Background: The clinical efficacy observed with inhibitors of programed cell death 1/programed cell death ligand 1 (PD-L1/PD-1) in cancer therapy has prompted studies to characterize the immune response in several tumor types, including lung cancer. However, the immunological profile of non–small cell lung carcinoma (NSCLC) treated with neoadjuvant chemotherapy (NCT) is not yet fully characterized, and it may be therapeutically important. The aim of this retrospective study was to characterize and quantify PD-L1/PD-1 expression and tumor-associated immune cells (TAICs) in surgically resected NSCLCs from patients who received NCT or did not receive NCT (non-NCT). Methods: We analyzed immune markers in formalin-fixed, paraffin-embedded tumor tissues resected from 112 patients with stage II/III NSCLC, including 61 non-NCT (adenocarcinoma [ADC] = 33; squamous cell carcinoma [SCC] = 28) and 51 NCT (ADC = 31; SCC = 20). We used multiplex immunofluorescence to identify and quantify immune markers grouped into two 6-antibody panels: panel 1 included AE1/AE3, PD-L1, CD3, CD4, CD8, and CD68; panel 2 included AE1/AE3, PD1, granzyme B, FOXP3, CD45RO, and CD57. (Continued on next page) * Correspondence: firstname.lastname@example.org; BSepesi@mdanderson.org; email@example.com Department of Translational Molecular Pathology, Unit 951, The University of Texas MD Anderson Cancer Center, 2130 West Holcombe Blvd, Houston, TX 77030, USA Department of Thoracic and Cardiovascular Surgery, Unit 1489, The University of Texas MD Anderson Cancer Center, 1400 Pressler St. Houston, Houston,, TX 77030, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 2 of 11 (Continued from previous page) Results: PD-L1 expression was higher (> overall median) in NCT cases(median,19.53%)thaninnon-NCT cases(median, 1.55%; P = 0.022). Overall, density of TAICs was higher in NCT-NSCLCs than in non-NCT-NSCLCs. Densities of CD3+ cells in the tumor epithelial compartment were higher in NCT-ADCs and NCT-SCCs than in non-NCT-ADCs and non-NCT-SCCs (P = 0.043). Compared with non-NCT-SCCs, NCT-SCCs showed significantly higher densities of CD3 + CD4+ (P = 0.019) and PD-1+ (P < 0.001) cells in the tumor epithelial compartment. Density of CD68+ tumor-associated macrophages (TAMs) washigherinNCT-NSCLCsthaninnon-NCT-NSCLCsand wassignificantly higher in NCT-SCCs than in non-NCT-SCCs. In NCT-NSCLCs, higher levels of epithelial T lymphocytes (CD3+ CD4+) andepithelialand stromalTAMs(CD68+) were associated with better outcome in univariate and multivariate analyses. Conclusions: NCT-NSCLCs exhibited higher levels of PD-L1 expression and T-cell subset regulationthannon-NCT-NSCLCs, suggesting that NCT activates specific immune response mechanisms in lung cancer. These results suggest the need for clinical trials and translational studies of combined chemotherapy and immunotherapy prior to surgical resection of locally advanced NSCLC. Keywords: Tumor compartments, Epithelial compartment, Stromal compartment, Adenocarcinoma, Squamous cell carcinoma, Survival, T cells Background suggesting that strategies to control and modify the im- Tumors grow by using a complex composite system that mune cell population are important approaches to can- includes epithelial and stromal cell activation, vessel pro- cer therapy. Although neoadjuvant chemotherapy (NCT) liferation, and inflammatory and immune cell activation for cancer has historically been considered immunosup- . In normal situations, T lymphocytes recognize ma- pressive, it is now accepted that certain chemotherapy lignant cells as abnormal and activate cytotoxic T lym- agents, such as paclitaxel, cisplatin, gemcitabine, and phocytes through helper T cells at the site, which carboplatin, can regulate and modulate antitumor im- infiltrate and kill the malignant cells. However, malig- munity [13–17]. Chemotherapy has a potential to trigger nant cells have developed sophisticated mechanisms and immune activation by inducing immunogenic cell death pathways through which they regulate negative and posi- and subsequent tumor-associated neoantigen release, tive signals, blocking cytotoxic T cell activation and which in turn activates antigen-presenting cells (APCs) regulatory T cells and thus promoting tumor growth and such as tumor-associated macrophages (TAMs) and eventual tumor metastasis . dendritic cells through Toll-like receptors [18–21]. For An increasing number of studies as well as clinical trials this study, therefore, we hypothesized that NCT influ- in the past few years have demonstrated the oncologic ef- ences anticancer response by favorably altering the im- fectiveness of antibody inhibitors of immune checkpoints; mune microenvironment. by inhibiting these checkpoints, these antibodies facilitate The aim of this retrospective study was to identify and release of inhibitory signals and augment the antitumor quantify chemotherapy-induced changes in the immune activity of the immune system. The remarkable clinical ef- microenvironment, including PD-L1/PD-1 expression, in ficacy observed with inhibitors of immune checkpoints the tumor and tumor-associated