharmacotherapeutic recommendation and were removed as drugs of enrolment. Additionally, CYP1A2 was removed from the panel since there remained no actionable drug-gene interactions related to this gene. In addition to updates of DPWG guidelines, new guideline for novel gene–drug interactions are also developed. For example, for CYP2D6 –brexpiprazole, HLA-B –allopurinol, HLA-B –lamotrigine, and HLA-B –oxcarbazepine. However, to avoid unequal number of patients on these drugs in study and control arms, these drugs were not added to the list of drugs of enrolment throughout the study. Indeed, removal of oxycodone and clozapine also resulted in an unequal number of patients on these drugs in study and control arms. However, the resulting nonactionability of the DPWG updates could not have been foreseen and therefore is substantiated.
The timing and methodology of delivering the relevant actionable DPWG guideline to treating clinicians may also have an effect on outcomes. To quantify the effectiveness of pharmacogenomics to prevent ADRs, one would prefer to initiate the ‘correct’ drug and dose, tailored to the patient’s pharmacogenomics profile. However, this would require patients to delay the initiation of their pharmacotherapy while awaiting the turn-around of their pharmacogenomics test result. We felt this would be unethical, especially for analgesic therapies. For this reason, we have imposed a maximum 7-day turn-around time in which the gene important to the drug of enrollment and the corresponding DPWG guideline should be reported to the treating physician. However, a limitation of this delay is the potential underestimation of the true effect of pharmacogenomics-guided pharmacotherapy, since patients start the index drug on an unoptimized dose or drug to bridge the turn-around time and the risk of ADRs may increase with an increasing number of days using the un-optimized dose. It will be possible to adjust for this in a per-protocol analysis.
Statistical analysis
The added value of testing a panel of pharmacogenomics markers as opposed to multiple single genes is the ability to reuse the pharmacogenomics-panel results in the future without the logistical hassle of turn-around time. However, this added value is not captured by the primary statistical analysis since it is limited to ADRs attributed to the index drug and excludes ADRs attributed to subsequent drugs. Our reasoning for this is to avoid selection bias since patients initiating subsequent drugs use more concomitant drugs and are therefore at higher risk of experiencing more ADRs. Nevertheless, there is a need to quantify the effectiveness of ADR prevention through pre-emptive panel testing. Since PREPARE enables data collection not only for the index drug but also for (multiple) subsequent drugs, we are able to address this need by re-running the primary analysis using ADRs attributed to subsequent drugs in a secondary analysis.
Gatekeeping analysis
Traditional RCTs use strict eligibility criteria to select only patients in whom a benefit is most likely to be observed from the assessed intervention. However, with pharmacogenomics testing, we are unable to perform such a selection because the genotype is not known prior to recruitment. Only those patients with an actionable gene–drug interaction for the index drug may benefit from the pharmacogenomics intervention for the drug of enrolment. Therefore, comparison of the entire study arm to the entire control arm would underestimate the effectiveness for the subgroup of patients who may benefit from the intervention. For example, a study by Coenen et al. [26 TPMT did not reduce the overall proportions of patients with hematologic ADRs during thiopurine treatment. However, within the subgroup carrying an actionable gene-drug interaction, there was a 10-fold reduction in hematologic ADRs. To minimize the risk of an unjust nonsignificant outcome when comparing arms, we decided upon a gatekeeping analysis (Fig. 4 ), where the first analysis quantifies the effect among those who have an actionable gene-drug interaction. However, when applied in a clinical setting, an entire population will need to be genotyped to be able to identify those who carry a variant genotype relevant for the drug to be used in the patient. This is addressed in the second analysis, where the effect of pharmacogenomics-guided pharmacotherapy among the entire screened population will be quantified.
Fig. 4: Primary statistical analysis: a two-step gatekeeping analysis. First, a logistic regression analysis will be performed among patients who had an actionable drug-genotype combination. This analysis is first performed per country, and the log-odds are pooled in a forest plot. Only if this is statistically significant a second analysis will be performed: a logistic regression analysis among all patients included in the study. Again, this is first performed per country, and log-odds are pooled in a forest-plot.
Statistical model and estimand
To account for the clustered nature of the data, the analysis will be stratified by country, correcting for study center and other relevant covariates on the patient level. Analyses will be pooled using a meta-analysis. The composite endpoint is binary representing – in general – ADE severity 0/1 in one group and ADE severities 2/3/4/5 in the other. We use logistic regression to assess the influence of the pharmacogenomics-intervention on the probability of transitions between these two groups. Transitions within the groups, for example between grades 3 and 4, are not considered by this analysis as not aligning with the implementation goal. The estimand of the primary analysis is, therefore, the odds ratio for transitioning between these two groups based on the intervention.
An ordered logistic regression (ordinal regression) is more powerful when the goal is to detect the influence of the intervention on lowering severity in general by choosing as an outcome the ordinal ADEs (not grouping ADE grade). As such an approach can give additional insight into mechanisms underlying the effect of pharmacogenomics-intervention, ordinal regression will be performed as a secondary analysis.
