Panitumumab plus trifluridine/tipiracil as anti-Epidermal Growth Factor Receptor rechallenge therapy in chemo-refractory RAS wild-type metastatic colorectal cancer: the randomized phase 2 VELO trial

doi:10.21203/rs.3.rs-2187013/v1

Current therapies for chemo-refractory metastatic colorectal cancer (mCRC) have limited efficacy. Rechallenge with epidermal growth factor receptor (EGFR) inhibitors in RAS wild-type (WT) mCRC could be valuable in this setting. In VELO, a randomized two-arm phase 2 trial, anti-EGFR monoclonal antibody panitumumab plus standard-of-care trifluridine/tipiracil (31 patients, arm B) was compared to trifluridine/tipiracil (31 patients, arm A) as third-line therapy (ClinicalTrials.gov Identifier NCT05468892). Primary endpoint, progression-free survival (PFS), was met. Median PFS was 4.0 months in arm B versus 2.5 months in arm A [hazard ratio (HR): 0.48; 95% CI 0.28–0.82; P = 0.007]. Baseline plasma RAS/BRAF WT circulating tumor DNA identified patients obtaining prolonged clinical benefit with panitumumab plus trifluridine/tipiracil as compared to trifluridine/tipiracil with PFS rates at 6 months of 38.5% versus 13% and at 12 months of 15.4% versus 0%, respectively. These findings warrant further development for liquid biopsy-guided anti-EGFR rechallenge combination strategies in chemo-refractory RAS WT mCRC.

Health sciences/Medical research/Translational research

Health sciences/Health care

In recent years, treatment of metastatic colorectal cancer (mCRC) has improved with the identification of key pathways, that are involved in various steps of cancer development and progression, and that can be blocked by selective anticancer drugs, which target molecules known to control cancer cell proliferation and survival¹. In this scenario, the epidermal growth factor receptor (EGFR) pathway plays a key role by promoting a variety of cancer functions, including proliferation, survival, invasion, and immune escape². Anti-EGFR monoclonal antibodies (cetuximab or panitumumab) in combination with standard cytotoxic drugs (FOLFIRI or FOLFOX) have been validated as options for treatment of mCRC patients with RAS (KRAS and NRAS) wild type (WT) disease^1,3,4. However, despite initial significant clinical efficacy by EGFR blockade, the clonal expansion of pre-existing EGFR-resistant cancer cells and/or the ability of cancer cells to develop acquired resistance mechanisms to anti-EGFR therapy will eventually concur to treatment failure and disease progression in all patients^5,6,7,8. In this respect, approximately 30 to 40% of mCRC patients with RAS WT tumor after treatment with cetuximab or panitumumab will develop KRAS or NRAS mutations, which are responsible for cancer cell resistance^7,8,9. Moreover, limited treatment options are available for RAS WT mCRC patients after failure of two lines of therapies with all available cytotoxic drugs (i.e., FOLFIRI and FOLFOX), with anti-EGFR drugs and with anti-angiogenic drugs (bevacizumab, aflibercept or ramucirumab)^1,3,4. In fact, for these chemo-refractory mCRC patients, the only two therapeutic options, whose use is supported by randomized phase 3 data, are regorafenib and trifluridine/tipiracil^10,11. Both drugs determine limited benefit in terms of overall survival (OS) and progression-free survival (PFS) as compared to placebo^10,11,12. However, so far these drugs are the only two approved treatments for chemo-refractory mCRC. Therefore, more effective therapeutic options are needed for the medical management of these patients.

Rechallenge therapy with EGFR inhibitors in RAS WT mCRC patients, that had a major clinical response to a previous therapy containing anti-EGFR drugs, has been proposed in chemo-refractory mCRC^7,13,14. There is a clear biologic rationale for this strategy. The majority of RAS WT cancer cells could be initially killed by treatment with chemotherapy plus cetuximab or panitumumab. At the same time, during anti-EGFR therapy, a genetic selection of RAS mutant cancer cells occurs. These cells become the prevalent cancer clones and will be responsible for tumor progression. However, a subsequent different treatment, such as chemotherapy plus anti-angiogenic drugs, could kill anti-EGFR resistant clones, causing the disappearance of RAS mutant clones and could potentially restore sensitivity to anti-EGFR monoclonal antibodies^14,15. In fact, it has been reported that, after stopping anti-EGFR treatment and introducing other anticancer therapies, a rapid decay in RAS mutant cancer cell clones occurs with half-life of approximately 4 months, whereas RAS WT cancer cell clones increase¹⁶. These findings support the concept that the genetic landscape of cancer cell clones is continuously evolving and changing over time due to the selective pressure of different therapies¹⁷.