immune cells (TAICs) such as programed cell death 1/programed cell death lig- using a multiplex immunofluorescence methodology . and 1 (PD-L1/PD-1) [3–5] has become increasingly im- We used this approach to compare surgically resected portant in studying the role of the immune cell system in non–small cell lung carcinoma (NSCLC) specimens from controlling tumor growth in various types of cancer. patients who received NCT with specimens from patients Various aspects of immune cells, such as type, func- who underwent primary surgical resection (non-NCT). tional polarization, and local distribution through the tumor, have been shown to influence clinical outcome Methods for cancer patients . Accumulating evidence shows Cases and specimens that high densities of mature T lymphocytes, in particu- Formalin-fixed, paraffin-embedded (FFPE) histologic sec- lar those with cytotoxic function such as CD8+ and nat- tions of NSCLCs were prospectively identified from ural killer cells, correlate with favorable prognosis, both primary tumors resected from 112 patients who under- in terms of recurrence-free survival (RFS) and overall went surgery with curative intent between January 1, survival (OS), in various cancer types, including lung 1997, and December 31, 2012, at The University of Texas cancer [7–11]. These findings strongly indicate that a MD Anderson Cancer Center. Of the 112 patients, 61 natural immune cell reaction controls the escape of underwent primary surgical resection (non-NCT group); metastatic cells and reduces cancer aggressiveness , 33 of the patients in this group had adenocarcinoma Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 3 of 11 (non-NCT-ADC) and 28 had squamous cell carcinoma fluorochrome used in the mIF to create the spectral li- (non-NCT-SCC). The comparison group consisted of brary in human tonsil FFPE tissues used in the multi- 51 patients who received NCT prior to surgical resec- spectral analysis . Human tonsil FFPE tissues were tion. This group comprised 31 with adenocarcinoma also used with and without primary antibodies as positive (NCT-ADC) and 20 with squamous cell carcinoma and negative (autofluorescence) controls, respectively. The (NCT-SCC). Tumor stage was classified according to mIF- and uniplex IF-stained slides were scanned with a the systems of the World Health Organization, 4th Vectra 3.0 microscope system (PerkinElmer) under fluores- edition , and the International Association for the cent illumination. From each slide, Vectra automatically cap- Study of Lung Cancer, 7th edition . Clinicopatho- tured the fluorescent spectra from 420 nm to 720 nm at logic information, including demographic data, smok- 20-nm intervals with thesameexposuretime and then com- ing status (current, former, or never), tumor size bined the captured images to create a single stack image that before NCT (according to image scanning tomog- retained the particular spectral signature of all IF markers raphy reports) and after NCT (pathologic report), . After the specimens were scanned at low magnification type of NCT used, adjuvant treatment, and follow-up (×10), five individual fields (669×500 μm each) in the tumor information (RFS and OS), was retrieved from patients’ area were examined with a Phenochart 1.0.4 (PerkinElmer) electronic medical records. The study received approval viewer so that they could be scanned at high resolution from the MD Anderson Cancer Center Institutional (×20) to capture various elements of tumor heterogeneity. Review Board; written informed consent was required of Histologic assessment of each tumor area ensured that clus- and obtained from all patients. ters of malignant cells were included in the selected area and that each area from panel 1 overlapped with the sequential Multiplex immunofluorescence staining tissue from panel 2. Manual multiplex immunofluorescence (mIF) staining was performed in 4-μm sequential histologic tumor sec- Multispectral analysis tions obtained from representative FFPE tumor blocks Tumor multispectral images containing PD-L1 and by using the Opal 7-Color fIHC Kit (PerkinElmer, Wal- TAICs, including tumor-infiltrating lymphocyte (TIL) tham, MA). The stained slides were scanned by a Vectra markers, were analyzed in two compartments: the epi- multispectral microscope (PerkinElmer) . The im- thelial compartment, defined as malignant cell nests, munofluorescence (IF) markers used were grouped into and the stromal compartment, characterized by the fi- two 6-antibody panels: panel 1 consisted of pancytokera- brous tissue present between malignant cells, as previ- tin AE1/AE3 (epithelial cell marker; dilution 1:300; ously described ; these compartments were identified Dako, Carpinteria, CA), PD-L1 (clone E1L3N, dilution by applying the tissue segmentation tool of the InForm 1:100; Cell Signaling Technology, Beverly, MA), CD3 (T 2.1.0 software (PerkinElmer) (Additional file 1:Figure S1). lymphocyte marker; dilution 1:100; Dako), CD4 (helper The individual cells (defined by nuclei [DAPI] staining) T cell marker; Novocastra clone 4B12, dilution 1:80; identified by the cell