Multiparametric immune profiling in HPV- oral squamous cell cancer - PubMed (original) (raw)

. 2017 Jul 20;2(14):e93652.

doi: 10.1172/jci.insight.93652.

Daniel Bethmann 1 3, Matthias Kappler 4, Carmen Ballesteros-Merino 1, Alexander Eckert 4, R Bryan Bell 1 5, Allen Cheng 5, Tuan Bui 5, Rom Leidner 1 5, Walter J Urba 1, Kent Johnson 6, Clifford Hoyt 6, Carlo B Bifulco 1 7, Juergen Bukur 8, Claudia Wickenhauser 3, Barbara Seliger 8, Bernard A Fox 1 9

Affiliations

Multiparametric immune profiling in HPV- oral squamous cell cancer

Zipei Feng et al. JCI Insight. 2017.

Abstract

Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV- OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.

Keywords: Immunology; Oncology.

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Conflict of interest statement

Conflict of interest: B.A. Fox serves on the PerkinElmer Inc. Health Division Science Advisory Board.

Figures

Figure 1

Figure 1. Differences in density and location of immune infiltrate in OSCC, as a typical example of squamous cell carcinoma, compared with colorectal cancer, as a typical example of intestinal adenocarcinoma.

(A) Representative example with demonstration of topographic position of CD8+ infiltrate in OSCC (original magnification, ×40). (B) Representative example of CD8+ infiltrate in colorectal cancer (CRC) (original magnification, ×40). (C) Enumeration of immune cell infiltrate using the Definiens platform. In OSCC, most CD8+ cells are located within the stromal side invasive margin (IM), while, in CRC, the majority of CD8+ cells are located within the tumor and tumor side IM. Data are represented as dot plots (mean ± SEM). Two-tailed unpaired t test was performed to test statistical significance. n = 55 for OSCC, n = 199 for CRC. CT, core of tumor.

Figure 2

Figure 2. Representative image of immune cell infiltrate in an OSCC tumor.

Tissue sections were simultaneously stained for 7 antigens. Images (original magnification, ×20) with a high density of immune cell infiltrate were taken from stromal and tumor side invasive margin. Red overlay, recognized by software as tumor; green overlay, recognized by the software as stroma.

Figure 3

Figure 3. Effect of immune infiltrate density at the invasive margin on overall survival.

Higher densities of CD8+, but not FoxP3+, T cells and PD-L1+ tumor cells at the invasive margin (IM) predict favorable OS, while this effect is more profound on the stromal side of the IM. Densities of CD8, FoxP3, and PD-L1 on both the tumor and stromal sides of the IM were enumerated using PerkinElmer inForm software. A median cutoff was used to separate high and low infiltrate. Log-rank statistics were performed to determine significance. n = 119.

Figure 4

Figure 4. Independent analysis and correlation of CD8+/FoxP3+ immune cell infiltrates at the tumor and stromal side of the invasive margins.

(A) Tumor side and (B) stromal side. Data are represented in a scattered plot with the best fit (solid line) and 95% confidence interval (dotted line) shown. Statistical test for P values was linear regression. n = 124 for both tumor and stroma.

Figure 5

Figure 5. Presentation of a relationship analysis.

A reference cell (star) was picked and surrounding cell types were enumerated within a certain distance. The overall relationship parameter is a function of an average of all certain cell types within the entire field (original magnification, ×20).

Figure 6

Figure 6. Optimization of the cutoff of the distance of FoxP3+ cells within specified distance of CD8+ T cells, normalized for CD8+ T cell number.

Cumulative survival for a test cohort of 34 patients characterized as being above (high) or below (low) the median for FoxP320μmCD8n (A), FoxP330μmCD8n (B), FoxP345μmCD8n (C), and total FoxP3 (D). The number of FoxP3+ T cells within 30 μm of a CD8+ T cell, normalized to number of CD8+ T cells (FoxP330μmCD8n) (B), provides the best statistical cutoff point and was therefore used for the analysis of the entire cohort of patients. Numbers of FoxP3+ T cells and distance were determined using PerkinElmer inForm and R script and were normalized to the number of CD8+ T cells (FoxP330μmCD8n). A median cutoff was used to separate high and low infiltrate. Kaplan-Meier survival plots were made, and statistics were generated using log-rank test. n = 34.

Figure 7

Figure 7. Effect of topographic distance of FoxP3+ T cells from CD8+ T cells on prognosis.

A high number of FoxP3+ cells within 30 μm of CD8+ T cells, normalized to CD8+ T cell numbers (FoxP330μmCD8n), on the tumor (A) and the stromal (B) side of the invasive margin present a significant prognostic marker for worse OS, while the ratio of FoxP3/CD8 T cells failed to provide a significant prognostic signature (C and D). Absolute numbers of T cells as well as distance relationship were determined using PerkinElmer inForm and R script and were normalized to the number of CD8+ T cells. A median cutoff was used to separate high and low distance relationships. Kaplan-Meier survival plots were made, and statistics were generated using log-rank test. n = 119.

Figure 8

Figure 8. Effect of topographic distance of PD-L1+ T cells from CD8+ T cells on prognosis.

A high number of PD-L1+ within 30 μm of CD8+ T cells (PD-L130μmCD8n) (A and B) as well as the ratio of PD-L1/CD8 T cells (C and D) on the tumor and the stromal side of the invasive margin present a significant prognostic marker for worse OS. Absolute numbers of T cells as well as distance relationship were determined using PerkinElmer inForm and R script and were normalized to the number of CD8+ T cells. A median cutoff was used to separate high and low distance relationships. Kaplan-Meier survival plots were made, and statistics were generated using log-rank test. n = 119.

Figure 9

Figure 9. Effect of a suppression index on prognosis.

