Key principles of the FOCUS4 trial design
The design of the FOCUS4 trial was based on seven key principles, as discussed below. These principles are also described by Kaplan et al.13
Key principle 1
Key principle 1 was to evaluate multiple treatments and biomarkers in the same protocol, including as many patients as possible with a given disease, with separate clinical questions for as many marker-defined subgroups as are supported by current evidence.
Incorporating multiple treatments across multiple population-enriched biomarker-defined trials fits into conventional clinical practice patterns, in which patients with one type of cancer are managed in a common manner with similar protocols. In CRC, this single treatment approach for all has evolved into two clinical pathways: first, for patients with KRAS wild-type tumours (for whom EGFR-targeted monoclonal antibodies may be planned) and, second, for those with KRAS-mutated tumours. As further individualisation of treatment approaches occurs, managing separately co-ordinated clinical research efforts, which often involve different collaborators and different research teams, becomes progressively more unwieldy and inefficient. The approach used in FOCUS4 filters all eligible patients into one overarching clinical trial programme; this design offers clear efficiency gains in both cost and time compared with running multiple individual trials to evaluate different treatments under separate protocols. This design also increases the likelihood that the investment leads to discovery of effective treatments and halts further development of ineffective treatments in this disease setting. Further efficiency is inherent in biomarker analysis being set up to include all diagnostic tests for the differing subgroups. So far as is scientifically feasible, an inclusive trial allows the maximum number of patients to participate and maximises the potential to recruit rare subtypes. It allows for maximum flexibility in refinement of the biomarker cohort definitions in response to developing clinical data from both within and outside the trial, and provides administrative and organisational efficiencies.
Key principle 2
For key principle 2, in the initial stages, assess each treatment in the presumptive biomarker-enriched subset (thus exploiting the putative link between biomarkers and novel treatments with corresponding mechanisms of action), but without assuming that this association would be confirmed in later stages.
In oncology, even when novel agents are found to be active, the expected biomarker selection may not apply.14 A key strength of the FOCUS4 protocol is that it neither assumes that any encouraging outcome results are limited to the biomarker selection nor expends numbers of biomarker-negative patients until there is a positive signal from the initial staged analyses (stages 1 to 2). Therefore, entry into the earlier phases of the evaluation of a novel treatment was restricted to those patients who were thought to be most likely to respond. Once the significance level associated with the activity of the experimental treatment fell to less than a given value, there was the option to open a similar efficacy evaluation among those patients who did not show the positivity of the biomarker in their tumour (i.e. off-target effect), using the same type of lack-of-activity assessments, to refute or confirm activity in this complementary population of patients. This approach builds on the putative link between the biomarker and drug efficacy but does not presume that this is a certainity.
Key principle 3
Key principle 3 was to use randomised evidence with a control group for each biomarker/treatment cohort evaluation (eliminating confounding resulting from prognostic biomarker effects).
Constructing the protocol as a set of parallel randomised comparisons ensured that the measured or unmeasured prognostic effects of different biomarkers did not confound the assessment of treatment efficacy, meaning that any benefits were ascribed to the new treatment and not the potential prognostic effect of the marker.
Key principle 4
Key principle 4 was to ensure rapid evaluation of each new treatment, which involves (1) incorporating the flexibility of Phase II and III components into each trial and (2) targeting a reasonably large treatment effect, with discontinuation of random assignment to treatments that are unpromising or overwhelmingly effective as early and reliably as possible.
For the individual trials within the protocol, a larger effect size could be targeted than might be chosen in a more traditional trial. This could be undertaken for two reasons. First, enrichment of the population meant that a somewhat larger effect could be expected, even if enrichment excluded only a proportion of those who did not benefit. Second, there are currently a large number of potential treatments available for evaluation and, therefore, it was reasonable to seek a larger treatment effect than if only a limited number of new treatments were available for testing.