segmentation tool were subjected to Leica Biosystems, Buffalo Grove, IL), CD8 (cytotoxic T the phenotyping pattern recognition learning algorithm cell marker; clone C8/144B, dilution 1:20; Thermo Fisher tool to characterize co-localization of the various cell pop- Scientific, Waltham, MA), and CD68 (macrophage ulations  using panel 1 and panel 2 labeling. Panel 1 marker; clone PG-M1, dilution 1:450; Dako). Panel 2 (Additional file 2: Figure S2) labeling was as follows: ma- consisted of pancytokeratin AE1/AE3 (dilution 1:300; lignant cells expressing PD-L1 (AE1/AE3 + PD-L1+); T Dako), PD-1 (clone EPR4877–2, dilution 1:250; Abcam, lymphocytes (CD3; pan T-cell marker including helper T Cambridge, MA), granzyme B (cytotoxic lymphocyte cells CD3 + CD4+, cytotoxic T cells CD3 + CD8+, and marker; clone F1, ready to use; Leica Biosystems), other CD3+ T cells); helper T cells (CD3 + CD4+); FOXP3 (regulatory T cell marker; clone 206D, dilution cytotoxic T cells (CD3 + CD8+); TAMs (CD68+); and 1:50; BioLegend, San Diego, CA), CD45RO (memory T TAMs expressing PD-L1 (CD68 + PD-L1+). Panel 2 cell marker; clone UCHL1, ready to use; Leica Biosystems), (Additional file 3: Figure S3) labeling was as follows: mem- and CD57 (natural killer cell marker; clone HNK-1, dilution ory cells (CD45RO; including memory/natural killer cells 1:40; BD Biosciences, San Jose, CA). CD45RO + CD57 + granzyme B−, memory/regulatory cells Primary antibody was visualized by using tyramide sig- CD45RO + FOXP3+, memory antigen experienced cells nal amplification linked to a specific fluorochrome from CD45RO + PD-1+, and other CD45RO+ cells); memory/ the Opal 7-Color fIHC Kit for each primary antibody. A regulatory cells (CD45RO + FOXP3+); memory antigen stripping procedure, based on the EZ Retriever micro- experienced cells (CD45RO + PD-1+); activated natural wave (BioGenex, Fremont, CA), was performed for each killer cells (CD57 + granzyme B + CD45RO−); and antigen consecutive antibody staining. In parallel, uniplex IF was experienced cells (PD-1; including PD-1 + CD45RO+ and used with each individual antibody and with the same other PD-1+ cells). The analysis with each panel created a Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 4 of 11 comprehensive cell-by-cell identification report of expres- interval from surgery to recurrence or last contact, and OS sion of the antibody markers in both compartments. The was defined as the interval from surgery to death or last individual cell report created by InForm was processed by contact. As described previously by Pataer and colleagues Spotfire software (TIBCO, PerkinElmer) to create a final , the hematoxylin and eosin–stained slides from NCT data report expressing the results as number of cells/mm patients were examined to determine the percent tumor from each individual cell phenotyping population as well viability and its influence on survival at a 10% cutoff. as the percentage of TAMs and malignant cells expressing Multivariate Cox proportional hazard regression models PD-L1 for the statistical analysis. and logistic regression models were utilized to study the variables significant in the univariate analysis and their as- Statistical methods sociation with outcome. Statistical analyses were carried out with the R software program (version 3.3.0, released May 2016; Vienna, Results Austria; URL https://www.R-project.org/). Expression Clinicopathologic characteristics greater than the median percentage of membranous Using mIF and image analysis approaches, we evaluated PD-L1 expression in malignant cells was considered the immune microenvironment of NSCLCs from pa- positive; based on this and on measured cell densities tients who did or did not receive NCT (Fig. 1). Clinico- per mm , we divided patients into two groups, high and pathologic features and chemotherapy treatment data low, relative to the median number of TAICs per mm . are summarized in Table 1. The median interval between Differences between groups for all parameters were de- completion of NCT and surgical resection was 35 days termined by using the Mann–Whitney U test (unpaired, (min/max, 17/75 days). The median numbers of malig- nonparametric, two-tailed), except for RFS and OS studies, nant cells expressing PD-L1+ and the TAIC densities in in which the log rank test was used. RFS was defined as the the non-NCT and NCT groups are shown in Table 2. Panel 1 Panel 2 P=0.008 DAPI Cytokeratin PD-L1 CD3 CD4 CD8 CD68 DAPI Cytokeratin PD-1 CD57 GranB CD45RO FOXP3 Panel 1 Panel 2 non-NCT NCT DAPI Cytokeratin PD-L1 CD3 CD4 CD8 CD68 DAPI Cytokeratin PD-1 CD57 GranB CD45RO FOXP3 Fig. 1 Representative multiplex immunofluorescences and PD-L1 expression in non-NCT and NCT. (Left) Multiplex immunofluorescence images of representative NSCLC tumor sections analyzed for panel 1 and panel 2 markers: upper images are from the group that did not receive neoadjuvant chemotherapy (non-NCT), while the lower images are from the group that did receive NCT. The images reflect the variations in cell phenotypes observed in these cases. (Right) Box plot showing that PD-L1 expression by