Use of a suppression index (SI) incorporating both the number of FoxP3+ cells and the number of PD-L1+ cells within 30 μm of a CD8+ T cell, normalized to CD8+ T cell number, on both the tumor (A) and the stromal (B) side of the invasive margin separates patients into 3 distinct prognostic groups, providing a highly significant biomarker. Here, the expressions based on median cutoff of FoxP330μmCD8n and PD-L130μmCD8n were added together and ranked from high (score of 2) to low (score of 0). Patients who were in the top 50% for both categories were in the high SI category and had the worst OS. Patients who were in the top 50% for one category were intermediate, and those in the lower 50% for both FoxP330μmCD8n and PD-L130μmCD8n had the lowest SI and the highest OS. Log-rank statistics were performed to determine significance. The top P value refers to all comparisons, the bottom P value refers to the difference between the lowest and highest score. n = 119.

Figure 10

Figure 10. Stage distribution and basic patient characteristics.

Kaplan-Meier survival plot of the cohort based on UICC stage.

Figure 11

Figure 11. Effect of the suppression index and distance relationships on OS in stage I–III OSCC patients_._

(A and B) Using a suppression index (SI) incorporating both FoxP330μmCD8n and PD-L130μmCD8n presents a significant prognostic marker for OS on the stromal side (B) but not on the tumor side of the invasive margin (IM) (A). (C–F) In stage I–III OSCC patients, only a high number of PD-L130μmCD8n cells on the stromal side of the IM (F) presents a significant biomarker, marking an unfavorable OS. For A and B, ranks were assigned as presented in Figure 9. For C–F, a median cutoff was used to separate high and low distance relationships. Kaplan-Meier survival plots were made, and statistics were generated using log-rank test. n = 59.

Figure 12

Figure 12. Effect of the suppression index and distance relationships on OS in stage IV OSCC patients_._

(A and B) Using a suppression index (SI) incorporating both the FoxP330μmCD8n and the PD-L130μmCD8n provides a significant prognostic marker for OS on both the tumor (A) and the stromal side of the invasive margin (IM) (B), with a more profound effect on the tumor side (A). (C–F) A high number of FoxP330μmCD8n (C) and PD-L130μmCD8n (E) cells on the tumor side of the IM provides a significant biomarker, identifying an unfavorable OS. For A and B, ranks were assigned as presented in Figure 9. For C–F, a median cutoff was used to separate high and low distance relationships. Kaplan-Meier survival plots were made, and statistics were generated using log-rank test. n = 60.

Figure 13

Figure 13. Effect of cytoplasmic expression of β2m on OS.

(A) Kaplan-Meier curve highlighting the strong yet insignificant tendency toward a prognostic effect of high levels of cytoplasmic expressed β2m (IRS 4–12 vs. 0–3), leading to a 1.64-fold increase (P = 0.08) in the relative risk (RR) of death, independent of T- and N-stage and grading. (B) This effect was predominantly accounted for by patients with T1–2 stage tumors (RR 2.77, P = 0.07), rather than in T3–4 stage tumors (RR 1.23, P = 0.54, data not shown).

Figure 14

Figure 14. The cumulative suppression index is a highly indicative prognostic marker superior to the prognostic index of the single markers.

(A) Kaplan-Meier curve showing the influence of the combined cytoplasmatic expression levels of β2m, HC, and LMP10 on OS. Patients were separated as above or below the median for each marker. A score of 0 represents below-median cutoff expression, and a score of 3 represents high expression for all three APM components. (B) Combining suppression indices (SI) from tumor and stroma to obtain cumulative suppression index (CSI). Each column represents FoxP330μmCD8n and PD-L130μmCD8n in tumor and stroma, with red indicating above-median cutoff, marking increased suppression. (C) Analysis of the entire cohort demonstrates the highly significant stepwise reduction of OS based on an increasing CSI, with 0 representing the lowest and 4 representing the most suppression relative to CD8+ T cells. (D) Kaplan-Meier curve for the CSI for patients with stage I–III disease. (E) Kaplan-Meier curve for the CSI for patients with stage IV disease. (F) Analysis of CSI in combination with the 3 APM components, demonstrating a highly significant stepwise reduction of OS based on an increasing score. A score of 0 represents low suppression and low cytoplasmatic APM expression (below-median cutoff); a score of 7 represents high (above-median) cutoff for all 7 categories. (A and C–F) Log-rank statistics were performed to determine significance. The top P value refers to all comparisons, the bottom P value refers to the difference between the lowest and highest score. n = 119 (A, C, and F); 59 (D); E = 60 (E).

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References

    1. Abreu LP, Kruger E, Tennant M. Oral cancer in Western Australia, 1982-2006: a retrospective epidemiological study. J Oral Pathol Med. 2010;39(5):376–381. doi: 10.1111/j.1600-0714.2009.00848.x. - DOI - PubMed
    1. Listl S, et al. Survival of patients with oral cavity cancer in Germany. PLoS One. 2013;8(1):e53415. doi: 10.1371/journal.pone.0053415. - DOI - PMC - PubMed
    1. Rogers SN, et al. Survival following primary surgery for oral cancer. Oral Oncol. 2009;45(3):201–211. doi: 10.1016/j.oraloncology.2008.05.008. - DOI - PubMed
    1. Zini A, Czerninski R, Sgan-Cohen HD. Oral cancer over four decades: epidemiology, trends, histology, and survival by anatomical sites. J Oral Pathol Med. 2010;39(4):299–305. - PubMed
    1. Pulte D, Brenner H. Changes in survival in head and neck cancers in the late 20th and early 21st century: a period analysis. Oncologist. 2010;15(9):994–1001. doi: 10.1634/theoncologist.2009-0289. - DOI - PMC - PubMed

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