Three phase 2 trials have recently provided evidence for anti-EGFR rechallenge therapies in chemo-refractory RAS WT mCRC^18,19,20. Antitumor activity was reported for anti-EGFR monoclonal antibodies as monotherapy (panitumumab in the CHRONOS trial)¹⁸, in combination with chemotherapy (cetuximab plus irinotecan in the CRICKET trial)¹⁹, or in combination with immunotherapy (cetuximab plus avelumab in the CAVE trial)²⁰. Further, these studies have highlighted the relevance of assessing plasma circulating tumor DNA (ctDNA) for RAS and BRAF mutations to select chemo-refractory RAS/BRAF WT mCRC patients, that could have clinical benefit from this approach. However, these three studies were proof-of-concept, single arm trials with relatively small sample size. Important questions are still not addressed, such as which is the best anti-EGFR rechallenge approach (anti-EGFR drug monotherapy or in combination with other anticancer agents, including chemotherapy or immunotherapy), and how anti-EGFR rechallenge compares to standard-of-care in terms of clinical activity in this disease setting¹⁴.

For this purpose, we conducted VELO, a multicenter, prospective, randomized phase 2 trial. To determine the contribution of anti-EGFR rechallenge to standard-of-care therapy, the addition of panitumumab to trifluridine/tipiracil was compared to trifluridine/tipiracil as third-line therapy in chemo-refractory RAS WT mCRC patients, that were treated in first-line with chemotherapy plus an anti-EGFR drug, progressed after having obtained a complete or a partial response from treatment and received a second-line therapy without EGFR inhibitors. Moreover, plasma samples were prospectively collected and analyzed for plasma ctDNA in order to define gene mutation profiles, which could allow better selection of patients that would benefit of anti-EGFR rechallenge.

Patient characteristics and enrollment in the VELO trial. Between March 2019 and April 2022, 68 RAS WT mCRC patients with disease progression after two lines of therapy were screened and assessed for eligibility. Sixty-two patients [intention to treat (ITT) patient population] were enrolled and randomly assigned (1:1) to receive panitumumab plus trifluridine/tipiracil (arm B, 31 patients) or trifluridine/tipiracil alone (arm A, 31 patients) (Fig. 1). Baseline demographic and disease characteristics were balanced between the two arms (Table 1). The median age was 66 years (range, 32–82) and 65 years (range, 39–81), in arms A and B, respectively. Performance status was 0 in the majority of patients (22/31, 71.0% and 21/31, 67.7%, in arms A and B, respectively). According to the site of primary tumor localization, the majority of cancers were in the left colon and rectum (27/31, 87.1%, and 28/31, 90.3%, in arms A and B, respectively). The primary tumor was resected in the majority of patients in both arms (25/31, 80.6% and 26/31, 83.9%, in arms A and B, respectively). In the majority of patients, metastatic disease was detected at the time of diagnosis (21/31, 67.7% in arm A and 22/31, 71.0% in arm B) (Table 1).

All 62 patients had received first-line treatment with chemotherapy (FOLFIRI or FOLFOX) plus an anti-EGFR monoclonal antibody [panitumumab in 23/31 (74.2%) patients in arm A and in 21/31 (67.7%) patients in arm B; or cetuximab in 8/31 (25.8%) patients in arm A and in 10/31 (32.3%) patients in arm B]. All 62 patients had achieved objective response (complete or partial response, CR or PR) as best response from first-line treatment. As per protocol, after progression during first-line treatment, all patients were treated with second-line therapy (FOLFOX or FOLFIRI plus bevacizumab or aflibercept) without EGFR inhibitors with an anti-EGFR drug-free interval of at least four months or more (Supplementary Materials Appendix 1 and 2).

Although results of liquid biopsy were not required for patient randomization and for treatment allocation, plasma was collected from all 62 patients at baseline and liquid biopsy analysis was performed by using Idylla™ Biocartis platform [threshold for positivity, variant allele fraction (VAF) ≥ 5%] for detecting in the plasma circulating tumor (ct) DNA activating hot spot mutations in KRAS and NRAS (exons 2, 3 and 4) genes and BRAFV600E mutation, that are known as major cancer cell resistance mechanisms to anti-EGFR therapies in mCRC^8,21. No BRAFV600E mutations were found. Baseline plasma RAS/BRAF WT ctDNA was observed in 23/31 (74.2%) patients in arm A and in 26/31 (83.9%) patients in arm B; whereas RAS mutations were found in 8/31 (25.8%) and in 5/31 (16.1%), respectively (Table 1).

Clinical activity of panitumumab plus trifluridine/tipiracil as compared to trifluridine/tipiracil monotherapy. In both arms, therapy was administered until treatment failure (disease progression). No patient stopped therapy for unacceptable toxicity. At the data cut off on September 16th, 2022, the median number of cycles that were administered was 4 cycles (range, 1–26) in arm B, while it was 2 cycles (range, 1–10) in arm A (Table 2A). One patient in arm A (from 9.5 months) and 2 patients in arm B (from 5.5 and 11 months) were still on treatment.

