onco13981-sup-0001-supinfo.xlsxExcel 2007 spreadsheet , 74 KB

Appendix: Supporting Information

onco13981-sup-0002-FigureS1.pdfPDF document, 209.1 KB

Figure S1. Correlation between disease status and the baseline counts or proportions of total PD-L1-positive CTCs.

CTC counts and PD-L1 status on CTCs from 148 patients before treatment with PD-1 or PD-L1 inhibitor (A). Objective response rate (ORR) and disease control rate (DCR) of patients with or without PD-L1-positive CTCs at baseline (B). Count distribution of total PD-L1-positive CTCs in the PR, SD and PD groups at baseline (C). Count distribution of PD-L1-positive CTCs in the DC and PD groups at baseline (D). Proportional distribution of PD-L1-positive CTCs relative to total CTCs in the PR, SD and PD groups at baseline (E). Proportional distribution of PD-L1-positive CTCs relative to total CTCs in the DC and PD groups at baseline (F). PR, partial response; SD, stable disease; PD, progressive disease, referring to patients who showed no response; DC: disease control, referring to patients who showed response (PR and SD). Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; **p < 0.01.

onco13981-sup-0003-FigureS2.pdfPDF document, 290.5 KB

Figure S2. Kaplan–Meier estimates for progression-free survival (PFS) and overall survival (OS) of patients based on different cutoff points for the PD-L1 levels on CTCs at baseline.

Kaplan–Meier estimates of PFS for patient subgroups with or without PD-L1-positive CTCs at baseline (A), patient subgroups with a ratio of PD-L1-positive expression relative to total CTCs above or below 50% at baseline (B), patient subgroups with a ratio of PD-L1-high expression relative to total CTCs above or below 20% at baseline (C), patient subgroups with a ratio of PD-L1-high expression relative to total CTCs above or below 30% at baseline (D) and patient subgroups with a total score of PD-L1-positive expression above or below 7 at baseline (E). Kaplan–Meier estimates of OS for patient subgroups with or without PD-L1-positive CTCs at baseline (F), patient subgroups with a ratio of PD-L1-positive expression relative to total CTCs above or below 50% at baseline (G), patient subgroups with a ratio of PD-L1-high expression relative to total CTCs above or below 20% at baseline (H), patient subgroups with a ratio of PD-L1-high expression relative to total CTCs above or below 30% at baseline (I), patient subgroups with a total score of PD-L1-positive expression above or below 7 at baseline (J), patients who received monotherapy in the subgroups with or without PD-L1-high expression on CTCs at baseline (K) and patients who received combined therapy in the subgroups with or without PD-L1-high expression on CTCs at baseline (L).

onco13981-sup-0004-FigureS3.pdfPDF document, 221.9 KB

Figure S3. Efficacy of positivity for PD-L1 expression in tumor samples.

Subgroup analysis of the objective response rate. Data are for patients (n=83) evaluable for PD-L1 expression in tumor samples. PD-L1 expression in at least 1% of tumor cells is considered PD-L1-positive (A). Kaplan–Meier estimate of PFS for patients with PD-L1-positive expression relative to those with PD-L1 negative expression in tumor samples (B). Kaplan–Meier estimate of OS for patients with PD-L1-positive expression relative to those with PD-L1 negative expression in tumor samples (C).

onco13981-sup-0005-FigureS4.pdfPDF document, 338 KB

Figure S4. Correlation between disease status and the baseline counts or proportions of total PD-L1-high CTCs for patients with HCC.

PD-L1 distribution on CTCs from patients with HCC before anti-PD-1/PD-L1 immunotherapy (at baseline). Patients are shown according to the percentage of PD-L1-high CTCs relative to total CTCs (A). ORR and DCR of patients with PD-L1-high CTCs at baseline (B). Count distribution of PD-L1-high CTCs in the PR, SD and PD groups at baseline (C). Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; **p < 0.01. Proportional distribution of PD-L1-high CTCs relative to total CTCs in the PR, SD and PD groups at baseline (D). Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; **p < 0.01. Subgroup analysis of objective responses in patients with HCC (E). Data are for patients with HCC (n=44) in the as-treated population. *p < 0.05, **p < 0.01. Kaplan–Meier estimates of PFS for HCC patients in the subgroups with or without PD-L1-high expression CTCs at T0 (F). Kaplan–Meier estimates of OS for HCC patients in the subgroups with or without PD-L1-high expression CTCs at T0 (G). Kaplan–Meier estimates of PFS for HCC patients in the subgroups with or without PD-L1-high expression CTCs at T1 (H).

onco13981-sup-0006-FigureS5.pdfPDF document, 135.6 KB

Figure S5. Correlation between disease status and the baseline counts or proportions of total PD-L1-high CTCs for patients received anti-PD-1/PD-L1 monotherapy.

