Article Figures & Data
Figures
- Figure 1.
A, H&E section of rectal cancer (original magnification, ×200) showing the tumor regions of interest; CT and IM. B, two spots immunostained for CD3 showing the heterogeneity of CD3+ cell infiltration within tumors among patients. C, establishment of an Immunoscore, based on the numeration of two lymphocyte populations (CD3 and CD8) in CT and in IM. The density of CD3+ cells and CD8+ cells is determined using an image analysis workstation. Each tumor is categorized into Hi or Lo density for each marker in each tumor region, according to a predetermined cutoff value (CD3 CT, 256 cells/mm2; CD3 IM, 144 cells/mm2; CD8 CT, 202 cells/mm2; CD8 IM, 50 cells/mm2) determined using the minimum P value approach. Patients are stratified according to a score ranging from I0 to I4 (8-10), depending on the total number of high densities observed (two markers assessed in CT, two markers assessed in IM). For example, I4 refers to a tumor with high densities of CD3+ and CD8+ cells in CT and IM regions of the tumor (4-Hi). I3 refers to tumors with three high densities. Patients with low densities of CD3 and CD8 in both tumor regions (0 Hi density) is classified I0. CT, core of the tumor; IM, invasive margin.
- Figure 2.
A, Kaplan–Meier curves for the duration in months of DFS according to T-cell (CD3+) density evaluated in combined tumor regions (CT and IM). B, Kaplan–Meier curves for the duration of DFS according to cytotoxic T-cell (CD8+) density evaluated in combined tumor regions (CT and IM). For each marker (CD3 and CD8), significant difference (P < 0.005) was observed for DFS times between patients with high densities in the CT and in the IM (HiHi; red line), and low densities in the CT and in the IM (LoLo; black line). Patients at risk at each interval in the Kaplan–Meier survival curves for the duration of DFS are shown.
- Figure 3.
Kaplan–Meier curves for the duration of DFS and OS according to the Immunoscore in patients with rectal cancer eligible for a primary surgery (log-rank statistical test P < 0.005 for all comparisons). Patients at risk at each interval in the Kaplan–Meier survival curves for the duration of DFS and OS according to the Immunoscore are shown in Supplementary Table S4. Patients with an Immunoscore of 0 or 1 experienced a very poor postoperative outcome and thus could be grouped together. Kaplan–Meier curves and associated statistics after grouping are shown in the Supplementary Fig. 2.
- Figure 4.
A, comparison of the mean (±SEM) densities of CD3+ cells in the CT and IM regions of tumor from patients eligible for a pCRT, without (white bars) or with (gray bars) tumor recurrence. B, biopsies immunostained were analyzed for the densities of CD3+ cells in tumor areas (in red in the small square) using a computerized image analysis system. The tumor area is divided into tiles for the analysis. A heat map view shows the densities of the stained cells in all tiles, from the minimum (green) to the maximum density (red). Right, illustration of the detection of CD3+ cells (in red) in a tile (magnification ×20). Bottom, distribution of high and low densities of CD3+ cells in biopsies of patients that will experience a complete/partial response or an absence of response to pCRT according to the ypTNM downstaging of the tumor. The categorization of the patient's densities for CD3+ cells (high vs. low) was performed at the median of the cohort. The Fisher exact test comparing the distribution of Hi and Lo infiltration in each group, P < 0.05 was considered significant.
Tables
- Table 1.