Treatment-emergent adverse events of any cause occurred in 30/31 (97%) patients in arm B and in 24/31 (77%) patients in arm A. Grade 3–4 treatment-related adverse events occurred in 16/31 (52%) patients in arm B and in 9/31 (29%) patients in arm A (Table 2B). There were no treatment-related deaths and no treatment discontinuation was reported. Hematologic and non-hematologic toxicities were similar in terms of occurrence and grade between the two arms (P = 0.80 and P = 0.45, respectively). However, anti-EGFR drug-related skin toxicities were only observed in the combination treatment arm as compared to trifluridine/tipiracil monotherapy (P = 0.0001). Dose reductions were necessary in 16/31 (52%) patients in arm B and in 9/31 (29%) patients in arm A (P = 0.07) (Table 2B).

The primary endpoint of the study was PFS in the 62 ITT patient population. The primary endpoint was met. In fact, treatment with panitumumab plus trifluridine/tipiracil determined a better PFS as compared to standard-of-care trifluridine/tipiracil monotherapy with 52% reduction in the risk of progression [hazard ratio (HR): 0.48; 95% confidence interval (CI) 0.28–0.82; P = 0.007]. Median PFS was 2.5 months (95% CI 1.36–3.63) in arm A, while it was 4.0 months (95% CI 2.75–5.26) in arm B (Fig. 2A).

Objective responses, such as PR and CR, as well as disease control rate (DCR: PR + CR + stable disease, SD, of 4 months or more) were secondary endpoints of the trial. In the 62 ITT patient population, no CR was reported. Confirmed PRs were observed only in arm B (3/31 patients; 9.7%). Disease control rates were 74.2% in arm B as compared to 38.7% in arm A (P = 0.009) (Fig. 3A). In addition, PFS rates at 6 and at 12 months were significantly higher in patients that were treated with panitumumab plus trifluridine/tipiracil (6 months PFS rate: 9.7% versus 35.5% in arms A and B, respectively; P = 0.016; 12 months PFS rate: 0% versus 12.9% in arm A and B, respectively; P = 0.040) (Fig. 3D). Unfortunately, disease progression was reported as the best response in the majority of patients in arm A (19/31, 61.3%); whereas it was observed in 8/31 (25.8%) patients in arm B.

Clinical activity of panitumumab plus trifluridine/tipiracil in baseline plasma RAS/BRAF WT ctDNA patients. We next evaluated if mutation profiling of baseline plasma ctDNA could allow to better identify patients that would benefit more of anti-EGFR rechallenge therapy with panitumumab plus triflirudine/tipiracil as compared to those treated with trifliridine/tipiracil alone. In a pre-specified subgroup analysis of clinical activity in patients with baseline plasma RAS/BRAF WT ctDNA disease, median PFS was 4.5 months (95% CI 2.21–6.78) in arm B (26 patients) versus 2.6 months (95% CI 0.98–4.27) in arm A (23 patients) (HR: 0.48; 95% CI 0.26–0.89; P = 0.019) (Fig. 2B). No RAS mutations were detected in baseline plasma ctDNA from the 3 patients that achieved a confirmed PR in arm B. Further, DCR was 80.7% in baseline plasma RAS/BRAF ctDNA WT patients that received panitumumab plus trifluridine/tipiracil compared to 47.8% in patients treated with trifluridine/tipiracil (Fig. 3B). The probability of being free from progression at landmark time points was also higher in baseline plasma ctDNA RAS/BRAF WT patients that were treated with panitumumab plus trifluridine/tipiracil (6 months PFS rate: 38.5% versus 13.0% in arms B and A, respectively; P = 0.047; 12 months PFS rate: 15.4% versus 0% in arm B and A, respectively; P = 0.052) (Fig. 3D and 3E). On the contrary, treatment was less active in both arms in patients with baseline plasma RAS mutant ctDNA disease with no advantage by adding panitumumab to trifluridine/tipiracil (HR: 0.72; 95% CI 0.15–1.75; P = 0.29) (Fig. 2C).

Extended molecular profiling by next generation sequencing analysis of baseline plasma ctDNA. The analysis of plasma ctDNA by using Idylla™ Biocartis platform suggested that patients that had no detectable plasma ctDNA RAS/BRAF mutations at the time of anti-EGFR rechallenge therapy would experience relevant clinical benefit by treatment with panitumumab plus trifluridine/tipiracil; thus highlighting the predictive value of liquid biopsy as a tool to inform therapeutic decisions in this setting. To explore if the use of a comprehensive genomic profiling by next generation sequencing (NGS) could help to identifying additional cancer cell resistance mechanisms to anti-EGFR drugs in order to better tailoring rechallenge treatment, a panel of 324 genes was evaluated with the use of FoundationOne Liquid CDx, that was performed on baseline plasma samples in 46/62 (74.2%) patients (Fig. 1). The complete list of gene alterations that were found is presented in Supplementary Table 1. Complete concordance for hot spot KRAS and NRAS mutations was observed between baseline plasma ctDNA analysis that was performed with Idylla™ Biocartis platform and FoundationOne Liquid CDx results. RAS WT ctDNA was confirmed in 39 patients and RAS mutant ctDNA was confirmed in the other 7 patients, in which FoundationOne Liquid CDx analysis was done. Collectively, extended molecular profiling by baseline NGS liquid biopsy was avalaible for 16/23 (69.6%) patients with RAS/BRAF WT ctDNA in arm A and for 23/26 (88.5%) patients with RAS/BRAF WT ctDNA in arm B. NGS analysis found that KRAS, PIK3CA, BRAF, MAP2K1, EGFR and ERBB2 were among the most frequently mutated genes within the EGFR pathway, whereas TP53, APC, ARID1A, and SMAD4 were the most frequently mutated tumor suppressor genes (Supplementary Table 1). APC and TP53 mutations were found in almost all patient samples (93% and 92% of cases, respectively).

