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. 2019 Mar 8;7(1):65.
doi: 10.1186/s40425-019-0540-1.

Expression and clinical significance of PD-L1, B7-H3, B7-H4 and TILs in human small cell lung Cancer (SCLC)

Daniel Carvajal-Hausdorf  1   2 , Mehmet Altan  3   4 , Vamsidhar Velcheti  5 , Scott N Gettinger  3 , Roy S Herbst  3 , David L Rimm  1   3 , Kurt A Schalper  6   7   8
Affiliations

Expression and clinical significance of PD-L1, B7-H3, B7-H4 and TILs in human small cell lung Cancer (SCLC)

Daniel Carvajal-Hausdorf et al. J Immunother Cancer. .

Abstract

Background: Small cell lung cancer (SCLC) accounts for 10-15% of all lung malignancies and its prognosis is dismal. Although early studies have shown promising clinical activity of immune checkpoint blockers, the immune composition and expression of potentially actionable immunostimulatory targets in this malignancy are poorly understood.

Methods: Using multiplexed quantitative immunofluorescence (QIF), we measured the levels of 3 different B7 family ligands PD-L1, B7-H3, B7-H4 and major tumor infiltrating lymphocyte (TIL) subsets in 90 SCLC samples represented in tissue microarray format. Associations between the marker levels, clinicopathological variables and survival were studied.

Results: PD-L1 protein was detected in 7.3%, B7-H3 in 64.9% and B7-H4 in 2.6% of SCLC cases. The markers showed limited co-expression and were not associated with the level of TILs, age, gender and stage. Elevated B7-H4 was associated with shorter 5-year overall survival. The levels of CD3+, CD8+ and CD20+ TILs and the ratio of total/effector T-cells were significantly lower in SCLC than in non-small cell lung cancer. High levels of CD3+, but not CD8+ or CD20+ TILs were significantly associated with longer survival.

Conclusions: Taken together, our study indicate variable expression and clinical role of B7-family ligands in SCLC with predominant expression of the candidate target B7-H3 and the presence of a limited cytotoxic anti-tumor immune response. These results support the evaluation of B7-H3 blockers and/or pro-inflammatory therapies in SCLC.

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Conflict of interest statement

Ethics approval and consent to participate

All tissue was used after approval from the Yale Human Investigation Committee protocols #9505008219 and #1608018220, which approved the patient consent forms or in some cases waiver of consent.

Consent for publication

Not applicable.

Competing interests

Kurt Schalper: Consultant or advisor for Celgene, Moderna Therapeutics, Shattuck Labs, AstraZeneca, Pierre-Fabre and Abbvie. Research funding by Navigate Biopharma, Vasculox/Tioma, Tesaro, Takeda, Moderna Therapeutics, Surface Oncology, Pierre-Fabre, Merck and Bristol-Myers Squibb.

David Rimm: Consultant or advisor for Amgen, AstraZeneca, Agendia, Biocept, BMS, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, InVicro/Konica/ Minolta, Merck, NanoString, Perkin Elmer, PAIGE.AI, and Ultivue. Equity holder in PixelGear (start-up company related to direct tissue imaging) Research funding from AstraZeneca, Cepheid, Navigate/Novartis, NextCure, Lilly, Ultivue, Ventana and Perkin Elmer/Akoya.

Mehmet Altan: Research funding from BMS and Lilly.

Vamsidhar Velcheti: Consultant or advisory role for Genentech, BMS, AstraZeneca, Merck, Nektar therapeutics, Reddy Labs, Celgene, Foundation Medicine and Takeda Oncology.

Roy Herbst: Consultant or advisory role for Abbvie Pharmaceuticals, AstraZeneca, Biodesix, Bristol-Myers Squibb , Eli Lilly and Company, EMD Serono, Genentech/Roche, Heat Biologics, Loxo Oncology, Merck and Company, Nektar, NextCure, Novartis, Pfizer, Sanofi, Seattle Genetics, Shire PLC, Spectrum Pharmaceuticals, Symphogen, Tesaro, Neon Therapeutics, Infinity Pharmaceuticals. Research support from AstraZeneca, Eli Lilly and Company, Merck and Company. Member of the board of directors (non-executive/ independent) for Junshi Pharmaceuticals.

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Figures

Fig. 1
Fig. 1
Detection of immune targets and TILs in SCLC using multiplex quantitative fluorescence. a Representative fluorescence pictures showing B7-H3 (upper panel) and PD-L1 (lower panel) protein expression in SCLC. The target signal (red fluorescence) is predominantly located in tumor cells. b Representative fluorescence pictures showing the signal for DAPI (blue), cytokeratin (CK, green), CD3 (red), CD8 (green) and CD20 (magenta) staining in SCLC. Bar = 100 um
Fig. 2
Fig. 2
Levels of different immune targets in SCLC. Distribution of PD-L1 (red), B7-H3 (green) and B7-H4 (magenta) QIF scores in SCLCs from Yale. The frequency of expression for each marker is indicated in parenthesis. The cut-point used to define expression was the signal detection threshold. AU = Arbitrary units of fluorescence
Fig. 3
Fig. 3
PD-L1, B7-H3 and B7-H4 are infrequently co-expressed in SCLC. A-C) Histograms showing the levels of PD-L1, B7-H3 and B7-H4 protein in small cell lung carcinomas from the Yale cohort. The linear regression coefficients (R2) of the scores between each marker pair are indicated within the charts
Fig. 4
Fig. 4
Levels of TIL subpopulations in SCLC. Distribution of CD3 (red), CD8 (green) and CD20 (magenta) QIF scores in SCLCs from the Yale cohort. Cases were stratified using the median score of each marker as stratification cut-point. AU = Arbitrary units of fluorescence
Fig. 5
Fig. 5
Levels of TIL subpopulations in SCLC and major NSCLC subtypes. a Chart showing the levels of CD3 (red), CD8 (green) and CD20 (magenta) in SCLC (left), primary lung adenocarcinomas (LADC, center) and lung squamous cell carcinomas (LSCC, right). Each bar depicts the median +/− SEM. The levels of TILs in NSCLC subtypes were obtained previously using the same multiplexing protocol [22]. b Chart showing the ratio of CD8/CD3 signal in SCLCs (left), LADCs (center) and LSCCs (right). The number of cases is indicated within each bar. *** = P < 0.001; ns = not significant. AU = Arbitrary units of fluorescence
Fig. 6
Fig. 6
Association between the levels of B7 family ligands, TIL subsets and survival in SCLC. Kaplan-Meier graphical analysis of the 5-year overall survival in patients with SCLC from the Yale cohort. a Survival based on the expression of the immune ligands PD-L1 (left), B7-H3 (center) and B7-H4 (right). b Survival based on the expression of the TIL markers CD3 (left), CD8 (center) and CD20 (right). The respective log-rank P values are indicated in the chart
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