malignant NSCLC cells was higher in the group that received NCT than in the non-NCT group. Images ×200 PD-L1 expression in malignant cells (%) Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 5 of 11 Table 1 Characteristics of NSCLC patients who received Table 2 Median densities of various immune marker–expressing neoadjuvant chemotherapy (NCT) or did not receive NCT cells in NSCLCs from patients who received neoadjuvant (non-NCT) (N = 112) chemotherapy (NCT) or did not receive NCT (non-NCT) (N = 112) N (%) Markers non-NCT NCT P (n = 61) (n = 51) Characteristic Category non-NCT NCT (n = 61) (n = 51) Median Cell Density Age Median 62 years 65 years 2 (cells/mm ) Sex Female 22 (36) 22 (43) Panel 1 Male 39 (64) 29 (57) MCs (AE1/AE3+) 3559.79 3269.35 0.615 Tobacco history No 3 (5) 6 (12) MCs PD-L1+ 34.37 574.58 0.022 Yes 58 (95) 45 (88) CD3+ 903.21 1501.99 0.021 Smoking status Never 3 (5) 6 (12) CD3 + CD4+ 671.00 1031.88 0.040 Former 28 (46) 14 (28) CD3 + CD8+ 156.38 276.08 0.588 Current 30 (49) 31 (60) CD68+ 298.80 609.36 0.059 Tumor size Median – 4.50 cm CD68 + PD-L1+ 194.46 307.32 0.122 pretreatment Panel 2 (CT scan) MCs (AE1/AE3+) 3536.02 2970.70 0.157 Tumor size Median 4.50 cm 4.10 cm posttreatment CD45RO+ 668.75 1180.26 0.290 (Pathology) CD45RO + CD57 + granzyme B− 679.09 965.58 0.147 Tumor status T 4 (6) 5 (10) CD45RO + PD-1+ 153.73 443.04 < 0.001 T 25 (41) 23 (44) CD45RO + FOXP3+ 5.38 8.37 0.427 T 23 (38) 13 (26) CD57 + granzymeB+ 6.87 20.32 < 0.001 T 9 (15) 10 (20) PD-1+ 336.02 795.21 < 0.001 Nodal status N 10 (16) 7 (16) MC malignant cells N 18 (29) 11 (18) a Mann Whitney U test N 33 (54) 33 (66) AJCC stage II 7 (12) 5 (8) PD-L1 expression by malignant cells higher in NCT- III 54 (88) 46 (92) treated tumors Density of malignant cells expressing PD-L1 (AE1/AE3 + Histology ADC 33 (54) 31 (60) PD-L1+) was higher in NCT-treated tumors (median, SCC 28 (46) 20 (40) 574.58 cells/mm ) than in non-NCT tumors (median, Neoadjuvant Carboplatin/paclitaxel – 23 (45) 34.37 cells/mm , P = 0.022) (Table 2). The percentage chemotherapy Carboplatin/pemetrexed – 10 (20) of malignant cells expressing PD-L1 also was higher in Cisplatin/others – 18 (35) NCT-treated tumors (median, 19.53%) than in non-NCT tu- Adjuvant therapy No 22 (36) 11 (20) mors (median, 1.55%; P=0.008) (Fig. 1). Although both ADCs andSCCs in theNCT groupshowedhigherPD-L1 Yes 39 (64) 32 (64) expression by malignant cells, only NCT-ADC showed sig- Vital status Alive 24 (39) 13 (26) nificantly higher density and percentage of malignant cells Dead 37 (61) 38 (74) expressing PD-L1 (median, 362.92 cells/mm , 11.10%) than Overall survival Median 34 months 21 months their non-NCT counterparts (median, 13.45 cells/mm , ADC, adenocarcinoma; SCC, squamous cell carcinoma 0.29%; P =0.007 and P = 0.008, respectively) (Table 3). by pathology report by International Association for the Study of Lung Cancer classification system TAIC densities higher in NCT-treated tumors Other chemotherapies such as gemcitabine or docetaxel d As showninTable 2, Fig. 2 and Additional file 4:FigureS4, Adjuvant therapy unknown in 8 cases from NCT group the densities of TAICs of various phenotypes were higher We identified no significant correlations between clini- overall in NCT tumors than in non-NCT tumors. The copathologic features and malignant cell expression of number of T lymphocytes (CD3+) was significantly higher PD-L1+ or TAIC density in either the non-NCT or the in NCT tumors than in non-NCT tumors (P=0.021). NCT group, nor did we observe differences related to Furthermore, the densities of T lymphocytes (CD3+), helper chemotherapy regimen or interval between surgical re- T cells (CD3 + CD4+), activated natural killer cells (CD57 + section and completion of NCT. granzyme B + CD45RO−), memory antigen experienced cells Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 6 of 11 Table 3 Median densities of various immune marker–expressing cells in NSCLCs from patients who received neoadjuvant chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor histology (N = 112) a a Markers ADC (n = 64) P SCC (n = 51) P non-NCT NCT non-NCT NCT 2 2 Median Cell Density (cells/mm ) Median Cell Density (cells/mm ) Panel 1 MCs (AE1/AE3+) 3135.42 3043.84 0.841 4125.37 3641.55 0.554 MCs PD-L1+ 13.45 362.92 0.007 317.91 944.99 0.593 CD3+ 916.14 1406.72 0.133 811.47 1825.85 0.070 CD3 + CD4+ 671.49 997.47 0.170 650.63 1360.68 0.072 CD3 + CD8+ 157.39 212.85 0.647 155.34 358.93 0.842 CD68+ 298.80 591.42 0.430 321.12 705.60 0.040 CD68 + PD-L1+ 196.71 222.96 0.600 172.72 357.54 0.090 Panel 2 MCs (AE1/AE3+) 3134.08 2596.71 0.299 4528.99 3703.28 0.352 CD45RO+ 623.31 595.51 0.484 723.46 1356.35 < 0.001 CD45RO + CD57 + granzyme B− 240.95 332.13 0.008 118.68 395.21 0.013 CD45RO + PD1+ 170.47 243.04 0.016 112.40 380.26 0.007 CD45RO + FOXP3+ 7.17 7.17 0.448 4.18 9.56 0.073 CD57 + granzyme B+ CD45RO− 4.26 40.65 < 0.001 9.86 9.86 0.925 PD-1+ 426.90 718.68 0.014 273.84 1314.49 < 0.001 ADC adenocarcinoma, SCC squamous cell carcinoma, MC malignant cells Mann Whitney U test Neoadjuvant chemotherapy Non-neoadjuvant chemotherapy CD45RO+ CD57+ CD45RO+ CD45RO+ CD45RO+ CD57+ PD-1+ PD-1+ CD3+CD4+ CD3+ CD3+CD4+ CD3+ CD45RO+ CD3+ MCs CD3+ PD-1+ CD8+ Panel 1 and Panel 2 CD8+ CD45RO+ MCs PD-1+ Panel 1and Panel 2 CD68+ PD-L1+ CD68+ CD68+ MCs PD-L1+ CD68+ PD-L1+ CD45RO+FOXP3+ CD57+Granzyme B+CD45RO- CD45RO+FOXP3+ MCs PD-L1+ CD57+Granzyme B+CD45RO- Panel 1 (co-expressions) Panel 2 (co-expressions) Malignant cells (MCs-AE1/AE3) Malignant cells (MCs-AE1/AE3) Malignant cells (MCs-AE1/AE3) PD-L1+ Memory cells CD45RO+ T-cells CD3+ Memory/regulatory cells CD45RO+FOXP3+ Helper T-cells CD3+CD4+ Memory/natural killer cells CD45RO+CD57+Granzyme B- Cytotoxic T-cells CD3+CD8+ Memory/Antigen experienced cells CD45RO+PD-1+ Macrophages CD68+ Activated natural killer cells CD57+Granzyme B+ CD45RO- Macrophages CD68+PD-L1+ Antigen experienced cells PD-1+ Fig. 2 Representative figure compared phenotypes between non-NCT and NCT. Graphic representation of relative densities of different cell phenotypes detected by analysis with panel 1 and 2 markers in NSCLCs that were treated or not treated with neoadjuvant chemotherapy (NCT). Overall, the numbers of various immune cell phenotypes were higher in the group that received NCT than in the non-NCT group Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 7 of 11 (CD45RO + PD-1+), and antigen experienced (PD-1+) cells cell densities were higher overall in both compartments were higher in NCT tumors than in non-NCT tumors (be- of NCT-ADCs and NCT-SCCs than in those of tween P= 0.040 and P < 0.001). Density of TAMs (CD68+) non-NCT-ADCs and non-NCT-SCCs. Important and was also higher in NCT tumors than in non-NCT tumors significant differences were observed in various cell (P = 0.059). Although the densities of TAICs overall phenotypes: in NCT-ADCs, the densities of activated were higher in NCT-ADCs and NCT-SCCs than in natural killer cells (CD57 + granzyme B + CD45RO−)in non-NCT-ADCs and non-NCT-SCCs, as shown in Table 3, both the epithelial and stromal compartments were signifi- the NCT-ADC tumors showed significantly higher cantly higher than those in non-NCT-ADCs (P =0.001 and densities of activated natural killer cells (CD57 + P = 0.001, respectively). However, densities of memory/regu- granzyme B + CD45RO−), memory/natural killer T-cells latory cells (CD45RO + FOXP3+) in both epithelial and stro- (CD45RO + CD57 + granzyme B−), memory antigen expe- mal compartments were lower in NCT-ADCs than in rienced cells (CD45RO + PD-1+), and antigen experienced non-NCT-ADCs (P = 0.085 and P = 0.001, respectively), but (PD-1+) cells than non-NCT-ADCs (P <0.001, P =0.008, thedifferencewas significantonlyinthe stromalcompart- P =0.016, P = 0.014, respectively), while the NCT-SCCs ment. In the epithelial compartments of SCCs, the densities of showed significantly higher densities of memory cells T lymphocytes (CD3+), helper T cells (CD3 + CD4+), antigen (CD45RO+), memory/natural killer T cells (CD45RO experienced (PD-1+) cells, and TAMs (CD68+) were +CD57+granzyme B−), memory antigen experienced higher in the NCT group than in the non-NCT group cells (CD45RO + PD-1+), antigen experienced (PD-1+) (P =0.023, P = 0.019, P < 0.001, and P = 0.016, respect- cells, and TAMs (CD68+) than non-NCT-SCCs (be- ively). In the stromal compartments of SCCs, the tween P =0.040 and P < 0.001). density of antigen experienced (PD-1+) cells was higher in NCT tumors than in non-NCT tumors (P =0.015). TAIC densities higher in both epithelial and stromal compartments of NCT tumors Inflammatory cell–modulated prognostic correlations in The TAIC density differences between non-NCT and NCT patients NCT tumors were independent of histology and of To identify the contribution of each immune cell sub- compartment. As shown in Additional file 5: Table S1 population to the biological behavior of NCT-treated and Additional file 6:FigureS5, thedensities of TAICswere lung tumors, we analyzed their impact on long-term sur- higher overall in the stromal compartments of non-NCT vival. The main observed differences were for epithelial and NCT tumors than in their respective epithelial compart- helper T cells (CD3 + CD4+) and epithelial/stromal ments. In the epithelial compartments, the densities of T TAMs (CD68+), which have been previously linked to lymphocytes (CD3+), helper T cells (CD3 + CD4+), activated better prognosis. At a tumor viability cutoff of 10% in natural killer cells (CD57 + granzyme B + CD45RO−), mem- NCT, no prognostic difference was observed. In the entire ory/natural killer T cells (CD45RO + CD57 + granzyme B−), cohort of NCT-treated patients (ADC and SCC), OS was memory antigen experienced cells (CD45RO + PD-1+), anti- longer, based on univariate analysis, in patients with higher gen experienced (PD-1+) cells, and TAMs (CD68+) were densities of helper T cells (CD3 + CD4+; P = 0.048) and significantly higher in the NCT group than in the TAMs (CD68+; P = 0.035) (Fig. 3). Logistic regression non-NCT group. Density of TAMs (CD68+) expressing multivariate models incorporating tumor stage corrobo- PD-L1+ was significantly higher in the epithelial compart- rated the association between survival and higher