El aumento del antígeno carcinoembrionario durante la quimioterapia basada en oxaliplatino predice el beneficio del nivolumab en el cáncer colorrectal metastásico con microsatélites estables: un análisis post hoc de los datos del ensayo METIMMOX.

The introduction of immune checkpoint inhibitors (ICIs) has significantly improved survival outcomes in many solid cancers, including local, locally advanced, and metastatic disease for the subgroup of colorectal cancer (CRC) patients with microsatellite-instable/mismatch-repair (MMR)-deficient tumors.[1–5] However, the large majority of CRC patients bear microsatellite-stable (MSS)/MMR-proficient disease with low tumor antigenicity and therefore low or absent ICI responsiveness. To overcome the inherent ICI resistance of MSS/MMR-proficient CRC, various approaches have been explored. These include combining ICI with radiation therapy^[6] or chemotherapy and an antiangiogenic agent,[7–9] or dual-checkpoint ICI combinations.^[10] In the first-line METIMMOX trial, patients with metastatic MSS/MMR-proficient CRC were randomly assigned to oxaliplatin-based chemotherapy (the Nordic FLOX regimen^[11]) alone or short-course FLOX alternating with the ICI nivolumab. The primary endpoint of improved progression-free survival (PFS) was not met for the intention-to-treat population of 38 subjects receiving the experimental regimen. However, we noted that 16% of experimental-arm patients achieved a radiologic complete response (CR) with eradication of all tumor manifestations, as opposed to none of control-arm patients.^[12]

Efforts are ongoing to identify biomarkers that predict ICI responsiveness. Some are well-established for certain tumor types and used clinically, such as programmed-death ligand-1 expression and tumor mutational burden.^[13] However, no universal utility of these biomarkers has been proven as yet.^[14] Pragmatic biomarkers for response prediction are needed to select more precisely the patients who will benefit but also to avoid treating patients who are prone to ICI failure. Dynamic on-treatment biological changes could represent an alternative to the static baseline biomarkers. Changes in the circulating C-reactive protein level early during ICI treatment have been associated with response in metastatic disease from several malignancies.[15–18] When administrating immunomodulating chemotherapy before ICI, as done in the METIMMOX trial, the initial adaptation reflected systemically might serve as a unique setting for dynamic biomarker discovery.

Carcinoembryonic antigen (CEA) belongs to a family of cell adhesion molecules.^[19] Circulating CEA is commonly elevated in advanced CRC. Although its predictive value is limited,^[20] serial measurements are used for surveiling recurrence risk after curative-intent treatment^[21] as well as in evaluation of therapy response in the metastatic setting.^[22] A transient increase, or flare, in CEA in patients with metastatic CRC responding to chemotherapy was first reported in two small studies in 2004 and 2006.^[23],^[24] In multivariable analysis, compared with steadily increasing CEA, flare was found to be an independent favorable predictive and prognostic factor for tumor response and survival in patients undergoing first-line chemotherapy.^[25] However, it remains unclear whether CEA flare also occurs when chemotherapy is combined with ICI, and whether such a flare may be predictive of ICI benefit in this experimental treatment setting.

In this post hoc analysis of METIMMOX data, we hypothesized that CEA flare may be a surrogate marker of immunogenicity incited by oxaliplatin-based chemotherapy, leading to ICI responsiveness in patients with previously untreated, unresectable metastatic MSS/MMR-proficient CRC, and that it may provide a pragmatic tool for early monitoring of treatment activity or failure.

Material and Methods

Ethics Approvals and Consent to Participate

Approvals were given by the Regional Committee for Medical and Health Research Ethics of South-East Norway (2017/1850), the Norwegian Medicines Agency (17/12,752), and the institutional review boards. The trial was conducted in accordance with the Helsinki Declaration. All patients provided written informed consent. The trial was registered with ClinicalTrials.gov, NCT03388190, by 2 January 2018.### Patients, Procedures, and Outcomes

Details have been published previously.^[12] To summarize for the current context, MSS/MMR-proficient CRC patients with unresectable infradiaphragmatic (liver, peritoneal, nodal) metastases were randomly assigned to first-line treatment with FLOX (oxaliplatin with bolus 5-fluorouracil/folinic acid) Q2W (control arm) or alternating two cycles of FLOX Q2W and nivolumab Q2W (experimental arm), for both trial arms with a break period after four months (eight cycles in a treatment sequence). During active treatment and breaks, radiologic response assessment was performed every eight weeks. If disease progression occurred during a break, a new treatment sequence was initiated. This go-and-stop schedule is depicted in Supplementary Figure S1. The primary endpoint PFS was defined as the time from study inclusion to the first disease progression on active therapy (progressive disease (PD)) by blinded independent central review according to Response Evaluation Criteria in Solid Tumors or iRECIST.^[26] For patients not experiencing PD, the time from study inclusion to an intolerable adverse event, death from causes other than cancer progression, consent withdrawal, or censoring was calculated. The best overall response (BOR) was defined as the best achieved radiologic response during study participation. A total of 80 patients were enrolled between 29 May 2018 and 22 October 2021. Data cut-off was set to 15 March 2024 when the last patient reached two years of disease-free (CR) follow-up, three years after starting METIMMOX treatment. Median follow-up time was 20.2 months (95% confidence interval (CI), 14.6–23.2).### Specimen Characteristics

