First-line durvalumab and tremelimumab with chemotherapy in RAS-mutated metastatic colorectal cancer: a phase 1b/2 trial

Abstract

Although patients with microsatellite instable metastatic colorectal cancer (CRC) benefit from immune checkpoint blockade, chemotherapy with targeted therapies remains the only therapeutic option for microsatellite stable (MSS) tumors. The single-arm, phase 1b/2 MEDITREME trial evaluated the safety and efficacy of durvalumab plus tremelimumab combined with mFOLFOX6 chemotherapy in first line, in 57 patients with RAS-mutant unresectable metastatic CRC. Safety was the primary objective of phase Ib; no safety issue was observed. The phase 2 primary objective of efficacy in terms of 3-month progression-free survival (PFS) in patients with MSS tumors was met, with 3-month PFS of 90.7% (95% confidence interval (CI): 79.2-96%). For secondary objectives, response rate was 64.5%; median PFS was 8.2 months (95% CI: 5.9-8.6); and overall survival was not reached in patients with MSS tumors. We observed higher tumor mutational burden and lower genomic instability in responders. Integrated transcriptomic analysis underlined that high immune signature and low epithelial-mesenchymal transition were associated with better outcome. Immunomonitoring showed induction of neoantigen and NY-ESO1 and TERT blood tumor-specific T cell response associated with better PFS. The combination of durvalumab-tremelimumab with mFOLFOX6 was tolerable with promising clinical activity in MSS mCRC. Clinicaltrials.gov identifier: NCT03202758 .

Fig. 1. Clinical characterization of patients.

a,b, Kaplan–Meier curves of PFS (a) and OS (b) in patients with MSS tumors with the median DOR (n = 48). c, Waterfall plot for target lesion tumor size for the whole cohort (n = 57), colored according to RECIST based on maximal percentage of tumor reduction from baseline. Patients with MSI tumors (n = 3) are identified with dashed bars. d, Kaplan–Meier curves of DOR in patients with MSS tumors (n = 48) with the median DOR. e, Swimmer plot of PFS times for the whole cohort (n = 57), colored according to RECIST. The length of the bars represent the time from randomization to disease progression. RECIST was not evaluable for one patient. Patients with MSI tumors (n = 3) are identified with dashed bars. NE, non-evaluable. f, Spider plot showing percent change from baseline in sum of diameter according to the evaluation times for the whole population (n = 57). Each line corresponds to one patient and is colored according to RECIST. Patients with MSI tumors are identified with stars. g, Forest plot representation of overall HR estimates with 95% CIs for the association of clinical variables with PFS in the MSS cohort (n = 48). The circle symbols represent the point estimates, and the whiskers represent the 95% CI. The vertical, dashed line is marking no change (a ratio of 1) compared to the reference level. h, Forest plot representation of odds ratio estimates with 95% CIs for the association of clinical variables with objective response rate in the MSS cohort (n = 48). The circle symbols represent the point estimates, and the whiskers represent the 95% CI. The vertical, dashed line is marking no change (a ratio of 1) compared to the reference level. Two-sided P value with significance level set at 0.05. For each Kaplan–Meier curve, s.d. interval is marked in gray. Source data

Fig. 2. Genomic characterization of patients.

a, Oncoplot representing genomic landscape of genes most frequently observed in the cohort. TMB and PFS times are also indicated for each patient at the bottom. b, Kaplan–Meier curves for PFS with patients stratified according to TMB, with a cutoff of 5.8 mutations per Mb (n = 35). Two-sided P value with significance level set at 0.05. c, Kaplan–Meier curves for PFS with patients stratified according to number of neopeptides with a cutoff on the median (n = 35). Two-sided P value with significance level set at 0.05. d, Forest plot representation of overall HR estimates with 95% CIs for the association of whole-exome-derived variables with PFS. e, Forest plot representation of odds ratio estimates with 95% CIs for the association of whole-exome-derived variables with objective response rate. The circle symbols represent the point estimates, and the whiskers represent the 95% CI (n = 35 patients (d,e)). The vertical, dashed line is marking no change (a ratio of 1) compared to the reference level. f, Kaplan–Meier curves for PFS with patients stratified according to the HRD score (n = 35). Two-sided P value with significance level set at 0.05. Survival distributions were compared using the log-rank test (b,c,f). Univariate Cox proportional hazard models were performed to estimate the HR and 95% CI (d,e). *P < 0.05, assessed using the two-sided Wald test (d,e). mut/Mb, mutations per megabase. Source data