epithelial ments of NCT tumors than in those of non-NCT tumors and stromal densities of TAMs (CD68+) (P =0.044; hazard (between P = 0.049 and P <0.001). ratio [HR], 0.506; 95% confidence interval [CI], 0.261–0.982) Density of memory/regulatory cells (CD45RO + FOXP3 and higher epithelial density of helper T cells (CD3 + CD4+) +) in the epithelial compartment was significantly lower in (P = 0.097; HR, 0.547; 95% CI, 0.269–1.114) (Additional file 8: NCT tumors than in non-NCT tumors (P =0.092). Table S3). However, densities of T lymphocytes (CD3+) and activated natural killer cells (CD57 + granzyme B + CD45RO−)inthe stromal compartment were significantly higher in the NCT Discussion tumors than in non-NCT tumors (P = 0.029 and P=0.002, In this study, mIF and image analysis were used to respectively). As in the epithelial compartment, dens- evaluate both PD-L1 expression and densities of TAIC ity of memory/regulatory cells (CD45RO + FOXP3+) populations via high-throughput analysis of tumor in the stromal compartment was lower in NCT tu- epithelial and stromal compartments in NSCLCs treated mors than in non-NCT tumors, but the difference or not treated with NCT. Utilizing the median PD-L1 was not significant (P = 0.060). expression value in malignant cells as the cut-off for When the analysis included both tumor histology and positive expression, we observed higher densities and compartment, as shown in Additional file 7: Table S2, percentages of PD-L1+ malignant cells in NCT-treated Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 8 of 11 markers in NSCLC tissues that had or had not been treated Tumor epithelial CD3+CD4+ (>median) E/N=16/25 with NCT. Similar to some studies in urothelial , thymic , ovarian , lung , and head and neck  cancers treated with NCT, we found higher densities and percentages of malignant cells expressing PD-L1 in NCT-treated tumors, independent of the NCT regimen, than in non-NCT tumors, suggesting that PD-L1 expres- sion can be increased by NCT, potentially through activa- tion of immune-related pathways such as IFNα,IFNγ, STAT3, and TNFα . Previous research has demon- strated that immunogenic death of malignant cells induced by various chemotherapy regimens  can enhance the activation of APCs, which in turn activates TILs and their production of IFN-γ, which subsequently increases the ex- P=0.048 pression of PD-L1 by malignant cells . Although expos- ure of tumor cell lines to various chemotherapy agents [35, 36] and some mechanisms of action of chemotherapy Months agents [37–39] can increase PD-L1 expression in cancer cells, the exact mechanisms by which chemotherapy drugs Epithelial/Stromal CD68+ (>median) E/N=17/25 induce this increase remain unknown. TAICs play an important role in anticancer immune surveillance . TAICs, including TILs and TAMs, are involved in the anticancer immune response, and it is known that TILs, which are generally represented by cytotoxic T cells (CD3 + CD8+) and helper T cells (CD3 + CD4+), along with natural killer cells [41, 42], have an important role in anticancer immunity. Malignant cells are usually killed by several pathways that induce cell apoptosis, orchestrated by a successful cytotoxic attack mediated by APCs such as TAMs or dendritic cells . The significantly higher numbers of T lymphocytes, helper T cells, activated natural killer cells, memory/nat- P=0.035 ural killer cells, and memory antigen experienced cells observed in NCT-treated specimens suggest that, in Months general, cytotoxic chemotherapy may induce tumor Fig. 3 Univariate analysis in NCT. Kaplan-Meier analysis of patients immunogenicity through the release of neoantigens from with NSCLC who received NCT showed longer survival among those apoptotic malignant cells . with a higher density of helper T cells (CD3 + CD4+) in the tumor It has been reported that tumors that otherwise ex- epithelial compartment and among those with a higher density of press low levels of antigens, when treated with chemo- CD68+ tumor-associated macrophages in the tumor epithelial and therapy, undergo sufficient release of antigens to stromal compartments sensitize stromal cells for destruction by cytotoxic T cells . In the NCT-treated group, amounts of specific tumors than in non-NCT tumors. Similarly, utilizing the TILs such as helper T cells (CD3 + CD4+) and activated median TAIC density as the cut-off value, we observed natural killer cells (CD57 + granzyme B + CD45RO−) higher densities of TAICs in NCT-treated tumors than were increased in both the epithelial and stromal com- in non-NCT tumors. Among NCT-treated patients, partments. This suggests that chemotherapy induces re- Kaplan-Meier analysis showed better prognosis for pa- cruitment of inflammatory cells against malignant cells, tients with higher-than-median density of helper T cells with especially significantly higher expression of helper (CD3 + CD4+) in the tumor epithelial compartment and T cells (CD3 + CD4+) in the epithelial compartment of higher-than-median densities of TAMs (CD68+) in the SCCs and activated natural killer cells (CD57 + granzyme tumor epithelial