We next focused on baseline plasma RAS/BRAF WT ctDNA mCRC patients in order to find additional resistance mechanisms which were not detectable by PCR-based analysis by Idylla™ Biocartis platform. A schematic representation of additional gene mutations, that were detected by FoundationOne liquid CDx, is illustrated in Fig. 4 for each individual patient according to PFS. For patients that were treated with trifluridine/tipiracil alone, no clear evidence of specific gene alterations that could be associated with cancer cell resistance was observed. Mutations and/or amplifications in the EGFR pathway were found independently of PFS duration (Fig. 4A). In contrast, for patients that were treated with panitumumab plus trifluridine/tipiracil, additional gene alterations, which could be responsible for cancer cell resistance to anti-EGFR drugs, were detected (Fig. 4B). In this respect, KRASQ61L mutation was found in patient 66. NGS analysis of baseline plasma ctDNA identified two mutations: KRASG12S (at low VAF) and ERBB2G776V in patient 60. ERBB2V777L gene amplification was found in patient 49. KRAS gene was amplified in patient 54. PIK3CA E542K mutation was found in patient 58. EGFRG465E mutation was detected in patient 52. Finally, BRAFV600E mutation (that was not detected by analysis with Idylla™ Biocartis platform since it was below its detection limit) was found together with MAP2K1K57T, PIK3CAE542K and ARID1A splice site 4101 + 1G > A mutations in patient 39. However, a long PFS (13.5 months) was observed in patient 3, in whose baseline plasma ctDNA KRASQ61R and PIK3CAG1049R mutations were found, suggesting that not all KRAS and PIK3CA mutations are necessarily correlated with cancer cell resistance to EGFR inhibitors (Fig. 4).

Extended molecular profiling by next generation sequencing analysis of plasma ctDNA before and after therapy. To detect cancer cell resistance mutations, which could raise during therapy, an exploratory analysis was performed on 24 plasma RAS/BRAF WT ctDNA patients (13 in arm A and 11 in arm B) for whom FoundationOne Liquid Cdx was available both at baseline and at disease progression (Fig. 5 and Supplementary Table 2). Of note, the acquisition of one or more mutations in the EGFR pathway were found in the post-progression liquid biopsy analysis of 6/11 patients treated with panitumumab plus trifluridine/tipiracil for the following genes: KRAS (4 patients), EGFR (3 patients), BRAF (3 patients), MAP2K1 (1 patient), and PIK3CA (1 patient).

To our knowledge, the VELO trial reported here is the first prospective, randomized phase 2 clinical study, in which the combination of an anti-EGFR drug (panitumumab) with a standard-of-care drug (trifluridine/tipiracil) has been evaluated as anti-EGFR rechallenge therapy for chemo-refractory RAS WT mCRC. The study met the primary endpoint. In fact, in the 62 ITT patient population significantly better PFS was observed for treatment with panitumumab plus trifluridine/tipiracil. The risk of disease progression was reduced of 52% and the disease control rate was almost doubled (74.2% versus 38.7%), as compared to patients that received trifluridine/tipiracil alone.

The findings of the VELO trial support the use of pre-treatment plasma ctDNA analysis for anti-EGFR rechallenge patient selection^14,18,19,20. In fact, chemo-refractory mCRC patients with baseline plasma RAS/BRAF WT ctDNA, that were treated with panitumumab plus trifluridine/tipiracil, had a significantly better outcome in terms of median PFS (4.5 versus 2.6 months), of reduced risk of disease progression (HR, 0.48), and of objective responses (PR, 11.5% versus 0%). Clinically relevant prolonged disease control was also obtained. In this respect, the 6 months probability of being on treatment without progression was almost three times higher for patients that received the combined treatment (38.5% versus 13.0%). Finally, progression-free times 12 months or more were reported only for patients treated with panitumumab plus trifluridine/tipiracil (15.4% of patients). The clinical benefit of panitumumab plus trifluridine/tipiracil was accompanied by an acceptable safety profile. No unexpected toxicities were reported. Hematologic adverse events were similar between the two treatment arms, while anti-EGFR drug-related skin toxicities were observed after panitumumab plus trifluridine/tipiracil therapy.