Fig. 3. Transcriptomic characterization of patients.

a, Forest plot of overall HR estimates with 95% CIs for the association of selected RNA-seq-derived signatures related to EMT and T cell infiltration with PFS (n = 32). b, Volcano plot describing differential analysis performed on RNA-seq data between complete responders and other patients. The log₂ FC indicates the mean expression level for each gene. Each dot represents one gene. c, Forest plots of overall HR estimates with 95% CIs for the association of immune cell populations derived from MCP-counter with PFS (n = 32). d, Forest plots of overall HR estimates with 95% CIs for the association of immune cell populations derived from ImmuCellAI with PFS (n = 32). e, Forest plots of overall HR estimates with 95% CIs for the association of immune cell populations derived from Kassandra with PFS (n = 32). The circle symbols represent the point estimates, and the whiskers represent the 95% CI (a,c–e). The vertical, dashed line is marking no change (a ratio of 1) compared to the reference level. *P < 0.05, log-rank test (a,c–e). f, Kaplan–Meier curves for PFS with patients stratified according to CTLA4 gene expression level with a cutoff at the median. Two-sided P value with significance level set at 0.05. g, Decision tree for PFS estimated with stromal and immune-related parameters. h, Kaplan–Meier curves with patients stratified according to groups created by the decision tree for PFS. Two-sided P value with significance level set at 0.05. Survival distributions were compared using the log-rank test (f–h). MAIT, mucosal-associated invariant T. Source data

Fig. 4. Immunological exploratory analysis.

a, Representative pictures of CD8 staining of CRC samples from a responder and a non-responder patient (scale bar, 100 µm) focusing on an area of the invasive (IF) front and an area of the tumor core (TC) (scale bar, 20 µm). b,c, Analysis of CD8⁺ cells in the TC (b) and the IF (c). Top panel, the correlation between TC (respectively invasive margin) CD8 number per mm³ and PFS was determined. Correlation was performed using the Spearman test. Middle panel, Kaplan–Meier curves for PFS with patients stratified according to high or low CD8 number in the TC (respectively invasive margin). The overall median was used as a threshold to distinguish the two groups. Two-sided P value with significance level set at 0.05. Bottom panel, bar plots showing the percentage of complete response and partial response (CR + PR) or stable disease and progressive disease (SD + PD) according to the number of CD8 in the tumor core (respectively invasive margin) (n = 37). NS, not significant; *P < 0.05, comparison using Fisher’s exact test. d, Decision tree for PFS estimated with immunohistochemistry variables. e, Kaplan–Meier curves with patients stratified according to groups created by the decision tree for PFS. Two-sided P value with significance level set at 0.05. f, Representative pictures of decorin/SATB2 staining of CRC samples from a patient with low expression of decorin (left) and a patient with high expression of decorin (right) (scale bar, 100 µm). g,h, Kaplan–Meier curves for PFS with patients stratified according to decorin protein expression level (g) and the combination of CD8 and decorin protein expression level (h) (n = 47). Two-sided P value with significance level set at 0.05. Survival distributions were compared using the log-rank test (e,g,h). Source data

Fig. 5. Immunological exploratory analysis.

a, Upper panel, parts of whole (black and gray) showing the distribution of patients according to their anti-tumor responses against zero or at least one antigen at baseline (n = 40) and at C2 (n = 45). Lower panel, parts of whole (red and blue) showing the percentage of positive (in red) or negative (in blue) anti-tumor responses against TERT or NY-ESO1 at baseline (n = 48), at C2 (n = 45) and at C5 (n = 42). b–d, Bar plots showing the number of complete response and partial response (CR + PR) or stable disease and progressive disease (SD + PD) according to TERT-specific T cell responses at C2 (b), at C5 (c) and NY-ESO1-specific T cell responses at C5 (d). Two-sided P value with significance level set at 0.05, comparison using Fisher’s exact test. e,f, Kaplan–Meier curves for PFS with patients stratified according to TERT-specific T cell responses at C2 (e) and at C5 (f) (n = 45). Two-sided P value with significance level set at 0.05. g,h Kaplan–Meier curves for PFS with patients stratified according to the combination of TERT and NY-ESO1-specific T cell responses at C2 (g) and at C5 (h) (n = 45). Two-sided P value with significance level set at 0.05. Survival distributions were compared using the log-rank test (e–h). *P < 0.05. NEG, negative; POS, positive. Source data