and stromal compartments than for pa- B + CD45RO−) in the epithelial compartment of ADCs. tients with lower-than-median densities of these cells. Helper T cells (CD3 + CD4+) are important for initiating To ourknowledge,thisisthe first study that used mIFto and maintaining anticancer immune responses; in their analyze and compare large panels of immune-profiling absence, specific cytotoxic T lymphocytes can become Overall Survival Overall Survival Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 9 of 11 lethargic  or depleted . Helper T cells are also es- surgical resection for stage III lung cancers. This group sential for inducing transformation of cytotoxic T cells of patients was likely clinically different from the group into long-lived functional effector cells . that received NCT. Further studies are needed to valid- Activated natural killer cells (CD57 + granzyme B+ ate our findings, to establish clinically meaningful refer- CD45RO−) have a high cytotoxic potential , suggest- ence values for TAICs in the tumor and peritumoral ing that the activation of these cells could have import- compartments based on mIF techniques, and to sort out ant therapeutic implications. Overall, quantities of the details of the mechanisms by which chemotherapy memory/regulatory T-cells (CD45RO + FOXP3+) and induces a potentially favorable microenvironment for the antigen experienced cells (PD-1+) also were increased in administration of checkpoint inhibitor immunotherapy. NCT-treated cases, predominantly SCCs. When we ana- lyzed data by tumor compartment, we found that quan- Conclusions tity of memory/regulatory T cells (CD45RO + FOXP3+) Patients with NSCLC treated with NCT exhibited higher was significantly lower in the stromal compartment of levels of PD-L1+ malignant cells and TAIC than patients NCT-ADCs than in the stromal compartment of who underwent upfront tumor resection without NCT. non-NCT-ADCs, suggesting that chemotherapy can In patients who underwent NCT, those with higher modulate phenotype by tumor tissue compartment. The abundance of helper T cells and TAMs survived longer, reasons for this discrepancy are still unclear, but the var- suggesting that these cells may important in chemother- iety of chemotherapy regimens cannot be ruled out, apy response. Together, our findings suggest that since immune effectiveness differs among different drugs chemotherapy may activate immune response mecha- and histologies. nisms such as IFNα, IFNγ, STAT3, and TNFα in lung Overall, NCT-treated tumors showed a higher density cancer, which may generate a favorable tumor micro- of TAMs than non-NCT tumors. TAMs (CD68+) were environment for subsequent response to checkpoint significantly more abundant in NCT-SCCs, particularly immunotherapy. in the epithelial compartment in direct contact with ma- lignant cells. Like Hiraoka and colleagues , we found Additional files that higher density of epithelial helper T cells (CD3 + CD4+) correlated with a better prognosis in univariate Additional file 1: Figure S1. Representative multiplex analysis; however, this association was not confirmed in immunofluorescence (mIF) workflow, showing different steps in the process of analysis. (A) Vectra 3.0 multispectral microscope used for the the multivariate analysis. Both our univariate and multi- analysis. (B) Phenochart 1.0.4 software showing an image scanning at ×10 variate analyses did show, though, that higher densities and selection of five intratumoral areas (669×500 μm each) for further of epithelial and stromal TAMs (CD68+) correlated with analysis. (C) mIF image scanning at ×20 and viewed in the InForm 2.1.3 image analysis software. (D) Composed image showing the different better survival in NCT-treated NSCLC patients. As sug- phenotypes with panel 1. (E) Tumor segmentation (epithelial and stromal gested by Blankenstein , tumor suppression factors compartments) using the InForm software. (E) Cell segmentation to are meditated by activation of TAM class M1 in the characterize individual cells using DAPI as a counterstaining marker, and cell phenotyping identifying different subgroups of cells supervised by a tumor epithelial and stromal compartments and helper pathologist. (PPTX 6362 kb) T cells (CD3 + CD4+) in the tumor epithelial compart- Additional file 2: Figure S2. Characterization of immune cell ment. To further define the importance of TAMs in phenotyping using marker panel 1. Cell phenotypes were defined as NCT-treated tumors, future studies should focus on the malignant cells expressing PD-L1 (AE1/AE3 + PD-L1+), T lymphocytes (CD3; pan T-cell marker including helper T cells CD3 + CD4+, cytotoxic T subtypes of TAMs, since recent studies  suggest that cells CD3 + CD8+, and other CD3+ T cells), helper T cells (CD3 + CD4+), TAMs polarize to M1 (anti-tumorigenic) or M2 (con- cytotoxic T cells (CD3 + CD8+), tumor-associated macrophages (TAMs; tributing to carcinogenesis) subtypes and thus can exert CD68+), and TAMs expressing PD-L1 (CD68 + PD-L1+). Each type of cell is shown as individual images by marker and by a composite image. differential effects in