A key aspect of the FOCUS4 protocol was the flexibility to have both Phase II and Phase III components for each randomised comparison, with the potential to move seamlessly from Phase II to Phase III. The aim of the Phase III component was to determine if, in the interval after standard first-line chemotherapy, the proposed novel agents improved PFS and potentially (in some of the larger cohorts) OS, compared with placebo, within the biomarker-defined populations. Use of PFS and OS could be particularly important when agents with different mechanisms of action are being tested. This is in preference to other earlier outcomes of treatment response (e.g. disease response by response evaluation criteria in solid tumours), which may not reliably translate into longer-term outcomes of importance to the patient. For each agent-versus-placebo comparison, FOCUS4 employed a maximum of four stages:
stage 1 – safety and screening for sufficient activity (relevant primary end point:
PFS)
stage 2 – screening for sufficient activity (relevant primary end point:
PFS)
stage 3 – efficacy (relevant primary end point:
PFS)
stage 4 – efficacy [relevant primary end point:
OS (for those cohorts with sufficient patients)].
Stages 1 and 2 together can be considered analogous to a traditional Phase II trial, whereas continuing into stages 3 and 4 can be considered functionally to complete a traditional Phase III trial. Such designs can be adapted for different end points at different stages and use different decision criteria for moving from one stage to another.
For all therapies that passed activity screening in stage 2, a number of paths were possible. If no major changes were to be made to the research arm (e.g. in biomarker selection criteria or agent dosing), a seamless move to stage 3 was possible, with outcome data on all patients entered during stages 1 and 2 being used in stages 3 and 4. Alternatively, if a major change to a research arm was made, the trial could still continue to the Phase III component (stages 3 and 4), but with outcome data from only newly entered patients from that time forward contributing to the final (Phase III) analysis. However, there was considerable efficiency even in this situation. This so-called new Phase III trial did not require a new protocol and could be initiated by amendment and activated rapidly at all of the sites already participating in the single FOCUS4 protocol. A final possibility was that, for reasons external to the trial itself, the sponsor or funder could decide not to support continuation to the Phase III stages. This could also happen in the face of a positive outcome in the activity screening stages (e.g. if testing of the agent in other settings was planned). Note that such an outcome would still be appropriately viewed as useful if it served to stimulate or facilitate additional trials. In such a situation, other novel agent(s) could then be tested in the relevant cohort of FOCUS4.
For each of the trials within FOCUS4, the overall power was maintained at 80%, allowing for multiple interim data looks, with a maximum 5% two-sided overall significance level (type I error rate). To maintain 80% power overall (for each trial), the power of each trial for the primary analysis varied from 85% to 95%, depending on the number and timing of the interim analyses.
Each biomarker-defined trial was considered separately in terms of the effect size (hazard ratio) to be detected to suit issues relevant to specific biomarker cohorts or agents. An important distinction between stage 2 (lack of sufficient activity) and stage 3 (efficacy) was the difference in type I error, which was set higher for the stage 2 interim PFS analysis (one-sided 10%) than for the stage 3 main PFS efficacy analysis (one-sided 2.5%).
Data for each of the trials were reviewed by the Independent Data Monitoring Committee (IDMC) at each interim analysis. The committee could advise early closure of a trial in the event of overwhelming evidence of efficacy, using a significance level of p = 0.001 as a guideline on the Phase III efficacy outcome measure. This was considered at approximately the halfway point in terms of accrued number of events for each biomarker-defined trial.
Key principle 5
Key principle 5 was to allow the possibility of refining any biomarkers throughout the course of the trial, from either internal data or, more typically, data emerging outside the trial.
Biomarker definitions are constantly evolving. Although, for example, KRAS mutation was a multiply confirmed predictive marker for use of anti-EGFR antibody therapy, it subsequently became clear that not all KRAS mutations had identical effects, with some perhaps not even carrying the same negative predictive value (e.g. KRAS G13D).3 Evolving data now suggest that expression of EGFR ligands, such as epiregulin and amphiregulin, may also modulate response to this class of agents.15 Other alterations elsewhere in the RASRAF–MEK–ERK and interacting pathways [e.g. phosphoinositide 3-kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR)] are almost certainly also of considerable importance. An example of biomarker refinement would be the introduction of a new platform for mutation assessment. Developments in molecular diagnostics are occurring at such a pace that older technologies are continually being outperformed by newer technologies in cost and sample requirements. Such refinements could be introduced into a continuing programme, such as FOCUS4, but with close attention to quality assurance and a pre-planned parallel evaluation using both platforms for a period of time to ensure comparability of assessment. If there proved a need to revise one or another biomarker assay during the first two (signal-seeking) analysis stages, patients assigned on the basis of the earlier assay would probably have had to be excluded from the definitive third- and fourth-stage analyses and their possible use towards registration. The third-stage sample size might need to be increased, but the overall time delay introduced in the stage 3 analysis would be minimal (months), because the trial would already be activated and recruiting from a large number of sites.
