Scientific rationale for FOCUS4-B
PIK3CA is one of the most commonly altered genes in CRC, with mutations found in 12–20% of cases depending on whether the sequencing is limited to hotspot exons or covers the entire gene (i.e. broader genetic testing of the entire gene identifies a higher number of mutations than testing limited to the most common sites of mutation).32
PIK3CA encodes the catalytic p110α subunit of PI3K, a key node in the PI3K–AKT pathway, which regulates cellular proliferation, apoptosis and metabolism.33 Approximately two-thirds of PIK3CA mutations cluster to codons 542 and 545 in the exon 9 helical domain or to codon 1047 in the exon 20 kinase domain. Although they act through different mechanisms, both helical and kinase domain substitutions cause constitutive activation of the PI3K–AKT pathway and are strongly oncogenic in preclinical models.34
PIK3CA mutations in CRC are often associated with KRAS-activating mutations35 (and also with NRAS- and BRAF-activating mutations) and are reported to correlate with other clinicopathological features, such as right-sided cancers and tumoural MMR deficiency.36,37 Importantly, in contrast to other molecular aberrations, such as MMR deficiency, the frequency of PIK3CA mutation in metastatic CRC is broadly similar (12.7%) to that observed in early-stage disease,18 consistent with data indicating that neither exon 9 nor exon 20 mutations are prognostic in isolation in early CRC.38 Similarly, PIK3CA mutation does not appear to be prognostic in metastatic disease.18 Although it has been suggested that CRCs with mutation of both PIK3CA exon 9 and PIK3CA exon 20 display poor outcome,35 the low frequency (approximately 0.6%) of such double mutants means that validation is required. Emerging data suggest that, similar to KRAS, BRAF and NRAS mutations, PIK3CA mutation may predict resistance to cetuximab in metastatic CRC; however, the current evidence is conflicting (perhaps because of the high frequency of concurrent activating mutations in KRAS and NRAS) and there are insufficient analyses to inform practice.39,40
Unsurprisingly, given the high frequency of PIK3CA mutation in CRC and other solid tumours, much attention has focused on the development of PI3K inhibitors. Although the high degree of conservation of PI3K with other kinases has made the design of specific agents challenging, compounds with nanomolar affinity have recently entered clinical practice.41 Disappointingly, emerging data from current early phase clinical trials42 indicate that these drugs lack significant activity against CRC (either alone or in combination with MEK inhibitors) and, therefore, are poor candidates for further development in this particular disease. Our initial efforts to obtain these agents were, therefore, unfruitful. Therefore, at present, no specific therapeutic strategies exist for the subgroup of patients with PIK3CA-mutant CRC.
Aspirin as an anti-cancer agent in colorectal cancer
Over the past two decades, multiple retrospective and prospective studies have unequivocally demonstrated that regular use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) protects against the development of colorectal adenoma and CRC.43–46 More recently, aspirin has also been shown to reduce the incidence of metastatic disease following CRC resection.47,48 In both cases, the antineoplastic activity of aspirin is not only seen with high, multiple-daily dosing (300–600 mg four times per day) required for its systemic anti-inflammatory effect owing to inhibition of cyclo-oxygenase 2 (COX-2), but also seen with low, once-daily dosing (70–100 mg once daily) at which the action of aspirin is thought to be limited to an antiplatelet effect from irreversible inhibition of cyclo-oxygenase 1.48,49 Platelets promote tumour growth and metastasis through multiple mechanisms, including protection of circulating tumour cells from immune destruction by microthrombi formation, facilitation of tumour cell extravasation at distant sites and secretion of tumourigenic growth factors, including platelet-derived growth factor and vascular endothelial growth factor.50 Thrombocytosis predicts poor outcome in patients with metastatic CRC,51 and platelet depletion inhibits tumour growth in animal models.52–54 These data, together with the recent demonstration that tumour self-seeding by circulating tumour cells is an important promoter of cancer growth in mice,55 suggest that aspirin may demonstrate anti-tumour activity in the context of advanced disease. Such an evaluation would complement the investigation of aspirin as adjuvant therapy in CRC, which is to be determined by large randomised trials that are currently recruiting (ASCOLT56) or in set-up (Add-Aspirin57).