These results favorably compare with single arm phase 2 trials of anti-EGFR rechallenge combination therapies, such as CRICKET¹⁹, in which treatment consisted of cetuximab plus irinotecan, and CAVE²⁰, in which patients received cetuximab plus avelumab. In CRICKET, PRs were recorded in 4/13 patients with baseline plasma RAS/BRAF WT ctDNA, with median PFS of 4.0 months; whereas in CAVE, one CR, 3 PRs, 21 SD ≥ 4 months were observed in 48 patients with baseline plasma RAS/BRAF WT ctDNA, with median PFS of 4.1 months. More recently, Sartore-Bianchi et al. have evaluated the antitumor activity of panitumumab monotherapy in CHRONOS, a proof-of concept, single arm phase 2 trial, in which baseline plasma ctDNA was used to guide patient selection for anti-EGFR rechallenge therapy¹⁸. Among the 52 patients that were screened, 36 patients had plasma RAS/BRAF and EGFR extracellular domain WT ctDNA and were eligible for treatment. In the 27 patients, that were subsequently treated with panitumumab monotherapy, 6 confirmed plus 2 non confirmed PRs were observed, with a global disease control rate (PR + SD ≥ 4 months) of 63%.

The most common mechanisms of either intrinsic or acquired cancer cell resistance to EGFR inhibitors are KRAS and NRAS mutations^{21, 22, 23, 24, 25}. The majority occurs within hot spot regions, such as codons 12 or 13 of exon 2, as single nucleotide changes with amino acid substitutions and functional changes in RAS protein²¹. However, several additional gene alterations, that affect the EGFR signaling pathways, could be responsible for anti-EGFR drug resistance, including BRAFV600E mutation, KRAS amplification, EGFR mutations, PIK3CA mutations, HER2 amplification and/or mutations, MET amplification and/or mutations^{1,2,6,7,8,23,24,25,26}. Since the PCR-based Idylla™ Biocartis platform^27,28, that we have used for measuring baseline plasma ctDNA in the 62 ITT patient population, would capture only known mutations in the hotspot regions of exons 2, 3, and 4 for KRAS and NRAS, as well as the BRAFV600E mutation, NGS-based 324 genomic profiling of plasma ctDNA by using FoundationOne Liquid CDx was performed in 46/62 (74.2%) patients at baseline, before receiving protocol treatment^29,30. There was a complete concordance between the PCR- and the NGS-based liquid biopsy analyses for detecting the known RAS resistance mutations. In addition, in one patient, NGS analysis allowed to detect BRAFV600E mutation with a VAF, that was below the detection level of the PCR-based technique. Moreover, extended genomic analysis of baseline plasma ctDNA identified one or more additional mutations and/or amplifications in different EGFR pathway genes, that correlated with lack of clinical activity of panitumumab plus trifluridine/tipiracil, in 6/23 (23.0%) patients with baseline plasma RAS/BRAF WT ctDNA. These results suggest that NGS-based analysis of baseline plasma ctDNA could allow a more precise selection of chemo-refractory mCRC patients that would benefit of anti-EGFR rechallenge therapies. Furthermore, NGS analysis of plasma ctDNA was explored in a subgroup of 24 patients in the VELO trial before treatment and at disease progression. No clear correlation was observed between the two measurements for 13 patients that were treated with trifluridine/tipiracil alone. Of note, in 6/11 patients, that received panitumumab plus trifluridine/tipiracil, one or more plasma ctDNA (KRAS, EGFR, BRAFV600E, MAP2K1, and/or PIK3CA) mutations were found at disease progression. This exploratory analysis suggests that the emergence of cancer cell resistance to therapy was associated with mutations in the EGFR pathway in the majority of patients treated with panitumumab plus trifluridine/tipiracil. These findings are in agreement with similar exploratory subgroup analysis of plasma ctDNA at disease progression in the CAVE and CHRONOS trials^18,20.