Fig. 6. Analysis of in situ tumor-specific CD8 response in responders.

a, Radar plot showing the proportion of T cell clusters from sampling at C1, at C5 and at the time of surgery in blood and at the time of surgery in TILs. b, Venn diagram showing the distribution of TCR clonotypes given the sampling origin. c, Bar plots showing the spread of number of cells observed for each T cell population given the sampling origin, for each of the three most frequent TCR clusters, d, Sample preference of each cluster estimated by the RO/E index; ++ (2 ≤ RO/E < 3, P < 0.05) represents enriched; + (1.2 ≤ RO/E < 2, P < 0.05) represents slightly enriched; +/− (0.8 ≤ RO/E < 1.2 or P > 0.05) represents non-significant; and − (0 < RO/E < 0.8, P < 0.05) represents deletion. e, Heat map showing the fraction of T cells with clonotypes belonging to a primary phenotype cluster (rows) that are shared with other secondary phenotype clusters (columns). f, Left panel, explanatory diagram of the analysis of the specific T response performed in this responder patient. From a blood and tumor sample, exome sequencing was performed, and, using bioinformatics analysis, neopeptides found only in the tumor were identified. These peptides were synthesized, and the specific T response against these peptides was tested using blood and tumor samples to analyze the appearance of the specific anti-tumor response. Right panel, representative picture of ex vivo IFN-γ ELISpot using PBMCs taken at the time of liver surgery. g, Dot plot representing the number of IFN-γ spots for each condition (negative control, peptide pool, single peptide 1–14 and positive control (CEF pool)) in PBMCs at the time of liver surgery. Each number corresponds to the tested neoantigen. Ø, dimethyl sulfoxide; CEF, peptides from cytomegalovirus. Epstein–Barr virus and influenza virus and pool corresponds to the pool of tested neoantigens. Dots represent technical replicates. Data are the mean ± s.d. h,i, Bar graph representing the number of IFN-γ spots for peptides 2, 4, 9 and 10 in TILs from liver metastasis (i) and in PBMCs taken at baseline (C1) and after four cycles of chemotherapy (C5) (j). j, Dot plot representing the number of IFN-γ spots in PBMCs from patients collected at baseline or after two cycles of chemotherapy (C2) after stimulation with a pool of calculating tumor neoantigens (n = 10). MAIT, mucosal-associated invariant T. Source data

Extended Data Fig. 1. Diagram and flowchart of study cohort.

a, The diagram shows the study design divided into 2 phases: In phase 1b, 9 patients were included and interim analysis was performed to validate the study design. Then, phase 2 could start, and 48 further patients were included. The diagram shows the treatment regimen tested with 6 consecutive courses of FOLFOX, 4 courses of tremelimumab every other week and durvalumab every week until progression. b, A total of 76 patients were assessed for eligibility and 57 were recruited into the study. Data and blood samples were processed from 57 patients and analyzed at baseline, V2, V5, V12 and end of treatment. Treatment efficacy was evaluated by objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) in all 57 patients. The primary endpoint was evaluated in 54 patients comprising the MSS population, and safety evaluation was performed in all 57 patients. Immunological effects of treatment were studied with whole exome sequencing in 37 patients, RNA sequencing in 36 patients, immunohistology in 46 patients and flow cytometry in 48 patients.

Extended Data Fig. 2. Exploratory analysis of genomic correlates.

a, Violin plots showing the median, variability, and probability density of TMB for each type of colorectal cancer localization. b, Violin plots showing the median, variability, and probability density of HLA-B (left) and HLA-DOB (right) gene counts for responder and non-responder patients. *p < 0.05, data were compared using an unpaired two-sided Mann-Whitney Wilcoxon test. c, Forest plots of overall hazard ratio (HR) estimates with 95% confidence intervals for the association of clinical variables with overall and progression-free survival for tumor mutational burden (TMB), number of neopeptides and homologous repair deficiency (HRD) estimated on the TCGA cohort (n = 337 patients). The circles represent the point estimates and the whiskers represent the 95% CI. The vertical, dashed line marks no change (a ratio of one), compared to the reference level. *p < 0.05, assessed using the two-sided Wald test. Source data

Extended Data Fig. 3. Exploratory analysis of immunological correlates.