The patients were monitored with standard blood tests, including CEA, by the laboratory facilities of the respective study hospitals. Serum samples were stored at 2–8 °C and analyzed within three days of venipuncture. CEA (in μg/L) was measured at baseline, with each treatment administration (every two weeks), and during the treatment breaks (every eight weeks) until censoring. All five study hospitals used the Cobas 8000 e801 analyzer (Roche Diagnostics, Mannheim, Germany) to quantify CEA using electrochemiluminescence technology. None of the patients received high doses of biotin (vitamin B7; >5 mg/day), which might have interfered with the assay.^[27],^[28]### CEA Kinetics

For this post hoc analysis, patients were categorized based on the CEA kinetics during the first sequence of active therapy, requiring at least four measurements. In cases of missing values (n = 3), the categorization was based on the available measurements. Delay of treatment administration owing to an adverse event was not considered when evaluating the kinetics. The reference limit was below 5.0 μg/L for both men and women, as used by all participating hospitals. The evaluation did not account for smoking status or comorbidities, which are known to slightly elevate CEA levels.[29–31] Flare was defined as at least 20% increase from baseline, consistent with previous studies.^[23],^[24],^[32] The three kinetics groups were 1—Flare: 20% increase above baseline followed by decrease at, at least, two subsequent measurements to 20% below baseline (for patients with baseline CEA within the reference limit, Flare was defined as 20% rise followed by decline to baseline); 2—Non-responding: Gradual increase at, at least, two subsequent measurements to 20% above baseline; 3—Stable/Responding: Levels fluctuating around baseline with occasional increases followed by normalization without clear flare (Stable) or gradual decrease below baseline (Responding). Stable and Responding were grouped together because of similar PFS to chemotherapy in the metastatic setting.^[25] Time until flare occurred was calculated from the start of treatment to the peak CEA value in the first treatment sequence.### Statistical Methods

Analyses were performed using STATA SE version 17 and SPSS version 29.0. Figures were generated using GraphPad Prism version 9.5.1. Continuous variables were presented as median with minimum (min)–maximum (max) values, while categorical variables were presented as frequency and percentage. Differences in PFS stratified by the kinetics groups were visualized using Kaplan-Meier curves with number-at-risk table and assessed with the Log rank test. Cox regression model was estimated to determine association between PFS and kinetics groups adjusted for age, sex, and treatment arm. An interaction between the kinetics group and treatment arm was included to assess differences between the two arms regarding association between PFS and the kinetics groups. The proportional hazards assumption was assessed by a global test and inspecting Schoenfeld residuals. It was found to be violated for the kinetics groups, which was thus entered into the model as a time-dependent variable. For variables with time-dependent effects, and for interaction terms involving such variables, a single hazard ratio (HR) is not directly interpretable; these effects were therefore presented as regression coefficients with standard errors. For covariables with proportional hazards, and for pairwise group comparisons that did not exhibit time-dependent behavior, HRs with 95% CIs were derived from the model. To aid interpretation of time-dependent associations, HRs over time within each trial arm were illustrated graphically. As a sensitivity analysis, we applied a flexible parametric survival model to assess the robustness of the Cox model results. Differences among CEA kinetics groups were assessed by Fisher’s exact test. A nominal significance level of 0.05 was used, but p-values in the Cox model should be interpreted with caution due to the limited sample size of some groups. Whenever possible, interpretation focuses on effect sizes expressed as HRs with 95% CIs, which more appropriately convey the magnitude and uncertainty of associations.

Ethics Approvals and Consent to Participate

Approvals were given by the Regional Committee for Medical and Health Research Ethics of South-East Norway (2017/1850), the Norwegian Medicines Agency (17/12,752), and the institutional review boards. The trial was conducted in accordance with the Helsinki Declaration. All patients provided written informed consent. The trial was registered with ClinicalTrials.gov, NCT03388190, by 2 January 2018.