In summary, with all the limitations of a relatively small size, randomized phase 2 trial, the results of the VELO trial are the first evidence of improved clinical activity of anti-EGFR rechallenge therapy in a subset of chemo-refractory mCRC patients (i.e., patients with baseline plasma RAS/BRAF WT ctDNA) as compared to standard-of-care, supporting panitumumab plus trifluridine/tipiracil treatment as a valid therapeutic option. Larger, randomized phase 3 studies will be required to confirm the clinical efficacy of this approach. In this respect, several randomized trials are currently ongoing to better define the role of anti-EGFR rechallenge strategies in chemo-refractory RAS/BRAF WT mCRC. A randomized phase 2 trial (PARERE, (ClinicalTrials.gov Identifier NCT04787341) is currently evaluating the best therapeutic sequence between panitumumab and regorafenib³¹. CAVE-2 (ClinicalTrials.gov Identifier NCT05291156), a randomized phase 2 study, is comparing cetuximab as monotherapy or in combination with avelumab as rechallenge therapy in mCRC patients with baseline plasma RAS/BRAF WT ctDNA³². Moreover, to better define the role of anti-EGFR therapies within the continuum of care of mCRC, the CAPRI-2 trial (ClinicalTrials.gov Identifier NCT05312398), in which treatment of RAS/BRAF WT mCRC patients is driven by the results of liquid biopsy-assessed plasma RAS/BRAF ctDNA status throughout three lines of therapy, is currently ongoing. In this study, patients with plasma RAS/BRAF WT ctDNA at the time of third-line treatment will receive cetuximab plus irinotecan. Finally, the combination of trifluridine/tipiracil with futuxumab/modotuximab (a mixture of two anti-EGFR monoclonal antibodies) is being currently explored in the randomized, phase 3 COLSTAR trial (ClinicalTrials.gov Identifier NCT05223673). Although the results of these studies will be needed to better understand which is the most effective treatment choice (anti-EGFR monoclonal antibody as monotherapy or in combination with different anticancer drugs), the results of the VELO trial clearly support the clinical validity of liquid-biopsy guided anti-EGFR rechallenge therapy in chemo-refractory mCRC.

Study Design and patient population. VELO is a non-profit, academic, multicenter, open label two-arms, randomized phase 2 study. Patients were recruited in 7 Italian Medical Oncology Units. The study has been registered at ClinicalTrials.gov (NCT05468892) and has received the Italian Drug Agency (AIFA) approval (The full study protocol is available online. See Supplementary Materials Appendix 1). The trial was conducted in accordance with the Declaration of Helsinki and adhered to the international Good Clinical Practice guidelines. The protocol was approved by the local ethics committees of the participating sites. All patients provided written informed consent to study procedures. Patients with RAS (NRAS and KRAS, exon 2, 3, 4) WT, histologically confirmed mCRC were enrolled in the trial. Patients should have obtained an objective response (CR or PR) during first-line chemotherapy (FOLFIRI or FOLFOX) plus an anti-EGFR monoclonal antibody (panitumumab or cetuximab) and should have progressed. Patients should have received a second-line therapy after first-line treatment failure. An interval of 4 months or more from the last dose of the anti-EGFR drug that was administered in the first-line treatment was mandatory for enrollment. Additional inclusion/exclusion criteria are detailed in the Supplementary Material Appendix 2.

Patients were randomized (1:1) to receive as third-line therapy trifluridine/tipiracil (standard-of-care treatment, arm A) or panitumumab plus trifluridine/tipiracil (experimental treatment, arm B). Stratification criteria for randomization were baseline Eastern Cooperative Oncology Group (ECOG) performance status (PS) (0 versus 1); prior anti-EGFR monoclonal antibody (panitumumab, at least in 70% of study population versus cetuximab); primary tumor localization (right colon vs left colon and rectum). Panitumumab was administered as 6 mg/kg intravenous infusion over 60 minutes, every 2 weeks of a 28-day cycle (Day 1 and Day 15). Trifluridine/tipiracil was administered at 35 mg/m² orally twice daily, with a glass of water within 1 hour after completion of morning and evening meals, 5 days a week, with 2 days of rest, for 2 weeks, followed by a 14-day rest period. Treatment was administered in 28-days cycles until disease progression, unacceptable toxicity, withdrawal of consent or death due to any cause. All patients were closely monitored for safety and tolerability during all cycles of therapy, at the treatment discontinuation visit, and during the follow-up period. The NCI/common toxicities adverse events (CTAE) v.5.0 were used to characterize the toxicity profile of the study treatments. Patients returned to the clinic at regular intervals for assessments. Tumor measurements by computed tomography (CT) scan or magnetic resonance imaging (MRI) were performed every 8 weeks. Responses were evaluated using the RECIST 1.1 criteria. Patients, that would discontinue treatment for reasons other than disease progression (e.g., toxicity), would have continued the scheduled tumor assessments until disease progression, withdrawal of consent, study termination, or death, whichever would occur first. In the absence of disease progression, tumor assessments should have continued regardless of whether patients start a new anti-cancer therapy, unless consent would be withdrawn.