a, Representative pictures of PD-L1 staining of colorectal cancer sample (scale bar 100µM) with a zoom on a part of the blade (scale bar 20 µM). b, Kaplan Meier curves for progression-free survival; patients were split into two groups: patients with CPS score < 1 (blue curve) or patients with CPS score > 1 (red curve) (Kaplan-Meier method and log rank tests). c, Kaplan Meier curves for progression-free survival; patients were split into two groups: patients with CPS score < 5 (blue curve) or patients with CPS score > 5 (red curve) (Kaplan-Meier method and log rank tests). d, Kaplan Meier curves for progression-free survival; patients were split into two groups: patients with CPS score < 10 (blue curve) or patients with CPS score > 10 (red curve) (Kaplan-Meier method and log rank tests). n.s, not significant. e, Box plots of COL1 (left), CD3 (middle) protein expression, and CD3/COL1 ratio (right). The center line indicates the median value, lower and upper hinges represent the 25th and 75th percentiles, respectively, and whiskers denote minimum and maximum. Each dot corresponds to one patient (n = 7 responders and n = 5 non-responders). Data were compared using an unpaired two-sided Mann-Whitney Wilcoxon test. f, Representative false colour images of COL1 (white) and CD3 (red) markers merged with a nuclear stain (blue) and cropped of colorectal cancer samples from a responder patient (top) and a non-responder patient (bottom) (scale bar 100µM). One area of each sample has been magnified to better appreciate the markers. Source data

Extended Data Fig. 4. Exploratory analysis of immunological correlates.

a, Plasma from metastatic colorectal cancer (mCRC) patients was recovered at C1, C2 and C5 and plasma from healthy volunteers (HV) was also recovered. A bioplex assay was performed to analyze the amounts of secreted cytokines. The heat map on the left corresponds to normalized cytokine amount, in the middle is the median of each cytokine between HV and mCRC and on the right is the p-value from statistical analysis. Statistical analysis was performed using the two-sided Wilcoxon t-test. b, Bar plots showing the percentage of complete and partial response (CR+PR) or stable and progression disease (SD+PD) according to the amount of IFNβ measured in the patients’ plasma at baseline. n.s, not significant; comparison using two-sided Fisher’s exact test. c, Box plots of the soluble PD-L1 assay in the plasma of patients at C1 (baseline), C2 (after one cycle of chemotherapy) and C5 (after 4 cycles of chemotherapy). The center line indicates the median value, lower and upper hinges represent the 25th and 75th percentiles, respectively, and whiskers denote minimum and maximum. Each dot corresponds to one patient (n = 46). Data were compared using an unpaired Mann-Whitney Wilcoxon test. d-f, Plasma was recovered at C1, C2 and C5 and a bioplex assay was performed to analyze the amounts of soluble PD-L1 secreted before and during treatment. Kaplan-Meier curves are shown for progression-free survival with patients stratified according to the amount of soluble PD-L1 at C1 (d), C2 (e) and C5 (f). The overall median for each time point was used as a threshold to distinguish the two groups. Survival distributions were compared using the log-rank test (d-f). Two-sided P value with significance level set at 0.05. g,h, Bar plots showing the percentage of complete and partial response (CR+PR) or stable and progression disease (SD+PD) according to the amount of IL-6 (g) or IL-8 (h) measured in the patients’ plasma at C2. Comparison was assessed using Fisher’s exact test. i,j, Kaplan-Meier curves for progression-free survival with patients stratified according to IL-6 (i) or IL-8 (j) secretion level at C2. The overall median was used as a threshold to distinguish the two groups. Two-sided P value with significance level set at 0.05. Source data

Extended Data Fig. 5. Exploratory analysis of immunological correlates.