Extreme phenotypes
In addition to the aim of quantifying the clinical utility of a pharmacogenomics-panel, the PREPARE study also aims to expand our understanding of genetic variation on drug response. The PREPARE study is a unique opportunity to identify and further investigate patients who express unexpected drug responses, known as extreme phenotypes. Three criteria for defining extreme phenotypes were devised to identify patients within PREPARE: (1) the physician considers the ADR important, (2) ADR is of severity grade 3 or above, or in the case of hematological toxicity grade 4 or above, and (3) patient experienced (re)hospitalization as a result of the ADR. A major problem as a result of these criteria is that they are applied differently across study centers. In some cases, it is difficult to stratify a true ADR from disease-induced adverse events. To overcome this issue, interesting cases are identified from the existing list of extreme phenotypes. These are further analyzed for possible novel genetic variants or combination of variants by an initial genome-wide association study encompassing all relevant pharmacogenes and followed in special cases by whole genome sequencing. Based on the multitude of rare genetic variants uniquely present in specific individuals, we believe this is an important step toward higher future predictability of genetically related ADRs.
Discussion
Future perspective of pharmacogenomics testing to optimize pharmacotherapy
The potential effectiveness of pharmacogenomics testing to reduce on-target ADRs is determined by a number of factors including the ability of the tested variants to accurately predict the patient’s phenotype, the extent to which genetic variation affects target protein functionality, the extent to which the target protein determines drug exposure and finally, the causal relationship between the drug of exposure and ADR risk. In the future, we expect a shift in the field regarding the first two determinants; phenotype prediction will become more accurate due to accumulating knowledge of the effects of individual variants on enzyme and transporter functionality. Currently, phenotypes are predicted using a categorical approach. However, enzyme activity is usually normally distributed within a population and therefore is better described by a continuous phenotype scale. We envision a future where phenotypes can be predicted more precisely by using all of an individual’s genetic variation (common and rare variants), as opposed to limiting the assessment to only those variants included in a tested panel. Although we expect the variants in the ubiquitous pharmacogenomics panel to correctly predict most phenotypes, it may not be able to do so correctly for all patients, since rare, untested variants may also affect phenotype. As our understanding of the effects of individual variants to inform phenotype prediction improves, we imagine that phenotype prediction will ultimately become substrate-specific as opposed to simply gene-specific. Thus, as our knowledge improves and increases resolution, we expect further improvements in the effectiveness of pharmacogenomics testing to reduce ADRs. Looking even further forward into the future pharmacogenomics profiles may be combined with other -omic profiles including the epigenome [48 49 50
Generating evidence for other precision medicine approaches
In this article, we have provided an overview of the challenges and respective solutions while designing the PREPARE study. We expect similar challenges to confront fellow investigators aiming to generate evidence for precision medicine approaches other than pharmacogenomics. Conventionally, evidence supporting novel interventions is generated by prospective trials. However, in an era where digitalization is driving data accumulation and a concomitant increase in stratification of patient groups, we are moving towards utilization of real-world data to support precision medicine. Several authors have pointed out that precision medicine, and genomic medicine, in particular, would benefit from a convergence of implementation science and a learning health system approach to measure outcomes and generate evidence simultaneously [51 , 52 21 , 53 54–57
Generating evidence for pharmacogenomics: the PREemptive Pharmacogenomic testing for prevention of Adverse drug Reactions study
In conclusion, we have undertaken detailed considerations to enable quantification of the collective clinical utility of a panel of pharmacogenomics-markers within one trial as a proof-of-concept for pharmacogenomics-guided pharmacotherapy across multiple actionable gene–drug interactions. Although PREPARE will enable quantification of the clinical utility of a pharmacogenomics panel, it may also potentially underestimate the effects due to delayed initiation of a pharmacogenomics-guided dose or drug choice because of our turn-around-time (<7 days), and thereby limit the primary endpoint which is the reduction of ADRs caused by the initiated drug. Further research is therefore needed to quantify the effectiveness of panel-based pharmacogenomics-guided pharmacotherapy in patients encountering multiple gene–drug interactions over a longer time-horizon. This can be achieved within a clinical trial, or a more practical approach may be to use real-world data to estimate long-term (cost-)effectiveness. PREPARE provides the framework not only for future efforts in this area but also in providing the evidence base for ubiquitous adoption of pharmacogenomics-guided pharmacotherapy [58
Acknowledgements
We would like to thank the Scientific Advisory Board (Prof. Russ Altman, Prof. Michel Eichelbaum, David Haerry, Prof. Mark Ratain, Prof. Mary Relling, and Prof. Dan Roden) for their insights in the development of the study protocol and statistical analysis plan.
The research leading to these results has received funding from the European Community’s Horizon 2020 Programme under grant agreement No. 668353 (Ubiquitous Pharmacogenomics). M.S. is in part supported by the Robert Bosch Stiftung, Stuttgart, Germany.
Conflicts of interest
G.P.P. is a full member and National Representative of the European Medicines Agency, Committee for Human Medicinal Products – Pharmacogenomics Working Party (Amsterdam, the Netherlands). There are no conflicts of interest for the remaining authors.
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