PIK3CA mutation as an aspirin biomarker
Two large, retrospective studies35,58 have suggested that mutations in exons 9 and 20 of PIK3CA strongly predict benefit of aspirin in CRC. The first, reported by Liao et al.,35 retrospectively analysed 964 patients with a mixture of disease stages (stages 1–4). The subgroup of patients with stage 4 CRC in this study was small (64 patients, 7% of the total of 964 patients). The study demonstrated that, among patients with PIK3CA-mutant tumours, reduction in CRC-specific mortality was significantly greater in those who were regular users of aspirin after diagnosis than in non-users of aspirin [multivariate adjusted hazard ratio (HR) 0.18, 95% confidence interval (CI) 0.06 to 0.61; p = 0.0001]. By contrast, patients with tumours lacking PIK3CA mutations did not appear to benefit from aspirin use (HR 0.96, 95% CI 0.69 to 1.32; p = 0.76). Statistical analysis demonstrated a significant interaction between PIK3CA mutation and aspirin use (p = 0.02).
These data were supported by analysis of the VICTOR study: a large, randomised controlled trial (RCT) that compared the cyclo-oxygenase-2-specific inhibitor rofecoxib with placebo following completion of adjuvant therapy for stage 2/3 CRC.58 It should be noted that there were no patients with stage 4 CRC in this study. Although the study protocol did not permit concomitant NSAID or high-dose aspirin use, low-dose (≤ 100 mg daily) aspirin was allowed and was recorded both at randomisation and during follow-up. As in the study by Liao et al.,35 the benefit of aspirin appeared to be restricted to patients with PIK3CA-mutant cancers, with no recurrences in the subgroup of regular aspirin users whose tumours harboured PIK3CA exon 9 or 20 mutations (multivariate adjusted HR 0.11, 95% CI 0.001 to 0.832; p = 0.027; p-interaction = 0.024), compared with similar outcome of aspirin users and non-users in those lacking tumour PIK3CA mutation (HR 0.92, 95% CI 0.60 to 1.42; p = 0.71).
Although these data are provocative, it is important to note that other retrospective analyses have not confirmed improved outcome with aspirin use in PIK3CA-mutant CRC and have indeed suggested other biomarkers that may predict benefit from aspirin ().60 Furthermore, these studies were all limited by their retrospective nature, variable aspirin dose used, differing end points (e.g. relapse-free survival, CRC-specific mortality, OS) and small numbers of patients with advanced CRC. Therefore, prospective evaluation in a randomised, controlled trial is essential to determine the efficacy of aspirin as an anti-cancer therapy in PIK3CA-mutant stage 4 CRC.
Studies reporting aspirin benefit in CRC according to PIK3CA mutation
Potential toxicities of aspirin
Although concerns regarding the potential toxicities of aspirin, particularly that of gastrointestinal haemorrhage, have limited the use of aspirin as a chemopreventative agent,61 the absolute increase in risk of significant toxicities from low-dose therapy for patients without established risk factors for such toxicity is modest. A meta-analysis62 of RCTs that investigated the benefits of low-dose aspirin (75–500 mg daily) for primary cardiovascular prevention, including over 95,000 partipants, demonstrated that aspirin increased the incidence of serious extracranial bleeding (usually defined as requiring transfusion or resulting in death) from 0.07% per year to 0.1% per year (HR 1.54, 95% CI 1.30 to 1.82). Furthermore, although participants in the recent CAPP2 study63 were perhaps younger than the anticipated population in FOCUS4-B, in the CAPP2 study aspirin at 600 mg daily for 25 months was not associated with an excess of adverse events compared with placebo.
To minimise potential harm to trial participants, several measures proven to reduce the risk of serious adverse events from aspirin were recommended,64 including exclusion of patients with a high risk of complications (e.g. previous peptic/duodenal ulcer or gastrointestinal bleed); careful management and treatment of symptoms, such as dyspepsia; blood pressure monitoring; and avoidance of concomitant NSAID use. Testing and eradication of Helicobacter pylori was also recommended and proton pump inhibitors were used where appropriate.
Aspirin dosing
Although evidence indicates that aspirin at doses of 75–100 mg daily exerts an antineoplastic effect, at present the mechanism of action of aspirin is unclear and potentially multifactorial. Taking into account the possibility that some or all of these mechanisms could be dose dependent, and the data suggesting that the impact on CRC mortality is greater for patients taking the highest aspirin doses (> 6× 325 mg tablets per week vs. 2–5 × 325 mg per week),65 while recognising that gastrointestinal toxicity is likely to increase with doses higher than 325 mg daily, the recommended aspirin dose for patients in FOCUS4-B is 300 mg daily. This dose permits the evaluation of both the antiplatelet effect of aspirin and possible additional mechanisms underlying its antineoplastic effect.
