Samples from a retrospectively collected SCLC cohort from Yale University represented in 2 tissue microarrays (TMAs) (YTMA57 and YTMA259) totaling 90 cases were used. Detailed clinico-pathological characteristics of the cohorts were collected form surgical pathology reports and clinical records. TMAs were prepared using 0.6 mm tissue cores, each in 2-fold redundancy using standard procedures [16, 17]. The actual number of samples analyzed for each marker is lower than the total samples in the cohort due to unavoidable loss of tissue, absence or limited tumor cells in some spots as is commonly seen in TMA studies or incomplete clinicopathologic annotation. 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.

We measured the levels of PD-L1 (E1L3N, Cell Signaling technology), B7-H3 (D9M2L, Cell Signaling Technology), B7-H4 (D1M8I, Cell Signaling Technology), CD3 (clone E272, Novus Biologicals), CD8 (clone C8/144B, DAKO), CD20 (clone L26, DAKO) and pancytokeratin (AE1/AE3, DAKO) using QIF in TMA slides containing the cohort cases. PD-L1, B7-H3 and B7-H4 were stained in serial sections from the TMA blocks using a previously described protocol with simultaneous detection of cytokeratin and 4′,6-diamidino-2-phenylindole (DAPI) [18, 19]. Briefly, antigen retrieval was with citrate buffer pH 6.0 for 20 min at 97 °C in a pressure-boiling container and blocking was performed with 0.3% bovine serum albumin in 0.05% Tween solution for 30 min. Primary antibodies were incubated overnight using a dilution of 1:1600 for PD-L1, 1:500 for B7-H3 and 1:200 for B7-H4. Stringent validation and optimization of these assays using cell line transfectants and endogenous human tissue controls has been reported by our group [18, 20, 21]. Secondary antibody for cytokeratin was Alexa 546-conjugated goat anti-mouse or anti-rabbit (Invitrogen Molecular Probes, Eugene, OR, USA). Cyanine 5 (Cy5) directly conjugated to tyramide (FP1117; Perkin-Elmer) at a 1:50 dilution was used for target antibody detection.

CD3, CD8, CD20 and cytokeratin were simultaneously stained using a sequential staining protocol, as previously described [16, 20, 22]. Briefly, TMA sections were deparaffinized and subjected to antigen retrieval using pH = 8.0 EDTA buffer (Sigma-Aldrich, St Louis, MO, USA) and boiled for 20 min at 97 °C in a pressure-boiling container (PT module, Lab Vision, Thermo Scientific, Waltham, MA, USA). Slides were then incubated with dual endogenous peroxidase block (DAKO #S2003, Carpinteria, CA, USA) for 10 min at room temperature and subsequently with a blocking solution containing 0.3% bovine serum albumin in 0.05% Tween solution for 30 min. Residual horseradish peroxidase activity between incubations with secondary antibodies was eliminated by exposing the slides twice for 7 min to a solution containing benzoic hydrazide (100 mM) and hydrogen peroxide (50 mM) in PBS. Isotype specific, fluorophore-conjugated secondary antibodies were used for signal detection and nuclei were highlighted using DAPI.

Quantitative measurement of the fluorescent signal was performed using the AQUA® method of QIF, as previously reported [18, 20, 23]. Briefly, the QIF score of each fluorescence channel was calculated by dividing the target marker pixel intensities by the area of the desired compartment. Scores were normalized to the exposure time and bit depth at which the images were captured, allowing scores collected at different exposure times to be comparable. The immune target scores and TIL markers considered the signal detected in the whole tissue compartment using an adjusted DAPI mask. Cases were considered as target expressers when the QIF score was above the signal detection threshold determined using the negative control preparations and visual inspection. For stratification, the marker levels were classified as high/low using the top 25-percentile of the cohort scores as stratification cut-point.

QIF signal differences between groups were analyzed using t-test for continuous variables and chi-square test for categorical variables. Linear regression coefficients were calculated to determine the association between continuous scores. Survival analysis based on marker expression was performed using Kaplan-Meier analyses with log rank test and overall survival as endpoint. Statistical significance was considered at P < 0.05 and analyses were performed using JMP® Pro software (version 9.0.0, 2010, SAS Institute Inc.) and GraphPad Prism v6.0 for Windows (GraphPad Software, Inc). All statistical tests were two-sided.

We previously validated and optimized assays for detection of PD-L1, B7-H3, B7-H4 and TIL markers using formalin-fixed, paraffin-embedded (FFPE) preparations from human tissue samples and cell line transfectants [17, 20–23]. As expected for SCLC, positive staining for cytokeratin was focal and frequently showed a perinuclear dot-like staining pattern (Fig. 1). PD-L1 and B7-H3 were predominantly recognized in tumor cells with cytoplasmic and membranous staining (Fig. 1a). Prominent B7-H4 positivity was infrequently recognized and showed relatively low signal with a focal staining pattern. Expression of TIL markers showed predominance of CD3+ T-cell staining with CD8+ and CD20+ cells displaying low levels in the cohort. Representative examples from cases with prominent CD3+ TILs or CD20+ B-cell infiltrates are shown in Fig. 1b. Fig1Fig. 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

To evaluate the TIL scores of SCLCs in the context of other lung cancer subtypes, we compared the marker levels with those obtained in retrospective cohorts of lung adenocarcinomas (LADC) and lung squamous cell carcinomas (LSCC) measured using the same assay and analysis platform [22]. As shown in Fig. 5a, SCLCs showed significantly lower levels of all TIL markers than LADC and LSCC (P = 0.01-P < 0.0001). The most prominent difference was in CD8 level that was 5.4 fold lower than in LADC and 6-fold lower than in LSCC. Notably, the CD3/CD8 ratio was also prominently lower in SCLC than in the major NSCLC subsets, suggesting the presence of a less cytotoxic T-cell profile in this malignancy (Fig. 5b, mean CD3/CD8 ratio of 0.37 vs 0.63 in LADC and 0.62 in LSCC, P < 0.001).Fig5Fig. 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

Elevated expression of PD-L1, B7-H3 or B7-H4 (scores within the top signal quartile) were not significantly associated with major clinicopathologic variables or TIL markers in the cohort (Table 1). As expected, the levels of CD3 were positively associated with CD8, but there was no relationship between CD3 or CD8 and CD20 in the tumors. High levels of CD20+ B-cells were more commonly seen in samples from female patients (14 of 23 [37.8%] vs 6 of 38 [13.6%], P = 0.01). High PD-L1 or B7-H3 protein levels were not significantly associated with 5-year overall survival (Fig. 6a-b). However, elevated expression of B7-H4 was associated with shorter survival in the cohort (Fig. 6c, log-rank P = 0.05). In addition, increased expression of the pan T-cell marker CD3- but not of CD8 or CD20 was significantly associated with longer overall survival (log-rank P = 0.03, Fig. 6d-f).Tab1

Association of PD-L1, B7-H3, B7-H4 and TIL subsets with major clinico-pathological characteristics and TILs in SCLC