Key principle 6
Key principle 6 was to allow the possibility of introducing a new biomarker and treatment pairing into the overall trial programme when evidence warranted.
The flexibility inherent in the FOCUS4 design created opportunities to further adapt the trial to accommodate additional findings, typically from other research outside the trial. For instance, it is well known that there is a cohort of patients with MMR deficiency within CRC. In earlier disease, these patients amount to 15% of the total, but they have an improved prognosis, and series among metastatic patients reveal only approximately 4% prevalence of MMR deficiency. At the time that FOCUS4 was designed, there was no convincing case for testing a specific class of agent in this cohort. However, during this period, the rapid emergence of immune checkpoint inhibitors transformed cancer therapy and it is in the MMR-deficient subgroup of CRC that these agents have made a major impact. In principle, if such evidence emerged during the course of the study, the protocol could be amended to open up a new biomarker/treatment cohort by identifying the MMR-deficient cohort and testing the appropriate novel agent(s) compared with placebo in this group. The introduction of such a new biomarker-defined cohort during the trial would remove such patients from the other cohorts, but the introduction of a new cohort would not compromise the study design, requiring only sample size adjustments.
Key principle 7
Key principle 7 was to investigate new treatments in the earliest and most likely responsive settings that are clinically feasible. With many agents being developed and recognition that drug development is a lengthy and expensive process, it is critical to seek strong positive signals as early as possible in testing. When new agents are tested against all-comers and late in the natural history of the disease, it is usually difficult to determine whether or not observed modest improvements are likely to hold up with further testing, especially in earlier stages of disease.
In the FOCUS4 protocol, a four-stage selection process of patients for each trial was used, which improves the chance of identifying clinical benefit from novel agents. First, patients with aggressive disease, as manifested in a raised baseline platelet count, were excluded; this exclusion was later removed following full analysis of the effect of raised baseline platelet count (thrombocytosis) in other trials. Second, only those patients with stable or responding disease during 16 weeks of first-line systemic therapy were included. This, therefore, specifically selected responding patients, in comparison with most study designs, which select patients resistant to evaluate novel agents. Third, by testing the novel agents first in the molecular cohort in which theoretically they should have the greatest benefit, the likelihood of success was maximised. Finally, new agents are used on their own or in novel–novel combinations, after standard treatment, thereby avoiding unpredictable negative pharmacological or toxicity interactions with conventional chemotherapy, which has been seen repeatedly in CRC chemotherapy. Consequently, randomisation occurs in a treatment window of opportunity or treatment break, which is a clinically reasonable and safe strategy on the basis of randomised data from the COIN trial.16 Although this strategy is somewhat unusual in CRC, there are many settings in the management of other tumours in which periods of observation of patients off treatment are standard and could be used for such window-of-opportunity trials. In FOCUS4, the setting has the advantage of allowing relatively new agents to be tested in patients before the onset of chemotherapy resistance and yet well before comprehensive data would become available with regard to combined administration along with chemotherapy.
Patient consent, registration and biomarker panel testing
Patients were approached to take part in FOCUS4 using a two-stage consent process. Initially, patient consent was obtained for registration and permission for biomarker testing of their tumour tissue; consent was also obtained when eligibility for particular comparisons had been established on the basis of the test results. Patient information sheets were provided for each stage of consent and signed consent forms were required prior to registration or randomisation.