Molecular analysis of plasma ctDNA. Blood samples were collected and analyzed for plasma ctDNA by using Idylla™ Biocartis platform and FoundationOne Liquid CDx (324 genes profiling)^27,28,29,30. In particular, all 62 patients (ITT population), that were enrolled in the trial, were prospectively assessed at baseline by using the Idylla™ Biocartis platform to detect known EGFR-inhibitor cancer cell resistance hot spot mutations in KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), as well as BRAFV600E mutation. This analysis was performed in the laboratory of the coordinating center (Department of Precision Medicine, Università degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy)^27,28. At least 6 ml of whole blood were collected by standard procedures with peripheral vein blood draw, using Vacutainer® with EDTA as anticoagulant (K2EDTA, purple cap, catalog #367863, Becton Dickinson). Plasma was separated through two different centrifugation steps (the first at room temperature for 10 min at 1500 g and the second at 2000 g for the same time and temperature). Plasma was stored at -80°C until analysis. The Idylla™ Biocartis platform was used as previously reported^27,28. Detection limit for positivity for RAS or BRAFV600E mutations was ≥ 5% VAF. In addition, plasma samples from 46/62 (74.2%) patients at baseline were collected and analyzed for the ctDNA mutation profiling of 324 genes by NGS, that was performed with Foundation One Liquid CDx, according to the Manufacturer’s procedures^29,30. For 24/46 patients plasma samples were also analyzed at disease progression with Foundation One Liquid CDx. Briefly, 2 anti-coagulated peripheral whole blood tubes (8.5 ml per tube) were collected and sent at room temperature to Foundation Medicine (Roche Foundation Medicine, INC, Penzberg, Germany). This test requires ≥ 25 ng of plasma DNA. Extracted DNA undergoes to whole-genome library construction. The libraries are sequenced with deep coverage using the NovaSeq® 6000 platform. Sequence data are processed using a custom analysis pipeline designed to detect genomic alterations, substitutions and indels, CNAs, genomic rearrangements, tumor fraction, blood tumor mutational burden and MSI-H status. This test identifies 324 cancer-related genes, of which 309 genes with complete exon (coding) coverage and 15 genes with also selected noncoding coverage.

Statistical analysis. The primary endpoint of this randomized phase 2 trial was PFS, which was defined as the time from randomization to the earliest documented disease progression or death due to any cause. The median period of follow-up was been calculated for the entire study cohort according to the reverse Kaplan–Meier method. Distributions of time-to-event variables was estimated with the use of the Kaplan–Meier product-limit method. The stratified log-rank test was used as the primary analysis for comparison of treatment groups. Cox proportional-hazards modeling was also performed as supportive analyses. The objective response rate and the incidence of adverse events in the different arms was compared with the use of the χ square test. All statistical tests were two-sided, and P values of 0.05 or less were considered to indicate statistical significance. Hazard ratios and 95% confidence intervals were estimated with the Cox proportional hazards model. Secondary endpoints were overall response rate (ORR), safety and overall survival (OS). Analysis of the primary endpoint as well as the secondary endpoints according to baseline RAS/BRAF mutation status was pre-specified. Additional exploratory endpoints included genomic analysis by NGS of plasma samples, that were collected at baseline and, if possible, at end of treatment in order to identify other ctDNA mutations, which could be related to cancer cell resistance mechanisms.

The study was designed to have 80% power to detect hazard ratio for progression of 0.56 (44% reduction in risk) in the panitumumab plus trifluridine/tipiracil arm as compared to the standard-of-care trifluridine/tipiracil arm, with a two-sided type I error rate of 0.1. Given the treatment assignment ratio of 1:1, 74 events (disease progression) would have been required for the primary analysis. Unfortunately, clinical trial activation in several centers as well as patient enrolment for this non-profit, academic study was severely affected by the COVID-19 pandemic. Therefore, the planned patient enrolment was not reached and in April 2022 it was decided to stop recruitment when 62 patients had entered the trial. Analysis was performed for the primary endpoint in September 2022, when 59 events (disease progression) occurred. However, as reported in Results, even with this reduction in events, the primary endpoint of the study was met with a reduction of the risk for PFS of 52%. In fact, the HR in favor of panitumumab plus trifluridine/tipiracil as compared to trifluridine/tipiracil monotherapy was 0.48 (95% CI 0.28–0.82; P = 0.007), that is inferior to the planned HR (0.56), that would have been originally required for considering the trial positive.

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Aknowledgements

This was an academic nonprofit study, that was conducted in seven Italian centers. Study drugs were kindly provided free of charge by Amgen (panitumumab) and Servier (trifluridine/tipiracil). A research grant, that partially covered the costs of the study, was provided by Regione Campania (I-Cure Research Project, grant number: Cup 21C17000030007) to FC and LA.

Author Contributions

Conception and design of the trial: FC, TT, and SN. Financial support: FC and LA. Acquisition, analysis and interpretation of the clinical data: all authors. Statistical analysis: VF, LE, and MDM. Translational research data analysis and interpretation: FC, TT, SN, DC, GM, ADL, LA, VF and LE. Writing of the manuscript: FC, TT, SN. Final approval of the manuscript: all authors.

Competing interests

FC, advisory board member for Amgen, Roche, Merck KGaA, Pfizer, Bayer, Servier, MSD, Pierre Fabre, Eisai. TT, advisory board member for Amgen, MSD, Pierre Fabre, Roche, Merck KGaA, Sanofi. SN, travel grants from Amgen and Merck KGaA. DC, travel grants from Sanofi and Merck KGaA. AA, advisory board member for Amgen and Servier. EM, advisory board member for AstraZeneca, Amgen, Bayer, Merck KGaA, Roche, Sanofi, Servier, Pierre Fabre. All other authors have no competing interests to declare.