FOCUS4-B research objectives
FOCUS4-B aims to determine whether or not regular, intermediate-dose aspirin improves PFS when used as maintenance therapy following initial disease stabilisation or response from first-line treatment in patients with PIK3CA exon 9/exon 20 mutant CRC.
Translational studies, including analysis of tumour blocks for other putative aspirin biomarkers and analysis of cell-free DNA, were planned. The purpose of the former was to increase our understanding of determinants of response to antiplatelet therapy in malignant disease, whereas the latter could generate insights into the dynamics of tumour clones under therapy if efficacy was demonstrated.
Study methods
Trial approvals, patient eligibility and recruitment
The trial and subsequent amendments were approved by the UK National Ethics Committee Oxford, panel C, and by the MHRA. Patients with newly diagnosed metastatic CRC were registered into the FOCUS4 trial programme from 88 UK hospitals (). Patients were randomised into the FOCUS4-B trial in all participating hospitals, starting in February 2016. Patients aged ≥ 18 years with newly diagnosed locally advanced or metastatic CRC were assessed for eligibility for FOCUS4-B if their tumour was confirmed to have a PIK3CA exon 9 or exon 20 mutation (using a NGS platform), and if they had remained stable or were responding after 16 weeks of first-line treatment. Patients were required to undergo baseline CT within 4 weeks prior to randomisation; a minimum 3-week washout period after the last dose of chemotherapy or biological therapy and before the first dose of aspirin or matched placebo; adequate renal (creatinine clearance of > 50 ml/minute) and liver function; and a WHO performance status of 0–2.
The aspirin 300-mg tablets and matched placebo were supplied by Bayer AG (Leverkusen, Germany). The packaging, labelling and distribution of aspirin were undertaken by Alcura UK Ltd (Northampton, UK).
Patients randomised to receive aspirin or placebo continued to take the drug until disease progression, death or intolerable toxicity. The patients received 300 mg of aspirin or matched placebo once daily. Patients underwent clinical evaluation after the first 4 weeks from day 1 of randomised trial treatment by a research nurse or doctor to determine if there were any toxicity or tolerability issues. Patients were assessed by CT every 8 weeks from randomisation, together with full reporting of safety outcomes. PFS was the primary outcome measure for FOCUS4-B and, therefore, clinicians were required to use a consistent approach to treatment duration. Trial treatment was planned to be continued until progressive disease was identified on radiological grounds (RECIST v1.1), the development of cumulative toxicity or the patient chose to stop treatment. Patients were considered to be off trial treatment (but still in the trial) if there was a continuous break in their trial treatment of more than 28 days. If this occurred, trial medication was permanently discontinued and this was appropriately documented.
However, patients who discontinued the trial drug for reasons other than objective disease progression were to be followed up with tumour assessments every 8 weeks until objective disease progression as assessed by RECIST v1.1, even if they had started subsequent anti-cancer therapies. Toxicity assessments were to be carried out until the patient stopped trial treatment. On disease progression, patients were to restart their first-line treatment or move onto a standard second-line therapy at the discretion of the treating clinician. After progression, a progress electronic case report form was to be completed at 3 months and then every 6 months until patient death or the end of the trial.
Statistical methods
Treatment allocation
Once patients had been consented and deemed eligible for FOCUS4-B, they were randomly assigned to one of the following treatment arms (in a 2 : 1 ratio for aspirin vs. placebo):
Both patients and treating clinicians were blinded to patient allocation to 300 mg aspirin once daily or placebo.
Outcome measures
The primary outcome for FOCUS4-B was PFS, defined as the time from randomisation to either disease progression (according to RECIST criteria) or death from any cause. OS was a secondary outcome, defined as the time from randomisation to death from any cause. Other secondary outcomes included safety, toxicity and tumour response.
Sample size calculation
For FOCUS4-B, a randomisation ratio of 2 : 1 was used in favour of the active arm. A summary of the operating characteristics for FOCUS4-B, with predicted timelines for the staged interim analyses, is presented in . It was anticipated that all data were to be released at the end of stage 3 to allow an open decision on whether or not to proceed to an assessment of OS.