Patients were registered via an online registration platform managed at the MRC Clinical Trials Unit (CTU) at University College London (UCL). Male or female patients aged ≥ 18 years with World Health Organization (WHO) status of 0, 1 or 2 were eligible for registration providing that they had histologically confirmed adenocarcinoma of the small bowel or colon or rectum, with an accessible diagnostic formalin-fixed paraffin-embedded (FFPE) tumour block taken prior to the commencement of standard first-line treatment. They were required to have inoperable metastatic or locoregional disease (synchronous or metachronous) that could be RECIST (Response Evaluation Criteria in Solid Tumours) reported (v1.1) with unidimensionally measurable disease identified by computed tomography (CT) no more than 6 weeks before registration.
The movement of samples was tracked by the MRC
CTU from the local site pathology department to one of two mutually quality-assured laboratories in Cardiff (Department of Cellular Pathology and All Wales Molecular Genomics Laboratory, Institute of Molecular Genetics, both located at University Hospital of Wales) or one in Leeds (Division of Pathology and Data Analytics, Leeds Institute of Medical Research at St James’s, University of Leeds). Laboratory testing initially comprised pyrosequencing of the mutation hotspots, then, from August 2017, the use of whole-gene next-generation sequencing (NGS), plus immunohistochemistry (IHC) for MMR proteins and PTEN. The technical components of the biomarkers and inter-laboratory quality assurance have been described previously.17
Initially, deoxyribonucleic acid (DNA) was extracted from FFPE tissue and analysed using pyrosequencing to obtain the tumour mutation profile across known mutation hotspots in KRAS, NRAS, PIK3CA and BRAF. Further sections were stained on a DAKO Autostainer Link 48™ (Agilent Technologies, Inc., Santa Clara, CA, USA) to determine the protein expression of four mismatch repair (MMR) markers (MLH1, MSH2, MHS6 and PMS2) and PTEN. From August 2017, when FOCUS4-C was opened, the sequencing methodology was changed to NGS to allow coverage of the full sequence of KRAS, NRAS, PIK3CA and BRAF, and the addition of TP53. The GeneRead Clinically Relevant Mutation panel (QIAGEN, Hilden, Germany) was used, adhering to the manufacturer’s instructions. Results were uploaded to the centralised trial database from each laboratory. Where FFPE tumour blocks contained insufficient tumour tissue, an alternative tumour block was requested. If an alternative was unavailable, the patient was still eligible for entry into FOCUS4-N.
Molecular assays
Details of the molecular assays used for molecular characterisation of the tumour samples are presented in the following sections. Parts of this section are reproduced by Richman et al.17 This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: https://creativecommons.org/licenses/by/4.0/. The text below includes minor additions and formatting changes to the original text.
In addition, a copy of the Biomarker Laboratory Manual is provided in Report Supplementary Material 1.
Sample processing
A series of 5-mm-thick sections were taken from each block, the first of which was used for haematoxylin and eosin staining, to identify the area of greatest tumour density, and the rest made available for DNA extraction and whole-section IHC. From the residual blocks, tissue microarrays (TMAs) were created, comprising four 0.6-mm tumour tissue cores and one core, if available, of ‘tumour-associated’ normal tissue. To reduce tissue use, the TMAs were prepared only once, in Cardiff, and then shipped to Leeds, where sections were cut and used for IHC.
DNA macrodissection and extraction
The spare sections from the resection blocks were marked out for the richest areas of neoplastic cell content, using the corresponding haematoxylin- and eosin-stained section as a guide, and were macrodissected. DNA was extracted in Leeds using the QIAGEN QIAamp DNA Extraction Kit and in Cardiff using the QIAGEN EZ1 following the manufacturer’s standard protocol.
Mutation detection
The analysis of mutation hotspots was carried out by pyrosequencing within KRAS codons 12, 13, 61 and 146 (exons 2, 3 and 4); BRAF codon 600 (exon 15); NRAS codons 12, 13 and 61 (exons 2 and 3); and PIK3CA codons 542, 545, 546 and 1047 (exons 9 and 20). Pyrosequencing was carried out in each laboratory using a PyroMark Q96 (QIAGEN, Hilden, Germany). A negative water control and a positive control for each assay were included in every sample run. Raw data files were used to generate pyrograms for interpretation by qualified personnel.