PD-L1 distribution on CTCs from patients received anti-PD-1/PD-L1 monotherapy at baseline. Patients are shown according to the percentage of PD-L1-high CTCs relative to total CTCs (A). ORR and DCR of patients with PD-L1-high CTCs at baseline (B). Count distribution of PD-L1-high CTCs in the PR, SD and PD groups at baseline (C). Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; **p < 0.01. Proportional distribution of PD-L1-high CTCs relative to total CTCs in the PR, SD and PD groups at baseline (D). Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; *p < 0.05, **p < 0.01. Kaplan–Meier estimates of PFS for patients received anti-PD-1/PD-L1 monotherapy in the subgroups with or without PD-L1-high expression CTCs at T0 (E). Kaplan–Meier estimates of OS for patients received anti-PD-1/PD-L1 monotherapy in the subgroups with or without PD-L1-high expression CTCs at T0 (F).

onco13981-sup-0007-FigureS6.pdfPDF document, 119 KB

Figure S6. Dynamic changes in the count or proportional distribution of positive PD-L1 expression on CTCs before and after anti-PD-1/PD-L1 therapy.

Changes in the total CTC count in PR (red), SD (orange) and PD (blue) patients at T0 and T1 (A). Changes in the count of PD-L1-positive CTCs in PR (red), SD (orange) and PD (blue) patients at T0 and T1 (B). Changes in the count of PD-L1-high CTCs in PR (red), SD (orange) and PD (blue) patients at T0 and T1 (C). Changes in the proportion of PD-L1-positive CTCs relative to total CTCs in PR (red), SD (orange) and PD (blue) patients at T0 and T1 (D). Changes in the proportion of PD-L1-positive CTCs relative to total CTCs in DC (red) and PD (blue) patients at T0 and T1 (E). T0: at baseline; T1: 4-10 weeks after immunotherapy, when the first response evolution was ascertained. Differences in growth were determined using Student's t-test and by calculating subsequent p-values. NS: not significant; **p < 0.01.

onco13981-sup-0008-FigureS7.pdfPDF document, 195.3 KB

Figure S7. Comparing circulating biomarkers with PD-L1 levels on CTCs for monitoring tumor dynamics. Each panel represents data from a different patient.

Case 55 (A): An HCC patient who received combined therapy had SD at the first response evolution, and SD occurred at 46 weeks after treatment. Case 40 (B): A pelvic neuroendocrine carcinoma patient who received monotherapy had PR at the first response evolution, and PD occurred 34 weeks after treatment. For cases 22 (C), 31 (D), 61 (E), 78 (F), 89 (G) and 108 (H), the patients had SD at the first response evolution, and PD occurred at the time when the third blood sample was collected. Among these cases, patients 22, 61 and 89 received monotherapy, and patients 31, 61 and 108 received combined therapy. Cases 32 (I) and 72 (J): The patients first had PD as defined by RECIST, but they did not have PD according to iRECIST; then, PD occurred at the time when the third blood sample was collected. Top panels, plasma levels of circulating biomarkers. Middle panels show the counts of total CTCs (black lines), PD-L1-positive CTCs (blue lines) and PD-L1-high CTCs (red lines). Bottom panels show the proportion of PD-L1-positive CTCs (blue lines) and PD-L1-high CTCs (red lines) relative to total CTCs. Vertical dotted lines show the tumor response evaluation assessed according to RECIST criteria. PD, progressive disease; SD, stable disease; PR, partial response. T0: at baseline; T1: 4-10 weeks after treatment, when the first response evolution was ascertained; T2: 10-52 weeks after treatment.

onco13981-sup-0009-FigureS8.pdfPDF document, 450.9 KB

Figure S8. Representative CTCs with different PD-L1 levels isolated from case 4.

The fluorescence intensity distribution of representative CTCs with different PD-L1 levels isolated from case 4 at T0 (A), T1 (B) and T2 (C). In order from top to bottom, it was PDL1 negative (PD-L1 neg), lowly expressed (PD-L1 low), medially expressed (PD-L1 medium) and strongly expressed (PD-L1 high) CTC, respectively. T0: at baseline; T1: 4-10 weeks after treatment, when the first response evolution was ascertained; T2: 52 weeks after treatment.

onco13981-sup-0010-Tables.pdfPDF document, 254.8 KB

Supplemental Tables