Operating characteristics for FOCUS4-B: PIK3CA exon 9 or exon 20 mutant cohort
Statistical analysis
All analyses were performed in accordance with a predefined SAP that was agreed before database lock and were undertaken using Stata statistical software, version 16.1 (Stata Corporation, TX, USA). Given that this was a Phase II efficacy signal-seeking study, the primary analysis was prespecified as the per-protocol analysis, which was defined by patients who had completed at least one cycle of trial treatment. Analyses were also performed according to intention to treat. Patients were censored according to the following criteria:
For survival status, patients were censored on the date that they were last known to be alive because they collected a prescription from their hospital pharmacy or attended a follow-up visit or a
CT appointment.
For
PFS, patients were censored without progression on the date of the last
CT scan, showing no progression.
For patients who died before any follow-up visit or
CT appointment, the date of death was used as the date of the event and assumed death without progression providing the death occurred within 3 months of randomisation or any previous
CT scan confirming no progression.
Kaplan–Meier curves were used to present survival data and Cox regression modelling to estimate HRs between randomised groups. Unadjusted HRs were estimated, as well as HRs adjusted for the stratification factors that were used to minimise patients into allocated groups (primary analysis). A further analysis also adjusted for resection status, timing of metastatic disease, alkaline phosphatase, white blood cell count, age of tumour sample and use of aspirin at baseline. Deviation from non-proportional hazards was assessed using regression of scaled Schoenfeld residuals against the log of time.
Study results
Recruitment and patient characteristics
By December 2017, a total of 47 PIK3CA-mutated patients had been identified. Of these patients, six entered FOCUS4-B, four entered FOCUS4-N, 12 progressed, nine refused owing to patient or clinician decision, two were not randomised owing to toxicity concerns, five were ineligible for various reasons, two died during their first-line therapy and seven were still in the registration phase. Between June 2016 and September 2017, six patients were randomised into FOCUS4-B using a 2 : 1 ratio: four pateints to aspirin and two patients to placebo. An overview of the baseline and demographic characteristics for the six patients randomised into FOCUS4-B is presented in .
Baseline and demographic characteristics of patients randomised into FOCUS4-B
Outcome data
All four patients allocated to aspirin were recorded as having commenced trial treatment within 1 week of randomisation. At the time of database lock (4 November 2020), three of the four patients were recorded as having stopped trial treatment (at 28 days, 56 days and 61 days after randomisation).
At the time of database lock, all six patients had progressed. Five patients progressed at between 53 and 56 days (approximately 8 weeks) after randomisation, and the sixth patient (allocated to aspirin) progressed at 84 days (approximately 12 weeks) after randomisation.
In addition, three of the six patients were recorded as having died: two patients allocated to aspirin (with death occurring at 6 months and 22 months after randomisation) and one patient allocated to placebo (with death occurring at 8 months after randomisation). Only one moderate toxicity symptom was recorded: a grade 3 hypertension occurring in a patient allocated to aspirin. No adverse events were recorded.
Early closure of FOCUS4-B
In view of the very poor recruitment for FOCUS4-B, discussions took place between the FOCUS4 TMG and the oversight committees (FOCUS4 TSC and IDMC) on whether or not to close FOCUS4-B on the grounds of recruitment futility. Both committees recommended in July 2018 that this comparison should be closed and that data cleaning and analysis of the data for these six patients should be completed.
Discussion and conclusions
This cohort in FOCUS4 investigated the use of aspirin in a molecular-defined cohort of patients in whom previous laboratory and considerable epidemiological research had suggested potential for considerable benefit. Unfortunately, our enthusiasm for this subtrial was not matched by the successful recruitment of patients; therefore, early closure was necessary after we had enrolled only six patients. We made repeated efforts to boost recruitment, but all of these were ultimately unsuccessful. We explored the reasons for this and they were very varied, but more common findings were:
Patients felt that they could buy aspirin themselves over the counter if they strongly believed our hypothesis and that it may benefit them.
Conversely, some patients and doctors felt that a common drug being repurposed like aspirin was highly unlikely to be beneficial despite all the supporting laboratory and epidemiological data.
Although hugely disappointing, the results are important in showing the challenges involved in using repurposed and freely available drugs in this experimental setting. Aspirin continues to be of considerable interest to the cancer research community and is being investigated in many other cancer trials, including in the adjuvant Add-Aspirin trial57 in earlier stage colorectal and other cancers.