Next-generation sequencing
Next-generation sequencing was used for the detection of mutations in the KRAS, NRAS, BRAF, PIK3CA and TP53 genes. All samples were analysed for mutations at specified codons/exons within the following genes: BRAF codons 599, 600 and 601 (exon 15); KRAS codons 12 and 13 (exon 2), 61 (exon 3), 117 and 146 (exon 4); NRAS codons 12 and 13 (exon 2), 61 (exon 3), 117 and 146 (exon 4); PIK3CA codons 542, 545, 546 (exon 10*), 1047 and 1049 (exon 21*); and TP53. *Note: PIK3CA numbering corrected from previous standard operating procedures versions in line with ref sequence NM_006218.2 (previously referred to as exons 9 and 20).
Any changes in these specific codons were reported, as long as they were not considered to be polymorphisms. Full details of the NGS methods can be found in the Laboratory Manual.
Mismatch repair status determination
All four immunohistochemical analyses were carried out on a DAKO Autostainer Link 48 (Ely, UK) using DAKO pre-programmed protocols, which were available with the Autostainer. Antigen retrieval was performed in the accompanying PT-Link chamber with high-pH DAKO target retrieval solution, in accordance with manufacturer’s instructions. Slides were rinsed with DAKO wash buffer prior to loading into the Autostainer. DAKO ready-to-use antibodies were used for MLH1 (IR079), MSH2 (IR085) and MSH6 (IR086). DAKO PMS2 (M3674) was used at a dilution of 1 : 40. Sections from the two validation TMAs were stained with each antibody, then corresponding whole sections were also stained in cases where the cores appeared negative or equivocal or for cases where all cores had been lost from the TMA section. Tumours were deemed positive if any proportion of the tumour nuclei was positively stained, or negative where all discernible tumour nuclei were negative in the local presence of positively staining stromal and infiltrating lymphocytic cells. Any samples appearing wholly negative with respect to both tumour and stromal components were deemed to be of indeterminate status.
Phosphatase and tensin homologue protein expression
Immunohistochemical staining was carried out using the DAKO Autostainer Link 48. Antigen retrieval was carried out in the accompanying PT-Link chamber with high-pH DAKO target retrieval solution, in accordance with manufacturer’s instructions. Slides were rinsed with DAKO wash buffer prior to loading into the Autostainer. DAKO PTEN antibody (M3627) was used at a predetermined dilution of 1 : 100. Both validation TMAs were stained, along with all corresponding whole sections. The presence and intensity grade of cytoplasmic staining in the tumour component was noted (0 = negative; 1 = weak cytoplasmic staining, less intense than the surrounding stroma; 2 = moderate cytoplasmic staining, where staining is equal in intensity to the adjacent stromal staining; and 3 = strong cytoplasmic staining, where staining is stronger in intensity to the adjacent stromal staining). For the purposes of randomised stratification of patients, any positive result was reported as ‘no loss’ of PTEN, whereas the negative result was reported as ‘absence’ of PTEN. Three FFPE cell lines (LNCaP, PTEN negative; ZR-75–1, a weak expresser of PTEN; and MCF7, which overexpresses PTEN) were used to create a mini control TMA, which was stained along with each section. A suspension was generated from each cell line, which was subsequently spun down, fixed in 10% neutral-buffered formalin, added to 12% Noble agar at a 1 : 1 ratio, processed and paraffin embedded. Three cores were taken from each and embedded into a new paraffin block to create the mini ‘control TMA’. A section of this was cut onto the same slide as each of the 97 validation samples.
Data validation
Each laboratory sent the results of all analyses directly to the MRC
CTU for independent cross-referencing. Any discrepant results were discussed between the biomarker teams from both laboratories until a final unanimous result was agreed on.
A copy of the master protocol and of the protocols for the comparison subtrials can be found on the FOCUS4 trial website (www.focus4trial.org/; accessed 28 October 2022).