the tumor microenvironment. (PPTX 102487 kb) Our study has several limitations. The number of Additional file 3: Figure S3. Characterization of immune cell NCT-treated patients is rather small, and the cohorts phenotyping using marker panel 2. Cell phenotypes were defined as consist mainly of patients with stage III disease. Al- memory cells (CD45RO, including memory/natural killer cells CD45RO + CD57 + granzyme B−, memory/regulatory cells CD45RO + FOXP3+, though the variability of the chemotherapy regimens is a memory antigen experienced cells CD45RO + PD-1+, and other CD45RO+ potential confounder of our results, no differences were cells), memory/regulatory cells (CD45RO + FOXP3+), memory antigen observed in the various regimens’ impacts on the im- experienced cells (CD45RO + PD-1+), activated natural killer cells (CD57 + granzyme B + CD45RO−), and antigen experienced cells (PD-1, including mune microenvironment. The cohort of stage III pa- PD-1 + CD45RO+ and other PD-1+ cells). Each type of cell is shown as tients who did not undergo induction chemotherapy individual images by marker and by a composite image. (non-NCT) likely comprised patients with incidentally (PPTX 103717 kb) found stage III (N2) lung cancer in pathologic speci- Additional file 4: Figure S4. Multiplex immunofluorescence images showing densities of various tumor-associated immune cell phenotypes mens, because our preference since publication of Roth as determined by panel 1 and panel 2 markers from representative et al.  has been to administer chemotherapy prior to Parra et al. Journal for ImmunoTherapy of Cancer (2018) 6:48 Page 10 of 11 Ethics approval and consent to participate NSCLCs treated with neoaduvant chemotherapy (NCT) or not treated The study was approved by the MD Anderson Institutional Review Board, with NCT (non-NCT). Numbers of T lymphocytes (CD3+), helper T cells and consent for participation and publication was obtained from all the (CD3 + CD4+), tumor-associated macrophages (TAM; CD68+), activated patients included in the study and is available for review at any time. natural killer cells (CD57 + granzyme B + CD45RO−), memory antigen experienced cells (CD45RO + PD-1+), and antigen experienced PD-1+ Competing interests cells as well as PD-L1+ malignant cells were higher in the NCT group The authors ERP, PV, CB, MJ, AP, SGS, WNN, JZ, JL, TC, JVH, M-AF, CB, CH, CM, than in the non-NCT group. Images ×200. (PPTX 29362 kb) JZ, AV, DLG, BS, and IIW declare that they have no competing interests. Additional file 5: Table S1. Median densities of tumor-associated immune cells in NSCLCs of patients who received neoadjuvant chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor Publisher’sNote compartment (N = 112) (DOCX 21 kb) Springer Nature remains neutral with regard to jurisdictional claims in Additional file 6: Figure S5. Multiplex immunofluorescence images published maps and institutional affiliations. showing densities of various tumor-associated immune cell phenotypes (TAICs) as determined by panel 1 and panel 2 markers from the stromal Author details and epithelial compartments of representative NSCLCs treated with Department of Translational Molecular Pathology, Unit 951, The University neoadjuvant chemotherapy (NCT) or not treated with NCT (non-NCT). In of Texas MD Anderson Cancer Center, 2130 West Holcombe Blvd, Houston, general, densities of TAICs were higher in the stromal compartments than TX 77030, USA. Department of Thoracic/Head and Neck Medical Oncology, in their respective epithelial compartments in both NCT and non-NCT The University of Texas MD Anderson Cancer Center, Houston, TX, USA. tumors. Overall, density of tumor-associated macrophages (TAMs; CD68+) Department of Thoracic and Cardiovascular Surgery, Unit 1489, The was higher in NCT tumors than in non-NCT tumors, and density of University of Texas MD Anderson Cancer Center, 1400 Pressler St. Houston, memory/regulatory cells (CD45RO + FOXP3+) was lower in the NCT Houston,, TX 77030, USA. Department of Bioinformatics and Computational group than in the non-NCT group. Images ×200. (PPTX 15137 kb) Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, Additional file 7: Table S2. Median densities of tumor-associated USA. Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Department of Melanoma Medical immune cells in NSCLCs from patients who received neoadjuvant Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor USA. Department of Anatomical Pathology, The University of Texas MD compartment and histology (N = 112). (DOCX 23 kb) Anderson Cancer Center, Houston, TX, USA. Additional file 8: Table S3. Multivariate survival analysis (Cox regression model) of effects on survival of (A) tumor-associated macrophages (TAMs; Received: 14 February 2018 Accepted: 25 May 2018 CD68+) and (B) helper T cells (CD3 + CD4+) controlled by tumor stage in NSCLCs from patients who received neoadjuvant chemotherapy (N = 51). (DOCX 18 kb) References 1. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917): 860–7. Abbreviations 2. Igney FH, Krammer PH. 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