Univariate analyses for DFS and OS among patients with rectal cancer eligible for primary surgery
DFS OS Characteristic No. of pts (%) 5-year % (95% CI) HR (95% CI)a Pb 5-year % (95% CI) HR (95% CI) P Age (y) 1.07 (0.72–1.58) 0.6502 1.55 (1.18–2.04) 0.0152 <65 46 (41.4) 70.3 (57.5–85.9) 1.0 (reference) 77.3 (65.8–90.7) 1.0 (reference) 65–75 32 (28.8) 73 (57.7–92.4) 0.79 (0.32–1.99) 0.7729 57.2 (41.9–78) 1.69 (0.84–3.41) 0.1359 75–85 25 (22.5) 59.7 (39.9–89.4) 1.47 (0.61–3.56) 0.6793 42.5 (26.6–68) 2.73 (1.34–5.55) 0.0038 85 8 (7.2) 83.3 (58.3–100) 0.68 (0.09–5.22) 0.4901 37.5 (15.3–91.7) 3.04 (1.09–8.46) 0.0249 UICC (TNM) stage 1.62 (1.11–2.38) 0.0590 1.69 (1.3–2.2) 0.0002 0–I 58 (52.3) 79 (68.7–90.9) 1.0 (reference) 74.9 (64.3–87.3) 1.0 (reference) II 27 (24.3) 65.9 (48.1–90.3) 1.34 (0.53–3.37) 0.5294 60.8 (44.4–83.1) 1.52 (0.76–3.04) 0.2339 III 16 (14.4) 52.5 (32.2–85.6) 2.54 (1.01–6.39) 0.0397 37.5 (19.9–70.6) 2.27 (1.09–4.74) 0.0244 IV 10 (9) 0 (NA-NA) 4.6 (1.02–20.81) 0.0294 20 (5.8–69.1) 5.7 (2.32–13.99) <0.0001 T stage 1.72 (1.12–2.64) 0.0244 1.62 (1.19–2.21) 0.0159 pTis-1 24 (21.6) 95.5 (87.1–100) 1.0 (reference) 87.5 (75.2–100) 1.0 (reference) pT2 41 (36.9) 67.2 (53.5–84.3) 9.71 (1.28–73.9) 0.0070 62.4 (49.1–79.5) 3.32 (1.14–9.7) 0.0198 pT3 39 (35.1) 53.7 (37.8–76.3) 12.34 (1.61–94.45) 0.0019 46.1 (32.5–65.5) 4.66 (1.6–13.52) 0.0019 pT4 7 (6.3) 75 (42.6–100) 5.37 (0.34–85.93) 0.1822 42.9 (18.2–100) 5.22 (1.39–19.53) 0.0062 N stagec 1.71 (0.98–2.98) 0.0714 2.07 (1.44–2.98) 0.0002 N0 88 (79.3) 74.2 (64.8–85) 1.0 (reference) 70.1 (60.9–80.7) 1.0 (reference) N1 13 (11.7) 42.4 (20.6–87.2) 2.7 (1.09–6.72) 0.0257 30.8 (13.6–69.5) 2.35 (1.16–4.77) 0.0150 N2 10 (9) 60 (29.3–100) 2.06 (0.48–8.8) 0.3183 20 (5.8–69.1) 3.91 (1.77–8.6) 0.0003 LNRd 2.13 (1.28–3.54) <0.0001 2.04 (1.51–2.74) <0.0001 0 88 (81.5) 72.8 (63.3–83.8) 1.0 (reference) 68.8 (59.5–79.6) 1.0 (reference) <0.33 10 (9.3) 60 (33.1–100) 1.54 (0.46–5.15) 0.4803 40 (18.7–85.5) 1.95 (0.82–4.66) 0.1248 0.33–0.66 4 (3.7) 66.7 (30–100) NA (NA-NA) NA 25 (4.6–100) NA (NA-NA) NR >0.66 6 (5.6) 0 (NA-NA) 56.73 (7.78–413.75) <0.0001 0 (NA-NA) 9.71 (3.57–26.36) <0.0001 VELIPI 1.55 (0.54–4.48) 0.4117 2.05 (1.05–4) 0.0323 No 92 (84.4) 71.7 (62.3–82.5) 1.0 (reference) 64.7 (55.4–75.6) 1.0 (reference) Yes 17 (15.6) 58.9 (34.6–100) 1.55 (0.54–4.48) 0.4117 35.3 (18.5–67.2) 2.05 (1.05–4) 0.0323 CD3 (CT/IM)e 2.0 (1.23–3.22) 0.0983 2.04 (1.33–3.12) 0.0019 LoLo 7 (7.7) 21.4 (3.8–100) 4.57 (1.94–10.75) 0.0030 28.6 (8.9–92.2) 5.18 (2.01–13.37) 0.0002 Het 25 (27.5) 65.5 (47.8–89.9) 1.15 (0.92–1.44) NA 50.3 (33.8–74.8) 1.88 (0.99–3.58) 0.0502 HiHi 59 (64.8) 71.9 (60.4–85.7) 1.0 (reference) 66.2 (54.8–79.9) 1.0 (reference) CD8 (CT/IM)e 2.27 (1.43–3.70) 0.0817 2.27 (1.47–3.45) 0.0004 LoLo 13 (15.3) 38.9 (16.9–89.7) 5.88 (2.16–15.97) 0.0040 30.8 (13.6–69.5) 6 (2.23–16.11) <0.0001 Het 40 (47.1) 56.3 (41.2–76.8) 2.98 (1.26–7.06) 0.2611 47.3 (33.8–66.3) 3.43 (1.57–7.47) 0.00101 HiHi 32 (37.6) 85.7 (73.6–99.7) 1.0 (reference) 83.3 (70.9–97.9) 1.0 (reference) NOTE: VELIPI denotes the presence of vascular emboli (VE), lymphatic invasion (LI), and perineural invasion (PI), alone or in combination (information not available for 2 patients).