Patients, Procedures, and Outcomes

Details have been published previously.^[12] To summarize for the current context, MSS/MMR-proficient CRC patients with unresectable infradiaphragmatic (liver, peritoneal, nodal) metastases were randomly assigned to first-line treatment with FLOX (oxaliplatin with bolus 5-fluorouracil/folinic acid) Q2W (control arm) or alternating two cycles of FLOX Q2W and nivolumab Q2W (experimental arm), for both trial arms with a break period after four months (eight cycles in a treatment sequence). During active treatment and breaks, radiologic response assessment was performed every eight weeks. If disease progression occurred during a break, a new treatment sequence was initiated. This go-and-stop schedule is depicted in Supplementary Figure S1. The primary endpoint PFS was defined as the time from study inclusion to the first disease progression on active therapy (progressive disease (PD)) by blinded independent central review according to Response Evaluation Criteria in Solid Tumors or iRECIST.^[26] For patients not experiencing PD, the time from study inclusion to an intolerable adverse event, death from causes other than cancer progression, consent withdrawal, or censoring was calculated. The best overall response (BOR) was defined as the best achieved radiologic response during study participation. A total of 80 patients were enrolled between 29 May 2018 and 22 October 2021. Data cut-off was set to 15 March 2024 when the last patient reached two years of disease-free (CR) follow-up, three years after starting METIMMOX treatment. Median follow-up time was 20.2 months (95% confidence interval (CI), 14.6–23.2).

Specimen Characteristics

The patients were monitored with standard blood tests, including CEA, by the laboratory facilities of the respective study hospitals. Serum samples were stored at 2–8 °C and analyzed within three days of venipuncture. CEA (in μg/L) was measured at baseline, with each treatment administration (every two weeks), and during the treatment breaks (every eight weeks) until censoring. All five study hospitals used the Cobas 8000 e801 analyzer (Roche Diagnostics, Mannheim, Germany) to quantify CEA using electrochemiluminescence technology. None of the patients received high doses of biotin (vitamin B7; >5 mg/day), which might have interfered with the assay.^[27],^[28]

CEA Kinetics

For this post hoc analysis, patients were categorized based on the CEA kinetics during the first sequence of active therapy, requiring at least four measurements. In cases of missing values (n = 3), the categorization was based on the available measurements. Delay of treatment administration owing to an adverse event was not considered when evaluating the kinetics. The reference limit was below 5.0 μg/L for both men and women, as used by all participating hospitals. The evaluation did not account for smoking status or comorbidities, which are known to slightly elevate CEA levels.[29–31] Flare was defined as at least 20% increase from baseline, consistent with previous studies.^[23],^[24],^[32] The three kinetics groups were 1—Flare: 20% increase above baseline followed by decrease at, at least, two subsequent measurements to 20% below baseline (for patients with baseline CEA within the reference limit, Flare was defined as 20% rise followed by decline to baseline); 2—Non-responding: Gradual increase at, at least, two subsequent measurements to 20% above baseline; 3—Stable/Responding: Levels fluctuating around baseline with occasional increases followed by normalization without clear flare (Stable) or gradual decrease below baseline (Responding). Stable and Responding were grouped together because of similar PFS to chemotherapy in the metastatic setting.^[25] Time until flare occurred was calculated from the start of treatment to the peak CEA value in the first treatment sequence.

Statistical Methods

Analyses were performed using STATA SE version 17 and SPSS version 29.0. Figures were generated using GraphPad Prism version 9.5.1. Continuous variables were presented as median with minimum (min)–maximum (max) values, while categorical variables were presented as frequency and percentage. Differences in PFS stratified by the kinetics groups were visualized using Kaplan-Meier curves with number-at-risk table and assessed with the Log rank test. Cox regression model was estimated to determine association between PFS and kinetics groups adjusted for age, sex, and treatment arm. An interaction between the kinetics group and treatment arm was included to assess differences between the two arms regarding association between PFS and the kinetics groups. The proportional hazards assumption was assessed by a global test and inspecting Schoenfeld residuals. It was found to be violated for the kinetics groups, which was thus entered into the model as a time-dependent variable. For variables with time-dependent effects, and for interaction terms involving such variables, a single hazard ratio (HR) is not directly interpretable; these effects were therefore presented as regression coefficients with standard errors. For covariables with proportional hazards, and for pairwise group comparisons that did not exhibit time-dependent behavior, HRs with 95% CIs were derived from the model. To aid interpretation of time-dependent associations, HRs over time within each trial arm were illustrated graphically. As a sensitivity analysis, we applied a flexible parametric survival model to assess the robustness of the Cox model results. Differences among CEA kinetics groups were assessed by Fisher’s exact test. A nominal significance level of 0.05 was used, but p-values in the Cox model should be interpreted with caution due to the limited sample size of some groups. Whenever possible, interpretation focuses on effect sizes expressed as HRs with 95% CIs, which more appropriately convey the magnitude and uncertainty of associations.

Results

CEA Features

Of the total of 80 randomized patients, 71 (n = 35, control arm; n = 36, experimental arm) had evaluable CEA kinetics in the first treatment sequence (CONSORT diagram; Supplementary Figure S2). The two arms were similar with regard to clinical characteristics (Supplementary Table S1). Of patients in the control and experimental arms, respectively, 8 (23%) and 9 (25%) were Non-responding, 17 (49%), 21 (58%) were Stable/Responding, and 10 (29%) and 6 (17%) had Flare, with no differences in distribution of baseline clinical characteristics among the three kinetics groups except slightly higher age of Flare patients (p = 0.042; Supplementary Table S2).

Among the total of 16 Flare, only one patient had baseline CEA within the reference limit (