a, Box plots showing the frequency of Th1 central memory (CD4+ CXCR3+ CCR6- CD45RA- CCR7+ PD1low CD28+) and of CD4+ CD25+ Foxp3- cells (count/µL) in patients (n = 45) according to the median of progression-free survival (PFS). b,c, Left panel: Bar plots showing the percentage of complete and partial response (CR+PR) or stable and progression disease (SD+PD) according to frequency of Th1 central memory (b) or CD4+ CD25+ Foxp3- (c). Right panel: Kaplan-Meier curves for progression-free survival with patients stratified according to the frequency of Th1 central memory (b) or CD4+ CD25+ Foxp3- (c). d,e, Box plots showing the frequency of monocytic MDSC (CD45+ CD3- CD19- CD20- CD56- CD15- CD14+ HLA-DRlow) (d) and of granulocytic MDSC (CD45+ CD3- CD19- CD20- CD56- CD15+ CD14+) (e) (count/µL) at C1, C2 and C5 (n = 46). f,g, Bar plots showing the percentage of complete and partial response (CR+PR) or stable and progression disease (SD+PD) according to low or high frequency of mMDSC (f) or gMDSC (g). h,i, Kaplan-Meier curves for progression-free survival with patients stratified according to the frequency of mMDSC (h) or gMDSC (i) at baseline. j, Bar plots showing the percentage of complete and partial response (CR+PR) or stable and progression disease (SD+PD) according to the delta between C5 or C2 and C1 for the frequency of mMDSC measured at baseline. k, Kaplan-Meier curves for progression-free survival with patients stratified according to the delta between C5 or C2 and C1 for the frequency of mMDSC. Analysis performed at baseline by flow cytometry in patient’s blood at baseline. For boxplots, center line indicates the median value, lower and upper hinges represent the 25th and 75th percentiles, respectively, and whiskers denote minimum and maximum. Each dot corresponds to one patient. Statistical analysis was performed by unpaired two-sided Mann-Whitney Wilcoxon test (a,d,e) or two-sided Fisher’s exact test (b,c,f,g,j), and Log-rank test (b,c,h, I, k). For continuous variables, the overall median was used as a threshold to distinguish patients into two groups. n.s, not significant. Source data

Extended Data Fig. 6. Exploratory analysis of immunological correlates.

a, Bar plots showing the number of complete and partial response (CR+PR) or stable and progressive disease (SD+PD) according to the antitumor response against 0 or at least 1 antigen at C1 (n = 48). Two-sided P value with significance level set at 0.05, comparison using Fisher’s exact test. b, Kaplan-Meier curves for progression-free survival with patients stratified according to the antitumor response against 0 or at least 1 antigen at C1 (n = 48). Two-sided P value with significance level set at 0.05. c, Bar plots showing the number of complete and partial response (CR+PR) or stable and progressive disease (SD+PD) according to the antitumor response against TERT-specific T-cell responses at C1 (n = 38). Two-sided P value with significance level set at 0.05, comparison using Fisher’s exact test. d, Kaplan-Meier curves for progression-free survival with patients stratified according to the antitumor response against TERT-specific T-cell responses at C1 (n = 38). Two-sided P value with significance level set at 0.05. e, Bar plots showing the number of complete and partial response (CR+PR) or stable and progressive disease (SD+PD) according to the antitumor response against NY-ESO1-specific T-cell responses at C1 (n = 47). Two-sided P value with significance level set at 0.05, comparison using Fisher’s exact test. f, Kaplan-Meier curves for progression-free survival with patients stratified according to the antitumor response against NY-ESO1-specific T-cell responses at C1 (n = 47). Two-sided P value with significance level set at 0.05. Survival distributions were compared using the log-rank test (b,d,f). Source data

Extended Data Fig. 7. Analysis of in situ tumor specific CD8 response in responders.

a, Experimental strategy for single cell RNA sequencing on responder patient PBMC and TIL samples. b, Contrast enhanced CT-scan showing liver metastases at baseline and before liver surgery. c, Upper left: Representative picture of HES staining of a liver metastasis with delimitation of the sterilisation area of the tumour; Upper right: Representative picture of a PD-L1 staining by immunohistochemistry; Lower left: Representative picture of a CD8 staining by immunohistochemistry; Lower right: Merge of the 3 stainings (HES, CD8 and PD-L1) to visualize the spatial distribution of PD-L1 at the tumor sterilisation zone and the presence of a large number of CD8 in the proximity of the tumor. (Scale bar indicates 0.25 mm). d, Number of CD8 cells/mm² and H-score of PD-L1 before (in liver biopsy at baseline) and after liver surgery (at the time of surgery). Source data

Extended Data Fig. 8. Analysis of in situ tumor specific CD8 response in responders.

a-d, For each sampling, T-SNE visualization of CD8 T cells analyzed using single-cell RNA-sequencing and colored by cluster identity. Each dot corresponds to one single cell and 9 clusters were selected: 0: Central memory; 1: Exhausted; 2: Polyfunctional; 3: Naïve; 4: Effector; 5: Exhausted precursor; 6: MAIT (Mucosal-Associated Invariant T cells); 7: Effector memory; 8: Resident. e-i, Relative expression of TCF7 (e), LAG3 (f), FAS (g), HAVCR2 (h) and PDCD1 (i) genes according to pseudotimes; points are colored by T-cell clusters for blood surgery sampling. Source data