Abbreviations: LNR, lymph node ratio; NA, not applicable.
- Table 2.
Multivariate Cox proportional hazard analysis for DFS and OS among patients with rectal cancer eligible for primary surgery
DFS OS HR (95% CI) P HR (95% CI) P Model before stepwise (stepAIC) selection Age 1.14 (0.74–1.75) 0.5646 1.77 (1.3–2.42) 0.0003 Tumor (T) stage 1.63 (0.95–2.8) 0.0750 1.23 (0.8–1.88) 0.3535 N stage 0.42 (0.08–2.28) 0.3174 1.24 (0.43–3.54) 0.6885 LNR 4.03 (0.88–18.46) 0.0727 1.69 (0.7–4.1) 0.2426 VELIPI + (tumor emboli) 0.79 (0.18–3.45) 0.7564 0.57 (0.19–1.74) 0.3244 Immunoscorea (I0 to I4) 0.62 (0.44–0.87) 0.0061 0.67 (0.5–0.89) 0.0053 Model after stepwise (stepAIC) selection Age 1.66 (1.25–2.22) 0.0005 Tumor (T) stage 1.59 (0.94–2.7) 0.0836 LNR 1.88 (1.02–3.46) 0.0414 1.89 (1.31–2.72) .0007 Immunoscorea (I0 to I4) 0.62 (0.44–0.88) 0.0069 0.65 (0.49–0.85) 0.0019 UICC TNM Staging 1.43 (0.94–2.19) 0.0977 1.41 (1.01–1.98) 0.0437 Immunoscorea (I0 to I4) 0.55 (0.39–0.79) 0.0009 0.62 (0.47–0.81) 0.0004 NOTE: All categorical covariates were transformed into numeric codes before they entered into the Cox model.
Abbreviation: AIC, Akaike information criterion.
-
↵aLeave-one-out method. Correction using C = 1 − (SE[coef]/coef)2; heuristic shrinkage factor corrected with Holländer et al. (20).
-
Supplementary Data
Files in this Data Supplement:
- Supplementary Tables 1 - 5 - Supplementary Table 1: characteristics of the HEGP cohort of rectal cancer patients Supplementary Table 2: Characteristics of the St Spiridon Hospital cohort of patients Supplementary Table 3: Immune infiltration of CD3+ and CD8+ cells in tumor regions and clinical outcome. Cohort of 111 patients eligible to primary surgery. Supplementary Table 4: Patients at risk at each interval in the Kaplan Meier survival curves for the duration of DFS and OS according to the Immunoscore (CD3-CD8). Supplementary Table 5: Repartition of the "surgery cohort" patient's according to Stage and Immunoscore
- Supplementary Figure 1 - Kaplan Meier curves for the duration of OS according A: to T cell (CD3+) density evaluated in combined tumor regions (CT and IM). B: to T cell (CD8+) density evaluated in combined tumor regions (CT and IM).
- Supplementary Figure 2 - To reinforce the confidence on the statistical analyses, patients with poor postoperative outcome (I0 and I1) were pooled. The Kaplan–Meier curves illustrate the DFS according to the Immunoscore and show significant differences between patient groups. The multivariate analysis shows the prognostic power of the Immunoscore and illustrates how the Immunoscore overcomes the TNM scoring system.
Volume 20, Issue 7