Table 1

BASELINE CHARACTERISTICS	ARM A n=31 (%)	ARM B n=31 (%)	P Value
Sex
Male	17 (54.8)	19 (61.3)	0.61
Female	14 (45.2)	12 (38.7)

Median Age	66 (32-82)	65 (39-81)

Age Groups
≤ 65	15 (48.4)	16 (51.6)	0.80
> 65	16 (51.6)	15 (48.4)

ECOG PS
0	22 (71.0)	21 (67.7)	0.78
1	9 (29.0)	10 (32.3)

Site of primary tumor
Right	4 (12.9)	3 (9.7)	0.71
Left	20 (64.5)	23 (74.2)
Rectum	7 (22.6)	5 (16.1)

First line anti-EGFR drug
Panitumumab	23 (74.2)	21 (67.7)	0.58
Cetuximab	8 (25.8)	10 (32.3)

Resected primary tumor
Yes	25 (80.6)	26 (83.9)	0.74
No	6 (19.4)	5 (16.1)

Synchronous metastases
Synchronous	21 (67.7)	22 (71.0)	0.78
Metachronous	10 (32.3)	9 (29.0)

Baseline ctDNA
*RAS* Wild-type	23 (74.2)	26 (83.9)	0.35
*RAS* Mutated	8 (25.8)	5 (16.1)
*BRAFV600E* Wild-type	31 (100)	31 (100)
*BRAFV600E* Mutated	-	-

Table 2

	ARM A n=31 (%)	ARM B n=31 (%)	P Value
Adverse Event (AE) grade
1-2	15 (48.4)	14 (45.2)	0.039
3-4	9 (29)	16 (51.6)
NO	7 (22.6)	1 (3.2)
Hematologic toxicities*
NO	14 (45.2)	15 (48.4)	0.80
YES	17 (54.8)	16 (51.6)
Non-Hematologic toxicities*
NO	17 (54.8)	14 (45.2)	0.45
YES	14 (45.2)	17 (54.8)
Skin toxicities
NO	31 (100)	7 (22.6)	0.0001
YES	0 (0)	24 (77.4)
Dose reduction
NO	22 (71.0)	15 (48.4)	0.07
YES	9 (29.0)	16 (51.6)
Median cycles	2 (1-10)	4 (1-26)

A

*Non-haematological toxicities: diarrhoea, hand-foot syndrome, nausea and stomatitis;

*Haematological toxicities: anaemia, neutropenia and febrile neutropenia, decreased platelet count.

B

	ARM A n=31 (%)		ARM B n=31 (%)
	G1-G2 (%)	G3-G4 (%)	G1-G2 (%)	G3-G4 (%)
Skin toxicities
Rash	-	-	14 (45)	6 (19)
Dry skin	-	-	7 (23)	-
Nail disordes	-	-	6 (19)	-
Pruritus	-	-	2 (6)	-
Conjunctivitis	-	-	1 (3)	-
Stomatitis	-	-	4 (13)	-
HFS	-	-	2 (6)	-
Gastrointestinal disorders
Diarrhea	4 (13)	-	6 (19)	-
Abdominal pain	2 (6)	-	1 (3)	1 (3)
Nausea	5 (16)	-	6 (19)	-
Vomiting	7 (23)	-	3 (10)	1 (3)
AST/ALT increase	-	-	1 (3)	-
Blood birilubin increase	1 (3)	-	2 (6)	-
Lipase and/or amylase increase	1 (3)	-	-	-
Anorexia	3 (10)	-	1 (3)	-
Hematologic adverse event
Neutropenia	7 (23)	8 (26)	5 (16)	8 (26)
Anemia	2 (6)	1 (3)	3 (10)	1 (3)
Piastrinopenia	2 (6)	-	3 (10)	1 (3)
General disorders
Asthenia	8 (26)	-	11 (35)	2 (6)
Hypomagnesemia	-	-	2 (6)	2 (6)
Hypocalcemia	-	-	1 (3)	-
Hypertension	-	-	-	1 (3)

HFS: hand-foot syndrome; AST: aspartate amminotrasferase; ALT: alanine amminotrasferase.

Yes there is potential Competing Interest. FC, advisory board member for Amgen, Roche, Merck KGaA, Pfizer, Bayer, Servier, MSD, Pierre Fabre, Eisai. TT, advisory board member for Amgen, MSD, Pierre Fabre, Roche, Merck KGaA, Sanofi. SN, travel grants from Amgen and Merck KGaA. DC, travel grants from Sanofi and Merck KGaA. AA, advisory board member for Amgen and Servier. EM, advisory board member for AstraZeneca, Amgen, Bayer, Merck KGaA, Roche, Sanofi, Servier, Pierre Fabre. All other authors have no competing interests to declare.

Panitumumab plus trifluridine/tipiracil as anti-Epidermal Growth Factor Receptor rechallenge therapy in chemo-refractory RAS wild-type metastatic colorectal cancer: the randomized phase 2 VELO trial

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